Preface

Gynaecological malignancies are a heterogeneous group of diseases composed of multiple types of cancer based on their organ-of-origin within the female genital tract; each type having their own distinct molecular and clinical sub-categorisation. Women with advanced gynaecological malignancy, in particular the rarer subtypes, face a formidable challenge as fatal resistance to therapies commonly occurs within a few years of diagnosis. The improvement in our ability to understand the tumour biology and to target the underlying drivers and vulnerabilities of these tumours is essential in order to develop effective treatments for women battling this disease.

This book aims to present a review of the significant advances in the understanding and management of gynaecological malignancies. Major areas of importance in this field will be covered, incorporating new knowledge that has arisen due to the advancements in molecular techniques and the ability to correlate these molecular changes with clinical behaviour of gynaecologic tumours. The therapeutic implications of molecular subtyping to match appropriate therapies and the appreciation of the use of up-to-date radiotherapy techniques will be explored.

> **Gwo Yaw Ho** Monash University, Australia

Walter and Eliza Hall Institute, Parkville, Australia

Peter MacCallum Cancer Centre, Melbourne, Australia

> **Sophia Frentzas** Monash University, Australia

**1**

Section 1

Advances in

Gynaecological

Malignancies

Section 1

Advances in Gynaecological Malignancies

**3**

**Chapter 1**

**Abstract**

gynecological cancers.

**1. Introduction**

Malignancies

*Neha Sharma and Deepti Sharma*

Immunotherapy in Gynecological

Cancer immunotherapy is one of the most upcoming treatment strategies emerging as a fascinating option in the management of advanced gynecological malignancies. The development of immune-based antitumor approaches has led to safer treatment options that give fruitful results in these malignancies. In this chapter we are focusing on immune-based treatment in the management of gynecological cancers like cervical cancer, endometrial cancer, ovarian cancer, and vaginal and vulvar cancer. We are also discussing the clinical studies that have been conducted or are currently underway which are exploring these immune strategies that are developing as a logical overture for the treatment of advanced cancers including

**Keywords:** gynecological malignancy, immunotherapy, immune checkpoint

Cancer immunotherapy is emerging as an attractive strategy among different therapeutic options over the past years, and also the treatment of many advanced malignancies has been revolutionized with the development of immune-based antitumor therapies. The advent of targeted immune therapies leading to successful outcomes in other malignancies has led to an increase in the number of clinical trials using these interventional strategies in patients with gynecological cancer. Generally, the role of immunotherapy is either to reactivate the immune response or

There are three stages of the dynamic process of immunoediting, also known as the three Es: an early elimination phase with the activation of an innate and adoptive immune response, an equilibrium phase where the isolated tumor cells are able to endure immune incursion, and an immune escape phase that the cancer cell variants can alter their genomic or antigenic phenotype or they are under the control of immunoregulatory phenomena to survive in the immunosuppressive medium. In order to activate tumor-directed immune responses, recent immune therapies have consisted of several approaches, including adoptive cell transfer (ACT), cancer

Cervical cancer is unique among gynecologic malignant tumors because of its wellestablished and causative risk factor, chronic HPV infection. The infectious etiology of cervical cancer has led to effective vaccines for prevention; however, advanced stage/ metastatic disease remains a principal cause of gynecologic cancer mortality in much of the world. The implementation of antiangiogenic therapy has greatly improved the

inhibitors, cervical cancer, ovarian cancer, endometrial cancer

to diminish the tumor-directed immune inhibition.

vaccines, and immune checkpoint inhibitors.

#### **Chapter 1**

## Immunotherapy in Gynecological Malignancies

*Neha Sharma and Deepti Sharma*

#### **Abstract**

Cancer immunotherapy is one of the most upcoming treatment strategies emerging as a fascinating option in the management of advanced gynecological malignancies. The development of immune-based antitumor approaches has led to safer treatment options that give fruitful results in these malignancies. In this chapter we are focusing on immune-based treatment in the management of gynecological cancers like cervical cancer, endometrial cancer, ovarian cancer, and vaginal and vulvar cancer. We are also discussing the clinical studies that have been conducted or are currently underway which are exploring these immune strategies that are developing as a logical overture for the treatment of advanced cancers including gynecological cancers.

**Keywords:** gynecological malignancy, immunotherapy, immune checkpoint inhibitors, cervical cancer, ovarian cancer, endometrial cancer

#### **1. Introduction**

Cancer immunotherapy is emerging as an attractive strategy among different therapeutic options over the past years, and also the treatment of many advanced malignancies has been revolutionized with the development of immune-based antitumor therapies. The advent of targeted immune therapies leading to successful outcomes in other malignancies has led to an increase in the number of clinical trials using these interventional strategies in patients with gynecological cancer. Generally, the role of immunotherapy is either to reactivate the immune response or to diminish the tumor-directed immune inhibition.

There are three stages of the dynamic process of immunoediting, also known as the three Es: an early elimination phase with the activation of an innate and adoptive immune response, an equilibrium phase where the isolated tumor cells are able to endure immune incursion, and an immune escape phase that the cancer cell variants can alter their genomic or antigenic phenotype or they are under the control of immunoregulatory phenomena to survive in the immunosuppressive medium. In order to activate tumor-directed immune responses, recent immune therapies have consisted of several approaches, including adoptive cell transfer (ACT), cancer vaccines, and immune checkpoint inhibitors.

Cervical cancer is unique among gynecologic malignant tumors because of its wellestablished and causative risk factor, chronic HPV infection. The infectious etiology of cervical cancer has led to effective vaccines for prevention; however, advanced stage/ metastatic disease remains a principal cause of gynecologic cancer mortality in much of the world. The implementation of antiangiogenic therapy has greatly improved the

treatment for relapsed/advanced disease over the last 5 years. Several clinical trials including CheckMate 358 and KEYNOTE-028 and KEYNOTE-158 are evaluating the role of immune checkpoint inhibitors in the treatment of cervical cancer.

In endometrial cancer, patients with advanced or disseminated recurrent disease have a poor prognosis, and most patients with peritoneal recurrence are considered incurable. Platinum and taxane chemotherapy produces response rates of 40–60%, which decreases to 20% for second-line drugs. So there is a need for development of more effective treatment for patients having advanced disease.

Approximately 25% of endometrial tumors are characterized by defects in the DNA mismatch repair system manifested by errors in DNA replication of trinucleotide repeat regions, commonly referred to as microsatellite instability. These defects in mismatch repair (MMR) also result in a high somatic mutation rate and accordingly increased number of neoantigens in these MMR-deficient tumors. In endometrial cancer, the presence of high microsatellite instability (MSI-H) has become an area of interest for use of immune checkpoint inhibitors.

For several reasons ovarian cancer is an ideal tumor type for which to consider an immunomodulatory management approach. Firstly, there is no negative impact of cancer itself on immunoregulatory cells that may be present within the bone marrow or other body locations. Secondly, while standard cytotoxic therapy of ovarian cancer can result in a depression in the number of immunoregulatory cell, these effects are generally modest in extent and short in duration. Lastly, it is common for patients with ovarian cancer to maintain a quite reasonable performance status and satisfactory nutrition.

A majority of ovarian cancer patients respond to cytotoxic chemotherapy and invariably are free from disease for periods varying from months to several years. This time interval can be exploited for required "activation" of immune defense mechanisms, either by using a tested vaccination strategy or any other form of immune modulation.

Multiple studies involving immune checkpoint inhibitors, conducted in advanced endometrial cancer, ovarian cancer, and cervical cancer, have shown promising preliminary results. But similar to that seen in other tumor types, continued work will need to focus on identifying those subsets of patients that will benefit from these therapies as these treatments are not without significant toxicities.

The immune system plays an important role in cancer pathogenesis. Numerous clinical trials and multiple researches dedicated to study therapies that involve the immune system to favorably impact the disease course in various malignancies have not only shown improved patient survival but also diversified the whole cancer management scenario by approval of the use of various immunotherapeutic agents in advanced malignancies [1].

Since cancer immunotherapy has emerged as an effective and appealing therapeutic option among other different therapeutic strategies and has been proven competent against multiple malignancies, it has led to an increase in research on immunomodulatory approaches in gynecological malignancies [2].

The ongoing research on the understanding of tumor biology and immunology has led to improved comprehension of mechanisms of immune recognition, regulation, and tumor escape that has provided new approaches for cancer immunotherapy [3].

#### **2. Role of immune system in cancer**

The principal role of the immune system is against foreign pathogens and infections. It is further classified as cellular and humoral immune systems, mediated by T and B lymphocytes and their products, respectively.

**5**

immunogens [14].

*Immunotherapy in Gynecological Malignancies DOI: http://dx.doi.org/10.5772/intechopen.90711*

cells, CD8+ cytotoxic T cells, as well as CD4+ helper T cell [5].

vaccines, and immune checkpoint inhibitors.

fallopian tubes [9].

**3. Cervical cancer**

The initial innate immunity is nonspecific, and the adaptive immune response is the specialized defense. Both the strategies work in different manner. They employ the cellular immunity which has a rather fast response in eradicating intracellular microbes through the recognition of antigens, activation of antigen-presenting cells (APCs), and activation and proliferation of T cells. They also need humoral immunity mediated via antibodies produced by B cells for neutralizing toxins and act against infections. Where innate immunity works by releasing signals essential to stimulate responses from both T cells and B cells [4], the adaptive immune system is mainly consists of B

The immune system in tumor cells has a dynamic relationship, in which either it can identify or control tumor cells in a process called cancer immunosurveillance or cause tumor progression through chronic inflammation, immunoselection of poorly immunogenic variants, and suppressing antitumor immunity [6]. There are three stages of this dynamic process called immunoediting. The first is the elimination phase in which innate and adaptive immunity works together to identify and eliminate the cancer cells before they become clinically apparent [7]. If the cancer cells are not eliminated, they enter the second phase which is equilibrium. It can last from months to years. Here the cancer cells persist, but outgrowth is prevented by the immune system. Lastly the escape phase is in which either the cancer cell variants survive in the immunosuppressive microenvironment by altering genetic or antigenic phenotype or under the control of immunoregulatory phenomena. [8] In order to activate tumor-directed immune responses, recent immune therapies have consisted of several approaches, including adoptive cell transfer (ACT), cancer

Gynecological cancers are a group of malignancies that involve different organs that comprise the female reproductive system. The most common types of gynecologic malignancies are cervical cancer, ovarian cancer, and endometrial cancer. Other less common gynecological malignancies arise from the vagina, vulva, and

Cervical cancer represents 6.6% of all female cancers. It is the fourth most

Approximately 90% of deaths from cervical cancer occur in underdeveloped and developing countries [10]. Cervical cancer has emerged as a preventable disease due to currently employed screening tests which have highlighted HPV infection as an etiological factor. Although significant progress has been made in screening and prevention of cervical cancer, the 5-year overall survival remains 66% [11]. For cases diagnosed at an early stage, the recurrence rates vary between 10 and 20%, but for advanced cases, the rate of recurrence reaches up to 70% [12]. There is a need to improve outcomes, and immunotherapy could offer this possibility. The recognition of human papilloma virus as an etiological agent has greatly improved the understanding of the disease and led to improved strategies in prevention of cervical cancer [13]. The infectious etiology of cervical cancer has led to effective vaccines for prevention; however, advanced stage/metastatic disease remains a principal cause of gynecologic cancer mortality. Currently there are three licensed HPV prophylactic vaccines, namely, bivalent vaccine cervarix against HPV16/18, Gardasil against HPV-6/11/16/18, and Gardasil9, a nonavalent HPV-6/11/16/18/31/33/45/52/58 vaccine. All are based on on-infectious recombinant type-specific L1 capsid proteins assembled into viral-like particles (VLPs) as

common cancer in women with an estimated 570,000 new cases in 2018.

#### *Immunotherapy in Gynecological Malignancies DOI: http://dx.doi.org/10.5772/intechopen.90711*

The initial innate immunity is nonspecific, and the adaptive immune response is the specialized defense. Both the strategies work in different manner. They employ the cellular immunity which has a rather fast response in eradicating intracellular microbes through the recognition of antigens, activation of antigen-presenting cells (APCs), and activation and proliferation of T cells. They also need humoral immunity mediated via antibodies produced by B cells for neutralizing toxins and act against infections. Where innate immunity works by releasing signals essential to stimulate responses from both T cells and B cells [4], the adaptive immune system is mainly consists of B cells, CD8+ cytotoxic T cells, as well as CD4+ helper T cell [5].

The immune system in tumor cells has a dynamic relationship, in which either it can identify or control tumor cells in a process called cancer immunosurveillance or cause tumor progression through chronic inflammation, immunoselection of poorly immunogenic variants, and suppressing antitumor immunity [6]. There are three stages of this dynamic process called immunoediting. The first is the elimination phase in which innate and adaptive immunity works together to identify and eliminate the cancer cells before they become clinically apparent [7]. If the cancer cells are not eliminated, they enter the second phase which is equilibrium. It can last from months to years. Here the cancer cells persist, but outgrowth is prevented by the immune system. Lastly the escape phase is in which either the cancer cell variants survive in the immunosuppressive microenvironment by altering genetic or antigenic phenotype or under the control of immunoregulatory phenomena. [8] In order to activate tumor-directed immune responses, recent immune therapies have consisted of several approaches, including adoptive cell transfer (ACT), cancer vaccines, and immune checkpoint inhibitors.

Gynecological cancers are a group of malignancies that involve different organs that comprise the female reproductive system. The most common types of gynecologic malignancies are cervical cancer, ovarian cancer, and endometrial cancer. Other less common gynecological malignancies arise from the vagina, vulva, and fallopian tubes [9].

#### **3. Cervical cancer**

Cervical cancer represents 6.6% of all female cancers. It is the fourth most common cancer in women with an estimated 570,000 new cases in 2018. Approximately 90% of deaths from cervical cancer occur in underdeveloped and developing countries [10]. Cervical cancer has emerged as a preventable disease due to currently employed screening tests which have highlighted HPV infection as an etiological factor. Although significant progress has been made in screening and prevention of cervical cancer, the 5-year overall survival remains 66% [11]. For cases diagnosed at an early stage, the recurrence rates vary between 10 and 20%, but for advanced cases, the rate of recurrence reaches up to 70% [12]. There is a need to improve outcomes, and immunotherapy could offer this possibility. The recognition of human papilloma virus as an etiological agent has greatly improved the understanding of the disease and led to improved strategies in prevention of cervical cancer [13]. The infectious etiology of cervical cancer has led to effective vaccines for prevention; however, advanced stage/metastatic disease remains a principal cause of gynecologic cancer mortality. Currently there are three licensed HPV prophylactic vaccines, namely, bivalent vaccine cervarix against HPV16/18, Gardasil against HPV-6/11/16/18, and Gardasil9, a nonavalent HPV-6/11/16/18/31/33/45/52/58 vaccine. All are based on on-infectious recombinant type-specific L1 capsid proteins assembled into viral-like particles (VLPs) as immunogens [14].

There is a huge unmet need for the treatment for women having advanced/recurrent cancer after standard chemotherapy and immunotherapy aims to fill that void, through therapies that harness a patient's own immune system to attack the cancer.

#### **4. Cancer vaccines in cervical cancer**

Cancer vaccines are used to mediate immune response by activating T cells which can specifically recognize cancer cells by tagging them with tumor-specific antigens E6 and E7. These antigen-tagged tumor cells are recognized by antigenpresenting cells and killed by cytotoxic T cells [15].

Live vector vaccines are highly immunogenic vaccines which can stimulate mucosal as well as humoral and/or cellular systemic immunity. They present E6 and E7 to APC to cause immune response through major histocompatibility complex MHC I [16]. Although they are attenuated vaccines, still care has to be taken before administering it in immunocompromised individuals. ADXS11-001 is a type of live attenuated vaccine that uses *Listeria monocytogenes* (Lm), a gram-positive intracellular bacterium as bacterial vector. It secretes HPV-16 E7 antigen fused to a nonhemolytic fragment of Lm protein listeriolysin O [17].


**7**

**Table 3.**

*Immunotherapy in Gynecological Malignancies DOI: http://dx.doi.org/10.5772/intechopen.90711*

> **Patient cohort**

*n* = 6 Stage IB1 and HPV16+

*n* = 31 Recurrent or metastatic disease

*Peptide-based vaccine in cervical cancer.*

**Study name Patient** 

Refer **Table 2**.

**Study name**

Welters et al. [22] Phase II adjuvant

Poelgeest et al. [23] Phase II

**Table 2.**

Refer **Table 3**.

Ramanathan et al. [24] Phase I

Ferrara et al. [25] Phase I

Santin et al. [26] Phase I

*Dendritic vaccine in cervical cancer.*

**4.1 Peptide-based vaccines in cervical cancer**

**Treatment schedule**

HPV16 E6 E7 SLP vaccine

HPV16 E6-E7 SLP vaccine 300 g for four doses every 21 day

**Response Toxicity**

**Treatment schedule Response Toxicity**

Serological response in 3 pts Cellular response in 4 pts No objective clinical response

CD4+ T-cell response in all patients

Arm 1: placebo three doses

Arm 2: unprimed DC three doses 1 × 106 cells every

Arm 3: primed DC three doses 1 × 106 cells every

Analogous dendritic cells pulsed with HPV E7

DL1: HPV16/18 E7 antigenpulsed DC5 × 106 for five doses every 21 days DL2: HPV16/18 E7 antigenpulsed DC10 × 106 for five doses every 21 days DL3: HPV16/18 E7 antigenpulsed DC15 × 106 for five doses every 21 days

every 14 days

14 days

14 days

protein

Grade 1 and Grade 2: local pain, fever, flu-like symptoms, swelling, itching,

Grade 1 and Grade 2: fever, fatigue, headache, flu-like symptoms, chills, nausea, swelling extremities, rash, vomiting, tingling extremities, and injection

burning eyes

site pain

SD in Arm 3 Grade 1 and

Grade 2: itching at injection site, fever, chills, abdominal discomfort, vomit, ALP increased

Mild swelling and erythema at the injection site

Vaccine-enhanced number and activity of HPV16 specific CD4+ and CD8+ cells

Median OS: 12.6 months no tumor regression or delay of progression

**4.2 Dendritic vaccines in cervical cancer**

**cohort**

*n* = 14 Recurrent or metastatic disease

*n* = 15 Recurrent or metastatic disease

*n* = 10 Stage IB or IIA

The following studies have been conducted (**Table 1**):

#### **Table 1.**

*Role of vaccination in HPV-associated cervical cancer.*

### **4.1 Peptide-based vaccines in cervical cancer**

#### Refer **Table 2**.


#### **Table 2.**

*Peptide-based vaccine in cervical cancer.*

#### **4.2 Dendritic vaccines in cervical cancer**

#### Refer **Table 3**.


#### **Table 3.** *Dendritic vaccine in cervical cancer.*

#### **5. Immune checkpoint inhibitors in cervical cancer**

#### **5.1 PD1/PDL1 inhibitors**

Programmed cell death protein-1/programmed death ligand-1 immunoregulatory axis is a promising target for cervical cancer treatment [27]. Pembrolizumab is a humanized monoclonal immunoglobulin G4 (IgG4) kappa isotype antibody targeting PD-1 (**Table 4**).

Other ongoing trials of pembrolizumab include PAPAYA Trial [30] which is a phase I study involving Stage Ib to Stage IV cervical cancer. The treatment schedule includes intravenous pembrolizumab followed by cisplatin-based chemoradiotherapy and brachytherapy and additional pembrolizumab after radiation. Another phase II trial with pembrolizumab followed by chemoradiotherapy and brachytherapy is also open for recruitment [31].

Nivolumab is a human IgG4 monoclonal antibody that causes stimulation of PD1 pathway-mediated immune response inhibition by binding to the PD-1 receptor and blocking its interaction with PD-L1 and PD-L2. [32] Checkmate 358 trial is a phase I/II trial by Hollebecque et al. in 19 patients of cervical cancer which studied nivolumab 240 mg every 2 weeks and showed ORR was 20.8% and disease control rate was 70.8%. Responses were observed regardless of PD-L1 expression, HPV status, and number of prior therapies [33].

Other trials of nivolumab include NRG-GY002, a phase II trial in recurrent or metastatic breast cancer [34]. A trial of nivolumab with HPV 16 SLp vaccine in HPV 16 positive cervical cancer is also underway [35].

Other checkpoint inhibitors under investigation include atezolizumab which is a fully humanized monoclonal antibody IgG1 isotype PD-L1. It is being studied to assess the safety and efficacy in combination with cyclophosphamide/carboplatin in gynecological cancer including cervical cancer in phase Ib PRO-LOG study [36]. Another phase II study is ongoing to study the synergistic action of antiangiogenic therapy with immunotherapy by combining bevacizumab with atezolizumab in women with recurrent or metastatic cervical cancer [37, 38],

Durvalumab is a human IgG1 monoclonal antibody that blocks the action of PD-L1 with PD1 and CD 80. It is being studied along with tremelimumab, which is an antibody against CTLA4 in patients who have failed to respond or relapsed to standard treatment [39].

#### **5.2 CTLA-4 inhibitors**

Ipilimumab is a fully human monoclonal IgG1κ antibody which acts against the cytotoxic T lymphocyte antigen-4 (CTLA-4). CTLA4 is an immune-inhibitory


**9**

cells [40] (**Table 5**).

*CTLA4 inhibitors in cervical cancer.*

**Table 5.**

patient [43].

**5.3 Adoptive cell transfer therapy**

*Immunotherapy in Gynecological Malignancies DOI: http://dx.doi.org/10.5772/intechopen.90711*

> *n* = 42 Recurrent or metastatic disease

*n* = 34 FIGO IB2/IIA or IIB/IIIB/IVA, positive nodes

**Study name**

Lheureux et al. [41] Phase I/II

GOG9929 study Mayadev et al. [42] Phase I

molecule which is expressed in activated T cells and in suppressor T regulatory

Adoptive cell transfer therapy using autologous tumor-infiltrating lymphocytes is emerging as a promising treatment modality in immunotherapy for various cancers. There are two types of adoptive cell therapy which includes chimeric antigen receptor T-cell (CAR T-cell) therapy and tumor-infiltrating lymphocyte (TIL) therapy. Chimeric antigen receptor (CAR) T-cell therapy involves genetically engineered patient's autologous T cells that causes them to express a CAR specific for a tumor antigen. These cells are extracted, further divided, and reinfused back into the

**Patient cohort Treatment schedule Response Toxicity**

Median PFS 2.5 months

1 year DFS 74%

Grade 3 toxicity: diarrhea, colitis

Grade 1 and Grade 2: rash, endocrinopaties, gastrointestinal toxicity

Grade 3: 16% including lipase increased, neutropenia, and rash

Phase I: ipilimumab 3 mg/kg every 21 days for four doses Phase II: ipilimumab 10 mg/kg every 21 days for four doses and four cycles (same dose) every

12 weeks

after

Weekly cisplatin 40 mg/m<sup>2</sup>

DL1: ipilimumab 3 mg/kg for four doses every 21 days DL2: ipilimumab 10 mg/kg for four doses every 21 days DL3: ipilimumab 10 mg/kg for four doses every 21 days

 during 6 weeks and extended field radiotherapy. If no progression 2–6 weeks

A trial was conducted by Lu et al. which evaluated adoptive CD4+ T-cell therapy in solid metastatic cancer. It had two patients of metastatic cervical cancer, out of

There is a trial ongoing to test the safety, feasibility, and efficacy of CAR T-cell immunotherapy in patients who have GD@, PSMA, Muc1, mesothelin, or positive

TIL therapy predates the CAR T-cell therapy, and the basic principle involves the ex vivo culture of tumor specimens which have been resected and expansion of tumor-infiltrating lymphocytes (TILs) with interleukin-2. Selected T cells of a preferred antigen specificity and phenotype can be identified in vitro and divided. The number of antigen-specific T cells in peripheral blood after this method usually exceeds by far that possible by current vaccine treatment strategies alone. In addition, adoptive T cells appear more effective in inducing tumor regression than lymphocytes generated by vaccines, suggesting greater ability to overcome tumor-

which one patient had objective complete response [44].

cervical cancer markers by Chang et al. [45].

mediated immune evasion mechanisms [46].

#### **Table 4.**

*PD1/PDL1 inhibitors in cervical cancer.*


*Immunotherapy in Gynecological Malignancies DOI: http://dx.doi.org/10.5772/intechopen.90711*

**Table 5.**

*CTLA4 inhibitors in cervical cancer.*

molecule which is expressed in activated T cells and in suppressor T regulatory cells [40] (**Table 5**).

#### **5.3 Adoptive cell transfer therapy**

Adoptive cell transfer therapy using autologous tumor-infiltrating lymphocytes is emerging as a promising treatment modality in immunotherapy for various cancers. There are two types of adoptive cell therapy which includes chimeric antigen receptor T-cell (CAR T-cell) therapy and tumor-infiltrating lymphocyte (TIL) therapy.

Chimeric antigen receptor (CAR) T-cell therapy involves genetically engineered patient's autologous T cells that causes them to express a CAR specific for a tumor antigen. These cells are extracted, further divided, and reinfused back into the patient [43].

A trial was conducted by Lu et al. which evaluated adoptive CD4+ T-cell therapy in solid metastatic cancer. It had two patients of metastatic cervical cancer, out of which one patient had objective complete response [44].

There is a trial ongoing to test the safety, feasibility, and efficacy of CAR T-cell immunotherapy in patients who have GD@, PSMA, Muc1, mesothelin, or positive cervical cancer markers by Chang et al. [45].

TIL therapy predates the CAR T-cell therapy, and the basic principle involves the ex vivo culture of tumor specimens which have been resected and expansion of tumor-infiltrating lymphocytes (TILs) with interleukin-2. Selected T cells of a preferred antigen specificity and phenotype can be identified in vitro and divided. The number of antigen-specific T cells in peripheral blood after this method usually exceeds by far that possible by current vaccine treatment strategies alone. In addition, adoptive T cells appear more effective in inducing tumor regression than lymphocytes generated by vaccines, suggesting greater ability to overcome tumormediated immune evasion mechanisms [46].

Stevanovic et al. [47] conducted a trial on 17 patients of metastatic cervical cancer who received high-dose lymphocyte-depleting chemotherapy followed by aldesleukin. Patients were treated with a single infusion of human papillomavirus (HPV) E6 and E7 reactivity (HPV-TILs). Three of nine patients experienced objective tumor responses (two complete responses and one partial response).

#### **6. Endometrial cancer**

Endometrial cancer is the 4th most commonly occurring cancer in women and the 15th most commonly occurring cancer overall. There were over 380,000 new cases in 2018 [48]. In women with advanced and recurrent cancer, the prognosis is considered very poor. Unfortunately, there are limited treatment options for advanced or recurrent endometrioid endometrial cancer. However, with the advent of immunotherapy, immune checkpoint inhibitors have shown promising results in these cases.


**11**

**Table 7.**

*Immunotherapy in Gynecological Malignancies DOI: http://dx.doi.org/10.5772/intechopen.90711*

**6.1 Anticancer vaccines in endometrial cancer**

**Study name Patient cohort Treatment** 

*n* = 12 WT1/human leukocyte antigen (HLA)- A\*2402-positive gynecological cancer

*n* = 6 Pretreated patients with uterine cancer

*Anticancer vaccines in endometrial cancer.*

The following studies have been conducted (**Table 7**).

MMR-deficient tumors [50].

low, and CN high [51].

**7. Ovarian cancer**

Ohno et al. [58], phase II

Coosemans et al. [59]

(**Table 6**).

Microsatellite instability-high (MSI-H) status, tumor mutation burden, and high PD-L1 expression have been associated with higher response rates to this therapy [49]. Approximately 25% of endometrial cancer show microsatellite instability which

is caused by defects in mismatch repair genes. These defective MMR genes lead to high somatic mutation rates, thereby increasing the number of neoantigens in

Endometrial cancer has been subdivided into four prognostically distinct molecular subgroups based on the findings of the cancer genome atlas, namely, polymerase epsilon (*POLE)* ultramutated, MSI hypermutated, copy-number (CN)

The ultramutated POLE subgroup and MSI hypermutated subgroup have immune-rich microenvironment and high mutation load. Evidence has supported over-expression of the PD-1/PD-L1 pathway in these molecular subtypes, and therefore, PD1/PD L1-targeted immunotherapy has a role in these tumors [52]

An ongoing phase II, two group trials are studying the role of avelumab in POLE-mutated endometrial cancer and MSS-mutated endometrial cancer. Avelumab is administered at 10 mg/kg as 1-hour IV infusion every 2 weeks until disease progression or unacceptable toxicity. Sixteen patients are enrolled in each cohort in the first stage. The preliminary results are yet to be published [57].

Ovarian cancer accounts for 2.5% of all malignancies among females but 5% of female cancer deaths because of low survival rates, largely driven by late-stage diagnoses [60]. There were nearly 300,000 new cases in 2018. Ovarian cancer is considered to be an ideal type of tumor which can be dealt with immunomodulatory

**Response Toxicity**

Local erythema occurred at the WT1 vaccine injection site

One patient had a local allergic reaction

Stable disease in three patients and progressive disease in nine patients. The disease control rate

was 25.0%

response

Three out of four human leucocyte antigen-A2 (HLA-A2)-positive patients showed an oncological response. Two HLA-A2 negative patients did not show an oncological or an immunological

**schedule**

Intradermal injections of a HLA-A\*2402 restricted, modified 9-mer WT1 peptide every week for 12 weeks

Four times weekly vaccines of autologous dendritic cells (DCs) electroporated with WT1 mRNA

#### **Table 6.**

*Immunotherapy in endometrial cancer.*

#### *Immunotherapy in Gynecological Malignancies DOI: http://dx.doi.org/10.5772/intechopen.90711*

Microsatellite instability-high (MSI-H) status, tumor mutation burden, and high PD-L1 expression have been associated with higher response rates to this therapy [49].

Approximately 25% of endometrial cancer show microsatellite instability which is caused by defects in mismatch repair genes. These defective MMR genes lead to high somatic mutation rates, thereby increasing the number of neoantigens in MMR-deficient tumors [50].

Endometrial cancer has been subdivided into four prognostically distinct molecular subgroups based on the findings of the cancer genome atlas, namely, polymerase epsilon (*POLE)* ultramutated, MSI hypermutated, copy-number (CN) low, and CN high [51].

The ultramutated POLE subgroup and MSI hypermutated subgroup have immune-rich microenvironment and high mutation load. Evidence has supported over-expression of the PD-1/PD-L1 pathway in these molecular subtypes, and therefore, PD1/PD L1-targeted immunotherapy has a role in these tumors [52] (**Table 6**).

An ongoing phase II, two group trials are studying the role of avelumab in POLE-mutated endometrial cancer and MSS-mutated endometrial cancer. Avelumab is administered at 10 mg/kg as 1-hour IV infusion every 2 weeks until disease progression or unacceptable toxicity. Sixteen patients are enrolled in each cohort in the first stage. The preliminary results are yet to be published [57].

#### **6.1 Anticancer vaccines in endometrial cancer**

The following studies have been conducted (**Table 7**).

### **7. Ovarian cancer**

Ovarian cancer accounts for 2.5% of all malignancies among females but 5% of female cancer deaths because of low survival rates, largely driven by late-stage diagnoses [60]. There were nearly 300,000 new cases in 2018. Ovarian cancer is considered to be an ideal type of tumor which can be dealt with immunomodulatory


#### **Table 7.** *Anticancer vaccines in endometrial cancer.*

approach as the disease does not negatively affect the immunoregulatory cells in the bone marrow or other locations of the body, and the patients suffering from ovarian cancer maintain a relatively good performance status even in later stages, so immunotherapy can be used as a potential treatment option in these patients. Cytotoxic chemotherapy given in ovarian cancer can negatively impact the immunoregulatory cells, but the effect is short lasting. Further the patients who are in advanced stages, if they respond to standard treatment of ovarian cancer, have a relatively long disease-free period which is substantial for the activation of immune defense mechanism either by cancer vaccines or by immunomodulator drugs [61].

#### **7.1 Immune checkpoint inhibitors in ovarian cancer**

The first published data supporting checkpoint inhibitors as a potentially valuable therapeutic option in ovarian cancer were observed in the trials of the anti-PD-1 antibody nivolumab and the anti-PD-L1 antibody BMS-93655 [62]. Other studies are as follows (**Table 8**).


**13**

patients [77].

*Immunotherapy in Gynecological Malignancies DOI: http://dx.doi.org/10.5772/intechopen.90711*

is a 6-month progression-free survival rate.

cancer [68].

Cancer Institute [72].

Ongoing trials include JAVELIN Ovarian 200 is the first phase III trial, which is a three-arm trial, comparing avelumab administered alone or in combination with pegylated liposomal doxorubicin versus pegylated liposomal doxorubicin alone in

NCT02839707 is undergoing trial which is comparing pegylated liposomal doxorubicin with atezolizumab and/or bevacizumab in refractory ovarian

A phase II study by Wenham et al. [69] is studying combination of weekly paclitaxel and an anti-PD-1 (pembrolizumab). The primary endpoint of this study

ATALANTE trial is an ongoing phase III study to assess the efficacy of atezolizumab in combination with platinum-based chemotherapy plus bevacizumab

CheckMate 032 study trial to study the safety and efficacy of nivolumab as a

Similar trial in which nivolumab with or without ipilimumab in treating patients

A phase II trial to determine the median immune-related progression-free survival (irPFS) in combination of an anti-CTLA-4 antibody (tremelimumab) with an anti-PD-L1 antibody (durvalumab) versus their sequential use in platinum-resistant

Multiple other trial are using immune checkpoint inhibitors in initial therapy to improve progression-free survival like durvalumab or pembrolizumab with standard paclitaxel and carboplatin therapy, where pembrolizumab is used as adjuvant therapy after surgery [74]. The role of immune checkpoint inhibitors as maintenance therapy is also under investigation with JAVELIN Ovarian 100 phase II study of avelumab (anti-PD-L1) as maintenance after standard therapy or in combination

Various types of cancer vaccines are studied for the treatment of ovarian cancer. The cancer testis antigen, NY ESO1, is most frequently expressed in epithelial ovarian cancer, and vaccine against it has shown induced T-cell-specific immunogenicity [76]. Since NY-ESO-1 is regulated by DNA methylation, it was hypothesized that DNA methyltransferase (DNMT) inhibitors may augment NY-ESO-1 vaccine therapy. Decitabine is a hypomethylating agent that inhibits DNA methyltransferase. A phase I trial was conducted to study dose escalation of decitabine in addition to NY-ESO-1 vaccine and doxorubicin liposome in 12 patients with relapsed epithelial ovarian carcinoma. The results showed stable disease or partial response in six

Sabbatini et al. conducted a phase I trial in 28 patients which showed that in order to enhance the immunogenic response to NY-ESO1, the addition of immune modulation agents to the vaccine preparation such as Montanide and immunostimulants such as the toll-like receptor (TLR) ligand poly-ICLC (polyinosinic-polycytidylic acid—stabilized by lysine and carboxymethylcellulose) can be considered [78]. Other antigen under investigation is Her/neu2, which is expressed in 90% of epithelial ovarian cancers. A phase I/II study conducted BY Chu et al. demonstrated a 90% 3-year overall survival response in patients with advanced ovarian cancer who were remission for vaccination with monocyte-derived dendritic cells (DC) loaded with Her2/neu, hTERT, and PADRE peptides, with or without low-dose

single agent or in combination with ipilimumab is currently underway [71].

with standard therapy and then continued as maintenance treatment [75].

with persistent or recurrent epithelial ovarian is being studied by the National

patients with platinum-resistant/refractory recurrent ovarian cancer [67].

administered concurrent to chemotherapy and in maintenance [70].

epithelial ovarian cancer is also currently ongoing [73].

**7.2 Cancer vaccines in ovarian cancer**

intravenous cyclophosphamide [79].

#### **Table 8.**

*Immune checkpoint inhibitors in ovarian cancer.*

#### *Immunotherapy in Gynecological Malignancies DOI: http://dx.doi.org/10.5772/intechopen.90711*

Ongoing trials include JAVELIN Ovarian 200 is the first phase III trial, which is a three-arm trial, comparing avelumab administered alone or in combination with pegylated liposomal doxorubicin versus pegylated liposomal doxorubicin alone in patients with platinum-resistant/refractory recurrent ovarian cancer [67].

NCT02839707 is undergoing trial which is comparing pegylated liposomal doxorubicin with atezolizumab and/or bevacizumab in refractory ovarian cancer [68].

A phase II study by Wenham et al. [69] is studying combination of weekly paclitaxel and an anti-PD-1 (pembrolizumab). The primary endpoint of this study is a 6-month progression-free survival rate.

ATALANTE trial is an ongoing phase III study to assess the efficacy of atezolizumab in combination with platinum-based chemotherapy plus bevacizumab administered concurrent to chemotherapy and in maintenance [70].

CheckMate 032 study trial to study the safety and efficacy of nivolumab as a single agent or in combination with ipilimumab is currently underway [71].

Similar trial in which nivolumab with or without ipilimumab in treating patients with persistent or recurrent epithelial ovarian is being studied by the National Cancer Institute [72].

A phase II trial to determine the median immune-related progression-free survival (irPFS) in combination of an anti-CTLA-4 antibody (tremelimumab) with an anti-PD-L1 antibody (durvalumab) versus their sequential use in platinum-resistant epithelial ovarian cancer is also currently ongoing [73].

Multiple other trial are using immune checkpoint inhibitors in initial therapy to improve progression-free survival like durvalumab or pembrolizumab with standard paclitaxel and carboplatin therapy, where pembrolizumab is used as adjuvant therapy after surgery [74]. The role of immune checkpoint inhibitors as maintenance therapy is also under investigation with JAVELIN Ovarian 100 phase II study of avelumab (anti-PD-L1) as maintenance after standard therapy or in combination with standard therapy and then continued as maintenance treatment [75].

#### **7.2 Cancer vaccines in ovarian cancer**

Various types of cancer vaccines are studied for the treatment of ovarian cancer.

The cancer testis antigen, NY ESO1, is most frequently expressed in epithelial ovarian cancer, and vaccine against it has shown induced T-cell-specific immunogenicity [76]. Since NY-ESO-1 is regulated by DNA methylation, it was hypothesized that DNA methyltransferase (DNMT) inhibitors may augment NY-ESO-1 vaccine therapy. Decitabine is a hypomethylating agent that inhibits DNA methyltransferase. A phase I trial was conducted to study dose escalation of decitabine in addition to NY-ESO-1 vaccine and doxorubicin liposome in 12 patients with relapsed epithelial ovarian carcinoma. The results showed stable disease or partial response in six patients [77].

Sabbatini et al. conducted a phase I trial in 28 patients which showed that in order to enhance the immunogenic response to NY-ESO1, the addition of immune modulation agents to the vaccine preparation such as Montanide and immunostimulants such as the toll-like receptor (TLR) ligand poly-ICLC (polyinosinic-polycytidylic acid—stabilized by lysine and carboxymethylcellulose) can be considered [78].

Other antigen under investigation is Her/neu2, which is expressed in 90% of epithelial ovarian cancers. A phase I/II study conducted BY Chu et al. demonstrated a 90% 3-year overall survival response in patients with advanced ovarian cancer who were remission for vaccination with monocyte-derived dendritic cells (DC) loaded with Her2/neu, hTERT, and PADRE peptides, with or without low-dose intravenous cyclophosphamide [79].

In a phase I/II study by Baek et al., 10 ovarian cancer patients with minimal residual disease were treated with dendritic cell vaccination with IL2. Three out of 10 patients showed maintenance of complete response, and one patient showed stable disease [80].

A phase II study was conducted to study the efficacy of personalized peptide vaccine (PPV) for recurrent ovarian cancer patients by Kawano et al. [81]. The patients enrolled in this study showed an overall survival (OS) of 39.3 months in platinum-sensitive cases and 16.2 months in platinum-resistant cases. This was attributed to be secondary to the stabilization of disease and the prolongation of tumor progression rather than disease regression.

#### **7.3 Adoptive cell transfer in ovarian cancer**

Adoptive cell transfer therapy is not widely studied in ovarian cancers. In a Japanese study by Fujita et al., 13 patients with epithelial ovarian cancer were treated with tumor-infiltrating lymphocyte therapy. Eleven patients served as control group who received only chemotherapy following primary operation. The estimated 3-year overall survival rate of disease-free patients in the TIL group and in the control group was 100 and 67.5%, respectively [82].

Vulvar and vaginal cancer: Immunotherapy has shown promising results in advanced gynecological cancer. Checkmate 358 trial has shown that nivolumab has encouraging clinical activity in cases of HPV-positive vulvar and vaginal malignancies. A lot of research is warranted to establish immunotherapy as emerging treatment option in these cancers.

#### **8. Conclusion**

Immunotherapy is emerging as a viable treatment modality in multiple cancers, and its safety and efficacy are under investigation in advanced gynecological malignancies. Immune checkpoint inhibitors have shown promising preliminary results in advanced ovarian, cervical, and endometrial cancer.

#### **Author details**

Neha Sharma1 and Deepti Sharma2 \*

1 Department of Radiation Oncology, Lady Hardinge Medical College and Associated SSK and KSC Hospital, New Delhi, India

2 Department of Radiation Oncology, Institute of Liver and Biliary Science, New Delhi, India

\*Address all correspondence to: drdeeptisharma16@gmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[66] Lee JM et al. Safety and clinical activity of the programmed deathligand 1 inhibitor durvalumab in combination with poly (ADP-ribose) polymerase inhibitor olaparib or vascular endothelial growth factor receptor 1-3 inhibitor cediranib in women's cancers: A dose-escalation, phase I study. Journal of Clinical Oncology. 2017;**35**(19):2193-2202

[67] Pujade-Lourraine E,

2016;**34**(15 suppl.):TPS5600

gov/ct2/show/NCT02839707

Apte SM, Shahzad MM, Lee JK, Dorman D, Chon HS. Phase II trial of dose dense (weekly) paclitaxel with pembrolizumab (MK-3475) in platinum-resistant recurrent ovarian cancer. Journal of Clinical Oncology.

2016;**34**(15 suppl.):TPS5612

[70] ATALANTE: Atezolizumab vs Placebo Phase III Study in Late Relapse Ovarian Cancer Treated With Chemotherapy Bevacizumab—Full Text View [Internet]. ClinicalTrials.gov. Available from: https://clinicaltrials. gov/ct2/show/NCT02891824

[71] A Study of Nivolumab by Itself or Nivolumab Combined With Ipilimumab in Patients With Advanced or Metastatic

Solid Tumors—Full Text View

[69] Wenham RM,

[68] Pegylated Liposomal Doxorubicin Hydrochloride With Atezolizumab and/ or Bevacizumab in Treating Patients With Recurrent Ovarian, Fallopian Tube, or Primary Peritoneal Cancer— Full Text View [Internet]. ClinicalTrials. gov. Available from: https://clinicaltrials.

Colombo N, Disis ML, Fujiwara K, Ledermann JA, Mirza MR, et al. Avelumab (MSB0010718C; anti-PD-L1) ± pegylated liposomal doxorubicin vs pegylated liposomal doxorubicin alone in patients with platinum-resistant/refractory ovarian cancer: The phase III JAVELIN Ovarian 200 trial. Journal of Clinical Oncology.

[60] Howlader N, Noone AM, Krapcho M, et al., editors. SEER Cancer Statistics Review, 1975-2014. Bethesda, MD: National Cancer Institute; 2017. Available from: seer.cancer.gov/csr/1975\_2014/.

[61] Markman M. Immunotherapy in ovarian cancer—where are we going? American Journal of Hematology/ Oncology. 2016. Available from: https:// www.gotoper.com/publications/ ajho/2016/2016feb/immunotherapy-inovarian-cancer-where-are-we-going

[62] Taneja SS. Re: safety and activity of anti-PD-L1 antibody in patients with advanced cancer. Journal of Urology.

[63] Hamanishi J, Mandai M, Ikeda T, Minami M, Kawaguchi A, Murayama T, et al. Safety and antitumor activity of anti-PD-1 antibody, nivolumab, in patients with platinum-resistant ovarian cancer. Journal of Clinical Oncology.

[64] Disis ML, Patel MR, Pant S, Hamilton EP, Lockhart AC, Kelly K, et al. Avelumab (MSB0010718C; anti-PD-L1) in patients with recurrent/ refractory ovarian cancer from the JAVELIN solid tumor phase Ib trial: Safety and clinical activity. Journal of Clinical Oncology. 2016;**34**:5533

[65] Varga A, Piha-Paul SA,

Oncology. 2015;**33**:5510

Ott PA, Mehnert JM, Berton-Rigaud D, Johnson EA, et al. Antitumor activity and safety of pembrolizumab in patients (pts) with PD-L1 positive advanced ovarian cancer: Interim results from a phase Ib study. Journal of Clinical

[Accessed: 01 March 2018]

2012;**188**:2148-2149

2015;**33**:4015-4022

*Immunotherapy in Gynecological Malignancies DOI: http://dx.doi.org/10.5772/intechopen.90711*

[59] Coosemans A, Vanderstraeten A, Tuyaerts S, Verschuere T, Moerman P, Berneman ZN, et al. Wilms' tumor gene 1 (WT1)-loaded dendritic cell immunotherapy in patients with uterine tumors: A phase I/II clinical trial. Anticancer Research. 2013;**33**:5495-5500

[60] Howlader N, Noone AM, Krapcho M, et al., editors. SEER Cancer Statistics Review, 1975-2014. Bethesda, MD: National Cancer Institute; 2017. Available from: seer.cancer.gov/csr/1975\_2014/. [Accessed: 01 March 2018]

[61] Markman M. Immunotherapy in ovarian cancer—where are we going? American Journal of Hematology/ Oncology. 2016. Available from: https:// www.gotoper.com/publications/ ajho/2016/2016feb/immunotherapy-inovarian-cancer-where-are-we-going

[62] Taneja SS. Re: safety and activity of anti-PD-L1 antibody in patients with advanced cancer. Journal of Urology. 2012;**188**:2148-2149

[63] Hamanishi J, Mandai M, Ikeda T, Minami M, Kawaguchi A, Murayama T, et al. Safety and antitumor activity of anti-PD-1 antibody, nivolumab, in patients with platinum-resistant ovarian cancer. Journal of Clinical Oncology. 2015;**33**:4015-4022

[64] Disis ML, Patel MR, Pant S, Hamilton EP, Lockhart AC, Kelly K, et al. Avelumab (MSB0010718C; anti-PD-L1) in patients with recurrent/ refractory ovarian cancer from the JAVELIN solid tumor phase Ib trial: Safety and clinical activity. Journal of Clinical Oncology. 2016;**34**:5533

[65] Varga A, Piha-Paul SA, Ott PA, Mehnert JM, Berton-Rigaud D, Johnson EA, et al. Antitumor activity and safety of pembrolizumab in patients (pts) with PD-L1 positive advanced ovarian cancer: Interim results from a phase Ib study. Journal of Clinical Oncology. 2015;**33**:5510

[66] Lee JM et al. Safety and clinical activity of the programmed deathligand 1 inhibitor durvalumab in combination with poly (ADP-ribose) polymerase inhibitor olaparib or vascular endothelial growth factor receptor 1-3 inhibitor cediranib in women's cancers: A dose-escalation, phase I study. Journal of Clinical Oncology. 2017;**35**(19):2193-2202

[67] Pujade-Lourraine E, Colombo N, Disis ML, Fujiwara K, Ledermann JA, Mirza MR, et al. Avelumab (MSB0010718C; anti-PD-L1) ± pegylated liposomal doxorubicin vs pegylated liposomal doxorubicin alone in patients with platinum-resistant/refractory ovarian cancer: The phase III JAVELIN Ovarian 200 trial. Journal of Clinical Oncology. 2016;**34**(15 suppl.):TPS5600

[68] Pegylated Liposomal Doxorubicin Hydrochloride With Atezolizumab and/ or Bevacizumab in Treating Patients With Recurrent Ovarian, Fallopian Tube, or Primary Peritoneal Cancer— Full Text View [Internet]. ClinicalTrials. gov. Available from: https://clinicaltrials. gov/ct2/show/NCT02839707

[69] Wenham RM,

Apte SM, Shahzad MM, Lee JK, Dorman D, Chon HS. Phase II trial of dose dense (weekly) paclitaxel with pembrolizumab (MK-3475) in platinum-resistant recurrent ovarian cancer. Journal of Clinical Oncology. 2016;**34**(15 suppl.):TPS5612

[70] ATALANTE: Atezolizumab vs Placebo Phase III Study in Late Relapse Ovarian Cancer Treated With Chemotherapy Bevacizumab—Full Text View [Internet]. ClinicalTrials.gov. Available from: https://clinicaltrials. gov/ct2/show/NCT02891824

[71] A Study of Nivolumab by Itself or Nivolumab Combined With Ipilimumab in Patients With Advanced or Metastatic Solid Tumors—Full Text View

[Internet]. ClinicalTrials.gov. Available from: https://clinicaltrials.gov/ct2/ show/NCT01928394

[72] Nivolumab With or Without Ipilimumab in Treating Patients With Persistent or Recurrent Epithelial Ovarian, Primary Peritoneal, or Fallopian Tube Cancer—Full Text View [Internet]. ClinicalTrials.gov. Available from: https://clinicaltrials.gov/ct2/ show/NCT02498600

[73] Durvalumab and Tremelimumab in Treating Participants With Recurrent or Refractory Ovarian, Primary Peritoneal, or Fallopian Tube Cancer—Full Text View [Internet]. ClinicalTrials.gov. Available from: https://clinicaltrials. gov/ct2/show/NCT03026062

[74] Pembrolizumab, Carboplatin, and Paclitaxel in Treating Patients With Stage III-IV Ovarian, Primary Peritoneal, or Fallopian Tube Cancer— Full Text View [Internet]. ClinicalTrials. gov. Available from: https://clinicaltrials. gov/ct2/show/NCT02520154

[75] Avelumab in Previously Untreated Patients With Epithelial Ovarian Cancer (JAVELIN OVARIAN 100)—Full Text View [Internet]. ClinicalTrials.gov. Available from: https://clinicaltrials. gov/ct2/show/NCT02718417

[76] Diefenbach CS, Gnjatic S, Sabbatini P, Aghajanian C, Hensley ML, Spriggs DR, et al. Safety and immunogenicity study of NY-ESO-1b peptide and montanide ISA-51 vaccination of patients with epithelial ovarian cancer in high-risk first remission. Clinical Cancer Research. 2008;**14**:2740-2748

[77] Odunsi K, Matsuzaki J, James SR, Mhawech-Fauceglia P, Tsuji T, Miller A, et al. Epigenetic potentiation of NY-ESO-1 vaccine therapy in human ovarian cancer. Cancer Immunology Research. 2014. Available from:

https://www.ncbi.nlm.nih.gov/ pubmed/24535937

**Chapter 2**

**Abstract**

**1. Introduction**

prevalence [3].

**21**

The Role of Epigenetics

*Rodrigo Cáceres-Gutiérrez, Marco A. Andonegui-Elguera,*

Cervical cancer is the fourth most common type of cancer among women worldwide resulting in 528,475 new cases and 268,224 deaths. The principal etiological factor of cervical cancer is the persistent infection with high-risk types of human papillomaviruses (HPV), however is not sufficient, other factors like age, smoking, oral contraceptives, and genetic background are implicated in the development of this neoplasia. Although the understanding of cervical carcinogenesis has been increasing in recent decades, the epigenetic modifications (DNA methylation, histone modification, miRNAs and long non-coding RNAs) and its contribution to the development of cervical cancer remain largely unknown. In the next chapter, we will recapitulate the described findings on the alteration of epigenetic factors that, together with the persistent infection of HPV, could contribute to the

**Keywords:** HPV, DNA methylation, histone modification, ncRNAs, therapy

Cervical cancer is the fourth most common type of cancer among women worldwide, resulting in 528,475 new cases per year with 268,224 deaths [1]. Cervical cancer represents 6.6% of all female cancers and nearly 90% of all deaths occur in both low- and middle-income countries, as the disease is detected in the advanced stages or when the treatment is inaccessible [2]. The principal etiological factor of cervical cancer is the persistent infection with high-risk types of human papillomaviruses (hr-HPV). In fact, the HPV prevalence among women with normal cytology worldwide was 11.7%. This estimate varies by geography being Sahara African regions (24%), Latin America and the Caribbean (16.2%), Eastern Europe (14.2%),

and Southeaster Asia (14%) the regions with the highest percentage of

a mean of 3 months in age-independent manner. Nonetheless, the cytological regression takes a longer time. This period depends in great manner on the grade of the lesion and if one or several hr-HPV are present. While mild and moderate/ severe premalignant lesions with no HPV presence takes a mean of 5–6 months to recovery; mild, moderated, or severe premalignant lesions with the presence of

Most of hr-HPV premalignant lesions have a spontaneously viral clearance with

*Guadalupe Dominguez-Gómez and José Díaz-Chávez*

in Cervical Cancer

*Yair Alfaro-Mora, Luis A. Herrera,*

malignant and invasive phenotype in cervical cancer.

[78] Sabbatini P, Tsuji T, Ferran L, Ritter E, Sedrak C, Tuballes K, et al. Phase I trial of overlapping long peptides from a tumor self-antigen and poly-ICLC shows rapid induction of integrated immune response in ovarian cancer patients. Clinical Cancer Research. 2012. Available from: https://www.ncbi.nlm.nih.gov/ pubmed/23032745

[79] Chu CS, Boyer J, Schullery DS, Gimotty PA, Gamerman V, Bender J, et al. Phase I/II randomized trial of dendritic cell vaccination with or without cyclophosphamide for consolidation therapy of advanced ovarian cancer in first or second remission. Cancer Immunology, Immunotherapy. 2012. Available from: https://www.ncbi.nlm. nih.gov/pubmed/22021066

[80] Baek S, Kim Y-M, Kim S-B, Kim C-S, Kwon S-W, Kim YM, et al. Therapeutic DC vaccination with IL-2 as a consolidation therapy for ovarian cancer patients: A phase I/II trial [Internet]. Cellular & Molecular Immunology. 2015 Available from: https://www.ncbi.nlm. nih.gov/pubmed/24976269

[81] Kawano K, Tsuda N, Matsueda S, Sasada T, Watanabe N, Ushijima K, et al. Feasibility study of personalized peptide vaccination for recurrent ovarian cancer patients. Immunopharmacology and Immunotoxicology. 2014. Available from: https://www.ncbi.nlm.nih.gov/ pubmed/24773550

[82] Fujita K, Ikarashi H, Takakuwa K, Kodama S, Tokunaga A, Takahashi T, et al. Prolonged disease-free period in patients with advanced epithelial ovarian cancer after adoptive transfer of tumor-infiltrating lymphocytes. Clinical Cancer Research. 1995. Available from: https://www.ncbi.nlm. nih.gov/pubmed/9816009

#### **Chapter 2**

## The Role of Epigenetics in Cervical Cancer

*Yair Alfaro-Mora, Luis A. Herrera, Rodrigo Cáceres-Gutiérrez, Marco A. Andonegui-Elguera, Guadalupe Dominguez-Gómez and José Díaz-Chávez*

#### **Abstract**

Cervical cancer is the fourth most common type of cancer among women worldwide resulting in 528,475 new cases and 268,224 deaths. The principal etiological factor of cervical cancer is the persistent infection with high-risk types of human papillomaviruses (HPV), however is not sufficient, other factors like age, smoking, oral contraceptives, and genetic background are implicated in the development of this neoplasia. Although the understanding of cervical carcinogenesis has been increasing in recent decades, the epigenetic modifications (DNA methylation, histone modification, miRNAs and long non-coding RNAs) and its contribution to the development of cervical cancer remain largely unknown. In the next chapter, we will recapitulate the described findings on the alteration of epigenetic factors that, together with the persistent infection of HPV, could contribute to the malignant and invasive phenotype in cervical cancer.

**Keywords:** HPV, DNA methylation, histone modification, ncRNAs, therapy

#### **1. Introduction**

Cervical cancer is the fourth most common type of cancer among women worldwide, resulting in 528,475 new cases per year with 268,224 deaths [1]. Cervical cancer represents 6.6% of all female cancers and nearly 90% of all deaths occur in both low- and middle-income countries, as the disease is detected in the advanced stages or when the treatment is inaccessible [2]. The principal etiological factor of cervical cancer is the persistent infection with high-risk types of human papillomaviruses (hr-HPV). In fact, the HPV prevalence among women with normal cytology worldwide was 11.7%. This estimate varies by geography being Sahara African regions (24%), Latin America and the Caribbean (16.2%), Eastern Europe (14.2%), and Southeaster Asia (14%) the regions with the highest percentage of prevalence [3].

Most of hr-HPV premalignant lesions have a spontaneously viral clearance with a mean of 3 months in age-independent manner. Nonetheless, the cytological regression takes a longer time. This period depends in great manner on the grade of the lesion and if one or several hr-HPV are present. While mild and moderate/ severe premalignant lesions with no HPV presence takes a mean of 5–6 months to recovery; mild, moderated, or severe premalignant lesions with the presence of

hr-HPV takes a mean of 17, 24, and 60 months, respectively [4, 5]. However, although hr-HPV persistent infection is necessary for the development of cervical cancer, the solely infection is not sufficient. The presence of factors like age [6, 7], smoking [8], oral contraceptives [9], alcohol usage [10], and host and viral genetic background are necessary to observe an accumulation of epithelial cell abnormalities like sustained proliferation and growth of new blood vessels. These abnormalities emerge due to genomic alteration, defects in the genome maintenance and repair, destabilization of the number of DNA copies, and/or somatic mutations. Then, the cells that harbor all these abnormalities can evolve progressively to a tumorigenic, and further, a malignant and invasive phenotype [11].

cancer types like vulvar, vaginal, anal, penile, and oropharyngeal in females and males, the Advisory Committee on Immunization Practices (ACIP) recommend the routine vaccination with one of the three commercial available vaccines against HPV (9-valent, 4-valent, and 2-valent HPV vaccines, (HPVV)) in females and males at age 11 or 12 years and females aged 11–26 years and males aged 13 through 21 years not vaccinated previously. 2vHPVV contains HPV 16,18 virus-like particles; 4vHPVV contains HPV 6, 11, 16, and 18 virus-like particles; and 9vHPVV 6, 11, 16, 18, 31, 33, 45, 52, and 58 virus-like particles. These vaccines show a CIN prevention efficacy of 98% [24, 25]. Based in the above observations, these data highlight the importance of vaccination against HPVs since it seems like the expression of the HPV genome is the first step for development of pre-cancer lesions and a possible malignant progression. In this chapter, we review activities of E6 and E7 modulating epigenetics in cervical cancer and how these modifications

Traditionally, cancer has been viewed as a multifactorial genetic disease that raise from an accumulation of mutations in tumor suppressor and/or oncogenes that cause loss or gain of function and an abnormal genetic expression. Although the understanding of cervical carcinogenesis has been increasing in recent decades, the epigenetic modifications (DNA methylation, histone modification and non-coding RNA (ncRNA)) and its contribution to the development of cervical cancer remain unknown. Nonetheless, in the past years, multiple epigenetic modifications have been associated with cancer initiation and proliferation [26]. The epigenetic are all the heritable changes in gene expression that are not due to changes in the nucleotide sequence of DNA. These modifications are established during embryonic development to bring cellular identity and are stably maintained during cellular replication in differentiated tissues. This is achieved by controlling the accessibility of transcription factors and by altering the capability of DNA packaging, having as result a temporal and spatial modulation in gene expression. Collectively, these modifications are referred as the epigenome. The epigenome comprises four main phenomena: Pos-translational histone modifications, DNA methylation, chromatin remodeling, and regulation by non-coding RNAs [26–28]. Recently, different works have been shown that hr-HPV E6 and E7 viral proteins have the capability of target

The DNA methylation is associated with gene silencing due the recruitment and/ or disassociation of DNA-binding proteins that can act as repressor complexes or transcription factors which generate a transcriptional silencing. Moreover, the methylation is necessary for a correct embryonic development [15], genome stability [16], X chromosome inactivation [17, 18], genomic imprinting [19], and silence of retrotransposons [20]. In mammals, the predominant form of DNA methylation occurs by a covalent addition of a methyl group in the fifth carbon of cytosine residues that are preceded by guanine nucleotides (CpG dinucleotides) in both DNA strands. This methyl group comes from a universal donor called S-adenosyl-L-

methionine (SAM) and the enzymatic reaction is controlled by 3 DNA

methyltransferases named DNMT1, DNMT3A, and DNMT3B, and the enzymatically inactive proteins DNMT2 and DNMT3L [21, 22]. Nearly 80% of all the DNA CpG dinucleotides in somatic tissues are methylated and comprises satellite DNAs, repetitive elements like transposons, non-repetitive intergenic DNA, and exons of genes [23]. From this DNA elements, there are CpG dinucleotides that are nonmethylated that can be detected in germ cells, early embryo, and in somatic tissues.

could contribute to the development of this neoplasia.

*The Role of Epigenetics in Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.89819*

key proteins which regulate epigenetic marks.

**3. DNA methylation**

**23**

#### **2. Papillomaviruses**

HPVs are DNA viruses that are able to infect the skin or mucosa of animal species. More than 200 human papillomavirus genotypes are known and have been categorized into phylogenetic genera as Alpha, Beta, Gamma, Mu, and Nu. The high-risk types of the Alpha genus are sexually transmitted being the types 16, 18, 52, 31, 58, 39, 51, and 56 the most common hr-HPV type found in women with apparent normal cytology. hr-HPV16 is the most frequently detected followed by hr-HPV18 and both are present in 70% of all the cervical cancers [12].

Papillomaviruses consist of a circular double-stranded DNA genome of approximately 8000 base pairs that harbor two main DNA structures: a long control region (LCR) which contains union sites for both, host cellular transcription factors and the viral proteins E1 and E2 that control viral replication and gene expression; and the open reading frames that codify to eight genes necessary for the maintenance and replication of the viral DNA. The high-risk alpha papillomaviruses present two well-characterized promoters: late promoter (LP or p670) which regulate gene expression of late proteins L1 and L2; and early promoter (PE or p97) which controls gene expression of early proteins E1, E2, E4, E5, E6, and E7. These genes are expressed by a complex pattern of mRNA splicing at different stages of the viral life cycle. The early and late viral proteins exert different function in the infected cell. E1 and E2 are involved in the viral genome replication, L1 and L2 orchestrate the virus assembly, and the E4, E5, E6, and E7 alter the replication machinery of the infected cell to facilitate the virus replication. Due to the target of the viral proteins E6 and E7 in the host cell, these proteins have been termed viral oncoproteins [13, 14].

The main interaction partner of HPV-E6 is the E3 ubiquitin ligase E6-asociated protein (E6AP) which in turn targets the tumor suppressor p53 and proteins with a PDZ domain to proteasomal degradation to promote de-differentiation, impairing apoptosis induction, and eliminate cell cycle checkpoints of the infected cell [15–17]. HPV-E7 binds to multiple proteins of the Rb family members, such as pRb, p107, and p130 (collectively referred as pocket proteins) that is more extensively studied. hr-HPV E7 uses a short stretch of residues known as LXCXE motif and residues in its N-terminus interact and target degradation of the three Rb family members. The proteasome-mediated destruction of E7/Rb pocket proteins is mediated by the recruitment of Cullin 2 E3 ubiquitin ligase complex, allowing the infected cell to remain in a proliferative state [18–20]. It has been observed that a correlation between viral DNA integration to host cell genomic material and a higher expression of E6 and E7 viral protein, provides an advantage in the cellular growing and oncogenic progression by promoting cell proliferation, abrogating the cell cycle checkpoints, and causes genomic instability [21–23]. Since HPV is considered the principal risk factor in cervical cancer, it is also associated with other

#### *The Role of Epigenetics in Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.89819*

hr-HPV takes a mean of 17, 24, and 60 months, respectively [4, 5]. However, although hr-HPV persistent infection is necessary for the development of cervical cancer, the solely infection is not sufficient. The presence of factors like age [6, 7], smoking [8], oral contraceptives [9], alcohol usage [10], and host and viral genetic background are necessary to observe an accumulation of epithelial cell abnormalities like sustained proliferation and growth of new blood vessels. These abnormalities emerge due to genomic alteration, defects in the genome maintenance and repair, destabilization of the number of DNA copies, and/or somatic mutations. Then, the cells that harbor all these abnormalities can evolve progressively to a

*Gynaecological Malignancies - Updates and Advances*

tumorigenic, and further, a malignant and invasive phenotype [11].

hr-HPV18 and both are present in 70% of all the cervical cancers [12].

HPVs are DNA viruses that are able to infect the skin or mucosa of animal species. More than 200 human papillomavirus genotypes are known and have been categorized into phylogenetic genera as Alpha, Beta, Gamma, Mu, and Nu. The high-risk types of the Alpha genus are sexually transmitted being the types 16, 18, 52, 31, 58, 39, 51, and 56 the most common hr-HPV type found in women with apparent normal cytology. hr-HPV16 is the most frequently detected followed by

Papillomaviruses consist of a circular double-stranded DNA genome of approximately 8000 base pairs that harbor two main DNA structures: a long control region (LCR) which contains union sites for both, host cellular transcription factors and the viral proteins E1 and E2 that control viral replication and gene expression; and the open reading frames that codify to eight genes necessary for the maintenance and replication of the viral DNA. The high-risk alpha papillomaviruses present two well-characterized promoters: late promoter (LP or p670) which regulate gene expression of late proteins L1 and L2; and early promoter (PE or p97) which controls gene expression of early proteins E1, E2, E4, E5, E6, and E7. These genes are expressed by a complex pattern of mRNA splicing at different stages of the viral life cycle. The early and late viral proteins exert different function in the infected cell. E1 and E2 are involved in the viral genome replication, L1 and L2 orchestrate the virus assembly, and the E4, E5, E6, and E7 alter the replication machinery of the infected cell to facilitate the virus replication. Due to the target of the viral proteins E6 and E7 in the host cell, these proteins have been termed viral

The main interaction partner of HPV-E6 is the E3 ubiquitin ligase E6-asociated protein (E6AP) which in turn targets the tumor suppressor p53 and proteins with a PDZ domain to proteasomal degradation to promote de-differentiation, impairing apoptosis induction, and eliminate cell cycle checkpoints of the infected cell [15–17]. HPV-E7 binds to multiple proteins of the Rb family members, such as pRb, p107, and p130 (collectively referred as pocket proteins) that is more extensively studied. hr-HPV E7 uses a short stretch of residues known as LXCXE motif and residues in its N-terminus interact and target degradation of the three Rb family members. The proteasome-mediated destruction of E7/Rb pocket proteins is mediated by the recruitment of Cullin 2 E3 ubiquitin ligase complex, allowing the infected cell to remain in a proliferative state [18–20]. It has been observed that a correlation between viral DNA integration to host cell genomic material and a higher expression of E6 and E7 viral protein, provides an advantage in the cellular growing and oncogenic progression by promoting cell proliferation, abrogating the cell cycle checkpoints, and causes genomic instability [21–23]. Since HPV is considered the principal risk factor in cervical cancer, it is also associated with other

**2. Papillomaviruses**

oncoproteins [13, 14].

**22**

cancer types like vulvar, vaginal, anal, penile, and oropharyngeal in females and males, the Advisory Committee on Immunization Practices (ACIP) recommend the routine vaccination with one of the three commercial available vaccines against HPV (9-valent, 4-valent, and 2-valent HPV vaccines, (HPVV)) in females and males at age 11 or 12 years and females aged 11–26 years and males aged 13 through 21 years not vaccinated previously. 2vHPVV contains HPV 16,18 virus-like particles; 4vHPVV contains HPV 6, 11, 16, and 18 virus-like particles; and 9vHPVV 6, 11, 16, 18, 31, 33, 45, 52, and 58 virus-like particles. These vaccines show a CIN prevention efficacy of 98% [24, 25]. Based in the above observations, these data highlight the importance of vaccination against HPVs since it seems like the expression of the HPV genome is the first step for development of pre-cancer lesions and a possible malignant progression. In this chapter, we review activities of E6 and E7 modulating epigenetics in cervical cancer and how these modifications could contribute to the development of this neoplasia.

Traditionally, cancer has been viewed as a multifactorial genetic disease that raise from an accumulation of mutations in tumor suppressor and/or oncogenes that cause loss or gain of function and an abnormal genetic expression. Although the understanding of cervical carcinogenesis has been increasing in recent decades, the epigenetic modifications (DNA methylation, histone modification and non-coding RNA (ncRNA)) and its contribution to the development of cervical cancer remain unknown. Nonetheless, in the past years, multiple epigenetic modifications have been associated with cancer initiation and proliferation [26]. The epigenetic are all the heritable changes in gene expression that are not due to changes in the nucleotide sequence of DNA. These modifications are established during embryonic development to bring cellular identity and are stably maintained during cellular replication in differentiated tissues. This is achieved by controlling the accessibility of transcription factors and by altering the capability of DNA packaging, having as result a temporal and spatial modulation in gene expression. Collectively, these modifications are referred as the epigenome. The epigenome comprises four main phenomena: Pos-translational histone modifications, DNA methylation, chromatin remodeling, and regulation by non-coding RNAs [26–28]. Recently, different works have been shown that hr-HPV E6 and E7 viral proteins have the capability of target key proteins which regulate epigenetic marks.

#### **3. DNA methylation**

The DNA methylation is associated with gene silencing due the recruitment and/ or disassociation of DNA-binding proteins that can act as repressor complexes or transcription factors which generate a transcriptional silencing. Moreover, the methylation is necessary for a correct embryonic development [15], genome stability [16], X chromosome inactivation [17, 18], genomic imprinting [19], and silence of retrotransposons [20]. In mammals, the predominant form of DNA methylation occurs by a covalent addition of a methyl group in the fifth carbon of cytosine residues that are preceded by guanine nucleotides (CpG dinucleotides) in both DNA strands. This methyl group comes from a universal donor called S-adenosyl-Lmethionine (SAM) and the enzymatic reaction is controlled by 3 DNA methyltransferases named DNMT1, DNMT3A, and DNMT3B, and the enzymatically inactive proteins DNMT2 and DNMT3L [21, 22]. Nearly 80% of all the DNA CpG dinucleotides in somatic tissues are methylated and comprises satellite DNAs, repetitive elements like transposons, non-repetitive intergenic DNA, and exons of genes [23]. From this DNA elements, there are CpG dinucleotides that are nonmethylated that can be detected in germ cells, early embryo, and in somatic tissues. These CpG dinucleotides are concentrated in short DNA stretches whit an overage length from 500 to 2000 base pairs (bp) that are known as CpG Islands (CGIs) [24]. The main characteristics of the CGIs are an elevated G + C base concentration, low CpG depletion, absence of DNA methylation, and are preferentially located at 50 end of genes, occupying approximately 60% of human gene promoters [25–27].

In general, DNA methylation of CpG around the Transcription Start Site (TSS) is negatively correlated with gene expression, whereas a low DNA methylation around TSS and a high DNA methylation in the gene body are positively correlated with gene expression [28]. It has been reported that DNMT3A is overexpressed in HPV positive tumors and that DNMT1 overexpression leads to an increased overall DNA methylation and transformation of NIH 3 T3 cells [29, 30]. Also, it has been shown an increase in DNMT1 protein levels in low-grade CIN and in SCC in comparison with normal epithelium [31]. These observations positioned DNMT1 as a regulator of tumor progression. Interestingly, the analysis of genome wide methylation in squamous carcinoma (SCC) cell lines reveals that in SCC cells HPV positive harbors higher CpG methylation in repetitive regions and in genic and nongenic non-repetitive regions in comparison to SCC HPV negative cells [30]. This HPV-mediated DNA methylation increase can be explained by the modulation of E6 and E7 proteins over the expression and activity of the DNA methylation machinery that is described as follow.

The DNMT1 is known as maintenance methyltransferase. During the DNA replication, DNMT1 ensures that hemi-methylated CpG sites in the newly synthesized DNA maintain the methylation patterns accurately using as template for parental strand [32], whereas Dnmt3A and Dnmt3b mediate the de novo DNA methylation and establish the pattern of methylation in embryonic development [33]. The DNMT1 gene expression is controlled by the complex conformed by the tumor suppressor p53, transcription factor Specificity Protein 1 (SP1), and the Histone Deacetylases 1 and 6 (p53-SP1-HDAC1/6). This complex binds to SP1 binding sites near the DNMT1 promoter [34]. When present, E6 oncoprotein collaborates to increase the DNMT1 expression. In vitro assays shown that HPV16-E6 increases DNA methylation levels by stimulating expression and activity of DNMT1 by p53 suppression [35, 36]. As p53 is targeted to degradation by hr-HPV-E6 and E3 ubiquitin ligase E6-asociated protein (E6AP) [37], the complex p53-SP1- HDAC1/6 could be disrupted increasing the levels of SP1 in the cell and leading to an SP1-mediated DNMT1 protein expression. Moreover, it has been shown that if SP1 protein levels increases, it is capable to target p53 to degradation by MDM2 mediated ubiquitination [34]. On the other hand, E7 oncoprotein binds directly to DNMT1 mediated by the C-terminal zinc-finger CR3 domain of E7, upregulating the methyltransferase activity and stabilizing the DNMT1 protein [38, 39]. This direct activation of DNMT1 by E7 could be potentiated in a positive feedback manner since the transcription of the gene is regulated by pRB/E2F1 [40]. Interestingly, Cicchini and colleagues shown that near E7-dependent hypermethylated clusters are an enrichment of EPAS1, FOXJ3, CDX2, IRF4, FOXF1, and GCR transcription factor binding motifs, suggesting that HPV16-E7 is capable to direct DNMT1 to silence gene promoters through an E7-transcription factor interaction [41]. Although it has been reported that the interaction of E7 with different transcription factors [42–44] and cells expressing hr-HPV viral DNA harbors a plethora of hypermethylated genes [30, 41, 45–54] (See **Table 1**), further experiments are needed to clarify this data.

The ability of HPV to maintain a persistent infection resides on mechanisms of immune host response evasion. The major histocompatibility complex (MHC-I) αsubunit HLA-E is significantly downregulated by hypermethylation in a distant regulatory CpG island by HPV16-E7 suggesting that E7 alters immune cell

recognition during early stages of persistent infection [41]. On the other hand, CxCL 14 is a chemokine that functions as a potent angiogenesis inhibitor and a chemotactic factor for dendritic and natural killer cells [69, 70]. It has been seen that E7 downregulates the chemokine CXCL14 by a direct hypermethylation of its promoter. If the CxCL14 expression is restored, an increase of the presence of natural killer and CD8+ T cells in tumor-draining lymph nodes is observed [65]. HPV also inhibit the ability of Langerhans cells (antigen presenting cells) to infiltrate into the virus infected area by reducing the E-cadherin expression on infected keratinocytes cell membrane [71]. It has been demonstrated that in oral tongue,

*Cervical cancer genes hypermethylated reported in literature.*

**Table 1.**

**25**

WDFY3 [54]

**Gene Reference Gene Reference** APC [55] MGMT [45, 48, 49] C8ORF4 [56] MRC2 [54] C13ORF18 [51] MT1G [57] CADM1 [50] NKX2-8 [54] CCNA1 [58, 59] NMES1 [56] CCND2 [60] NPTX-1 [54] CDH1 [46, 56, 61] p16 [46, 48] CDH13 [60] P73 [62] CDKN2A [49] PHACTR3 [54] CLIC3 [54] PRDM14 [54] CNNA1 [51, 58, 59] PTEN [63] CREB3LI [54] RAR-62 [64] CxCL 14 [65] RARB [60] DAPK [45, 46, 49, 60] RASSF1A [66] DDK3 [53] RASSF2 [52] E-cadherin [67] RRAD [56] H-cadherin [67] SFRP1 [56] EPB41L3 [52] SFRP2 [53] FAM19A4 [54] SFRP4 [53] FHIT [47, 49] SFRP5 [53] HLA-E [41] SLCA4 [54] FLJ36166 [56] SOST [54] FN1 [56] SOX17 [53] GPNMB [56] SPARC [56] HSPA2 [56] SSX4 [56] hTERT [45, 48, 49, 51] TFPI2 [56] INK4A [48] TIMP-3 [46] LFNG [54] TNFSF13 [54] LHX1 [54] TSCL1 [68] MAL [50] TWIST1 [51, 60]

*The Role of Epigenetics in Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.89819*

#### *The Role of Epigenetics in Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.89819*

These CpG dinucleotides are concentrated in short DNA stretches whit an overage length from 500 to 2000 base pairs (bp) that are known as CpG Islands (CGIs) [24]. The main characteristics of the CGIs are an elevated G + C base concentration, low CpG depletion, absence of DNA methylation, and are preferentially located at 50

In general, DNA methylation of CpG around the Transcription Start Site (TSS) is

of genes, occupying approximately 60% of human gene promoters [25–27].

negatively correlated with gene expression, whereas a low DNA methylation around TSS and a high DNA methylation in the gene body are positively correlated with gene expression [28]. It has been reported that DNMT3A is overexpressed in HPV positive tumors and that DNMT1 overexpression leads to an increased overall DNA methylation and transformation of NIH 3 T3 cells [29, 30]. Also, it has been shown an increase in DNMT1 protein levels in low-grade CIN and in SCC in comparison with normal epithelium [31]. These observations positioned DNMT1 as a regulator of tumor progression. Interestingly, the analysis of genome wide methylation in squamous carcinoma (SCC) cell lines reveals that in SCC cells HPV positive harbors higher CpG methylation in repetitive regions and in genic and nongenic non-repetitive regions in comparison to SCC HPV negative cells [30]. This HPV-mediated DNA methylation increase can be explained by the modulation of E6 and E7 proteins over the expression and activity of the DNA methylation

The DNMT1 is known as maintenance methyltransferase. During the DNA replication, DNMT1 ensures that hemi-methylated CpG sites in the newly synthesized DNA maintain the methylation patterns accurately using as template for parental strand [32], whereas Dnmt3A and Dnmt3b mediate the de novo DNA methylation and establish the pattern of methylation in embryonic development [33]. The DNMT1 gene expression is controlled by the complex conformed by the tumor suppressor p53, transcription factor Specificity Protein 1 (SP1), and the Histone Deacetylases 1 and 6 (p53-SP1-HDAC1/6). This complex binds to SP1 binding sites near the DNMT1 promoter [34]. When present, E6 oncoprotein collaborates to increase the DNMT1 expression. In vitro assays shown that HPV16-E6 increases DNA methylation levels by stimulating expression and activity of

DNMT1 by p53 suppression [35, 36]. As p53 is targeted to degradation by hr-HPV-E6 and E3 ubiquitin ligase E6-asociated protein (E6AP) [37], the complex p53-SP1- HDAC1/6 could be disrupted increasing the levels of SP1 in the cell and leading to an SP1-mediated DNMT1 protein expression. Moreover, it has been shown that if SP1 protein levels increases, it is capable to target p53 to degradation by MDM2 mediated ubiquitination [34]. On the other hand, E7 oncoprotein binds directly to DNMT1 mediated by the C-terminal zinc-finger CR3 domain of E7, upregulating the methyltransferase activity and stabilizing the DNMT1 protein [38, 39]. This direct activation of DNMT1 by E7 could be potentiated in a positive feedback manner since the transcription of the gene is regulated by pRB/E2F1 [40]. Interestingly, Cicchini and colleagues shown that near E7-dependent hypermethylated clusters are an enrichment of EPAS1, FOXJ3, CDX2, IRF4, FOXF1, and GCR transcription factor binding motifs, suggesting that HPV16-E7 is capable to direct DNMT1 to silence gene promoters through an E7-transcription factor interaction [41]. Although it has been reported that the interaction of E7 with different transcription factors [42–44] and cells expressing hr-HPV viral DNA harbors a plethora of hypermethylated genes [30, 41, 45–54] (See **Table 1**), further experiments are

The ability of HPV to maintain a persistent infection resides on mechanisms of immune host response evasion. The major histocompatibility complex (MHC-I) αsubunit HLA-E is significantly downregulated by hypermethylation in a distant regulatory CpG island by HPV16-E7 suggesting that E7 alters immune cell

machinery that is described as follow.

*Gynaecological Malignancies - Updates and Advances*

needed to clarify this data.

**24**

end


#### **Table 1.**

*Cervical cancer genes hypermethylated reported in literature.*

recognition during early stages of persistent infection [41]. On the other hand, CxCL 14 is a chemokine that functions as a potent angiogenesis inhibitor and a chemotactic factor for dendritic and natural killer cells [69, 70]. It has been seen that E7 downregulates the chemokine CXCL14 by a direct hypermethylation of its promoter. If the CxCL14 expression is restored, an increase of the presence of natural killer and CD8+ T cells in tumor-draining lymph nodes is observed [65]. HPV also inhibit the ability of Langerhans cells (antigen presenting cells) to infiltrate into the virus infected area by reducing the E-cadherin expression on infected keratinocytes cell membrane [71]. It has been demonstrated that in oral tongue,

breast, and prostate cell lines as well as breast and prostate tumors that Enhancer of Zeste Homolog 2 (EZH2), Embryonic Ectoderm Development (EED), and Suppressor of Zeste 12 (ZUS12), components of the Polycomb Repressive Complex 2 (PCR2) along with Histone Deacetylase 1 (HDAC1) are responsible of E-cadherin silencing by Histone 3 lysine 27 trimethylation (H3K27me3) on E-cadherin promoter [72, 73]. Since it has been reported that HPV16-E6 and E7 induce a decrease in the transcription levels of E-cadherin gene without targeting E-cadherin to proteasome degradation or methylation of the E-cadherin promoter [36, 39], this PRC2 silencing mechanism could be the responsible of E7-mediated E-cadherindownregulation due E7 can induce EZH2 expression via liberation of E2F transcription factors from the inhibitory activity of pRB, p107, and p130 [74]. EZH2 increase expression could arise the formation of PRC2 that, in turn, can recruit and hyperactivate type 1 Histone Deacetylases (HDAC-1) leading to histone deacetylation and a subsequent trimethylation in H3K27 at the E-cadherin promoter silencing its expression [75, 76]. In addition, it has been shown that hr-HPV16 E7 can block HDAC-HIF-1α interaction [77] leading to a possible increase in HDAC free levels that can interact with PCR2. Moreover, HPV16/18 E6 and E7 oncoproteins increase the expression of thymopoietin pseudogene 2 (TMPOP2; lncRNA-EBIC) a long non-coding RNA that is repressed in cis by p53 transcription factor (see below). This lncRNA-EBIC can interact with EZH2 generating a TMPOP2-EZH2 complex that has been postulated as a PRC2-recruit facilitator to Ecadherin promoter region silencing these gene [78, 79].

Interestingly, HPV16 DNA is an efficient target for DNA methylation by host cell DNA methylation machinery. The viral DNA is organized into nucleosomes in equal form that eukaryote DNA [85, 86]. This viral DNA organization can modulate the viral gene expression by DNA methylation and histone modifications. The E2 viral protein is the master regulator of E6 and E7 expression by binding into four conserved E2-binding sites (E2BS) that are located in the LCR close to DNA binding sites of several cellular transcription factors like TATA-binding protein, AP-1, Sp1, GPS2/AMF-1, TopoBP1, CDP, and YY1. These E2BS have a consensus DNA

protein can activate or repress viral transcription in a dose dependent manner. At low concentrations E2 binds to E2BS4 due its great affinity, leaving the E6 promoter active. When E2 rises, the low affinity binding sites E2BS1 and E2BS2 are occupied by E2 blocking the binding of transcription factors and the recruitment of transcriptional repressors at the E6 promoter, preventing E6 and E7 transcription [87–91]. In addition, E2 is able to bind the double bromodomain protein Brd4, through of its C-terminal region and the bromodomain-containing region BDR4 recruits E2 viral protein by its N-terminal and C-terminal DNA binding domain region to E2BS-4, thus preventing the Transcription Factor II D (TFIID) and polymerase II interaction with TATA box and E6 promotor region, respectively [92]. The E2-BDR4 complex also represses the interaction between BDR4 and the Positive Transcription Elongation Factor b (P-TEFb) which is necessary to E6 and E7 expression [93]. In this way, the loss of regulation of the E2 viral protein deregulate the expression of E6 and E7 viral proteins, which can in turn contribute to further malignant transformation. HPV genome integration usually occurs in the E1 and E2

ORF regions generating a loss of E2 negative expression control allowing

environment for viral replication.

**27**

**4. Pos-translational histone modifications**

unregulated transcription of E6 and E7 viral genes [90, 94]. The viral integration has been shown to occur in two different ways: as a single genome and a head-to-tail multiple tandem repeats correlating positively the amount of CpG methylation with the number of integrated viral genome copies [95–97]. If multiple viral DNA copies are integrated in host genome, only one copy is transcriptionally active due a extensively methylation of the other integrated genome viral copies [95]. Otherwise, has been shown in vitro that E2 viral protein E2BSs binding capability is impaired by CpG methylation being more prevalent E2BS1 site methylated. These E2BSs methylation in the HPV16 LCR trigger the overexpression of E6 and E7 viral proteins [95, 97–99]. Moreover, the grade of methylation in E2BSs and in LCR varies in great manner depending of the differentiated status of the host cell, being highly methylated in less well differentiated cells and hypomethylated in LCR of viral genomes in more highly differentiated epithelial cells, correlating with the E6 and E7 course expression in infecting cells [100]. In addition to disruption of E2 ORF, the methylation of specific CpG present in hr-HPV LCR leads to an increase expression of E6 and E7 viral genes even if E2 viral protein still expressing in the host cell. All these observations underscore the combined mechanisms conducted by E6 and E7 in the methylation and hypomethylation to achieve an optimum

It is importantly to note that the E6 and E7 capability of altering gene expression can occur by interaction with a subset of chromatin-modifying enzymes that are flanking target genes. In higher eukaryotes and double-stranded DNA viruses, the DNA is tightly wrapping around a heterogeneous multi-unit structure termed nucleosome. The nucleosome is the core unit of chromatin which is 146-bp length


sequence 50

*The Role of Epigenetics in Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.89819*

Although the hypermethylation gene status is predominant in the hr-HPV host cell genome, there are works that demonstrate a hypomethylation in promotor genes (See **Table 2**). Yin et al., analyzed the expression and promoter methylation status of STK31 gene in cell lines and cervical tumors expressing hr-HPV. They found an increased expression and a hypomethylation of STK31 CpG islands in HPV16/18-positive HeLa, SiHa, and CaSki cervical cancer cell lines and HPV16/18 positive pre-malignant lesion Cervical Intraepithelial Neoplasia grade 3 (CIN3) and Cervical Cancer (CC) biopsies compared with HPV-negative C33A and HT-3 cervical cancer cell lines and HPV-negative CIN3 and CC. In addition, the authors reported that STK31 promotor were hypermethylated in all normal, CIN1, and CIN2 biopsies analyzed. However, STK3 promoter were hypomethylated in all CIN3 and CC biopsies analyzed being found more often hypomethylated in CIN3 than in CC [82]. Other genes found to be hypomethylated were Rap guanidine Nucleotide Exchange Factor (RAPGEF1) and Cancer Antigen Gene (CAGE). Samuelsson and colleagues shown that 48% of cervical squamous carcinomas analyzed present no methylation in CGI near RAPGEF1 promoter and hypomethylation on a CGI present in the first intron of these gene [80]. Lee and colleagues analyzed the methylation status of CAGE promotor gene in 40 cervical cancer patients finding that 87.5% of the samples where hypomethylated in comparison of control nonneoplastic tissues [81].


#### **Table 2.**

*Cervical cancer genes hypomethylated reported in literature.*

#### *The Role of Epigenetics in Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.89819*

breast, and prostate cell lines as well as breast and prostate tumors that Enhancer of Zeste Homolog 2 (EZH2), Embryonic Ectoderm Development (EED), and Suppressor of Zeste 12 (ZUS12), components of the Polycomb Repressive Complex 2 (PCR2) along with Histone Deacetylase 1 (HDAC1) are responsible of E-cadherin silencing by Histone 3 lysine 27 trimethylation (H3K27me3) on E-cadherin promoter [72, 73]. Since it has been reported that HPV16-E6 and E7 induce a decrease in the transcription levels of E-cadherin gene without targeting E-cadherin to proteasome degradation or methylation of the E-cadherin promoter [36, 39], this PRC2 silencing mechanism could be the responsible of E7-mediated E-cadherindownregulation due E7 can induce EZH2 expression via liberation of E2F transcription factors from the inhibitory activity of pRB, p107, and p130 [74]. EZH2 increase

expression could arise the formation of PRC2 that, in turn, can recruit and hyperactivate type 1 Histone Deacetylases (HDAC-1) leading to histone

free levels that can interact with PCR2. Moreover, HPV16/18 E6 and E7

**Gene Reference** RAPGEF1 [80] CAGE [81] STK31 [82] COL17A1 [83] Ribosomal DNA [84]

cadherin promoter region silencing these gene [78, 79].

*Gynaecological Malignancies - Updates and Advances*

neoplastic tissues [81].

*Cervical cancer genes hypomethylated reported in literature.*

**Table 2.**

**26**

deacetylation and a subsequent trimethylation in H3K27 at the E-cadherin promoter silencing its expression [75, 76]. In addition, it has been shown that hr-HPV16 E7 can block HDAC-HIF-1α interaction [77] leading to a possible increase in HDAC

oncoproteins increase the expression of thymopoietin pseudogene 2 (TMPOP2; lncRNA-EBIC) a long non-coding RNA that is repressed in cis by p53 transcription factor (see below). This lncRNA-EBIC can interact with EZH2 generating a

TMPOP2-EZH2 complex that has been postulated as a PRC2-recruit facilitator to E-

Although the hypermethylation gene status is predominant in the hr-HPV host cell genome, there are works that demonstrate a hypomethylation in promotor genes (See **Table 2**). Yin et al., analyzed the expression and promoter methylation status of STK31 gene in cell lines and cervical tumors expressing hr-HPV. They found an increased expression and a hypomethylation of STK31 CpG islands in HPV16/18-positive HeLa, SiHa, and CaSki cervical cancer cell lines and HPV16/18 positive pre-malignant lesion Cervical Intraepithelial Neoplasia grade 3 (CIN3) and Cervical Cancer (CC) biopsies compared with HPV-negative C33A and HT-3 cervical cancer cell lines and HPV-negative CIN3 and CC. In addition, the authors reported that STK31 promotor were hypermethylated in all normal, CIN1, and CIN2 biopsies analyzed. However, STK3 promoter were hypomethylated in all CIN3 and CC biopsies analyzed being found more often hypomethylated in CIN3 than in CC [82]. Other genes found to be hypomethylated were Rap guanidine Nucleotide Exchange Factor (RAPGEF1) and Cancer Antigen Gene (CAGE). Samuelsson and colleagues shown that 48% of cervical squamous carcinomas analyzed present no methylation in CGI near RAPGEF1 promoter and hypomethylation on a CGI present in the first intron of these gene [80]. Lee and colleagues analyzed the methylation status of CAGE promotor gene in 40 cervical cancer patients finding that 87.5% of the samples where hypomethylated in comparison of control non-

Interestingly, HPV16 DNA is an efficient target for DNA methylation by host cell DNA methylation machinery. The viral DNA is organized into nucleosomes in equal form that eukaryote DNA [85, 86]. This viral DNA organization can modulate the viral gene expression by DNA methylation and histone modifications. The E2 viral protein is the master regulator of E6 and E7 expression by binding into four conserved E2-binding sites (E2BS) that are located in the LCR close to DNA binding sites of several cellular transcription factors like TATA-binding protein, AP-1, Sp1, GPS2/AMF-1, TopoBP1, CDP, and YY1. These E2BS have a consensus DNA sequence 50 -ACCG(n)4CGGT-30 upstream of the p97 early promoter. The E2 viral protein can activate or repress viral transcription in a dose dependent manner. At low concentrations E2 binds to E2BS4 due its great affinity, leaving the E6 promoter active. When E2 rises, the low affinity binding sites E2BS1 and E2BS2 are occupied by E2 blocking the binding of transcription factors and the recruitment of transcriptional repressors at the E6 promoter, preventing E6 and E7 transcription [87–91]. In addition, E2 is able to bind the double bromodomain protein Brd4, through of its C-terminal region and the bromodomain-containing region BDR4 recruits E2 viral protein by its N-terminal and C-terminal DNA binding domain region to E2BS-4, thus preventing the Transcription Factor II D (TFIID) and polymerase II interaction with TATA box and E6 promotor region, respectively [92]. The E2-BDR4 complex also represses the interaction between BDR4 and the Positive Transcription Elongation Factor b (P-TEFb) which is necessary to E6 and E7 expression [93]. In this way, the loss of regulation of the E2 viral protein deregulate the expression of E6 and E7 viral proteins, which can in turn contribute to further malignant transformation. HPV genome integration usually occurs in the E1 and E2 ORF regions generating a loss of E2 negative expression control allowing unregulated transcription of E6 and E7 viral genes [90, 94]. The viral integration has been shown to occur in two different ways: as a single genome and a head-to-tail multiple tandem repeats correlating positively the amount of CpG methylation with the number of integrated viral genome copies [95–97]. If multiple viral DNA copies are integrated in host genome, only one copy is transcriptionally active due a extensively methylation of the other integrated genome viral copies [95]. Otherwise, has been shown in vitro that E2 viral protein E2BSs binding capability is impaired by CpG methylation being more prevalent E2BS1 site methylated. These E2BSs methylation in the HPV16 LCR trigger the overexpression of E6 and E7 viral proteins [95, 97–99]. Moreover, the grade of methylation in E2BSs and in LCR varies in great manner depending of the differentiated status of the host cell, being highly methylated in less well differentiated cells and hypomethylated in LCR of viral genomes in more highly differentiated epithelial cells, correlating with the E6 and E7 course expression in infecting cells [100]. In addition to disruption of E2 ORF, the methylation of specific CpG present in hr-HPV LCR leads to an increase expression of E6 and E7 viral genes even if E2 viral protein still expressing in the host cell. All these observations underscore the combined mechanisms conducted by E6 and E7 in the methylation and hypomethylation to achieve an optimum environment for viral replication.

#### **4. Pos-translational histone modifications**

It is importantly to note that the E6 and E7 capability of altering gene expression can occur by interaction with a subset of chromatin-modifying enzymes that are flanking target genes. In higher eukaryotes and double-stranded DNA viruses, the DNA is tightly wrapping around a heterogeneous multi-unit structure termed nucleosome. The nucleosome is the core unit of chromatin which is 146-bp length

DNA wound around octameric of the four highly conserved histone proteins (H3, H4, H2A, and H2B). Each nucleosome is linked one to other by a stretch of DNA called DNA linker with a length of 40–55 bp. The chromatin gives DNA structure and regulates the gene transcription via post-translational modifications (PTM). This PTM are modifications such acetylation, methylation, phosphorylation, ubiquitination, sumoylation, glycosylation, homocysteinylation, crotonylation, propionylation, and butyrylation in the amino-terminal and carboxy-terminal tail of histones that are mediated by diverse histone modifying enzymes. These PTM regulate gene expression by affecting the nucleosome stability and structure [101, 102].

correct NF-κB transcription, it is necessary the recruitment and interaction with different transcriptional coactivators like CREB binding protein (CBP), p300, Steroid Receptor-Coactivator-1 (SRC-1), or Nuclear receptor CoAtivator-1 (NCoA-1) [111]. This interaction is mediated by Protein Kinase A (PKA) phosphorylation in p65/Rel A serine 276 residue unmasking the CPB-interaction domain present in p65/ Rel A. This phosphorylation generates a conformational change that permits a bivalent interaction; first with CBP KIX domain (450–679 aa) and 276 phosphorylated p65-serine and last with CBP region comprised by 313–450 aa CBP and p65 region flanked by 477–504 aa [112]. The transcription of multiple p53-regulated genes is mediated by cyclic-AMP-regulated enhancer (CRE) transcription factor (CREB) and the HAT CBP, p300, and HMT PRMT1, CARM1, and SET7 coactivators that modulate the methylation and acetylation of histones surrounding p53 target genes [113, 114]. The complex CREB–CBP can bind to specific transcription factors where recruit and bind with histone binding factor RbAp48. This CREB–CBP-RbAp48 complex allows the interaction and subsequent CBP/p300 acetylation of target genes histones leading to a chromatin structure rearrange and recruitment of transcription machinery [115–120]. Moreover, An and coworkers demonstrated that in vivo and in vitro PRMT1 and CARM1 interacts directly with p53 trough Nterminal (1–43 aa) and C-terminal (370–393 aa), respectively. Also, they shown that are a cooperatively functions in p53 transcription by p300, PRMT1, and CARM1 coactivators for an optimal p53 transcription activity, being necessary the ordered recruitment to p53-responsive genes: first PRMT1 is recruited and methylate H4R3, then a p300 accumulation and H4 acetylation, and last a subsequent CARM1 accumulation and H3R17 methylation [114]. Like phosphorylation, it has been shown in vitro and in vivo that p53 can be activated and stabilized against ubiquitinmediated degradation by SET7-mediated mono-methylation in residue 372 (p53- K372me1) and, presumably, a subsequent CBP/p300-mediated acetylation [121, 122]. The CBP/p300-p53 complex can interact with multiple p300 and p53 domains. It has been shown that p300 domains like N-terminal Taz1 domain (CH1 domain; 302–451 aa), KIX domain (588–683 aa), C-terminal Taz2 domain (CH3 domain; 1514–1737 aa), and nuclear receptor coactivator binding domain (NCBD; 2059–2117 aa) can interact with p53 TAD (1–61 aa) and DNA-binding Core Domain (90–160 aa) [123–127]. This CBP/p300-p53 interaction promotes p53 C-terminal domain (363–393 aa) acetylation leading to increase in p53-DNA binding and tran-

*The Role of Epigenetics in Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.89819*

scription activity in vivo and in vitro [123, 124, 128, 129].

**29**

Lee and coworkers demonstrated that p53 TAD multisite phosphorylation enhances p53 affinity for Taz1, Taz2, and KIX domains of CBP leading to a graded p53 response to genotoxic stress [130]. On other side, in vivo and in vitro experiments shown that the second zinc finger present in C-terminal region of HPV 16/18- E6 (aa 100–107) interact with CBP/p300 via its Transcriptional Adapter Motif (TRAM), a 19-aa sequence present in CBP II domain, competing with the CBP/ p300-p53 interaction [131]. Also, has been shown that E6 interacts with p300 CH1 domain (340–413 aa) and NCBD domain (1970–2220 aa) generating a E6-p53-p300 complex without E6AP participation. This trimeric complex inhibits both p300 mediated acetylation of p53 and nucleosomal core histones abrogating the p53 dependent transcription activated by CBP/p300. In addition to a p53-E6-E6AP, in vitro and in vivo, HPV18-E6 promotes p53 degradation by direct association and

inhibition of SET7 methyltransferase activity that stabilizes p53 by monomethylation in K372 residue. Whereas not all p53 is promoted to degradation due loss of K372me1, HPV18-E6 can abolishes the p53-dependent remnant gene transcription by direct interaction and downregulation of coactivators CARM1, PRMT1, and SET7 methyltransferase activities, generating a reduced p53 DNA binding and loss of p53 gene expression [122]. Notably, DNMT1 is associated and mono-

The E6 and E7 viral proteins can alter the chromatin structure by association and/or modifying the enzymatic activity and/or altering the expression of chromatin-remodeling enzymes. HPV16-E7 modulates the immune host response downregulating a subset of proteins by methylation. Viral nucleic acids are sensed by a pathogen recognition receptor (PRR) called toll-like receptor 9 (TLR9) that are expressed in keratinocytes. This receptor allows the recognition of unmethylated double-stranded DNA CpG motifs present in the HPV DNA and initiate a signaling cascade that leads to the production of type I Interferon (INF) and proinflammatory cytokines which in turn activates host immune defenses against the infection. Nonetheless, in vitro experiments have been shown that HPV16-E7 suppress TLR9 transcription by inducing the formation of a repressive chromatin modification complex witch is formed by ERα, HDAC1, JARID1B, and NF-kB p50 p65 at specific NF-kB element (site B) of TLR9 promoter. Recruited by ERα, JARID1B prevents the trimethylation of histone 3 at lysin 4 (H3K4me3) and HDAC-1 prevents the acetylation of histone 4 (AcH4) from the site B until the transcription start site of the TLR9 promoter in C33A cells with HPV16 [103]. However, two different reports observed that TLR9 expression was only expressed in fully differentiated keratinocytes and in different layers of HPV-positive cervical epithelia neoplasia and that TLR9 expression is primary intracellular in cervical epithelium [104, 105]. Another study conducted by Canella and collaborators observed that TLR9 expression under presence of low-risk or high-risk HPV and an increase in the TLR9 protein expression in patients with persistent HPV infection. The authors argue that the discrepancies in the TLR9 expression in HPV infected cells reside in a balance between the strength of TLR9 inhibition by HPV and the subject capability to drive proper TLR9 activation [106]. However, further studies are needed to elucidate this data discrepancy.

HPV16-E7 also interferes with downstream signaling of TLRs. It has been seen that E7 interacts in vivo and in vitro with the Interferon Regulatory Factor-1 (IRF-1). IRF-1 is a transcription factor how belong to a family of 9 DNA-binding factors are called from IRF-1 to IRF-9. IRF-1 recognizes a central 11–13 nucleotide core region denominated INF stimulated response elements (ISREs) [107]. These regulatory elements are present in the promoters of INF-β and some INF-inducible genes [108]. HPV16-E7 interacts directly with its CR1/2 domains and the carboxylterminal transactivation domain of IRF-1, eliminating its transactivation function of IRF-1 both in vitro and in vivo. Moreover, the Nucleosome remodeling and deacetylase (NuRD) complex could be implicated since HPV16-E7 interacts directly with Mi2β (a subunit of the NuRD complex) via C-terminal zinc-finger CR3 domain leading to a chromatin deacetylation and silencing IRF-1-dependent transcription suppressing cellular immune response due viral infection [109, 110].

E6 and E7 viral proteins can alter the activity of histone acetyltransferases (HAT) and histone deacetylases (HDAC). NF-κB is a transcription factor composed of homodimers or heterodimers complexes of five subunits named p50, p52, p65/ Rel A, c-Rel, and Rel B; being p50/p65 the most common dimmer. To achieve a

#### *The Role of Epigenetics in Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.89819*

DNA wound around octameric of the four highly conserved histone proteins (H3, H4, H2A, and H2B). Each nucleosome is linked one to other by a stretch of DNA called DNA linker with a length of 40–55 bp. The chromatin gives DNA structure and regulates the gene transcription via post-translational modifications (PTM). This PTM are modifications such acetylation, methylation, phosphorylation, ubiquitination, sumoylation, glycosylation, homocysteinylation, crotonylation, propionylation, and butyrylation in the amino-terminal and carboxy-terminal tail of histones that are mediated by diverse histone modifying enzymes. These PTM regulate gene expression by affecting the nucleosome stability and structure

*Gynaecological Malignancies - Updates and Advances*

The E6 and E7 viral proteins can alter the chromatin structure by association

chromatin-remodeling enzymes. HPV16-E7 modulates the immune host response downregulating a subset of proteins by methylation. Viral nucleic acids are sensed by a pathogen recognition receptor (PRR) called toll-like receptor 9 (TLR9) that are expressed in keratinocytes. This receptor allows the recognition of unmethylated double-stranded DNA CpG motifs present in the HPV DNA and initiate a signaling

proinflammatory cytokines which in turn activates host immune defenses against the infection. Nonetheless, in vitro experiments have been shown that HPV16-E7 suppress TLR9 transcription by inducing the formation of a repressive chromatin modification complex witch is formed by ERα, HDAC1, JARID1B, and NF-kB p50 p65 at specific NF-kB element (site B) of TLR9 promoter. Recruited by ERα, JARID1B prevents the trimethylation of histone 3 at lysin 4 (H3K4me3) and HDAC-1 prevents the acetylation of histone 4 (AcH4) from the site B until the transcription start site of the TLR9 promoter in C33A cells with HPV16 [103]. However, two different reports observed that TLR9 expression was only expressed in fully differentiated keratinocytes and in different layers of HPV-positive cervical epithelia neoplasia and that TLR9 expression is primary intracellular in cervical epithelium [104, 105]. Another study conducted by Canella and collaborators observed that TLR9 expression under presence of low-risk or high-risk HPV and an increase in the TLR9 protein expression in patients with persistent HPV infection. The authors argue that the discrepancies in the TLR9 expression in HPV infected cells reside in a balance between the strength of TLR9 inhibition by HPV and the subject capability to drive proper TLR9 activation [106]. However, further studies are needed to

HPV16-E7 also interferes with downstream signaling of TLRs. It has been seen

that E7 interacts in vivo and in vitro with the Interferon Regulatory Factor-1 (IRF-1). IRF-1 is a transcription factor how belong to a family of 9 DNA-binding factors are called from IRF-1 to IRF-9. IRF-1 recognizes a central 11–13 nucleotide core region denominated INF stimulated response elements (ISREs) [107]. These regulatory elements are present in the promoters of INF-β and some INF-inducible genes [108]. HPV16-E7 interacts directly with its CR1/2 domains and the carboxylterminal transactivation domain of IRF-1, eliminating its transactivation function of

IRF-1 both in vitro and in vivo. Moreover, the Nucleosome remodeling and

suppressing cellular immune response due viral infection [109, 110].

deacetylase (NuRD) complex could be implicated since HPV16-E7 interacts directly with Mi2β (a subunit of the NuRD complex) via C-terminal zinc-finger CR3 domain leading to a chromatin deacetylation and silencing IRF-1-dependent transcription

E6 and E7 viral proteins can alter the activity of histone acetyltransferases (HAT) and histone deacetylases (HDAC). NF-κB is a transcription factor composed of homodimers or heterodimers complexes of five subunits named p50, p52, p65/ Rel A, c-Rel, and Rel B; being p50/p65 the most common dimmer. To achieve a

and/or modifying the enzymatic activity and/or altering the expression of

cascade that leads to the production of type I Interferon (INF) and

[101, 102].

elucidate this data discrepancy.

**28**

correct NF-κB transcription, it is necessary the recruitment and interaction with different transcriptional coactivators like CREB binding protein (CBP), p300, Steroid Receptor-Coactivator-1 (SRC-1), or Nuclear receptor CoAtivator-1 (NCoA-1) [111]. This interaction is mediated by Protein Kinase A (PKA) phosphorylation in p65/Rel A serine 276 residue unmasking the CPB-interaction domain present in p65/ Rel A. This phosphorylation generates a conformational change that permits a bivalent interaction; first with CBP KIX domain (450–679 aa) and 276 phosphorylated p65-serine and last with CBP region comprised by 313–450 aa CBP and p65 region flanked by 477–504 aa [112]. The transcription of multiple p53-regulated genes is mediated by cyclic-AMP-regulated enhancer (CRE) transcription factor (CREB) and the HAT CBP, p300, and HMT PRMT1, CARM1, and SET7 coactivators that modulate the methylation and acetylation of histones surrounding p53 target genes [113, 114]. The complex CREB–CBP can bind to specific transcription factors where recruit and bind with histone binding factor RbAp48. This CREB–CBP-RbAp48 complex allows the interaction and subsequent CBP/p300 acetylation of target genes histones leading to a chromatin structure rearrange and recruitment of transcription machinery [115–120]. Moreover, An and coworkers demonstrated that in vivo and in vitro PRMT1 and CARM1 interacts directly with p53 trough Nterminal (1–43 aa) and C-terminal (370–393 aa), respectively. Also, they shown that are a cooperatively functions in p53 transcription by p300, PRMT1, and CARM1 coactivators for an optimal p53 transcription activity, being necessary the ordered recruitment to p53-responsive genes: first PRMT1 is recruited and methylate H4R3, then a p300 accumulation and H4 acetylation, and last a subsequent CARM1 accumulation and H3R17 methylation [114]. Like phosphorylation, it has been shown in vitro and in vivo that p53 can be activated and stabilized against ubiquitinmediated degradation by SET7-mediated mono-methylation in residue 372 (p53- K372me1) and, presumably, a subsequent CBP/p300-mediated acetylation [121, 122]. The CBP/p300-p53 complex can interact with multiple p300 and p53 domains. It has been shown that p300 domains like N-terminal Taz1 domain (CH1 domain; 302–451 aa), KIX domain (588–683 aa), C-terminal Taz2 domain (CH3 domain; 1514–1737 aa), and nuclear receptor coactivator binding domain (NCBD; 2059–2117 aa) can interact with p53 TAD (1–61 aa) and DNA-binding Core Domain (90–160 aa) [123–127]. This CBP/p300-p53 interaction promotes p53 C-terminal domain (363–393 aa) acetylation leading to increase in p53-DNA binding and transcription activity in vivo and in vitro [123, 124, 128, 129].

Lee and coworkers demonstrated that p53 TAD multisite phosphorylation enhances p53 affinity for Taz1, Taz2, and KIX domains of CBP leading to a graded p53 response to genotoxic stress [130]. On other side, in vivo and in vitro experiments shown that the second zinc finger present in C-terminal region of HPV 16/18- E6 (aa 100–107) interact with CBP/p300 via its Transcriptional Adapter Motif (TRAM), a 19-aa sequence present in CBP II domain, competing with the CBP/ p300-p53 interaction [131]. Also, has been shown that E6 interacts with p300 CH1 domain (340–413 aa) and NCBD domain (1970–2220 aa) generating a E6-p53-p300 complex without E6AP participation. This trimeric complex inhibits both p300 mediated acetylation of p53 and nucleosomal core histones abrogating the p53 dependent transcription activated by CBP/p300. In addition to a p53-E6-E6AP, in vitro and in vivo, HPV18-E6 promotes p53 degradation by direct association and inhibition of SET7 methyltransferase activity that stabilizes p53 by monomethylation in K372 residue. Whereas not all p53 is promoted to degradation due loss of K372me1, HPV18-E6 can abolishes the p53-dependent remnant gene transcription by direct interaction and downregulation of coactivators CARM1, PRMT1, and SET7 methyltransferase activities, generating a reduced p53 DNA binding and loss of p53 gene expression [122]. Notably, DNMT1 is associated and monomethylated in K142 residue (DNMT1-K142) by SET7 causing its degradation [132]. Thus, it is possible that the presence of E6 abrogates the SET7-dependent degradation of DNMT1 increasing the free protein levels that can interact with E7 viral protein, generating an increased activity earlier described of DNMT1-E7 protein complex. Further experiments needed to demonstrate this hypothesis.

Also, hr-HPV 16-E6 disrupt the NF-κB-dependent transactivation by binding competition on N-terminus CH1 domain and C-terminus of CBP that are recognition sites of RelA/p65 and SCR-1, respectively. Furthermore, HPV16-E7 also suppresses the NF-κB-dependent transactivation. The N terminal (1–51 aa) region of E7 viral protein interact both in vitro and in vivo with TAZ2 domain of transcriptional coactivator CBP/p300. Notably, this interaction increases if HPV16-E7 CKII site (Ser31 and Ser32) is phosphorylated [129, 133–137]. hr-HPV16-E7 also can bind to P/CAF HAT domain (352–658 aa) via E7-leucine 67 residue diminishing P/CAF acetyltransferase activity [135].

#### **5. HPV RNA targets**

It has been described that, in humans, less than 3% of genome encodes to protein-coding exons while more than 85% of genome is transcribed into noncoding RNAs (ncRNAs) [138, 139]. These ncRNAs can be classified accordingly by their size as short or long ncRNAs. Micro RNAs (miRNAs) are a group of small noncoding single-strand RNA of 19–24 nucleotides that play key roles in differentiation and development by post-transcriptional regulation of cellular genes. Their main function is to repress the expression of target mRNA by cleavage or translational silencing depending of the degree of miRNA sequence complementation with the 30 -UTR of target mRNAs [140]. The HPV viral proteins can target different RNA species modifying their expression (See **Tables 3**–**5**). For example, HPV16 E2 and E6 viral proteins interact with RNA molecules and reduce the pre-RNA splice efficiency. The N-terminal trans activation domain and the hinge region of HPV16- E2 (1–220 aa and 221–259 aa respectively) and the central region of HPV16-E6 (42– 102 aa) are the responsibly of splicing suppression; whereas the E2 C-terminal DNA-binding domain (260–365 aa) and the E6 C-terminal Nuclear Localization Signal (NLS3) domain (115–124 aa) are the protein portions responsible for protein-RNA interaction. Moreover, HPV16-E2 can interact with splicing factors SRp30, SRp40, SRp55, and SRp75 and HPV16-E6 interacts with SRp30, SRp55, and SRp75 via C-terminal of both viral proteins [173]. miRNA-23b is located in the intron 14 of the host gene C9ORF3 on chromosome 9. This miRNA regulates c-MET gene which mediates cellular apoptosis via AKT signaling pathway. When HPV16-E6 is present, C9ORF3 and the intronic miRNA-23b is downregulated by DNMT1-mediated CGI hypermethylation located 1 kb upstream from the transcription start site of C9ORF3 gene [174].

described early. Moreover, miR-124 and miR-375 mediated a reciprocal regulation with long non-coding RNA MALAT1. If miR-375 is overexpressed a significant reduction in MALAT1 expression is observed. This regulation could be by direct interaction between miR-375 and MALAT1 due miR-375 has two putative MALAT1 binding sites whereas MALAT1 harbors two putative binding sites with miR-124 [169, 178]. Future experiments are necessary to elucidate which factors influence the downregulation of both, cellular and viral gene expression and the molecular factors are involved in HPV E6 and E7 interaction with these miRNAs and

**Gene up-regulated Reference Gene down-regulated** *Reference* AC007879.7 [144] MEG3 [153,154] CCAT [160, 161] MIR205HG [144] CCEPR [163] OIS1 [155] CCHE1 [142, 143] PVT1 [156] FAM83H [144] RP3-510D11.2 [144] GAS5 [144] RP6-65G23.3 [144] GS1-600G8.5 [144] RP11-479G22.8 [144] H19 [144] RP13.463N16.6 [144] HOTAIR [149] RSU1P2 [157] HOXC-As5 [144] SFTA1P [144] LINC00963 [144] SNHG15 [144] LINC01057 [144] SPRY4-IT1 [159] lncRNA LET [151] TMPOP2 (lncRNA-EBIC) [79] MAFG-AS1 [144] XIST [162] MALAT1 [152] XLOC\_010588 [164]

Otherwise, the long non-coding RNAs (lncRNAs) are transcripts of more than 200 nucleotides in length. These RNAs possess structural characteristics of messenger RNAs (mRNAs) like that are transcribed by RNA Polymerase II, spliced, harbor

native splicing, mRNA stability, mRNA translation and chromatin remodeling by bind to RNA, DNA, or a subset of proteins. Interestingly, Khalil and colleagues showed that the mammalian genome encodes nearly 4500 lncRNAs and approximately 24% of these lncRNAS interact with chromatin-modifying proteins like the repressive complex PRC2, CoREST, and SCMX [179]. Due their role in distinct cellular processes, HPV viral proteins can modulate multiple host's lncRNAs [140]. As described earlier, the long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was associated with cell proliferation and invasion in HPV positive cervical cancer cells [152, 169, 180]. Also, in CaSki cell line, the transfection of MALAT1 increases the expression of cyclin D1, cyclin E and cyclin-dependent kinase 6 (CDK6). When HPV16 E6 and E7 are downregulated, MALAT1 expression is downregulated too, indicating that these viral proteins are involved in the MALAT1 expression [152]. However, further studies are needed to

Barr and colleagues identify a subset of lncRNAs upper and downregulated in primary human foreskin keratinocytes which express HPV16-E6 viral protein. The

elucidate the mechanism of MALAT1 regulation by HPV.


MALAT1.

**31**

**Table 3.**

a poly adenylated tail, and a 5<sup>0</sup>

*lncRNAs reported up- and down-regulated in literature.*

*The Role of Epigenetics in Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.89819*

The miR-375 has been shown to regulate the HPV viral gene expression in vitro and in vivo. miR-375 can downregulates E6 and E7 viral transcription due the presence of two putative binding sites present in the E7 region (677–698 aa; 687–708 aa) and three in the E1 region (1236–1258 aa; 1259–1280 aa; 1862–1884 aa) of the HPV genome. Also, this miRNA in vivo and in vitro can bind directly the 3<sup>0</sup> UTR of E6AP and the transcription factor SP1 diminishing E6AP and SP1 mRNA and protein. As a result of E6AP and SP1 proteins degradation mediated by miR-375, an increase in p21, p53, and Rb proteins can be observed [175–177]. However, in vitro assays demonstrated that HPV16-E6 can hypermethylate DNMT1-mediated miR-375 promoter region [178] downregulating miR-375 and leading an increase in SP1 transcription factor levels, thereby, contributing to DNMT1-positive loop feedback

#### *The Role of Epigenetics in Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.89819*


#### **Table 3.**

methylated in K142 residue (DNMT1-K142) by SET7 causing its degradation [132]. Thus, it is possible that the presence of E6 abrogates the SET7-dependent degradation of DNMT1 increasing the free protein levels that can interact with E7 viral protein, generating an increased activity earlier described of DNMT1-E7 protein

Also, hr-HPV 16-E6 disrupt the NF-κB-dependent transactivation by binding competition on N-terminus CH1 domain and C-terminus of CBP that are recognition sites of RelA/p65 and SCR-1, respectively. Furthermore, HPV16-E7 also suppresses the NF-κB-dependent transactivation. The N terminal (1–51 aa) region of E7 viral protein interact both in vitro and in vivo with TAZ2 domain of transcriptional coactivator CBP/p300. Notably, this interaction increases if HPV16-E7 CKII site (Ser31 and Ser32) is phosphorylated [129, 133–137]. hr-HPV16-E7 also can bind to P/CAF HAT domain (352–658 aa) via E7-leucine 67 residue diminishing P/CAF

It has been described that, in humans, less than 3% of genome encodes to protein-coding exons while more than 85% of genome is transcribed into noncoding RNAs (ncRNAs) [138, 139]. These ncRNAs can be classified accordingly by their size as short or long ncRNAs. Micro RNAs (miRNAs) are a group of small noncoding single-strand RNA of 19–24 nucleotides that play key roles in differentiation and development by post-transcriptional regulation of cellular genes. Their main function is to repress the expression of target mRNA by cleavage or translational silencing depending of the degree of miRNA sequence complementation with the


The miR-375 has been shown to regulate the HPV viral gene expression in vitro

UTR of

and in vivo. miR-375 can downregulates E6 and E7 viral transcription due the presence of two putative binding sites present in the E7 region (677–698 aa; 687–708 aa) and three in the E1 region (1236–1258 aa; 1259–1280 aa; 1862–1884 aa) of the HPV genome. Also, this miRNA in vivo and in vitro can bind directly the 3<sup>0</sup>

E6AP and the transcription factor SP1 diminishing E6AP and SP1 mRNA and protein. As a result of E6AP and SP1 proteins degradation mediated by miR-375, an increase in p21, p53, and Rb proteins can be observed [175–177]. However, in vitro assays demonstrated that HPV16-E6 can hypermethylate DNMT1-mediated miR-375 promoter region [178] downregulating miR-375 and leading an increase in SP1 transcription factor levels, thereby, contributing to DNMT1-positive loop feedback

complex. Further experiments needed to demonstrate this hypothesis.

*Gynaecological Malignancies - Updates and Advances*

acetyltransferase activity [135].

**5. HPV RNA targets**

30

gene [174].

**30**

*lncRNAs reported up- and down-regulated in literature.*

described early. Moreover, miR-124 and miR-375 mediated a reciprocal regulation with long non-coding RNA MALAT1. If miR-375 is overexpressed a significant reduction in MALAT1 expression is observed. This regulation could be by direct interaction between miR-375 and MALAT1 due miR-375 has two putative MALAT1 binding sites whereas MALAT1 harbors two putative binding sites with miR-124 [169, 178]. Future experiments are necessary to elucidate which factors influence the downregulation of both, cellular and viral gene expression and the molecular factors are involved in HPV E6 and E7 interaction with these miRNAs and MALAT1.

Otherwise, the long non-coding RNAs (lncRNAs) are transcripts of more than 200 nucleotides in length. These RNAs possess structural characteristics of messenger RNAs (mRNAs) like that are transcribed by RNA Polymerase II, spliced, harbor a poly adenylated tail, and a 5<sup>0</sup> -caping. lncRNAs can modulate transcription, alternative splicing, mRNA stability, mRNA translation and chromatin remodeling by bind to RNA, DNA, or a subset of proteins. Interestingly, Khalil and colleagues showed that the mammalian genome encodes nearly 4500 lncRNAs and approximately 24% of these lncRNAS interact with chromatin-modifying proteins like the repressive complex PRC2, CoREST, and SCMX [179]. Due their role in distinct cellular processes, HPV viral proteins can modulate multiple host's lncRNAs [140].

As described earlier, the long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was associated with cell proliferation and invasion in HPV positive cervical cancer cells [152, 169, 180]. Also, in CaSki cell line, the transfection of MALAT1 increases the expression of cyclin D1, cyclin E and cyclin-dependent kinase 6 (CDK6). When HPV16 E6 and E7 are downregulated, MALAT1 expression is downregulated too, indicating that these viral proteins are involved in the MALAT1 expression [152]. However, further studies are needed to elucidate the mechanism of MALAT1 regulation by HPV.

Barr and colleagues identify a subset of lncRNAs upper and downregulated in primary human foreskin keratinocytes which express HPV16-E6 viral protein. The


high expression of FAM83H-AS1 correlates with worse overall survival compared

**Gene Reference Gene Reference** let-7a-c [145] miR-218 [99, 166, 167] let-7b [145] miR-23b [145, 166] let-7c [145] miR-26a [141] miR-100 [168] miR-29a [167] miR-101 [166] miR-328 [168] miR-10b [167] miR-34a [166] miR-124 [169] miR-368 [170] miR-125b [167, 168, 171] miR-370 [171] miR-126 [167, 170] miR-375 [167, 168] miR-139-3p [168] miR-379 [168] miR-139-5p [168] miR-381 [168] miR-143 [166, 170] miR-424 [166, 167] miR-145 [166, 168, 170] miR-433 [172] miR-149 [168] miR-494 [171] miR-188 [171] miR-497 [168, 170] miR-193b [171] miR-513 [141] miR-195 [167, 168, 170] miR-572 [171] miR-196b [145] miR-574-3p [168] miR-199a [141] miR-575 [171] miR-199a-5p [168] miR-617 [168] miR-199b-5p [168] miR-638 [171] miR-203 [171] miR-99a [167, 168]

and recruits the PRC2 to repress transcription of multiple gene loci in trans.

HPV16-E7 protein levels too, generating a regulatory loop [183, 184].

The lncRNA HOX Transcript Antisense Intergenic RNA (HOTAIR) can binds to

HOTAIR expression is downregulated in earlier stages of cervical cancer. However, in HPV16 positive cervical carcinomas and in HPV positive cell lines which harbor a higher HPV16-E7 protein expression, the lncRNA HOTAIR is upregulated correlating with high HPV16-E7 expression level. Moreover, HPV16-E7 interacts with HOTAIR. This interaction could impair the formation of the PCR2 complex generating diminish of H3K27me3 repression mark and thus increasing the expression of a large number of genes [149, 181, 182]. Interestingly, the HPV16-E7-HOTAIR interaction generates an autoregulatory loop between HOTAIR, miR-331-3p and Neuropilin 2 (NRP2). It has been shown that HOTAIR is a competitive endogenous RNA (ceRNA) showing a sponge effect over miR-331-3p and that miR-331-3p directly regulates NRP2. So, when is present, HPV16-E7 interacts and diminishes HOTAIR expression generating an increase of miR-331-3p levels due the lack of HOTAIR sponge effect over miR-331-3p. The miR-331-3p induce a decrease of NRP2 levels by binding through 3'UTR of NRP. Being NRP2 a HPV16-E7 transcription regulator, the downregulation of NRP2 protein levels lead to a diminished

with normal cervix samples [144].

*miRNAs reported down-regulated in literature.*

*The Role of Epigenetics in Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.89819*

**Table 5.**

**33**

#### **Table 4.**

*miRNAs reported up-regulated in literature.*

authors found that FAM83H-AS1 is overexpressed by HPV16-E6 viral protein mediated by p300, and its inhibition decrease proliferation, migration, and resistance to apoptosis in vitro, whereas in pre-malignant and cervical cancer tissues the

#### *The Role of Epigenetics in Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.89819*


#### **Table 5.**

*miRNAs reported down-regulated in literature.*

high expression of FAM83H-AS1 correlates with worse overall survival compared with normal cervix samples [144].

The lncRNA HOX Transcript Antisense Intergenic RNA (HOTAIR) can binds to and recruits the PRC2 to repress transcription of multiple gene loci in trans. HOTAIR expression is downregulated in earlier stages of cervical cancer. However, in HPV16 positive cervical carcinomas and in HPV positive cell lines which harbor a higher HPV16-E7 protein expression, the lncRNA HOTAIR is upregulated correlating with high HPV16-E7 expression level. Moreover, HPV16-E7 interacts with HOTAIR. This interaction could impair the formation of the PCR2 complex generating diminish of H3K27me3 repression mark and thus increasing the expression of a large number of genes [149, 181, 182]. Interestingly, the HPV16-E7-HOTAIR interaction generates an autoregulatory loop between HOTAIR, miR-331-3p and Neuropilin 2 (NRP2). It has been shown that HOTAIR is a competitive endogenous RNA (ceRNA) showing a sponge effect over miR-331-3p and that miR-331-3p directly regulates NRP2. So, when is present, HPV16-E7 interacts and diminishes HOTAIR expression generating an increase of miR-331-3p levels due the lack of HOTAIR sponge effect over miR-331-3p. The miR-331-3p induce a decrease of NRP2 levels by binding through 3'UTR of NRP. Being NRP2 a HPV16-E7 transcription regulator, the downregulation of NRP2 protein levels lead to a diminished HPV16-E7 protein levels too, generating a regulatory loop [183, 184].

authors found that FAM83H-AS1 is overexpressed by HPV16-E6 viral protein mediated by p300, and its inhibition decrease proliferation, migration, and resistance to apoptosis in vitro, whereas in pre-malignant and cervical cancer tissues the

miR-146a [166] miR-223 [166] miR-146b-5p [168] miR-224 [167] miR-148a [141] miR-25 [165] miR-150 [165] miR-26a [165] miR-151 [165] miR-26b [165] miR-155 [166–168] miR-28 [165] miR-15 [165, 166, 168] miR-29a [165] miR-15b [166, 167, 171] miR-29b [165] miR-16 [167, 171, 172] miR-301 [165] miR-17 [168] miR-301b [172] miR-181a [165] miR-302b [141] miR-181b [165] miR-30a-3p [165]

**Table 4.**

**32**

*miRNAs reported up-regulated in literature.*

**Gene up-regulated Reference Gene up-regulated Reference Gene up-regulated Reference** let-7e [165] miR-181c [165, 172] miR-30b [165] let-7i [165] miR-182 [170] miR-30d [165] miR-106a [167, 168] miR-183 [170] miR-30e [165] miR-106b [168, 171, 172] miR-185 [168] miR-326 [165] miR-10 [165] miR-186 [165] miR-339-5p [168] miR-10b [168] miR-187 [165] miR-340 [165] miR-1224-5p [168] miR-192 [172] miR-342 [165] miR-124 [172] miR-194 [165] miR-34a [165] miR-126 [165] miR-195 [165] miR-34c [165] miR-127 [165] miR-196a [141] miR-374 [165] miR-129 [165] miR-199a [165] miR-449a [172] miR-130a [165] miR-199b [165] miR-449b [172] miR-130b [165, 168] miR-199s [165] miR-512-3p [172] miR-132 [141, 165] miR-19a [165] miR-517a [172] miR-133a [165] miR-20 [165] miR-517c [172] miR-133b [165] miR-200a [165] miR-518f [172] miR-134 [165] miR-200c [170] miR-542-3p [172] miR-135a [165] miR-205 [170] miR-545 [172] miR-135b [165, 172] miR-20a [158, 167] miR-625 [168] miR-139 [165] miR-20b [168] miR-7g [165] miR-140 [165] miR-21 [145, 165, 168, 171] miR-886-5p [167] miR-141 [172] miR-210 [170] miR-9 [165] miR-142-3p [165] miR-213 [165] miR-92a [167] miR-142-5p [165] miR-214 [165] miR-93 [167, 168] miR-145 [165] miR-215 [165] miR-941 [168] miR-146 [165] miR-218 [165] miR-98 [165]

*Gynaecological Malignancies - Updates and Advances*

As described early, thymopoietin pseudogene 2 (TMPOP2, lncRNA-EBIC) is a lncRNA that interact with EZH2 to repress E-cadherin gene expression. Interestingly, this lncRNA regulates the expression of HPV viral genes in cervical cancer cells. Several miRNAs, like miR-375 and miR-139, can target to degradation the HPV16/18 E6 and E7 mRNA. However, lncRNA-EBIC also acts as a ceRNA, sequestering miR-375 and miR-139 increasing the E6 and E7 viral gene expression. Moreover, the upregulation of E6 and E7 by lncRNA-EBIC lead to p53 degradation which is a transcriptional repressor of lncRNA-EBIC, generating a positive loop feedback [79].

they observed a restoration of APC gene expression in HeLa and CaSki cervical cancer cells. This gene re-expression was due to APC promoter region demethylation [55]. In 2005, Zambrano and colleagues mounted a phase 1 study of hydralazine employing different dosages (from 25 mg/8 h to 50 mg/8 h) for a 10 days period. They found that employing any hydralazine concentration tested, eight tumor suppressors genes were demethylate and re-expressed in untreated cervical

Another compound capable to restore gene expression of tumor suppressor genes hypermethylated is Trichosanthin (TCS). TCS is a 237 aa type I ribosomeinactivating protein extracted from the root tubers of the Chinese medical herb *Trichocanthes kirilowi*. Huang and colleagues reported increases mRNA and protein levels of APC and TSLC1 due demethylation in the CpG islands in the promoter region in HeLa and CaSki cervical cancer cells treated with 20, 40 and 80 μg/ml for 48 h presumable mediated by DNMT1 since its mRNA, protein levels, and enzyme activity decreases following the treatment in a dose-dependent manner [68]. However, until these data shown a likely useful as a demethylating agent for treatment, this work does not report the toxicity effects over non-transformed cell lines. In another study, hydralazine was proved in combination with the HDAC inhibitor valproate acid. After 5 days of Hydralazine at 10 μM and magnesium Valproate at 1 mM treatment, SiHa, CasKi, and HeLa cervical cancer cells lead to a small increase HPV gene expression due demethylation and acetylated H4 enrichment at 5'region of LCR. However, a p53 gene expression and protein levels were increased after treatment whit Hydralazine, Valproate, or in combination in CasKi, HeLa, and SiHa cell lines being p53 stability likely due 373 and 382 lysine p53 hyperacetylation that protects from E6-mediated degradation. Also, the hydralazine/valproate phase II trial with treatment of Hydralazine at 182 or 83 mg and magnesium Valproate at 40 mg/kg shown that E6 and E7 transcripts remains unchanged in primary tumors of patients with cervical cancer, suggesting that epigenetic therapy cannot facilitate increase of viral oncogene activation [186]. On the other hand, apicidin, an inhibitor of histone deacetylases, induces downregulation of DNMT1 and increase p21WAF1/Cip1 expression in HeLa cervical cancer cell line. The Apicidin-mediated DNMT1 downregulation is achieved by a significant H3 and H4 hypoacetylation, depletion of H3K4me3 gene transcription mark, and enriched H3K9me3 and H3K27me3 repressive marks in the nucleosomes on DNMT1 transcriptional initiation site. Moreover, Apicidin treatment lead to a decreased Pol II presence on the transcription initiation site and the recruitment of co-repressors pRB and HDAC1 and dissociation of activators P/CAF and HAT from the E2F consensus-binding site on the DNMT1 promoter site. However, HeLa cells treated solely with Apicidin does not induce apoptosis of HeLa cells in comparison of DNMT1 knock down which cause an apoptotic effect, indicating that other

cancer patients without affecting global DNA methylation [185].

*The Role of Epigenetics in Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.89819*

targets are needed to achieve Apicidin therapeutic effect [187].

**35**

Quercetin a flavonoid found in fruits and vegetables also have epigenetics effects, it has been reported that quercetin induces attenuating lipid peroxidation, platelet aggregation, capillary permeability, anti-proliferative, anti-migratory, and proapoptotic effect in HeLa cervical carcinoma cells [188]. Employing doses of 25 and 50 μM, Quercetin can inhibit the activity of DNMT1, HDACs, H3K9 HMT activity, in a dose-dependent manner. Using the same Quercetin concentrations was observed a decreased methylation percentage and increase APC, CDH1, CDH13, DAPK1, FHTI, GSTP1, MGMT, MLH1, PTEN, RARB, RASSF1, SOC51, TIMP3, and VHL expression and a global DNA methylation in a dose-dependent manner. Also, Quercetin modulates the expression of several enzymes and chromatin modifiers like HDAC2, HDAC1, DNMT1, HDAC3, HAT1, DNMT3B, HDAC7, HDAC6, HDAC11, DNMT3A, and HDAC5 in a dose-dependent manner [189]. Interestingly,

The lncRNA LET [151], GAS5 [146], and MEG3 [153, 154] expression is downregulated in cervical cancer tissues and is associated with poor prognosis, malignant status, lymph node metastasis, invasion, and shorter overall survival. The expression of MEG3 leads to an increase in cell apoptosis, increased levels of p53 and cleaved caspase 3 in cervical cancer cells. Also, this lncRNA can regulate the expression levels of miR-21-5p [153, 154].

On the contrary, the lnc Ras Suppressor Protein 1 Pseudogene 2 (RSU1P2) expression is upregulated in cervical cancer tissues and promotes proliferation, invasion, and migration of cervical cancer cell lines. Moreover, in vitro and in vivo assays demonstrated that RSU1P2 acts as ceRNA binding directly to and downregulating let7a expression, leading to an increase of Let-7a target genes as IGF1R, N-myc, and EphA4. Interestingly, let-7a can target the 3-UTR of N-Myc inhibiting its mRNA and protein production, whereas N-Myc can bind to RSU1P2 promoter region and increase its transcription. Therefore, N-Myc can forms a positive loop feedback with RSU1P2 increasing its oncogenic activity [157]. If any HPV viral protein can modulate this pathway is currently unknown.

The lncRNA Plasmacytoma Variant Translocation 1 (PVT1) expression is upregulated in cervical cancer tissues and correlates positively with poor overall survival. If PVT1 expression is inhibit a decrease in cellular proliferation, migration, and invasion is observed whereas apoptosis and cisplatin toxicity increase in cervical cancer cell lines [156].

There are numerous lncRNAs that have been poorly investigated in their molecular mechanism in HPV-infected cervical carcinoma cells. However, some studies described the correlations between lncRNAS expression and clinical characteristics of cervical cancer patients. For example, the lncRNA Colon Cancer-Associated Transcript 2 (CCAT2) [160, 161], SPRY4-IT1 [159], and CCHE1 [142] are highly expressed and positively associated with cell proliferation and survival of cervical cancer cells as well malignant status and poor prognosis of cervical cancer patients. CCHE1 high expression promotes cell proliferation of cervical cancer cells. Interestingly, CCHE1 physically interacts with Proliferating Cell Nuclear Antigen (PCNA) mRNA increasing the PCNA gene expression. This PCNA expression is necessary for the proliferation effect of CCHE1 [143].

#### **6. Therapeutic approaches**

The balance alteration of oncogenes and tumor-suppressor genes creates an advantage to cancer cells. Many of these alterations are due epigenetic alterations such DNA methylation, histone modification, and/or non-coding RNAs expression/ repression. However, this cancer cells advantage can serve also as therapeutic targets to counterattack cancer pathogenesis and progression. Currently, there are some studies describing drugs that alter these epigenetic changes present in cervical cancer cells.

A study employs a peripheral vasodilator drug and DNA methylation inhibitor called Hydralazine. The authors employed hydralazine at 40 μmol/L for 72 h and

#### *The Role of Epigenetics in Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.89819*

As described early, thymopoietin pseudogene 2 (TMPOP2, lncRNA-EBIC) is a lncRNA that interact with EZH2 to repress E-cadherin gene expression. Interestingly, this lncRNA regulates the expression of HPV viral genes in cervical cancer cells. Several miRNAs, like miR-375 and miR-139, can target to degradation the HPV16/18 E6 and E7 mRNA. However, lncRNA-EBIC also acts as a ceRNA, sequestering miR-375 and miR-139 increasing the E6 and E7 viral gene expression. Moreover, the upregulation of E6 and E7 by lncRNA-EBIC lead to p53 degradation which is a transcriptional repressor of lncRNA-EBIC, generating a positive loop feedback [79]. The lncRNA LET [151], GAS5 [146], and MEG3 [153, 154] expression is downregulated in cervical cancer tissues and is associated with poor prognosis, malignant status, lymph node metastasis, invasion, and shorter overall survival. The expression of MEG3 leads to an increase in cell apoptosis, increased levels of p53 and cleaved caspase 3 in cervical cancer cells. Also, this lncRNA can regulate the

On the contrary, the lnc Ras Suppressor Protein 1 Pseudogene 2 (RSU1P2) expression is upregulated in cervical cancer tissues and promotes proliferation, invasion, and migration of cervical cancer cell lines. Moreover, in vitro and in vivo

downregulating let7a expression, leading to an increase of Let-7a target genes as IGF1R, N-myc, and EphA4. Interestingly, let-7a can target the 3-UTR of N-Myc inhibiting its mRNA and protein production, whereas N-Myc can bind to RSU1P2 promoter region and increase its transcription. Therefore, N-Myc can forms a positive loop feedback with RSU1P2 increasing its oncogenic activity [157]. If any

The lncRNA Plasmacytoma Variant Translocation 1 (PVT1) expression is upregulated in cervical cancer tissues and correlates positively with poor overall survival. If PVT1 expression is inhibit a decrease in cellular proliferation, migration, and invasion is observed whereas apoptosis and cisplatin toxicity increase in cervi-

There are numerous lncRNAs that have been poorly investigated in their molecular mechanism in HPV-infected cervical carcinoma cells. However, some studies described the correlations between lncRNAS expression and clinical characteristics of cervical cancer patients. For example, the lncRNA Colon Cancer-Associated Transcript 2 (CCAT2) [160, 161], SPRY4-IT1 [159], and CCHE1 [142] are highly expressed and positively associated with cell proliferation and survival of cervical cancer cells as well malignant status and poor prognosis of cervical cancer patients. CCHE1 high expression promotes cell proliferation of cervical cancer cells. Interestingly, CCHE1 physically interacts with Proliferating Cell Nuclear Antigen (PCNA) mRNA increasing the PCNA gene expression. This PCNA expression is

The balance alteration of oncogenes and tumor-suppressor genes creates an advantage to cancer cells. Many of these alterations are due epigenetic alterations such DNA methylation, histone modification, and/or non-coding RNAs expression/ repression. However, this cancer cells advantage can serve also as therapeutic targets to counterattack cancer pathogenesis and progression. Currently, there are some studies describing drugs that alter these epigenetic changes present in cervical

A study employs a peripheral vasodilator drug and DNA methylation inhibitor called Hydralazine. The authors employed hydralazine at 40 μmol/L for 72 h and

assays demonstrated that RSU1P2 acts as ceRNA binding directly to and

HPV viral protein can modulate this pathway is currently unknown.

necessary for the proliferation effect of CCHE1 [143].

expression levels of miR-21-5p [153, 154].

*Gynaecological Malignancies - Updates and Advances*

cal cancer cell lines [156].

**6. Therapeutic approaches**

cancer cells.

**34**

they observed a restoration of APC gene expression in HeLa and CaSki cervical cancer cells. This gene re-expression was due to APC promoter region demethylation [55]. In 2005, Zambrano and colleagues mounted a phase 1 study of hydralazine employing different dosages (from 25 mg/8 h to 50 mg/8 h) for a 10 days period. They found that employing any hydralazine concentration tested, eight tumor suppressors genes were demethylate and re-expressed in untreated cervical cancer patients without affecting global DNA methylation [185].

Another compound capable to restore gene expression of tumor suppressor genes hypermethylated is Trichosanthin (TCS). TCS is a 237 aa type I ribosomeinactivating protein extracted from the root tubers of the Chinese medical herb *Trichocanthes kirilowi*. Huang and colleagues reported increases mRNA and protein levels of APC and TSLC1 due demethylation in the CpG islands in the promoter region in HeLa and CaSki cervical cancer cells treated with 20, 40 and 80 μg/ml for 48 h presumable mediated by DNMT1 since its mRNA, protein levels, and enzyme activity decreases following the treatment in a dose-dependent manner [68]. However, until these data shown a likely useful as a demethylating agent for treatment, this work does not report the toxicity effects over non-transformed cell lines.

In another study, hydralazine was proved in combination with the HDAC inhibitor valproate acid. After 5 days of Hydralazine at 10 μM and magnesium Valproate at 1 mM treatment, SiHa, CasKi, and HeLa cervical cancer cells lead to a small increase HPV gene expression due demethylation and acetylated H4 enrichment at 5'region of LCR. However, a p53 gene expression and protein levels were increased after treatment whit Hydralazine, Valproate, or in combination in CasKi, HeLa, and SiHa cell lines being p53 stability likely due 373 and 382 lysine p53 hyperacetylation that protects from E6-mediated degradation. Also, the hydralazine/valproate phase II trial with treatment of Hydralazine at 182 or 83 mg and magnesium Valproate at 40 mg/kg shown that E6 and E7 transcripts remains unchanged in primary tumors of patients with cervical cancer, suggesting that epigenetic therapy cannot facilitate increase of viral oncogene activation [186].

On the other hand, apicidin, an inhibitor of histone deacetylases, induces downregulation of DNMT1 and increase p21WAF1/Cip1 expression in HeLa cervical cancer cell line. The Apicidin-mediated DNMT1 downregulation is achieved by a significant H3 and H4 hypoacetylation, depletion of H3K4me3 gene transcription mark, and enriched H3K9me3 and H3K27me3 repressive marks in the nucleosomes on DNMT1 transcriptional initiation site. Moreover, Apicidin treatment lead to a decreased Pol II presence on the transcription initiation site and the recruitment of co-repressors pRB and HDAC1 and dissociation of activators P/CAF and HAT from the E2F consensus-binding site on the DNMT1 promoter site. However, HeLa cells treated solely with Apicidin does not induce apoptosis of HeLa cells in comparison of DNMT1 knock down which cause an apoptotic effect, indicating that other targets are needed to achieve Apicidin therapeutic effect [187].

Quercetin a flavonoid found in fruits and vegetables also have epigenetics effects, it has been reported that quercetin induces attenuating lipid peroxidation, platelet aggregation, capillary permeability, anti-proliferative, anti-migratory, and proapoptotic effect in HeLa cervical carcinoma cells [188]. Employing doses of 25 and 50 μM, Quercetin can inhibit the activity of DNMT1, HDACs, H3K9 HMT activity, in a dose-dependent manner. Using the same Quercetin concentrations was observed a decreased methylation percentage and increase APC, CDH1, CDH13, DAPK1, FHTI, GSTP1, MGMT, MLH1, PTEN, RARB, RASSF1, SOC51, TIMP3, and VHL expression and a global DNA methylation in a dose-dependent manner. Also, Quercetin modulates the expression of several enzymes and chromatin modifiers like HDAC2, HDAC1, DNMT1, HDAC3, HAT1, DNMT3B, HDAC7, HDAC6, HDAC11, DNMT3A, and HDAC5 in a dose-dependent manner [189]. Interestingly,

those therapeutic approaches described here where tested employing cervical cancer models. However, it would be interesting explore the effectiveness of these approaches on HPV-infected anus and oral models where HPV is associated with malignant transformation [150, 190–192].

**References**

rvical-cancer/en/

1789-1799

1782-1783

2016;**16**(1):70

**37**

[1] Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians. 2018;**68**(6):394-424

*The Role of Epigenetics in Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.89819*

> persistence in Guanacaste, Costa Rica. The Journal of infectious diseases. 2005;

> [8] International Collaboration of Epidemiological Studies of Cervical Cancer, Appleby P, Beral V, Berrington de Gonzalez A, Colin D, Franceschi S, et al. Carcinoma of the cervix and tobacco smoking: Collaborative reanalysis of individual data on 13,541 women with carcinoma of the cervix and 23,017 women without carcinoma of the cervix from 23 epidemiological studies. International Journal of Cancer.

**191**(11):1808-1816

2006;**118**(6):1481-1495

1505-1513

**143**(7):1442-1450

**121**(3):621-632

(Suppl 5):F55-F70

[9] Marks M, Gravitt PE, Gupta SB, Liaw KL, Tadesse A, Kim E, et al. Combined oral contraceptive use increases HPV persistence but not new HPV detection in a cohort of women from Thailand. The Journal of Infectious Diseases. 2011;**204**(10):

[10] Oh HY, Kim MK, Seo S, Lee DO, Chung YK, Lim MC, et al. Alcohol consumption and persistent infection of high-risk human papillomavirus. Epidemiology and Infection. 2015;

[11] Hanahan D, Weinberg RA. Hallmarks of cancer: The next

generation. Cell. 2011;**144**(5):646-674

[12] Smith JS, Lindsay L, Hoots B, Keys J, Franceschi S, Winer R, et al. Human papillomavirus type distribution in invasive cervical cancer and high-grade cervical lesions: A meta-analysis update. International Journal of Cancer. 2007;

[13] Doorbar J, Quint W, Banks L, Bravo IG, Stoler M, Broker TR, et al. The

biology and life-cycle of human papillomaviruses. Vaccine. 2012;**30**

[2] WHO. Cervical Cancer. Geneva: World Health Organization; 2018. Available from: http://www.who.int/ca ncer/prevention/diagnosis-screening/ce

[3] Bruni L, Diaz M, Castellsague X, Ferrer E, Bosch FX, de Sanjose S. Cervical human papillomavirus prevalence in 5 continents: Metaanalysis of 1 million women with normal cytological findings. The Journal of Infectious Diseases. 2010;**202**(12):

[4] Nobbenhuis MA, Helmerhorst TJ, van den Brule AJ, Rozendaal L, Voorhorst FJ, Bezemer PD, et al. Cytological regression and clearance of high-risk human papillomavirus in women with an abnormal cervical smear. Lancet. 2001;**358**(9295):

[5] Schiffman M, Doorbar J,

Wentzensen N, de Sanjose S, Fakhry C, Monk BJ, et al. Carcinogenic human papillomavirus infection. Nature Reviews. Disease Primers. 2016;**2**:16086

[6] Pirtea L, Grigoras D, Matusz P, Pirtea M, Moleriu L, Tudor A, et al. Age and HPV type as risk factors for HPV persistence after loop excision in

patients with high grade cervical lesions: An observational study. BMC Surgery.

[7] Castle PE, Schiffman M, Herrero R,

Bratti MC, et al. A prospective study of

Hildesheim A, Rodriguez AC,

age trends in cervical human papillomavirus acquisition and

### **7. Conclusions**

Here we describe the epigenetic regulation mechanisms observed when hr-HPV is present in cervical cancer. The viral oncoproteins expression from hr-HPV induce genetic and epigenetic changes in the cells that contribute to malignant transformation and development of cervical cancer. These modifications could be used as biomarkers and new therapeutic molecules that could help in the treatment of cervical cancer.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Yair Alfaro-Mora<sup>1</sup> , Luis A. Herrera<sup>2</sup> , Rodrigo Cáceres-Gutiérrez<sup>2</sup> , Marco A. Andonegui-Elguera<sup>2</sup> , Guadalupe Dominguez-Gómez<sup>2</sup> and José Díaz-Chávez<sup>2</sup> \*

1 Department of Genetics and Molecular Biology, Center for Research and Advanced Studies (CINVESTAV-IPN), Mexico City, México

2 Biomedical Research Unit in Cancer, Biomedical Research Institute, UNAM/ National Cancer Institute (INCan), Mexico City, México

\*Address all correspondence to: josediaz030178@hotmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*The Role of Epigenetics in Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.89819*

#### **References**

those therapeutic approaches described here where tested employing cervical cancer models. However, it would be interesting explore the effectiveness of these approaches on HPV-infected anus and oral models where HPV is associated with

Here we describe the epigenetic regulation mechanisms observed when hr-HPV is present in cervical cancer. The viral oncoproteins expression from hr-HPV induce genetic and epigenetic changes in the cells that contribute to malignant transformation and development of cervical cancer. These modifications could be used as biomarkers and new therapeutic molecules that could help in the treatment of

, Rodrigo Cáceres-Gutiérrez<sup>2</sup>

, Guadalupe Dominguez-Gómez<sup>2</sup>

1 Department of Genetics and Molecular Biology, Center for Research and

2 Biomedical Research Unit in Cancer, Biomedical Research Institute, UNAM/

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

,

malignant transformation [150, 190–192].

*Gynaecological Malignancies - Updates and Advances*

The authors declare no conflict of interest.

, Luis A. Herrera<sup>2</sup>

Advanced Studies (CINVESTAV-IPN), Mexico City, México

\*Address all correspondence to: josediaz030178@hotmail.com

National Cancer Institute (INCan), Mexico City, México

\*

provided the original work is properly cited.

**7. Conclusions**

cervical cancer.

**Author details**

Yair Alfaro-Mora<sup>1</sup>

**36**

and José Díaz-Chávez<sup>2</sup>

Marco A. Andonegui-Elguera<sup>2</sup>

**Conflict of interest**

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[2] WHO. Cervical Cancer. Geneva: World Health Organization; 2018. Available from: http://www.who.int/ca ncer/prevention/diagnosis-screening/ce rvical-cancer/en/

[3] Bruni L, Diaz M, Castellsague X, Ferrer E, Bosch FX, de Sanjose S. Cervical human papillomavirus prevalence in 5 continents: Metaanalysis of 1 million women with normal cytological findings. The Journal of Infectious Diseases. 2010;**202**(12): 1789-1799

[4] Nobbenhuis MA, Helmerhorst TJ, van den Brule AJ, Rozendaal L, Voorhorst FJ, Bezemer PD, et al. Cytological regression and clearance of high-risk human papillomavirus in women with an abnormal cervical smear. Lancet. 2001;**358**(9295): 1782-1783

[5] Schiffman M, Doorbar J, Wentzensen N, de Sanjose S, Fakhry C, Monk BJ, et al. Carcinogenic human papillomavirus infection. Nature Reviews. Disease Primers. 2016;**2**:16086

[6] Pirtea L, Grigoras D, Matusz P, Pirtea M, Moleriu L, Tudor A, et al. Age and HPV type as risk factors for HPV persistence after loop excision in patients with high grade cervical lesions: An observational study. BMC Surgery. 2016;**16**(1):70

[7] Castle PE, Schiffman M, Herrero R, Hildesheim A, Rodriguez AC, Bratti MC, et al. A prospective study of age trends in cervical human papillomavirus acquisition and

persistence in Guanacaste, Costa Rica. The Journal of infectious diseases. 2005; **191**(11):1808-1816

[8] International Collaboration of Epidemiological Studies of Cervical Cancer, Appleby P, Beral V, Berrington de Gonzalez A, Colin D, Franceschi S, et al. Carcinoma of the cervix and tobacco smoking: Collaborative reanalysis of individual data on 13,541 women with carcinoma of the cervix and 23,017 women without carcinoma of the cervix from 23 epidemiological studies. International Journal of Cancer. 2006;**118**(6):1481-1495

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[168] Lajer CB, Garnaes E, Friis-Hansen L, Norrild B, Therkildsen MH, Glud M, et al. The role of miRNAs in human papilloma virus (HPV)-associated cancers: Bridging between HPV-related head and neck cancer and cervical cancer. British Journal of Cancer. 2012; **106**(9):1526-1534

[169] Liu S, Song L, Zeng S, Zhang L. MALAT1-miR-124-RBG2 axis is involved in growth and invasion of HR-HPV-positive cervical cancer cells. Tumour Biology: Journal of the International Society for Oncodevelopmental Biology and Medicine. 2016;**37**(1):633-640

[170] Martinez I, Gardiner AS, Board KF, Monzon FA, Edwards RP, Khan SA. Human papillomavirus type 16 reduces the expression of microRNA-218 in cervical carcinoma cells. Oncogene. 2008;**27**(18):2575-2582

[171] Xu Z, Zhou Y, Shi F, Cao Y, Dinh TLA, Wan J, et al. Investigation of differentially-expressed microRNAs and genes in cervical cancer using an

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integrated bioinformatics analysis. Oncology Letters. 2017;**13**(4):2784-2790

[158] Kang HW, Wang F, Wei Q, Zhao YF, Liu M, Li X, et al. miR-20a promotes migration and invasion by regulating TNKS2 in human cervical cancer cells. FEBS Letters. 2012;**586**(6):

*Gynaecological Malignancies - Updates and Advances*

[165] Lee JW, Choi CH, Choi JJ, Park YA,

Kim SJ, Hwang SY, et al. Altered microRNA expression in cervical carcinomas. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research. 2008;

[166] Wang X, Tang S, Le SY, Lu R, Rader JS, Meyers C, et al. Aberrant expression of oncogenic and tumorsuppressive microRNAs in cervical cancer is required for cancer cell growth. PLoS One. 2008;**3**(7):e2557

[167] Li Y, Wang F, Xu J, Ye F, Shen Y, Zhou J, et al. Progressive miRNA expression profiles in cervical carcinogenesis and identification of HPV-related target genes for miR-29. The Journal of pathology. 2011;**224**(4):

[168] Lajer CB, Garnaes E, Friis-Hansen L, Norrild B, Therkildsen MH, Glud M, et al. The role of miRNAs in human papilloma virus (HPV)-associated cancers: Bridging between HPV-related head and neck cancer and cervical cancer. British Journal of Cancer. 2012;

[169] Liu S, Song L, Zeng S, Zhang L. MALAT1-miR-124-RBG2 axis is involved in growth and invasion of HR-HPV-positive cervical cancer cells.

Tumour Biology: Journal of the International Society for

Oncodevelopmental Biology and Medicine. 2016;**37**(1):633-640

[171] Xu Z, Zhou Y, Shi F, Cao Y, Dinh TLA, Wan J, et al. Investigation of differentially-expressed microRNAs and

genes in cervical cancer using an

2008;**27**(18):2575-2582

[170] Martinez I, Gardiner AS, Board KF, Monzon FA, Edwards RP, Khan SA. Human papillomavirus type 16 reduces the expression of microRNA-218 in cervical carcinoma cells. Oncogene.

**14**(9):2535-2542

484-495

**106**(9):1526-1534

[159] Cao Y, Liu Y, Lu X, Wang Y, Qiao H, Liu M. Upregulation of long noncoding RNA SPRY4-IT1 correlates with tumor progression and poor prognosis in cervical cancer. FEBS Open

[160] Chen X, Liu L, Zhu W. Upregulation of long non-coding RNA CCAT2 correlates with tumor metastasis and poor prognosis in cervical squamous cell cancer patients. International Journal of Clinical and Experimental Pathology. 2015;**8**(10):13261-13266

[161] Wu L, Jin L, Zhang W, Zhang L. Roles of long non-coding RNA CCAT2 in cervical cancer cell growth and apoptosis. Medical Science Monitor: International Medical Journal of Experimental and Clinical Research.

[163] Sharma S, Munger K. Expression of the cervical carcinoma expressed PCNA regulatory (CCEPR) long noncoding

papillomavirus E6 protein and modulates cell proliferation independent of PCNA.

[164] Liao LM, Sun XY, Liu AW, Wu JB, Cheng XL, Lin JX, et al. Low expression of long noncoding XLOC\_010588 indicates a poor prognosis and promotes proliferation through upregulation of c-Myc in cervical cancer. Gynecologic Oncology. 2014;**133**(3):616-623

Bio. 2016;**6**(9):954-960

2016;**22**:875-879

[162] Kawakami T, Zhang C, Taniguchi T, Kim CJ, Okada Y, Sugihara H, et al. Characterization of loss-of-inactive X in Klinefelter syndrome and female-derived cancer cells. Oncogene. 2004;**23**(36):6163-6169

RNA is driven by the human

Virology. 2018;**518**:8-13

**48**

897-904

[172] Gardiner AS, McBee WC, Edwards RP, Austin M, Lesnock JL, Bhargava R, et al. MicroRNA analysis in human papillomavirus (HPV) associated cervical neoplasia and cancer. Infectious Agents and Cancer. 2010; **5**(1):A55

[173] Bodaghi S, Jia R, Zheng ZM. Human papillomavirus type 16 E2 and E6 are RNA-binding proteins and inhibit in vitro splicing of pre-mRNAs with suboptimal splice sites. Virology. 2009;**386**(1):32-43

[174] Yeung CL, Tsang TY, Yau PL, Kwok TT. Human papillomavirus type 16 E6 suppresses microRNA-23b expression in human cervical cancer cells through DNA methylation of the host gene C9orf3. Oncotarget. 2017; **8**(7):12158-12173

[175] Jung HM, Phillips BL, Chan EK. miR-375 activates p21 and suppresses telomerase activity by coordinately regulating HPV E6/E7, E6AP, CIP2A, and 14-3-3zeta. Molecular Cancer. 2014; **13**:80

[176] Morel A, Baguet A, Perrard J, Demeret C, Jacquin E, Guenat D, et al. 5azadC treatment upregulates miR-375 level and represses HPV16 E6 expression. Oncotarget. 2017;**8**(28): 46163-46176

[177] Wang F, Li Y, Zhou J, Xu J, Peng C, Ye F, et al. miR-375 is down-regulated in squamous cervical cancer and inhibits cell migration and invasion via targeting transcription factor SP1. The American Journal of Pathology. 2011;**179**(5): 2580-2588

[178] Liu S, Song L, Yao H, Zhang L, Xu D, Gao F, et al. MiR-375 is epigenetically downregulated by HPV-16 E6 mediated DNMT1 upregulation

and modulates EMT of cervical cancer cells by suppressing lncRNA MALAT1. PLoS One. 2016;**11**(9):e0163460

[179] Khalil AM, Guttman M, Huarte M, Garber M, Raj A, Rivea Morales D, et al. Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proceedings of the National Academy of Sciences of the United States of America. 2009;**106**(28): 11667-11672

[180] Lu H, He Y, Lin L, Qi Z, Ma L, Li L, et al. Long non-coding RNA MALAT1 modulates radiosensitivity of HR-HPV+ cervical cancer via sponging miR-145. Tumour Biology: Journal of the International Society for Oncodevelopmental Biology and Medicine. 2016;**37**(2):1683-1691

[181] Rinn JL, Kertesz M, Wang JK, Squazzo SL, Xu X, Brugmann SA, et al. Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell. 2007;**129**(7):1311-1323

[182] Gupta RA, Shah N, Wang KC, Kim J, Horlings HM, Wong DJ, et al. Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature. 2010; **464**(7291):1071-1076

[183] Zhang M, Song Y, Zhai F. ARFHPV E7 oncogene, lncRNA HOTAIR, miR-331-3p and its target, NRP2, form a negative feedback loop to regulate the apoptosis in the tumorigenesis in HPV positive cervical cancer. Journal of Cellular Biochemistry. 2018;**119**(6):4397-4407

[184] Fujii T, Shimada K, Asano A, Tatsumi Y, Yamaguchi N, Yamazaki M, et al. MicroRNA-331-3p suppresses cervical cancer cell proliferation and E6/ E7 expression by targeting NRP2. International Journal of Molecular Sciences. 2016;**17**(8)

[185] Zambrano P, Segura-Pacheco B, Perez-Cardenas E, Cetina L, Revilla-Vazquez A, Taja-Chayeb L, et al. A phase I study of hydralazine to demethylate and reactivate the expression of tumor suppressor genes. BMC Cancer. 2005;**5**:44

[186] de la Cruz-Hernandez E, Perez-Cardenas E, Contreras-Paredes A, Cantu D, Mohar A, Lizano M, et al. The effects of DNA methylation and histone deacetylase inhibitors on human papillomavirus early gene expression in cervical cancer, an in vitro and clinical study. Virology Journal. 2007;**4**:18

[187] You JS, Kang JK, Lee EK, Lee JC, Lee SH, Jeon YJ, et al. Histone deacetylase inhibitor apicidin downregulates DNA methyltransferase 1 expression and induces repressive histone modifications via recruitment of corepressor complex to promoter region in human cervix cancer cells. Oncogene. 2008;**27**(10):1376-1386

[188] Li Y, Yao J, Han C, Yang J, Chaudhry MT, Wang S, et al. Quercetin, inflammation and immunity. Nutrients. 2016;**8**(3):167

[189] Kedhari Sundaram M, Hussain A, Haque S, Raina R, Afroze N. Quercetin modifies 5'CpG promoter methylation and reactivates various tumor suppressor genes by modulating epigenetic marks in human cervical cancer cells. Journal of Cellular Biochemistry. 2019

[190] Lewis A, Kang R, Levine A, Maghami E. The new face of head and neck cancer: The HPV epidemic. Oncology. 2015;**29**(9):616-626

[191] Kim SM. Human papilloma virus in oral cancer. Journal of the Korean Association of Oral and Maxillofacial Surgeons. 2016;**42**(6):327-336

[192] Hernandez BY, McDuffie K, Zhu X, Wilkens LR, Killeen J, Kessel B, et al. Anal human papillomavirus infection in women and its relationship with cervical infection. Cancer Epidemiology, Biomarkers and Prevention: A Publication of the American Association for Cancer Research, Cosponsored by the American Society of Preventive Oncology. 2005; **14**(11 Pt 1):2550-2556

**51**

**Chapter 3**

**Abstract**

with possible HGMOC.

**1. Introduction**

**Keywords:** mucinous ovarian cancer, glucagonoma, genomic

Primary mucinous epithelial ovarian cancer (mEOC) is a rare subset, 2.7–11.9%, of epithelial ovarian cancer. The incidence for high grade mucinous ovarian cancer (HGMOC) is even lower [1]. More than two-thirds of primary HGMOC cases are misdiagnoses, which has huge implications for the outcome of these patients [2]. The overall 5-year survival outcome for localised primary mucinous ovarian cancer is over 95%, whereas the life expectancy of women with metastatic mucinous cancer ranges from months to years depending on the

Glucagonoma Masquerading as a

Mucinous Cancer of the Ovary:

*Gwo Yaw Ho, Sumitra Ananda, Cassandra J. Vandenberg,* 

*Orla McNally, Jeanne Tie, Kylie Gorringe, David Bowtell,* 

High-grade mucinous ovarian cancer (HGMOC) is often a misnomer as the majority of cases are metastatic disease with a gastro-intestinal origin. The standard platinum-based ovarian cancer (OC) chemotherapy regimens are often ineffective, and there are insufficient data to support the use of colorectal cancer (CRC) chemotherapy regimens due to the rarity of HGMOC. We described a cohort of four consecutive suspected HGMOC cases treated at the Royal Women's Hospital, Melbourne in 2012. Two cases were treated as primary MOC, whereas the other two were considered to be metastatic CRC based on histopathological and clinical evidence. From the RNAseq analysis, we identified two cases of HGMOC whose gene expression profiles were consistent with mucinous epithelial OC, one case that was treated as metastatic CRC with gene expression profile correlated with CRC and one case with neuroendocrine (NET) gene expression features. Interestingly, glucagon was over-expressed in this tumor that was subsequently confirmed by immunohistochemistry. These findings suggest a rare glucagonoma-like NET appendiceal tumor that had metastasized to the surface of ovary and were unresponsive to CRC chemotherapy regimens. In summary, a carefully curated panel of expression markers and selected functional genomics could provide diagnosis and treatment guidance for patients

*Jan Pyman, Matthew J. Wakefield and Clare L. Scott*

Lessons from Cell Biology

#### **Chapter 3**

[185] Zambrano P, Segura-Pacheco B, Perez-Cardenas E, Cetina L, Revilla-Vazquez A, Taja-Chayeb L, et al. A phase I study of hydralazine to demethylate and reactivate the expression of tumor suppressor genes.

*Gynaecological Malignancies - Updates and Advances*

et al. Anal human papillomavirus infection in women and its relationship

with cervical infection. Cancer Epidemiology, Biomarkers and Prevention: A Publication of the American Association for Cancer Research, Cosponsored by the American Society of Preventive Oncology. 2005;

**14**(11 Pt 1):2550-2556

[186] de la Cruz-Hernandez E, Perez-Cardenas E, Contreras-Paredes A, Cantu D, Mohar A, Lizano M, et al. The effects of DNA methylation and histone

[187] You JS, Kang JK, Lee EK, Lee JC,

downregulates DNA methyltransferase 1 expression and induces repressive histone modifications via recruitment of corepressor complex to promoter region in human cervix cancer cells. Oncogene.

Lee SH, Jeon YJ, et al. Histone deacetylase inhibitor apicidin

2008;**27**(10):1376-1386

2016;**8**(3):167

[188] Li Y, Yao J, Han C, Yang J,

and reactivates various tumor suppressor genes by modulating epigenetic marks in human cervical cancer cells. Journal of Cellular

[190] Lewis A, Kang R, Levine A, Maghami E. The new face of head and neck cancer: The HPV epidemic. Oncology. 2015;**29**(9):616-626

[192] Hernandez BY, McDuffie K, Zhu X, Wilkens LR, Killeen J, Kessel B,

[191] Kim SM. Human papilloma virus in oral cancer. Journal of the Korean Association of Oral and Maxillofacial Surgeons. 2016;**42**(6):327-336

Biochemistry. 2019

**50**

Chaudhry MT, Wang S, et al. Quercetin, inflammation and immunity. Nutrients.

[189] Kedhari Sundaram M, Hussain A, Haque S, Raina R, Afroze N. Quercetin modifies 5'CpG promoter methylation

deacetylase inhibitors on human papillomavirus early gene expression in cervical cancer, an in vitro and clinical study. Virology Journal. 2007;**4**:18

BMC Cancer. 2005;**5**:44

## Glucagonoma Masquerading as a Mucinous Cancer of the Ovary: Lessons from Cell Biology

*Gwo Yaw Ho, Sumitra Ananda, Cassandra J. Vandenberg, Orla McNally, Jeanne Tie, Kylie Gorringe, David Bowtell, Jan Pyman, Matthew J. Wakefield and Clare L. Scott*

### **Abstract**

High-grade mucinous ovarian cancer (HGMOC) is often a misnomer as the majority of cases are metastatic disease with a gastro-intestinal origin. The standard platinum-based ovarian cancer (OC) chemotherapy regimens are often ineffective, and there are insufficient data to support the use of colorectal cancer (CRC) chemotherapy regimens due to the rarity of HGMOC. We described a cohort of four consecutive suspected HGMOC cases treated at the Royal Women's Hospital, Melbourne in 2012. Two cases were treated as primary MOC, whereas the other two were considered to be metastatic CRC based on histopathological and clinical evidence. From the RNAseq analysis, we identified two cases of HGMOC whose gene expression profiles were consistent with mucinous epithelial OC, one case that was treated as metastatic CRC with gene expression profile correlated with CRC and one case with neuroendocrine (NET) gene expression features. Interestingly, glucagon was over-expressed in this tumor that was subsequently confirmed by immunohistochemistry. These findings suggest a rare glucagonoma-like NET appendiceal tumor that had metastasized to the surface of ovary and were unresponsive to CRC chemotherapy regimens. In summary, a carefully curated panel of expression markers and selected functional genomics could provide diagnosis and treatment guidance for patients with possible HGMOC.

**Keywords:** mucinous ovarian cancer, glucagonoma, genomic

#### **1. Introduction**

Primary mucinous epithelial ovarian cancer (mEOC) is a rare subset, 2.7–11.9%, of epithelial ovarian cancer. The incidence for high grade mucinous ovarian cancer (HGMOC) is even lower [1]. More than two-thirds of primary HGMOC cases are misdiagnoses, which has huge implications for the outcome of these patients [2]. The overall 5-year survival outcome for localised primary mucinous ovarian cancer is over 95%, whereas the life expectancy of women with metastatic mucinous cancer ranges from months to years depending on the organ site of the primary tumour. Primary mEOC is a unique subtype of ovarian neoplasm, which tends to occur in younger women, is confined to the ovaries and has a more indolent natural history. Primary mEOC is unlike metastatic mucinous epithelial cancer, which tends to occur in older women with multiple sites of metastasis (often both ovaries involved) and retains the biological behaviour of the primary tumour [3].

The poor outcome of patients with HGMOC is largely due to two main factors. Firstly, the majority of these patients have incurable advanced stage (stage IV) disease at diagnosis. Secondly, these tumours are largely unresponsive to the ovarian cancer chemotherapy regimen, in particular platinum-based chemotherapy regimen, as first-line and subsequent-line treatment [4]. Historically, mucinous ovarian cancers are treated as a single entity together with epithelial ovarian cancer, as seen in large clinical trials such as ICON3 [5], ICON5 [6] and ICON7 [7].

The distinction between primary and metastatic mucinous adenocarcinoma of the ovary has become a major focus given its importance in predicting outcomes and also to allow appropriate tumour workup and treatment planning. The diagnosis of primary HGMOC and metastatic mucinous epithelial cancer remains challenging although there is now a better recognition by pathologists in distinguishing both subsets of cancer. Advances in imaging techniques and the involvement of multidisciplinary discussions are aiding in differentiating between primary and metastatic mEOC. In a recent retrospective analysis of patients enrolled into the ICON5 trial, where the patients were screened by a panel of experts and treated as ovarian cancer, 68% of stage III and IV HGMOC cases were redefined as metastasis to the surface of ovaries [8]. This was reflected in the poor outcomes of these patients because they had received standard ovarian cancer treatment as part of their adjuvant and palliative treatment. In general, patients with advanced mEOC should be treated as a separate entity requiring an alternative therapeutic approach, such as fluorouracil (5FU) based chemotherapy regimen [9]. Despite strong preliminary support for a change in regimen there is still a universal lack of evidence in directing treatment for this subset of cancer due to the rarity of HGMOC. A recent phase II trial comparing the use of platinum-based chemotherapy versus 5FU-based chemotherapy with or without the use of an anti-angiogenic agent (Bevacizumab) failed due to poor patient accrual. Interestingly, upon specialist pathology review of all cases (n = 36), 52% of mEOC were actually metastatic disease from elsewhere, highlighting again the diagnostic difficulties [10].

The molecular events leading to the development of HGMOC are largely unknown. Gene and protein expression analyses have been performed on wellcurated mucinous ovarian cancers to elucidate the key molecular processes allowing a better understanding of the tumour biology and development of biomarkers [11]. In a study published in 2006 by Heinzelmann-Schwarz et al., the gene expression profile of mEOC was distinct, compared with other subtypes of ovarian cancer, in particular, with serous and endometrioid ovarian cancer. mEOC was shown to express genes associated with mucin production and intestinal cell surface adhesion (e.g. LGALS4), demonstrating molecular similarity to malignant intestinal type epithelial cells but with key differences in gene expression, for example, lack of KRAS activity at the transcriptional level [11]. Perhaps surprisingly given earlier reports [12], mutations in p53 are observed in 64% of true primary mEOC [13]. HGMOC were distinguished by having more chromosomal copy number events, although still not as extensively genomically unstable as High Grade Serous Ovarian Cancer (HGSOC) [13].

**53**

*Glucagonoma Masquerading as a Mucinous Cancer of the Ovary: Lessons from Cell Biology*

We describe in our mini-series four of nine consecutive cases who were referred to The Royal Women's Hospital, Melbourne in 2012 and initially treated as primary HGMOC. These cases were annotated with the initial diagnostic work up, surgical procedure and subsequent management, which include follow-up investigations and systemic treatments. We performed RNAseq analysis on fresh frozen tumour samples from four patients who had consented for tumour tissue bio-banking under the Australian Ovarian Cancer Study (AOCS) platform. Within our metastatic HGMOC cohort, we identified one case with a gene and protein expression profile suggestive of a glucagonoma-like NET gastro-intestinal tumour, which was largely unresponsive to 5FU-based chemotherapy. This report highlights the genomic diversity of HGMOC that might account for a variable outcome to treatment and also the potential clinical application of functional genomics in curating a panel of mutation

The study group consisted of patients referred to and assessed for mEOC at the Department of Gynaecology, Royal Women's Hospital (RWH) in Melbourne, between December 2011 and March 2013. For all patients, the diagnosis of mEOC was confirmed histologically and slides were reviewed by the RWH pathologists. The Australian Ovarian Cancer Study (AOCS) was approved by Human Research Ethics Committees at the Peter MacCallum Cancer Centre, Queensland Institute of Medical Research, University of Melbourne and all participating hospitals. Additional approval was obtained from the Human Research Ethics Committees at the Royal Women's Hospital and the Walter and Eliza Hall Institute. Case data were obtained via the CONTRO-engined gemma database, Royal Women's Hospital and the following parameters were collected: histology, age, date of diagnosis, stage of disease, grade, primary surgery (and outcomes), tumour markers (CA-125 and carcinoembryonic antigen) before and after chemotherapy, chemotherapy regimen, clinical outcome of patient following treatments (initial

HGMOC cases (Grade 2 or 3) were selected for RNAseq analysis based on the availability of fresh frozen tumour sample collected at the time of surgery and

Fresh frozen tumour tissue was obtained from the bio-bank (AOCS) facility. Total RNA was isolated using the RNeasy kit (Qiagen), and Illumina polyA RNAseq performed according to standard protocols at Australian Genome Research Facility. Libraries were 50 bp single end sequenced in multiplexed pools to an average depth

The resulting reads were mapped with Bowtie2 to the human reference genome with local alignment and discarding multi-mapped reads. Reads were summarised to genes using HTSeq and ENSEMBL v69. Differential expression analysis was performed in edgeR [14], comparing the four HGMOC cases as a group (to identify gene expression common to all cases), and each case individually (to allow for high levels of heterogeneity between cases) to a panel of 16 High Grade Serous Ovarian

*DOI: http://dx.doi.org/10.5772/intechopen.92554*

and expression markers to improve diagnostic accuracy.

and subsequent lines), and date of death or last follow-up.

patient consent to the AOCS study.

**2.2 RNAseq**

of 50 million reads.

Cancer (HGSOC) cases.

**2. Patients and methods**

**2.1 Patient selection**

*Glucagonoma Masquerading as a Mucinous Cancer of the Ovary: Lessons from Cell Biology DOI: http://dx.doi.org/10.5772/intechopen.92554*

We describe in our mini-series four of nine consecutive cases who were referred to The Royal Women's Hospital, Melbourne in 2012 and initially treated as primary HGMOC. These cases were annotated with the initial diagnostic work up, surgical procedure and subsequent management, which include follow-up investigations and systemic treatments. We performed RNAseq analysis on fresh frozen tumour samples from four patients who had consented for tumour tissue bio-banking under the Australian Ovarian Cancer Study (AOCS) platform. Within our metastatic HGMOC cohort, we identified one case with a gene and protein expression profile suggestive of a glucagonoma-like NET gastro-intestinal tumour, which was largely unresponsive to 5FU-based chemotherapy. This report highlights the genomic diversity of HGMOC that might account for a variable outcome to treatment and also the potential clinical application of functional genomics in curating a panel of mutation and expression markers to improve diagnostic accuracy.

#### **2. Patients and methods**

#### **2.1 Patient selection**

*Gynaecological Malignancies - Updates and Advances*

the primary tumour [3].

ICON7 [7].

difficulties [10].

Cancer (HGSOC) [13].

organ site of the primary tumour. Primary mEOC is a unique subtype of ovarian neoplasm, which tends to occur in younger women, is confined to the ovaries and has a more indolent natural history. Primary mEOC is unlike metastatic mucinous epithelial cancer, which tends to occur in older women with multiple sites of metastasis (often both ovaries involved) and retains the biological behaviour of

The poor outcome of patients with HGMOC is largely due to two main factors. Firstly, the majority of these patients have incurable advanced stage (stage IV) disease at diagnosis. Secondly, these tumours are largely unresponsive to the ovarian cancer chemotherapy regimen, in particular platinum-based chemotherapy regimen, as first-line and subsequent-line treatment [4]. Historically, mucinous ovarian cancers are treated as a single entity together with epithelial ovarian cancer, as seen in large clinical trials such as ICON3 [5], ICON5 [6] and

The distinction between primary and metastatic mucinous adenocarcinoma of the ovary has become a major focus given its importance in predicting outcomes and also to allow appropriate tumour workup and treatment planning. The diagnosis of primary HGMOC and metastatic mucinous epithelial cancer remains challenging although there is now a better recognition by pathologists in distinguishing both subsets of cancer. Advances in imaging techniques and the involvement of multidisciplinary discussions are aiding in differentiating between primary and metastatic mEOC. In a recent retrospective analysis of patients enrolled into the ICON5 trial, where the patients were screened by a panel of experts and treated as ovarian cancer, 68% of stage III and IV HGMOC cases were redefined as metastasis to the surface of ovaries [8]. This was reflected in the poor outcomes of these patients because they had received standard ovarian cancer treatment as part of their adjuvant and palliative treatment. In general, patients with advanced mEOC should be treated as a separate entity requiring an alternative therapeutic approach, such as fluorouracil (5FU) based chemotherapy regimen [9]. Despite strong preliminary support for a change in regimen there is still a universal lack of evidence in directing treatment for this subset of cancer due to the rarity of HGMOC. A recent phase II trial comparing the use of platinum-based chemotherapy versus 5FU-based chemotherapy with or without the use of an anti-angiogenic agent (Bevacizumab) failed due to poor patient accrual. Interestingly, upon specialist pathology review of all cases (n = 36), 52% of mEOC were actually metastatic disease from elsewhere, highlighting again the diagnostic

The molecular events leading to the development of HGMOC are largely unknown. Gene and protein expression analyses have been performed on wellcurated mucinous ovarian cancers to elucidate the key molecular processes allowing a better understanding of the tumour biology and development of biomarkers [11]. In a study published in 2006 by Heinzelmann-Schwarz et al., the gene expression profile of mEOC was distinct, compared with other subtypes of ovarian cancer, in particular, with serous and endometrioid ovarian cancer. mEOC was shown to express genes associated with mucin production and intestinal cell surface adhesion (e.g. LGALS4), demonstrating molecular similarity to malignant intestinal type epithelial cells but with key differences in gene expression, for example, lack of KRAS activity at the transcriptional level [11]. Perhaps surprisingly given earlier reports [12], mutations in p53 are observed in 64% of true primary mEOC [13]. HGMOC were distinguished by having more chromosomal copy number events, although still not as extensively genomically unstable as High Grade Serous Ovarian

**52**

The study group consisted of patients referred to and assessed for mEOC at the Department of Gynaecology, Royal Women's Hospital (RWH) in Melbourne, between December 2011 and March 2013. For all patients, the diagnosis of mEOC was confirmed histologically and slides were reviewed by the RWH pathologists.

The Australian Ovarian Cancer Study (AOCS) was approved by Human Research Ethics Committees at the Peter MacCallum Cancer Centre, Queensland Institute of Medical Research, University of Melbourne and all participating hospitals. Additional approval was obtained from the Human Research Ethics Committees at the Royal Women's Hospital and the Walter and Eliza Hall Institute.

Case data were obtained via the CONTRO-engined gemma database, Royal Women's Hospital and the following parameters were collected: histology, age, date of diagnosis, stage of disease, grade, primary surgery (and outcomes), tumour markers (CA-125 and carcinoembryonic antigen) before and after chemotherapy, chemotherapy regimen, clinical outcome of patient following treatments (initial and subsequent lines), and date of death or last follow-up.

HGMOC cases (Grade 2 or 3) were selected for RNAseq analysis based on the availability of fresh frozen tumour sample collected at the time of surgery and patient consent to the AOCS study.

#### **2.2 RNAseq**

Fresh frozen tumour tissue was obtained from the bio-bank (AOCS) facility. Total RNA was isolated using the RNeasy kit (Qiagen), and Illumina polyA RNAseq performed according to standard protocols at Australian Genome Research Facility. Libraries were 50 bp single end sequenced in multiplexed pools to an average depth of 50 million reads.

The resulting reads were mapped with Bowtie2 to the human reference genome with local alignment and discarding multi-mapped reads. Reads were summarised to genes using HTSeq and ENSEMBL v69. Differential expression analysis was performed in edgeR [14], comparing the four HGMOC cases as a group (to identify gene expression common to all cases), and each case individually (to allow for high levels of heterogeneity between cases) to a panel of 16 High Grade Serous Ovarian Cancer (HGSOC) cases.

The resulting list of up-regulated genes present in HGMOC was filtered for genes that are expressed in less than 10 anatomical systems in the eGenetics expression resource using ENSEMBL biomart [15].

#### **3. Results**

#### **3.1 Patient characteristics**

Nine patients with a histologically confirmed diagnosis of high-grade mucinous ovarian cancer presented at the Gynaecology Department of RWH between December 2011 and March 2013 (**Figure 1**). Three patients declined consent to AOCS and were therefore excluded from this study. Of the six patients who consented to AOCS, one did not have fresh frozen tumour tissue stored during the original surgery and another case was excluded due to subsequent diagnosis of pseudomyxoma peritonei. RNAseq analysis was performed on the remaining four cases using tumour tissue snap frozen at surgery. The patients' characteristics were summarised as per **Table 1**. Representative histology images are shown in **Figure 2**.

#### **3.2 Case reports**

#### *3.2.1 Tumour 1*

Patient #32, a 31-year-old woman with no significant family history of malignancy, presented with a short history of increasing right iliac fossa abdominal pain. She previously had a CT scan 1 month earlier, which showed a large 16 cm complex left ovarian mass. This mass was confirmed by her pre-operative pelvic MRI scan with enlarged para-aortic lymph nodes below the renal artery and no other obvious

#### **Figure 1.**

*Patients screened at Royal Women's hospital during 2012/13 being treated as high grade mucinous epithelial ovarian cancer for RNA sequencing analysis.*

**55**

and well at 5-year follow-up.

*3.2.2 Tumour 2*

**Table 1.**

*Patient characteristics.*

*Glucagonoma Masquerading as a Mucinous Cancer of the Ovary: Lessons from Cell Biology*

lesion identified. This patient underwent total abdominal hysterectomy (TAH), bilateral salpingo-oophorectomy (BSO) and para-aortic lymph node sampling. At surgery, her bowels and intra-peritoneal cavity looked normal. Her tumour histology was reviewed at a multi-disciplinary meeting and was diagnosed as grade 2 primary mEOC stage IA. She received no further systemic treatment. For completion of her cancer assessment, the patient underwent upper gastro-intestinal endoscopy and colonoscopy, which were both normal and subsequently had a PET/ CT scan that showed no evidence of metastatic disease. The patient remained alive

Patient #35 was a 34-year-old woman with no previous significant background medical history and presented to her general practitioner with 1-month history of intermittent lower abdominal pain. Her initial ultra-sound scan organised by her general practitioner showed a large left ovarian cyst and pre-operative MRI scan confirmed a 18 cm complex mixed cystic lesion with a 5 cm solid component associated with moderate ascites. The patient underwent up-front surgery with TAH and BSO. Her peritoneum, abdominal organs and diaphragm appeared to be normal during surgery. The histopathology result confirmed high-grade mucinous adenocarcinoma of the ovary with no surface spread and negative lymph node

*DOI: http://dx.doi.org/10.5772/intechopen.92554*

*Glucagonoma Masquerading as a Mucinous Cancer of the Ovary: Lessons from Cell Biology DOI: http://dx.doi.org/10.5772/intechopen.92554*


#### **Table 1.**

*Gynaecological Malignancies - Updates and Advances*

sion resource using ENSEMBL biomart [15].

**3. Results**

**3.2 Case reports**

*3.2.1 Tumour 1*

**3.1 Patient characteristics**

The resulting list of up-regulated genes present in HGMOC was filtered for genes that are expressed in less than 10 anatomical systems in the eGenetics expres-

Nine patients with a histologically confirmed diagnosis of high-grade mucinous ovarian cancer presented at the Gynaecology Department of RWH between December 2011 and March 2013 (**Figure 1**). Three patients declined consent to AOCS and were therefore excluded from this study. Of the six patients who consented to AOCS, one did not have fresh frozen tumour tissue stored during the original surgery and another case was excluded due to subsequent diagnosis of pseudomyxoma peritonei. RNAseq analysis was performed on the remaining four cases using tumour tissue snap frozen at surgery. The patients' characteristics were summarised as per **Table 1**. Representative histology images are shown in **Figure 2**.

Patient #32, a 31-year-old woman with no significant family history of malignancy, presented with a short history of increasing right iliac fossa abdominal pain. She previously had a CT scan 1 month earlier, which showed a large 16 cm complex left ovarian mass. This mass was confirmed by her pre-operative pelvic MRI scan with enlarged para-aortic lymph nodes below the renal artery and no other obvious

*Patients screened at Royal Women's hospital during 2012/13 being treated as high grade mucinous epithelial* 

**54**

**Figure 1.**

*ovarian cancer for RNA sequencing analysis.*

*Patient characteristics.*

lesion identified. This patient underwent total abdominal hysterectomy (TAH), bilateral salpingo-oophorectomy (BSO) and para-aortic lymph node sampling. At surgery, her bowels and intra-peritoneal cavity looked normal. Her tumour histology was reviewed at a multi-disciplinary meeting and was diagnosed as grade 2 primary mEOC stage IA. She received no further systemic treatment. For completion of her cancer assessment, the patient underwent upper gastro-intestinal endoscopy and colonoscopy, which were both normal and subsequently had a PET/ CT scan that showed no evidence of metastatic disease. The patient remained alive and well at 5-year follow-up.

#### *3.2.2 Tumour 2*

Patient #35 was a 34-year-old woman with no previous significant background medical history and presented to her general practitioner with 1-month history of intermittent lower abdominal pain. Her initial ultra-sound scan organised by her general practitioner showed a large left ovarian cyst and pre-operative MRI scan confirmed a 18 cm complex mixed cystic lesion with a 5 cm solid component associated with moderate ascites. The patient underwent up-front surgery with TAH and BSO. Her peritoneum, abdominal organs and diaphragm appeared to be normal during surgery. The histopathology result confirmed high-grade mucinous adenocarcinoma of the ovary with no surface spread and negative lymph node

#### **Figure 2.**

*Histopathology of the four cases: Representative haematoxylin and eosin stained slides presented at 10× and 20× magnification.*

involvement. The tumour was stage IC given that the peritoneal washing was positive for malignant cells. Patient received adjuvant ovarian cancer chemotherapy, consisting of carboplatin and paclitaxel, at her local medical oncology centre. She

**57**

of systemic treatment.

*3.2.4 Tumour 4*

*Glucagonoma Masquerading as a Mucinous Cancer of the Ovary: Lessons from Cell Biology*

also underwent upper gastro-intestinal endoscopy and colonoscopy as completion of her tumour assessment, which were normal. She remained well and alive at her

Patient #49 was a 64-year-old woman with known type II diabetes mellitus who presented to her local hospital with increasing abdominal pain, nausea, vomiting, and urinary frequency. Her initial CT scan showed a right ovarian mass associated with peritoneal deposits. This was confirmed by her diagnostic laparoscopy that showed a 14 cm ovarian mass adherent to the left adnexa and pouch of Douglas associated with macroscopic tumour deposits on her anterior abdominal wall and omentum. The original biopsy confirmed adenocarcinoma favouring gastro-intestinal tumour. She underwent TAH, BSO, omentectomy and appendectomy. Bilateral ovarian masses were resected during her surgery together with appendiceal and omental nodules. The histopathology confirmed metastatic mucinous adenocarcinoma on both ovaries with evidence of similar tumour effacement of the appendix suggestive of appendiceal origin. It was noted by the pathologist that there was NET differentiation of her mucinous adenocarcinoma with immunohistochemistry staining for chromogranin and synaptophysin positive. She was discharged from hospital following recovery of her surgery to the care of the gastro-intestinal (GI) team. Her case was discussed at the GI tumour board meeting and the expert opinion was to treat this as advanced stage (Stage IV) colorectal cancer with palliative fluorouracil (5FU) based chemotherapy following her surgical debulking procedure. The patient had minimal residual disease prior to commencing her palliative chemotherapy. Her gastroscopy and colonoscopy performed post-operatively showed significant pathology. She completed 8 cycles of FOLFOX (5FU with oxaliplatin) following by single agent 5FU until late 2014. The patient had an interval PET/CT scan performed a year later that showed minimal metabolic activity in known low volume metastatic peritoneal disease. She subsequently presented in 4–6 months later with incomplete bowel obstruction and radiological evidence of slow peritoneal disease progression. Her bowel obstruction resolved with conservative management and she declined further lines of systemic treatment. She received palliative radiation therapy to her peritoneal metastasis with some relief of abdominal symptom. She had multiple admissions to her local hospital in the following 12 months, with bowel-related complications and subsequently passed away in that year, 4 years following the diagnosis of her cancer having only effectively completed one line

Patient #60 was a 67-year-old woman who was diagnosed with metastatic appendiceal mucinous adenocarcinoma of her right ovary 2 years prior to her re-referral with a left ovarian mass. Her initial cancer was treated with surgical removal of the right ovarian and appendiceal mass. Her surgery was complicated with extensive venous thrombo-embolic (VTE) events. She received no systemic treatment following her initial surgery and represented with a 12 cm mixed cystic/ solid mass arising from the left ovary based on initial imaging. She underwent second de-bulking surgery following insertion of an inferior vena cava filter for her VTE. This involved the removal of the dense left pelvic tumour mass that was adherent to her bowel, ureter and bladder requiring cystotomy and colostomy. The histopathology report confirmed evidence of adenocarcinoma with focal

*DOI: http://dx.doi.org/10.5772/intechopen.92554*

last follow-up assessment 5 years later.

*3.2.3 Tumour 3*

also underwent upper gastro-intestinal endoscopy and colonoscopy as completion of her tumour assessment, which were normal. She remained well and alive at her last follow-up assessment 5 years later.

#### *3.2.3 Tumour 3*

*Gynaecological Malignancies - Updates and Advances*

**56**

**Figure 2.**

*magnification.*

involvement. The tumour was stage IC given that the peritoneal washing was positive for malignant cells. Patient received adjuvant ovarian cancer chemotherapy, consisting of carboplatin and paclitaxel, at her local medical oncology centre. She

*Histopathology of the four cases: Representative haematoxylin and eosin stained slides presented at 10× and 20×* 

Patient #49 was a 64-year-old woman with known type II diabetes mellitus who presented to her local hospital with increasing abdominal pain, nausea, vomiting, and urinary frequency. Her initial CT scan showed a right ovarian mass associated with peritoneal deposits. This was confirmed by her diagnostic laparoscopy that showed a 14 cm ovarian mass adherent to the left adnexa and pouch of Douglas associated with macroscopic tumour deposits on her anterior abdominal wall and omentum. The original biopsy confirmed adenocarcinoma favouring gastro-intestinal tumour. She underwent TAH, BSO, omentectomy and appendectomy. Bilateral ovarian masses were resected during her surgery together with appendiceal and omental nodules. The histopathology confirmed metastatic mucinous adenocarcinoma on both ovaries with evidence of similar tumour effacement of the appendix suggestive of appendiceal origin. It was noted by the pathologist that there was NET differentiation of her mucinous adenocarcinoma with immunohistochemistry staining for chromogranin and synaptophysin positive. She was discharged from hospital following recovery of her surgery to the care of the gastro-intestinal (GI) team. Her case was discussed at the GI tumour board meeting and the expert opinion was to treat this as advanced stage (Stage IV) colorectal cancer with palliative fluorouracil (5FU) based chemotherapy following her surgical debulking procedure. The patient had minimal residual disease prior to commencing her palliative chemotherapy. Her gastroscopy and colonoscopy performed post-operatively showed significant pathology. She completed 8 cycles of FOLFOX (5FU with oxaliplatin) following by single agent 5FU until late 2014. The patient had an interval PET/CT scan performed a year later that showed minimal metabolic activity in known low volume metastatic peritoneal disease. She subsequently presented in 4–6 months later with incomplete bowel obstruction and radiological evidence of slow peritoneal disease progression. Her bowel obstruction resolved with conservative management and she declined further lines of systemic treatment. She received palliative radiation therapy to her peritoneal metastasis with some relief of abdominal symptom. She had multiple admissions to her local hospital in the following 12 months, with bowel-related complications and subsequently passed away in that year, 4 years following the diagnosis of her cancer having only effectively completed one line of systemic treatment.

#### *3.2.4 Tumour 4*

Patient #60 was a 67-year-old woman who was diagnosed with metastatic appendiceal mucinous adenocarcinoma of her right ovary 2 years prior to her re-referral with a left ovarian mass. Her initial cancer was treated with surgical removal of the right ovarian and appendiceal mass. Her surgery was complicated with extensive venous thrombo-embolic (VTE) events. She received no systemic treatment following her initial surgery and represented with a 12 cm mixed cystic/ solid mass arising from the left ovary based on initial imaging. She underwent second de-bulking surgery following insertion of an inferior vena cava filter for her VTE. This involved the removal of the dense left pelvic tumour mass that was adherent to her bowel, ureter and bladder requiring cystotomy and colostomy. The histopathology report confirmed evidence of adenocarcinoma with focal

intracytoplasmic mucin consistent with mucinous adenocarcinoma similar with the original diagnosis 2 years ago. The CK20 was strongly positive and associated with negative staining for CK7. The patient was discharged back to her original colorectal team for further management.

#### **3.3 Transcriptome analysis by RNAseq**

Due to the high level of heterogeneity in expression within the HGMOC group, significantly differentially expressed genes were not able to be detected in the group comparison. However, the individual tumour analyses identified a large number of differentially expressed genes. This large number of differentially expressed genes is an expected limitation of this type of analysis, as variance can only be estimated from the control group and there is no suppression of random variability as would be seen in a group of replicates. Because many of these genes were minimally informative, the differentially expressed genes were filtered to identify upregulated genes that are annotated as having organ specific expression and may be informative for the organ of origin. The RNAseq analysis identified 18 genes with a restricted tissue/organ expression pattern that were differentially up regulated in the four tumour samples. These genes were enriched for expression in colon, stomach, pancreas, lung, kidney and skeletal muscle. Only two of the genes, LGALS4 and ERN2, are annotated as expressed in gynaecological tissues and both are also expressed in colonic tissue (**Figure 3**).

#### *3.3.1 Primary mucinous ovarian epithelial carcinoma exhibits a gene expression profile distinct from metastatic mucinous epithelial carcinoma and high-grade serous ovarian cancer*

The variable genes identified by transcript profiling revealed that the two primary HGMOC tumours #32 and #35, could be clearly distinguished from the two metastatic mEOC, tumours #49 and #60. A cluster of genes including PGC (encodes a digestive gastric protein), ANAX10 (encodes a calcium- and phospholipid-binding gastric protein), DOUX2 (encodes an oxidase enzyme common in thyroid and GI system) and C12orf36 (non-protein encoding RNA) were up regulated in both tumour #32 and tumour #35. Tumour #49 and tumour #60 had CDH17 (encodes a cadherin superfamily glycoprotein common in gastro-intestinal and pancreatic cells), GUCY2C (encodes for guanylyl cyclase enzyme found in intestinal epithelium) and SCGN (encodes a secretory calcium binding protein in cell cytoplasm) genes up regulated. All four tumours shared in common high expression of seven genes not seen in HGSOC, in particular LGALS4, an intestinal surface cell adhesion molecule that is overexpressed in intestinal carcinomas [16]. LGALS4 had previously been shown to be specifically expressed in mEOC [11]. However, in our cohort, this gene was universally expressed in all four tumours rendering it as a non-distinguishing gene. Interestingly, the two primary HGMOC (tumour #32 and tumour #35) retained some expression of PAX8 and WT1 together with KRT7/CK7 expression as also seen in the HGSOC control panel. The expression of PAX8 in mucinous epithelial ovarian cancer, and the lack of its expression in appendiceal cancers, has been previously described and this further supports the relevance of this gene expression in differentiating the organ of origin of the tumour [17]. With only two mEOC cases this analysis is weakly powered and heavily influenced by the individual cases. Analysis of a larger cohort and validation will be required to identify robust clinical markers.

**59**

**Figure 3.**

*FGT: female genital tract; FT; fallopian tube.*

*Glucagonoma Masquerading as a Mucinous Cancer of the Ovary: Lessons from Cell Biology*

*Heat map of the most differentially expressed genes in the four tumours analysed compared to HGSOC (top panel), and expression comparison of four commonly used markers (lower panel). The tissue specific expression of the listed genes: GCG: pancreas; REGA: GIT (D, Sm, C, R) + appendix; GUCY2C: GIT (D, Sm, C, R); CDH17: GIT (S, D, Sm, C, R) + appendix; SCGN: GIT (S, D, Sm, C, R) + pancreas; HNF4A: GIT (S, D, Sm, C, R) + liver + pancreas + appendix; VIL2: GIT + FGT; PDX1: GIT (D, S) + pancreas; LGALS4: GIT (S, D, S) + gallbladder + appendix; ERN2: GIT (S, D, S, C, R) + appendix; GPX2: GIT + liver + kidney; MUC17: GIT (D, Sm); PGC: S; ANAX10: S; DUOX2: thyroid + stomach; C12ord36: S; CLDN18: S; APOBEC1: Sm; KRT7/CK7: FT. cervix, uterine, liver, gallbladder, pancreas; KRT20/CK20: GIT (D, S, C, R); WT1: FGT; PAX8: FGT. GIT: gastro-intestinal tract; D: duodenum; S: stomach; Sm: small intestine; C: caecum; R: rectum;* 

*DOI: http://dx.doi.org/10.5772/intechopen.92554*

*Glucagonoma Masquerading as a Mucinous Cancer of the Ovary: Lessons from Cell Biology DOI: http://dx.doi.org/10.5772/intechopen.92554*

#### **Figure 3.**

*Gynaecological Malignancies - Updates and Advances*

team for further management.

**3.3 Transcriptome analysis by RNAseq**

are also expressed in colonic tissue (**Figure 3**).

*high-grade serous ovarian cancer*

intracytoplasmic mucin consistent with mucinous adenocarcinoma similar with the original diagnosis 2 years ago. The CK20 was strongly positive and associated with negative staining for CK7. The patient was discharged back to her original colorectal

Due to the high level of heterogeneity in expression within the HGMOC group,

significantly differentially expressed genes were not able to be detected in the group comparison. However, the individual tumour analyses identified a large number of differentially expressed genes. This large number of differentially expressed genes is an expected limitation of this type of analysis, as variance can only be estimated from the control group and there is no suppression of random variability as would be seen in a group of replicates. Because many of these genes were minimally informative, the differentially expressed genes were filtered to identify upregulated genes that are annotated as having organ specific expression and may be informative for the organ of origin. The RNAseq analysis identified 18 genes with a restricted tissue/organ expression pattern that were differentially up regulated in the four tumour samples. These genes were enriched for expression in colon, stomach, pancreas, lung, kidney and skeletal muscle. Only two of the genes, LGALS4 and ERN2, are annotated as expressed in gynaecological tissues and both

*3.3.1 Primary mucinous ovarian epithelial carcinoma exhibits a gene expression profile distinct from metastatic mucinous epithelial carcinoma and* 

The variable genes identified by transcript profiling revealed that the two primary HGMOC tumours #32 and #35, could be clearly distinguished from the two metastatic mEOC, tumours #49 and #60. A cluster of genes including PGC (encodes a digestive gastric protein), ANAX10 (encodes a calcium- and phospholipid-binding gastric protein), DOUX2 (encodes an oxidase enzyme common in thyroid and GI system) and C12orf36 (non-protein encoding RNA) were up regulated in both tumour #32 and tumour #35. Tumour #49 and tumour #60 had CDH17 (encodes a cadherin superfamily glycoprotein common in gastro-intestinal and pancreatic cells), GUCY2C (encodes for guanylyl cyclase enzyme found in intestinal epithelium) and SCGN (encodes a secretory calcium binding protein in cell cytoplasm) genes up regulated. All four tumours shared in common high expression of seven genes not seen in HGSOC, in particular LGALS4, an intestinal surface cell adhesion molecule that is overexpressed in intestinal carcinomas [16]. LGALS4 had previously been shown to be specifically expressed in mEOC [11]. However, in our cohort, this gene was universally expressed in all four tumours rendering it as a non-distinguishing gene. Interestingly, the two primary HGMOC (tumour #32 and tumour #35) retained some expression of PAX8 and WT1 together with KRT7/CK7 expression as also seen in the HGSOC control panel. The expression of PAX8 in mucinous epithelial ovarian cancer, and the lack of its expression in appendiceal cancers, has been previously described and this further supports the relevance of this gene expression in differentiating the organ of origin of the tumour [17]. With only two mEOC cases this analysis is weakly powered and heavily influenced by the individual cases. Analysis of a larger cohort and validation will be required to

**58**

identify robust clinical markers.

*Heat map of the most differentially expressed genes in the four tumours analysed compared to HGSOC (top panel), and expression comparison of four commonly used markers (lower panel). The tissue specific expression of the listed genes: GCG: pancreas; REGA: GIT (D, Sm, C, R) + appendix; GUCY2C: GIT (D, Sm, C, R); CDH17: GIT (S, D, Sm, C, R) + appendix; SCGN: GIT (S, D, Sm, C, R) + pancreas; HNF4A: GIT (S, D, Sm, C, R) + liver + pancreas + appendix; VIL2: GIT + FGT; PDX1: GIT (D, S) + pancreas; LGALS4: GIT (S, D, S) + gallbladder + appendix; ERN2: GIT (S, D, S, C, R) + appendix; GPX2: GIT + liver + kidney; MUC17: GIT (D, Sm); PGC: S; ANAX10: S; DUOX2: thyroid + stomach; C12ord36: S; CLDN18: S; APOBEC1: Sm; KRT7/CK7: FT. cervix, uterine, liver, gallbladder, pancreas; KRT20/CK20: GIT (D, S, C, R); WT1: FGT; PAX8: FGT. GIT: gastro-intestinal tract; D: duodenum; S: stomach; Sm: small intestine; C: caecum; R: rectum; FGT: female genital tract; FT; fallopian tube.*

#### **Figure 4.**

*A. Adenocarcinoma seeding in the ovary; normal ovarian tissue (arrow), mucinous glandular component of adenocarcinoma (\*); prominent stromal desmoplasia can be typically seen in tumours that secondarily involve the ovary (5× magnification); B. Adenocarcinoma in the ovary (20× magnification); C. Chromogranin immunohistochemical staining shows strong and diffuse reactivity (20× magnification); D. Glucagon immunohistochemical staining shows strong reactivity in tumour cells (20× magnification); E. Adenocarcinoma infiltrating the appendix (5× magnification); lumen of appendix (arrow); adenocarcinoma (\*); F. Adenocarcinoma in the appendix (20× magnification); G. Adenocarcinoma in the appendix (20× magnification); H. Adenocarcinoma in the appendix (20× magnification).*

**61**

**4. Discussion**

of the tumour.

in treatment selection.

*Glucagonoma Masquerading as a Mucinous Cancer of the Ovary: Lessons from Cell Biology*

*3.3.2 Identification of tumour #49 as a glucagonoma-like neuroendocrine tumour* 

The RNAseq analysis identified up regulation of GCG, a gene that encodes for glucagon, in tumour #49. GCG accounted for ~5% of transcriptional output indicating a high level of glucagon expression. The original histopathology report on the resected tumour confirmed evidence of NET differentiation within the mucinous adenocarcinoma, with positive IHC staining for chromogranin and synaptophysin. Our findings were returned to the original pathologist at RWH and further IHC for glucagon protein expression was performed. Strong glucagon staining was seen in the tumour cells by IHC, confirming the RNAseq findings (**Figure 4**). This "glucagonoma"-like tumour may have either a pancreatic origin or may have

This patient's case was discussed at the GI tumour board meeting, and despite the finding of our RNAseq analysis, it was treated as a standard colorectal cancer given the rarity of NET differentiated mucinous adenocarcinoma of the appendix. It was difficult to ascertain the full effect of CRC/5FU-based chemotherapy regimen on this patient given the limited line of treatment received and perceived minimal residual disease post-surgery. Unfortunately, the patient declined further chemotherapy at first progression but survived for a further 2 years receiving only pallia-

True mucinous epithelial ovarian carcinomas are a rare subtype of ovarian cancer. In our limited case cohort, half of the mEOC seen in our institute at a given period of time were re-diagnosed as metastatic mucinous epithelial carcinoma. This posed a challenge for both the pathologists and surgical team to provide an accurate and timely diagnosis of the cancer and enable the delivery of optimal treatment. Clinical and radiological information, such as patient age, laterality of tumour, tumour stage and to some extent tumour marker CA125 can guide diagnosis prior surgery [3]. Ultimately, it is the histology of the resected tumour that allows accurate assessment of tumour origin based on the pattern of protein expression seen by IHC and morphology [8]. However, in patient #60 case, a previous history of appendiceal tumour should have raised the suspicious for metastatic recurrence

Our pilot RNAseq study indicated that tumours initially diagnosed as mEOC can be a diverse collection of disease, and that gene expression analysis has the potential to identify prognostically useful subsets. Categorising based on gene expression and identifying genetic aberrations is likely to greatly assist in selection of the optimal treatment for each individual patient. While RNAseq for each individual patient is an impractical method for tumour identification, the observations from this study contributed to the design of a larger study, GAMuT—Genomic Analysis of Mucinous Tumours, which will compare HGMOC to low grade and borderline cases to identify prognostic and therapeutically useful gene expression signatures (Australian National Health and Medical Research (NH&MRC) Funded Study— APP1045783). This study will allow the selection of a panel of mutation and expression markers to elucidate the tumour organ of origin, thus providing some guidance

We highlighted the identification of a very rare "glucagonoma-like" NET appendiceal tumour in our series of mEOC to indicate the reliability of functional genomics in identifying rare conditions. This diagnosis is in context with the

*of likely appendiceal origin by transcriptome analysis*

originated from the appendix as clinically implicated (**Figure 4**).

tive radiation treatment to problematic intra-abdominal lesions.

*DOI: http://dx.doi.org/10.5772/intechopen.92554*

*Glucagonoma Masquerading as a Mucinous Cancer of the Ovary: Lessons from Cell Biology DOI: http://dx.doi.org/10.5772/intechopen.92554*

*3.3.2 Identification of tumour #49 as a glucagonoma-like neuroendocrine tumour of likely appendiceal origin by transcriptome analysis*

The RNAseq analysis identified up regulation of GCG, a gene that encodes for glucagon, in tumour #49. GCG accounted for ~5% of transcriptional output indicating a high level of glucagon expression. The original histopathology report on the resected tumour confirmed evidence of NET differentiation within the mucinous adenocarcinoma, with positive IHC staining for chromogranin and synaptophysin. Our findings were returned to the original pathologist at RWH and further IHC for glucagon protein expression was performed. Strong glucagon staining was seen in the tumour cells by IHC, confirming the RNAseq findings (**Figure 4**). This "glucagonoma"-like tumour may have either a pancreatic origin or may have originated from the appendix as clinically implicated (**Figure 4**).

This patient's case was discussed at the GI tumour board meeting, and despite the finding of our RNAseq analysis, it was treated as a standard colorectal cancer given the rarity of NET differentiated mucinous adenocarcinoma of the appendix. It was difficult to ascertain the full effect of CRC/5FU-based chemotherapy regimen on this patient given the limited line of treatment received and perceived minimal residual disease post-surgery. Unfortunately, the patient declined further chemotherapy at first progression but survived for a further 2 years receiving only palliative radiation treatment to problematic intra-abdominal lesions.

#### **4. Discussion**

*Gynaecological Malignancies - Updates and Advances*

**60**

**Figure 4.**

*A. Adenocarcinoma seeding in the ovary; normal ovarian tissue (arrow), mucinous glandular component of adenocarcinoma (\*); prominent stromal desmoplasia can be typically seen in tumours that secondarily involve the ovary (5× magnification); B. Adenocarcinoma in the ovary (20× magnification); C. Chromogranin immunohistochemical staining shows strong and diffuse reactivity (20× magnification); D. Glucagon immunohistochemical staining shows strong reactivity in tumour cells (20× magnification); E. Adenocarcinoma* 

*infiltrating the appendix (5× magnification); lumen of appendix (arrow); adenocarcinoma (\*); F. Adenocarcinoma in the appendix (20× magnification); G. Adenocarcinoma in the appendix* 

*(20× magnification); H. Adenocarcinoma in the appendix (20× magnification).*

True mucinous epithelial ovarian carcinomas are a rare subtype of ovarian cancer. In our limited case cohort, half of the mEOC seen in our institute at a given period of time were re-diagnosed as metastatic mucinous epithelial carcinoma. This posed a challenge for both the pathologists and surgical team to provide an accurate and timely diagnosis of the cancer and enable the delivery of optimal treatment. Clinical and radiological information, such as patient age, laterality of tumour, tumour stage and to some extent tumour marker CA125 can guide diagnosis prior surgery [3]. Ultimately, it is the histology of the resected tumour that allows accurate assessment of tumour origin based on the pattern of protein expression seen by IHC and morphology [8]. However, in patient #60 case, a previous history of appendiceal tumour should have raised the suspicious for metastatic recurrence of the tumour.

Our pilot RNAseq study indicated that tumours initially diagnosed as mEOC can be a diverse collection of disease, and that gene expression analysis has the potential to identify prognostically useful subsets. Categorising based on gene expression and identifying genetic aberrations is likely to greatly assist in selection of the optimal treatment for each individual patient. While RNAseq for each individual patient is an impractical method for tumour identification, the observations from this study contributed to the design of a larger study, GAMuT—Genomic Analysis of Mucinous Tumours, which will compare HGMOC to low grade and borderline cases to identify prognostic and therapeutically useful gene expression signatures (Australian National Health and Medical Research (NH&MRC) Funded Study— APP1045783). This study will allow the selection of a panel of mutation and expression markers to elucidate the tumour organ of origin, thus providing some guidance in treatment selection.

We highlighted the identification of a very rare "glucagonoma-like" NET appendiceal tumour in our series of mEOC to indicate the reliability of functional genomics in identifying rare conditions. This diagnosis is in context with the

patient's clinical findings and also with IHC proving glucagon protein expression only apparent after the RNA sequencing results were available. In hindsight, it is hard to predict if this patient would have benefited from repeated surgical resection of recurrent tumour [18], or to NET based treatment regimens, such as mTOR inhibition (everolimus) [19] or multiple tyrosine kinase inhibitor (sunitinib, pazopanib) [20, 21]. Furthermore, the patient did not exhibit glucagon syndrome and her glucagon serum level was never tested. Nevertheless, clinically tumour #49 behaved like a NET tumour with slow indolent progression and localised complication. Unfortunately, in this case, the problematic tumour caused repeated bowel obstructive symptoms requiring multiple hospital admissions in the months leading up to the patient's death.

The recognition of diversity of tumour subtypes even within a rare tumour population is important especially in designing clinical trials. Given the small number of patients available for accruement, it is vital that we accurately stratify patients into treatment arms and identify robust biomarkers early. A very rare tumour within a rare tumour subtype can pose a challenging issue in terms of being an outlier that would skew the outcome in a clinical trial and also in optimising treatment for this patient based on available evidence (which is lacking). These issues will need to be addressed in any clinical trials pertaining to rare cancer.

#### **Acknowledgements**

We thank Margot Osinski (Royal Women's Hospital) for database assistance and AOCS: The Australian Ovarian Cancer Study Group was supported by the U.S. Army Medical Research and Materiel Command under DAMD17-01-1-0729. The AOCS also acknowledges the cooperation of the participating institutions in Australia and acknowledges the contribution of the study nurses, research assistants and all clinical and scientific collaborators to the study. The complete AOCS Study Group can be found at www.aocstudy.org. We would like to thank the women who participated in these research programs.

**63**

**Author details**

Orla McNally7

*Glucagonoma Masquerading as a Mucinous Cancer of the Ovary: Lessons from Cell Biology*

Gwo Yaw Ho1,2,3,4,7\*, Sumitra Ananda1,3,4,8, Cassandra J. Vandenberg1,4,

Matthew J. Wakefield1,4 and Clare L. Scott1,3,4,5,6,7

2 Monash University, Melbourne, Australia

1 Walter and Eliza Hall Institute, Parkville, Australia

3 Peter MacCallum Cancer Centre, Melbourne, Australia

4 The University of Melbourne, Melbourne, Australia

5 Royal Melbourne Hospital, Parkville, Australia

6 Monash Medical Centre, Clayton, Australia

7 Royal Women's Hospital, Parkville, Australia

\*Address all correspondence to: ho.g@wehi.edu.au

8 Western Health, Melbourne, Australia

provided the original work is properly cited.

, Jeanne Tie1,3,4, Kylie Gorringe3,4, David Bowtell3

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Jan Pyman7

,

*DOI: http://dx.doi.org/10.5772/intechopen.92554*

*Glucagonoma Masquerading as a Mucinous Cancer of the Ovary: Lessons from Cell Biology DOI: http://dx.doi.org/10.5772/intechopen.92554*

#### **Author details**

*Gynaecological Malignancies - Updates and Advances*

up to the patient's death.

**Acknowledgements**

participated in these research programs.

patient's clinical findings and also with IHC proving glucagon protein expression only apparent after the RNA sequencing results were available. In hindsight, it is hard to predict if this patient would have benefited from repeated surgical resection of recurrent tumour [18], or to NET based treatment regimens, such as mTOR inhibition (everolimus) [19] or multiple tyrosine kinase inhibitor (sunitinib, pazopanib) [20, 21]. Furthermore, the patient did not exhibit glucagon syndrome and her glucagon serum level was never tested. Nevertheless, clinically tumour #49 behaved like a NET tumour with slow indolent progression and localised complication. Unfortunately, in this case, the problematic tumour caused repeated bowel obstructive symptoms requiring multiple hospital admissions in the months leading

The recognition of diversity of tumour subtypes even within a rare tumour population is important especially in designing clinical trials. Given the small number of patients available for accruement, it is vital that we accurately stratify patients into treatment arms and identify robust biomarkers early. A very rare tumour within a rare tumour subtype can pose a challenging issue in terms of being an outlier that would skew the outcome in a clinical trial and also in optimising treatment for this patient based on available evidence (which is lacking). These issues will need to be addressed in any clinical trials pertaining to rare cancer.

We thank Margot Osinski (Royal Women's Hospital) for database assistance and AOCS: The Australian Ovarian Cancer Study Group was supported by the U.S. Army Medical Research and Materiel Command under DAMD17-01-1-0729. The AOCS also acknowledges the cooperation of the participating institutions in Australia and acknowledges the contribution of the study nurses, research assistants and all clinical and scientific collaborators to the study. The complete AOCS Study Group can be found at www.aocstudy.org. We would like to thank the women who

**62**

Gwo Yaw Ho1,2,3,4,7\*, Sumitra Ananda1,3,4,8, Cassandra J. Vandenberg1,4, Orla McNally7 , Jeanne Tie1,3,4, Kylie Gorringe3,4, David Bowtell3 , Jan Pyman7 , Matthew J. Wakefield1,4 and Clare L. Scott1,3,4,5,6,7


\*Address all correspondence to: ho.g@wehi.edu.au

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **References**

[1] Schiavone MB et al. Natural history and outcome of mucinous carcinoma of the ovary. American Journal of Obstetrics and Gynecology. 2011;**205**:480.e1-480.e8

[2] Perren TJ. Mucinous epithelial ovarian carcinoma. Annals of Oncology. 2016;**27**:i53-i57

[3] Khunamornpong S et al. Primary and metastatic mucinous adenocarcinomas of the ovary: Evaluation of the diagnostic approach using tumor size and laterality. Gynecologic Oncology. 2006;**101**:152-157

[4] Pignata S et al. Activity of chemotherapy in mucinous epithelial ovarian cancer: A retrospective study. BMC Cancer. 2008;**8**:252

[5] Investigators, I.T. Paclitaxel plus carboplatin versus standard chemotherapy with either single-agent carboplatin or cyclophosphamide, doxorubicin, and cisplatin in women with ovarian cancer: The ICON3 randomised trial. The Lancet. 2002;**360**:505-515

[6] Bookman MA. GOG0182-ICON5: 5-arm phase III randomized trial of paclitaxel (P) and carboplatin (C) vs combinations with gemcitabine (G), PEG-lipososomal doxorubicin (D), or topotecan (T) in patients (pts) with advanced-stage epithelial ovarian (EOC) or primary peritoneal (PPC) carcinoma. Journal of Clinical Oncology. 2006:**24**(18\_suppl):5002-5002. DOI: 10.1200/jco.2006.24.18\_suppl.5002

[7] Oza AM et al. Standard chemotherapy with or without bevacizumab for women with newly diagnosed ovarian cancer (ICON7): Overall survival results of a phase 3 randomised trial. Lancet Oncology. 2015;**16**:928-936

[8] Zaino RJ et al. Advanced stage mucinous adenocarcinoma of the ovary is both rare and highly lethal. Cancer. 2010;**117**:554-562

[9] Hess V. Mucinous epithelial ovarian cancer: A separate entity requiring specific treatment. Journal of Clinical Oncology. 2004;**22**:1040-1044

[10] Gore ME et al. Multicentre trial of carboplatin/paclitaxel versus oxaliplatin/capecitabine, each with/ without bevacizumab, as first line chemotherapy for patients with mucinous epithelial ovarian cancer (mEOC). Journal of Clinical Oncology. 2015. DOI: 10.1200/ jco.2015.33.15\_suppl.5528

[11] Heinzelmann-Schwarz VA et al. A distinct molecular profile associated with mucinous epithelial ovarian cancer. 2006:1-10. DOI: 10.1038/sj.bjc.6603003

[12] Shih I-M, Kurman RJ. Ovarian tumorigenesis. The American Journal of Pathology. 2010;**164**:1511-1518

[13] Cheasley D et al. The molecular origin and taxonomy of mucinous ovarian carcinoma. Nature Communications. 2019:1-11. DOI: 10.1038/s41467-019-11862-x

[14] Robinson MD, McCarthy DJ, Smyth GK. edgeR: A bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2009;**26**:139-140

[15] Magali R et al. Ensembl core software resources: Storage and programmatic access for DNA sequence and genome annotation. Database. 2017:1-11. DOI: 10.1093/database

[16] Grotzinger C et al. LI-cadherin: A marker of gastric metaplasia and neoplasia. Gut. 2001;**49**:78-81

**65**

*Glucagonoma Masquerading as a Mucinous Cancer of the Ovary: Lessons from Cell Biology*

*DOI: http://dx.doi.org/10.5772/intechopen.92554*

[17] Laury A et al. A comprehensive analysis of PAX8 expression in human epithelial tumors. The American Journal

Zayadeen A. Case report glucagonoma and glucagonoma syndrome: A case report with review of recent advances in management. Case Reports in Surgery. 2016:1-3. DOI: 10.1155/2016/1484089

[19] Yao JC et al. Everolimus for the treatment of advanced, non-functional neuroendocrine tumours of the lung or gastrointestinal tract (RADIANT-4): A randomised, placebo-controlled, phase 3 study. The Lancet. 2016;**387**:968-977

[20] Dahan L et al. Sunitinib malate for the treatment of pancreatic

[21] Grande E et al. Pazopanib in pretreated advanced neuroendocrine tumors: A phase II, open-label trial of the Spanish task force Group for Neuroendocrine Tumors (GETNE). Annals of Oncology. 2015;**26**:1987-1993

neuroendocrine tumors. New England Journal of Medicine. 2011;**364**:501-513

of Pathology. 2011;**35**:816

[18] Al-Faouri A, Ajarma K, Alghazawi S, Al-Rawabdeh S, *Glucagonoma Masquerading as a Mucinous Cancer of the Ovary: Lessons from Cell Biology DOI: http://dx.doi.org/10.5772/intechopen.92554*

[17] Laury A et al. A comprehensive analysis of PAX8 expression in human epithelial tumors. The American Journal of Pathology. 2011;**35**:816

[18] Al-Faouri A, Ajarma K, Alghazawi S, Al-Rawabdeh S, Zayadeen A. Case report glucagonoma and glucagonoma syndrome: A case report with review of recent advances in management. Case Reports in Surgery. 2016:1-3. DOI: 10.1155/2016/1484089

[19] Yao JC et al. Everolimus for the treatment of advanced, non-functional neuroendocrine tumours of the lung or gastrointestinal tract (RADIANT-4): A randomised, placebo-controlled, phase 3 study. The Lancet. 2016;**387**:968-977

[20] Dahan L et al. Sunitinib malate for the treatment of pancreatic neuroendocrine tumors. New England Journal of Medicine. 2011;**364**:501-513

[21] Grande E et al. Pazopanib in pretreated advanced neuroendocrine tumors: A phase II, open-label trial of the Spanish task force Group for Neuroendocrine Tumors (GETNE). Annals of Oncology. 2015;**26**:1987-1993

**64**

*Gynaecological Malignancies - Updates and Advances*

[8] Zaino RJ et al. Advanced stage mucinous adenocarcinoma of the ovary is both rare and highly lethal. Cancer.

[9] Hess V. Mucinous epithelial ovarian cancer: A separate entity requiring specific treatment. Journal of Clinical

Oncology. 2004;**22**:1040-1044

[10] Gore ME et al. Multicentre trial of carboplatin/paclitaxel versus oxaliplatin/capecitabine, each with/ without bevacizumab, as first line chemotherapy for patients with mucinous epithelial ovarian cancer (mEOC). Journal of Clinical Oncology. 2015. DOI: 10.1200/ jco.2015.33.15\_suppl.5528

[11] Heinzelmann-Schwarz VA et al. A distinct molecular profile associated with mucinous epithelial ovarian cancer. 2006:1-10. DOI: 10.1038/sj.bjc.6603003

[12] Shih I-M, Kurman RJ. Ovarian tumorigenesis. The American Journal of

[13] Cheasley D et al. The molecular origin and taxonomy of mucinous ovarian carcinoma. Nature Communications. 2019:1-11. DOI: 10.1038/s41467-019-11862-x

[14] Robinson MD, McCarthy DJ, Smyth GK. edgeR: A bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2009;**26**:139-140

[15] Magali R et al. Ensembl core software resources: Storage and

programmatic access for DNA sequence and genome annotation. Database. 2017:1-11. DOI: 10.1093/database

[16] Grotzinger C et al. LI-cadherin: A marker of gastric metaplasia and neoplasia. Gut. 2001;**49**:78-81

Pathology. 2010;**164**:1511-1518

2010;**117**:554-562

[1] Schiavone MB et al. Natural history and outcome of mucinous carcinoma of the ovary. American Journal of Obstetrics and Gynecology.

[2] Perren TJ. Mucinous epithelial ovarian carcinoma. Annals of Oncology.

of the ovary: Evaluation of the diagnostic approach using tumor size and laterality. Gynecologic Oncology.

[4] Pignata S et al. Activity of

[5] Investigators, I.T. Paclitaxel plus carboplatin versus standard chemotherapy with either single-agent carboplatin or cyclophosphamide, doxorubicin, and cisplatin in women with ovarian cancer: The ICON3 randomised trial. The Lancet.

BMC Cancer. 2008;**8**:252

chemotherapy in mucinous epithelial ovarian cancer: A retrospective study.

[6] Bookman MA. GOG0182-ICON5: 5-arm phase III randomized trial of paclitaxel (P) and carboplatin (C) vs combinations with gemcitabine (G), PEG-lipososomal doxorubicin (D), or topotecan (T) in patients (pts) with advanced-stage epithelial ovarian (EOC) or primary peritoneal (PPC) carcinoma. Journal of Clinical Oncology. 2006:**24**(18\_suppl):5002-5002. DOI: 10.1200/jco.2006.24.18\_suppl.5002

[7] Oza AM et al. Standard chemotherapy with or without bevacizumab for women with newly diagnosed ovarian cancer (ICON7): Overall survival results of a phase 3 randomised trial. Lancet Oncology.

2015;**16**:928-936

[3] Khunamornpong S et al. Primary and metastatic mucinous adenocarcinomas

2011;**205**:480.e1-480.e8

2016;**27**:i53-i57

**References**

2006;**101**:152-157

2002;**360**:505-515

**Chapter 4**

Model

**Abstract**

siRNA delivery tool even *in vivo*.

**1. Introduction**

**67**

**Keywords:** siRNA, ovarian, Glypican-3, micelle, PLGA

Therapeutic Effect of Glypican-3

Murine Peritoneal Dissemination

*Tomoyo Kawakubo-Yasukochi and Manabu Nakashima*

Ovarian cancer is known to be the most lethal gynecologic cancer. It has been reported that Glypican-3 (Gpc3) expression induces immune responses, promotes the progression in ovarian cancer. Then, we focused on this Gpc3 gene silencing, tried to prepare siRNA delivery system. In this chapter, we introduce one of the therapeutic proposals in terms of novel drug delivery system using siRNA as a targeting medicine. This chapter introduces our works about preparation of siRNA-PLGA hybrid micelles to deliver the siRNA into the ovarian cancer cells and to evaluate gene silencing effects in mice model. As a result, siRNA-PLGA hybrid micelles were shown to effectively inhibit Gpc3 expression *in vitro*. In addition, siRNA-PLGA hybrid micelles also decreased the number of tumor nodes in the mesentery *in vivo.* These results suggested that Gpc3 could be a target molecule for ovarian cancer treatment and siRNA-PLGA hybrid micelles could be an effective

Epithelial ovarian carcinoma (EOC) is the most lethal gynecological malignancy. EOC accounts for about 90% of all ovarian cancers and distributed over the most common histotypes: high-grade serous (HGSC, 70%), low-grade serous (LGSC, < 5%), endometrioid (EC, 10%), mucinous (MC, 3–4%) and clear cell ovarian carcinoma (CCC, 10%) [1]. Five-year survival rates differ significantly across the histotypes, with drastically lower survival rates for serous carcinoma (SC (HGSC and LGSC), 43%) compared with EC (82%), MC (71%) and CCC (66%) in the USA. CCC is a comparatively rare tumor, depending on the geographic location. In west countries, OCCC represents <10% of all EOC. In contrast, the incidence of CCC

Gene Silencing Using siRNA

for Ovarian Cancer in a

*Mai Hazekawa,Takuya Nishinakagawa,*

### **Chapter 4**

## Therapeutic Effect of Glypican-3 Gene Silencing Using siRNA for Ovarian Cancer in a Murine Peritoneal Dissemination Model

*Mai Hazekawa,Takuya Nishinakagawa, Tomoyo Kawakubo-Yasukochi and Manabu Nakashima*

### **Abstract**

Ovarian cancer is known to be the most lethal gynecologic cancer. It has been reported that Glypican-3 (Gpc3) expression induces immune responses, promotes the progression in ovarian cancer. Then, we focused on this Gpc3 gene silencing, tried to prepare siRNA delivery system. In this chapter, we introduce one of the therapeutic proposals in terms of novel drug delivery system using siRNA as a targeting medicine. This chapter introduces our works about preparation of siRNA-PLGA hybrid micelles to deliver the siRNA into the ovarian cancer cells and to evaluate gene silencing effects in mice model. As a result, siRNA-PLGA hybrid micelles were shown to effectively inhibit Gpc3 expression *in vitro*. In addition, siRNA-PLGA hybrid micelles also decreased the number of tumor nodes in the mesentery *in vivo.* These results suggested that Gpc3 could be a target molecule for ovarian cancer treatment and siRNA-PLGA hybrid micelles could be an effective siRNA delivery tool even *in vivo*.

**Keywords:** siRNA, ovarian, Glypican-3, micelle, PLGA

#### **1. Introduction**

Epithelial ovarian carcinoma (EOC) is the most lethal gynecological malignancy. EOC accounts for about 90% of all ovarian cancers and distributed over the most common histotypes: high-grade serous (HGSC, 70%), low-grade serous (LGSC, < 5%), endometrioid (EC, 10%), mucinous (MC, 3–4%) and clear cell ovarian carcinoma (CCC, 10%) [1]. Five-year survival rates differ significantly across the histotypes, with drastically lower survival rates for serous carcinoma (SC (HGSC and LGSC), 43%) compared with EC (82%), MC (71%) and CCC (66%) in the USA. CCC is a comparatively rare tumor, depending on the geographic location. In west countries, OCCC represents <10% of all EOC. In contrast, the incidence of CCC

was reportedly 25% of EOC in Japan. The high number of patients (80%) with SC is diagnosed at advanced stages (stages III and IV). While, CCC which has the second number of patients (25%) after SC, is predominantly diagnosed at stage I (65%) [2]. Thus, CCC has different character compared with SC. Five-year survival rate at stage I for SC and CCC is same (80%). While, five-year survival rate at stage IV for SC is 40% and stage I of CCC is 25%. CCC has a very poor prognosis. One of the reasons is that CCC is associated with greater chemoresistance and a poorer prognosis compared with other EOC subtypes. Particularly for recurrent CCC, the response rate (RR) to salvage chemotherapy was extremely low. Previous studies have indicated that high L-type amino acid transporter 1 (LAT1), which belongs to system L, a Na<sup>+</sup> -independent carrier that transports large neutral amino acids, expression was associated with poorer prognosis and chemoresistance in CCC [3]. Furthermore, hepatocyte nuclear factor 1β (HNF1β) and glutaminolysis contribute for the chemoresistance to platinum-based antineoplastic agents of CCC through the intrinsically increased glutathione (GSH) bioavailability [4]. Therefore, novel and innovative strategies are required to improve outcomes for patients with CCC that is refractory to chemotherapy.

In this chapter, we report the therapeutic effect of Gpc3 gene silencing in ovarian cancer, and introduce the finding about a novel siRNA delivery system of

*Therapeutic Effect of Glypican-3 Gene Silencing Using siRNA for Ovarian Cancer in a Murine…*

**2. Effect of anti-metastasis in ovarian cancer caused by Glypican-3 gene**

GPC3, 55–65 kDa protein consisting of 580 amino acids, is a heparan sulfate chain proteoglycan (HSPGs) bound to cell membrane by a glycosylphosphatidylinositol (GPI) anchor. This protein is expressed in the liver and kidney of healthy fetuses but is hardly expressed in adults, except in the placenta. Loss of function mutations of GPC3 leads to Simpson-Golabi-Behmel syndrome (SGBS), a rare Xlinked disorder (X chromosome, Xq26) with significant overgrowth [5], which has also been observed in GPC3-null mice [30] because the gene shows high homology between humans and mice. GPC3 is expressed ubiquitously in the embryo but is reduced in the central nervous system (CNS) in adults [31]. Thus, GPC3 is considered to be one of the factors affecting prenatal development and metabolism originally. On the other hand, GPC3 is especially overexpressed in HCC [10, 11], CCC [32, 33], melanoma [34], and lung cancer [35]. Although the precious function of GPC3 remains unclear, it has been strongly suggested that it is related to the malignant transformation, accelerating cell growth and increasing inflammatory

The Wnt/Frizzled/β-catenin pathway is activated in about 50% of HCCs. Wt3a has been shown to mediate the GPC3-induced growth of HCCs via the canonical Wnt/β-catenin pathway [6, 37]. Sulfated heparan sulfate glycosaminoglycan (HSGAG) chains of GPC3 and other HSPGs are potential substrates for desulfation at the 6-O position by human sulfate 2 (SULF2). It has been reported that SULF2 activates Wnt/β-catenin signaling in HCC cells, and this process is GPC3-dependent and can be independent of exogenous Wnts [38]. In a previous study, a human monoclonal antibody against GPC3 inhibited Wnt3a/β-catenin signaling in HCC cells and antitumor activity *in vivo* [39]. Furthermore, blocking the heparan sulfate chains on GPC3 with human monoclonal antibody against GPC3 also reduced c-Met activation in hepatocyte growth factor (HGF)-treated HCC cells and 3D-cultured spheroids. GPC3 is involved in HCC cell migration and motility through HS chain-

Although the role of GPC3 in HCC has been reported little by little, the role of

Based on these, we focused on knocking down of GPC3 gene therapy for ovarian cancer using siRNA which can be expected to be effective in clinical practice. Then, we evaluated the efficiency of siRNA-PLGA hybrid micelles targeted to Gpc3 on

GPC3 in ovarian cancer, especially CCC expressed GPC3, has been remained unclear. So recurrent or persistent CCC has been reported as having a potentially chemoresistant phenotype against conventional cytotoxic agents, leading to poorer prognosis. Thus, novel treatment approaches must be adopted for CCC. With com-

pelling evidence that EOC is an immunogenic tumor, immunotherapeutic approaches are currently being evaluated and should be optimized based on histology-specific features. Previous research also suggested that GPC3 peptide vaccinations may hold a significant impact to prolong survival of patients with refractory CCC, allowing them to maintain quality of life with no serious

micelles for nucleic acid therapy based on our data [29].

mediated cooperation with the HGF/Met pathway [40].

**2.1 Role of Grypican-3 in ovarian cancer**

*DOI: http://dx.doi.org/10.5772/intechopen.90311*

**silencing**

reaction [36].

toxicities [41].

**69**

Glypican-3 (GPC3) is a member of the glypican family of heparan sulfate proteoglycans. GPC3 regulates cell proliferation signals by binding growth factors such as Wnt, fibroblast growth factor, and insulin-like growth factor and plays an important role in the proliferation and differentiation of embryonic cells [5–7]. GPC3 is expressed in various fetal tissues (liver, lung, kidney, and placenta) but is not detected in normal postnatal tissue due to DNA methylation-induced epigenetic silencing [8, 9]. While, previous studies showed that GPC3 was overexpressed in several malignant tumors, including hepatocellular carcinoma (HCC), CCC and melanoma. Particularly, GPC3 is detected in ≥80% of patients with HCC caused by hepatitis B or C [10, 11]. The function of membraneanchored GPC3 in these cancers is unknown, but it is likely involved in the neoplastic transformation of HCC [12]. Membrane-bound GPC3 can be cleaved and secreted into the blood. Mammalian GPC family members are cleaved at GPI anchor level by endogenous GPI phospholipase D [13]. Thus, various forms of GPC3 protein are present in blood, although their functions remain unclear. Given these features, GPC3 is useful not only as a target for cancer immunotherapy but also as a novel tumor marker.

Small interfering or silencing RNA (siRNA) technologies are based on the inhibition of gene expression or translation by siRNAs targeting messenger RNA selectively [14]. Gene interference therapy using siRNA has great potential for treatment of wide variety of diseases [15], ranging from cancer [16–19] to viral infection [20, 21] and brain disorder [22, 23]. The benefit of applying this technology to cancer therapy is that siRNA can target genes which are specific for tumor cells, leaving healthy, non-tumor tissue unaffected. Despite their medical potential, the clinical translation of siRNA technologies has up to now been limited. This limited progress is due to the difficulties of delivering siRNA *in vivo*. Unprotected siRNAs are easily degraded in the bloodstream, and siRNAs alone do not translocate across cell membrane [24]. In addition, it has been reported that siRNAs can be immunogenic [25]. Therefore, safe and efficient carriers must be developed for siRNA delivery to protect siRNA from nuclease action and at the same time triggers intracellular uptake *in vivo* [26, 27].

In our previous study, we prepared slow release formulation using biodegradable polymer (poly(lactide-co-glycolide), PLGA) such as micro-/nano particles [28]. Recently, we engaged to prepare the siRNA delivery system using PLGA for anti-metastasis therapy.

*Therapeutic Effect of Glypican-3 Gene Silencing Using siRNA for Ovarian Cancer in a Murine… DOI: http://dx.doi.org/10.5772/intechopen.90311*

In this chapter, we report the therapeutic effect of Gpc3 gene silencing in ovarian cancer, and introduce the finding about a novel siRNA delivery system of micelles for nucleic acid therapy based on our data [29].

#### **2. Effect of anti-metastasis in ovarian cancer caused by Glypican-3 gene silencing**

#### **2.1 Role of Grypican-3 in ovarian cancer**

was reportedly 25% of EOC in Japan. The high number of patients (80%) with SC is diagnosed at advanced stages (stages III and IV). While, CCC which has the second number of patients (25%) after SC, is predominantly diagnosed at stage I (65%) [2]. Thus, CCC has different character compared with SC. Five-year survival rate at stage I for SC and CCC is same (80%). While, five-year survival rate at stage IV for SC is 40% and stage I of CCC is 25%. CCC has a very poor prognosis. One of the reasons is that CCC is associated with greater chemoresistance and a poorer prognosis compared with other EOC subtypes. Particularly for recurrent CCC, the response rate (RR) to salvage chemotherapy was extremely low. Previous studies have indicated that high L-type amino acid transporter 1 (LAT1), which belongs to


expression was associated with poorer prognosis and chemoresistance in CCC [3]. Furthermore, hepatocyte nuclear factor 1β (HNF1β) and glutaminolysis contribute for the chemoresistance to platinum-based antineoplastic agents of CCC through the intrinsically increased glutathione (GSH) bioavailability [4]. Therefore, novel and innovative strategies are required to improve outcomes for patients with CCC

Glypican-3 (GPC3) is a member of the glypican family of heparan sulfate proteoglycans. GPC3 regulates cell proliferation signals by binding growth factors such as Wnt, fibroblast growth factor, and insulin-like growth factor and plays an important role in the proliferation and differentiation of embryonic cells [5–7]. GPC3 is expressed in various fetal tissues (liver, lung, kidney, and placenta) but is not detected in normal postnatal tissue due to DNA methylation-induced epige-

netic silencing [8, 9]. While, previous studies showed that GPC3 was

overexpressed in several malignant tumors, including hepatocellular carcinoma (HCC), CCC and melanoma. Particularly, GPC3 is detected in ≥80% of patients with HCC caused by hepatitis B or C [10, 11]. The function of membraneanchored GPC3 in these cancers is unknown, but it is likely involved in the neoplastic transformation of HCC [12]. Membrane-bound GPC3 can be cleaved and secreted into the blood. Mammalian GPC family members are cleaved at GPI anchor level by endogenous GPI phospholipase D [13]. Thus, various forms of GPC3 protein are present in blood, although their functions remain unclear. Given these features, GPC3 is useful not only as a target for cancer immunotherapy but

Small interfering or silencing RNA (siRNA) technologies are based on the inhibition of gene expression or translation by siRNAs targeting messenger RNA selectively [14]. Gene interference therapy using siRNA has great potential for treatment of wide variety of diseases [15], ranging from cancer [16–19] to viral infection [20, 21] and brain disorder [22, 23]. The benefit of applying this technology to cancer therapy is that siRNA can target genes which are specific for tumor cells, leaving healthy, non-tumor tissue unaffected. Despite their medical potential, the clinical translation of siRNA technologies has up to now been limited. This limited progress is due to the difficulties of delivering siRNA *in vivo*. Unprotected siRNAs are easily degraded in the bloodstream, and siRNAs alone do not translocate across cell membrane [24]. In addition, it has been reported that siRNAs can be immunogenic [25]. Therefore, safe and efficient carriers must be developed for siRNA delivery to protect siRNA from nuclease action and at the same time triggers

In our previous study, we prepared slow release formulation using biodegradable polymer (poly(lactide-co-glycolide), PLGA) such as micro-/nano particles [28]. Recently, we engaged to prepare the siRNA delivery system using PLGA for

system L, a Na<sup>+</sup>

that is refractory to chemotherapy.

*Gynaecological Malignancies - Updates and Advances*

also as a novel tumor marker.

intracellular uptake *in vivo* [26, 27].

anti-metastasis therapy.

**68**

GPC3, 55–65 kDa protein consisting of 580 amino acids, is a heparan sulfate chain proteoglycan (HSPGs) bound to cell membrane by a glycosylphosphatidylinositol (GPI) anchor. This protein is expressed in the liver and kidney of healthy fetuses but is hardly expressed in adults, except in the placenta. Loss of function mutations of GPC3 leads to Simpson-Golabi-Behmel syndrome (SGBS), a rare Xlinked disorder (X chromosome, Xq26) with significant overgrowth [5], which has also been observed in GPC3-null mice [30] because the gene shows high homology between humans and mice. GPC3 is expressed ubiquitously in the embryo but is reduced in the central nervous system (CNS) in adults [31]. Thus, GPC3 is considered to be one of the factors affecting prenatal development and metabolism originally. On the other hand, GPC3 is especially overexpressed in HCC [10, 11], CCC [32, 33], melanoma [34], and lung cancer [35]. Although the precious function of GPC3 remains unclear, it has been strongly suggested that it is related to the malignant transformation, accelerating cell growth and increasing inflammatory reaction [36].

The Wnt/Frizzled/β-catenin pathway is activated in about 50% of HCCs. Wt3a has been shown to mediate the GPC3-induced growth of HCCs via the canonical Wnt/β-catenin pathway [6, 37]. Sulfated heparan sulfate glycosaminoglycan (HSGAG) chains of GPC3 and other HSPGs are potential substrates for desulfation at the 6-O position by human sulfate 2 (SULF2). It has been reported that SULF2 activates Wnt/β-catenin signaling in HCC cells, and this process is GPC3-dependent and can be independent of exogenous Wnts [38]. In a previous study, a human monoclonal antibody against GPC3 inhibited Wnt3a/β-catenin signaling in HCC cells and antitumor activity *in vivo* [39]. Furthermore, blocking the heparan sulfate chains on GPC3 with human monoclonal antibody against GPC3 also reduced c-Met activation in hepatocyte growth factor (HGF)-treated HCC cells and 3D-cultured spheroids. GPC3 is involved in HCC cell migration and motility through HS chainmediated cooperation with the HGF/Met pathway [40].

Although the role of GPC3 in HCC has been reported little by little, the role of GPC3 in ovarian cancer, especially CCC expressed GPC3, has been remained unclear. So recurrent or persistent CCC has been reported as having a potentially chemoresistant phenotype against conventional cytotoxic agents, leading to poorer prognosis. Thus, novel treatment approaches must be adopted for CCC. With compelling evidence that EOC is an immunogenic tumor, immunotherapeutic approaches are currently being evaluated and should be optimized based on histology-specific features. Previous research also suggested that GPC3 peptide vaccinations may hold a significant impact to prolong survival of patients with refractory CCC, allowing them to maintain quality of life with no serious toxicities [41].

Based on these, we focused on knocking down of GPC3 gene therapy for ovarian cancer using siRNA which can be expected to be effective in clinical practice. Then, we evaluated the efficiency of siRNA-PLGA hybrid micelles targeted to Gpc3 on

ovarian cancer *in vitro* and examined its antitumor effects *in vivo* in a mouse peritoneal dissemination model.

#### **2.2 Effect of anti-metastasis caused by knocking down of Grypican-3 using LPEI coating siRNA-PLGA hybrid micelles** *in vivo*

The synthesis of siRNA-PLGA hybrid was described briefly as follows. PLGA was activated by DCC and NHS. Activated PLGA reacted with 3-(2-pyridyldithio) propionyl hydrazide (PDPH) as a cross-linker. After PDPH activated, PLGA (PLGA-PDPH) was used for siRNA conjugation. A thiol-modified double-strand siRNA was reacted with PLGA-PDPH, siRNA-PLGA hybrid was synthesized via a disulfide exchange reaction. The synthesized siRNA-PLGA hybrid conjugates spontaneously formed self-assembled micelles in aqueous solutions, resulting to form micelle with siRNA side facing the outer shell as shown in **Figure 1A** and **C**. Furthermore, we also prepared liner polyethylenimine (LPEI)-coated siRNA-PLGA micelles, its surface was positive charged by cationic polymer, to increase the efficiency of intracellular uptake as shown in **Figure 1D**.

Measurement of critical micelle concentration (**Figure 2**) and distribution of particle (**Figure 3**) were performed to evaluate the physical properties of micelles. The mean diameter and zeta potential of siRNA-PLGA hybrid micelles were about 110 nm and about 40 mV, respectively. The zeta potentials of siRNA-PLGA hybrid micelle were changed from negative charge to positive charge by LPEI coating.

Until now, the best agents for siRNA delivery are cationic lipids and polycations, i.e. polyelectrolytes bearing multiple positive charges to increase intracellular uptake *in vivo* [42, 43]. From these previous data, LPEI coating micelle can be expected its clinical potential *in vivo* because positive charge caused by LPEI makes micelles easy to be taken into the cell.

The GPC3 levels in HM-1 cell line, which is mouse ovarian cancer cell line, treated with siRNA-PLGA hybrid micelles were then evaluated by western blotting.

As shown in **Figure 4**, siRNA-PLGA hybrid micelles significantly suppressed GPC3

*Critical micelle concentration (CMC) detected by measuring the relative excitation intensity ratio of pyrene at*

*Therapeutic Effect of Glypican-3 Gene Silencing Using siRNA for Ovarian Cancer in a Murine…*

Assessment of antitumor effects of these micelles in a murine peritoneal dissemination model was performed by intraperitoneal (i.p.) injection as topical treatment. In general, topical administration is often more effective because it is easy to react since the medicine is close to the disease lesion [44]. The number of disseminated nodules and the peritoneal fluid volumes were evaluated at 15 days after injection of the HM-1 cells. As shown in **Figure 5**, the number of disseminated nodules and the volume of peritoneal fluid siRNA-PLGA hybrid micelle-treated groups were significantly low compared with the control. Next, GPC3 levels in the cell lysates of peritoneal cells collected from the peritoneal fluid were evaluated by

As shown in **Figure 6**, the levels of IFN-γ, IL-6, and TNF-α in mice treated with

suppressed compared with the control. GPC3 expression in the lymphocytes such as B cells, T cells and macrophages in the peritoneal fluid of mice, was detected by western blotting. From these results, there is a possibility that the therapeutic effect was induced by GPC3 gene knockdown of not only cancer cell but also lymphocytes

uncoated and LPEI-coated siRNA-PLGA hybrid micelles were significantly

expression compared with the control.

*Size distribution of siRNA-PLGA hybrid micelles.*

*emission of 329 nm and 338 nm (*I*338/*I*329).*

*DOI: http://dx.doi.org/10.5772/intechopen.90311*

in the peritoneal fluid as the additive effects.

western blotting.

**71**

**Figure 2.**

**Figure 3.**

**Figure 1.**

*(A) and (B) Structure of siRNA-PLGA hybrid and Fab'-PLGA hybrid via a cleavable disulfide linkage. (C)–(E) Schematic diagram for siRNA-PLGA hybrid micelle structure in an aqueous environment.*

*Therapeutic Effect of Glypican-3 Gene Silencing Using siRNA for Ovarian Cancer in a Murine… DOI: http://dx.doi.org/10.5772/intechopen.90311*

#### **Figure 2.**

ovarian cancer *in vitro* and examined its antitumor effects *in vivo* in a mouse

**2.2 Effect of anti-metastasis caused by knocking down of Grypican-3 using**

The synthesis of siRNA-PLGA hybrid was described briefly as follows. PLGA was activated by DCC and NHS. Activated PLGA reacted with 3-(2-pyridyldithio) propionyl hydrazide (PDPH) as a cross-linker. After PDPH activated, PLGA (PLGA-PDPH) was used for siRNA conjugation. A thiol-modified double-strand siRNA was reacted with PLGA-PDPH, siRNA-PLGA hybrid was synthesized via a disulfide exchange reaction. The synthesized siRNA-PLGA hybrid conjugates spontaneously formed self-assembled micelles in aqueous solutions, resulting to form micelle with siRNA side facing the outer shell as shown in **Figure 1A** and **C**. Furthermore, we also prepared liner polyethylenimine (LPEI)-coated siRNA-PLGA micelles, its surface was positive charged by cationic polymer, to increase the

Measurement of critical micelle concentration (**Figure 2**) and distribution of particle (**Figure 3**) were performed to evaluate the physical properties of micelles. The mean diameter and zeta potential of siRNA-PLGA hybrid micelles were about 110 nm and about 40 mV, respectively. The zeta potentials of siRNA-PLGA hybrid micelle were changed from negative charge to positive charge by LPEI

Until now, the best agents for siRNA delivery are cationic lipids and polycations,

i.e. polyelectrolytes bearing multiple positive charges to increase intracellular uptake *in vivo* [42, 43]. From these previous data, LPEI coating micelle can be expected its clinical potential *in vivo* because positive charge caused by LPEI makes

The GPC3 levels in HM-1 cell line, which is mouse ovarian cancer cell line, treated with siRNA-PLGA hybrid micelles were then evaluated by western blotting.

*(A) and (B) Structure of siRNA-PLGA hybrid and Fab'-PLGA hybrid via a cleavable disulfide linkage. (C)–(E) Schematic diagram for siRNA-PLGA hybrid micelle structure in an aqueous environment.*

**LPEI coating siRNA-PLGA hybrid micelles** *in vivo*

efficiency of intracellular uptake as shown in **Figure 1D**.

micelles easy to be taken into the cell.

peritoneal dissemination model.

*Gynaecological Malignancies - Updates and Advances*

coating.

**Figure 1.**

**70**

*Critical micelle concentration (CMC) detected by measuring the relative excitation intensity ratio of pyrene at emission of 329 nm and 338 nm (*I*338/*I*329).*

**Figure 3.**

*Size distribution of siRNA-PLGA hybrid micelles.*

As shown in **Figure 4**, siRNA-PLGA hybrid micelles significantly suppressed GPC3 expression compared with the control.

Assessment of antitumor effects of these micelles in a murine peritoneal dissemination model was performed by intraperitoneal (i.p.) injection as topical treatment. In general, topical administration is often more effective because it is easy to react since the medicine is close to the disease lesion [44]. The number of disseminated nodules and the peritoneal fluid volumes were evaluated at 15 days after injection of the HM-1 cells. As shown in **Figure 5**, the number of disseminated nodules and the volume of peritoneal fluid siRNA-PLGA hybrid micelle-treated groups were significantly low compared with the control. Next, GPC3 levels in the cell lysates of peritoneal cells collected from the peritoneal fluid were evaluated by western blotting.

As shown in **Figure 6**, the levels of IFN-γ, IL-6, and TNF-α in mice treated with uncoated and LPEI-coated siRNA-PLGA hybrid micelles were significantly suppressed compared with the control. GPC3 expression in the lymphocytes such as B cells, T cells and macrophages in the peritoneal fluid of mice, was detected by western blotting. From these results, there is a possibility that the therapeutic effect was induced by GPC3 gene knockdown of not only cancer cell but also lymphocytes in the peritoneal fluid as the additive effects.

#### *Gynaecological Malignancies - Updates and Advances*

**2.3 Recognition of cancer cell using Fab**<sup>0</sup>

*DOI: http://dx.doi.org/10.5772/intechopen.90311*

**micelle** *in vitro*

**Figure 6.**

**Figure 7.**

**73**

*Efficiency of intracellular uptake of Fab*<sup>0</sup>

*in vitro by flow cytometry analysis.*

*permission from Elsevier.*

**-PLGA/siRNA-PLGA hybrid mixed**

In previous study, we reported that Gpc3 knocking down using siRNA-PLGA hybrid micelle by intraperitoneal injection was effective to suppress the metastasis in peritoneal dissemination of ovarian cancer mice model [29]. However, it is

*Therapeutic Effect of Glypican-3 Gene Silencing Using siRNA for Ovarian Cancer in a Murine…*

*Effect of GPC3 knockdown caused by treatment with siRNA-PLGA micelles on the secretion of IFN-γ, IL-6, TNF-α in the peritoneal fluid in a mouse peritoneal dissemination model. Data represent the mean* � *SD (n = 5). \*\*p < 0.01 versus the control group (Bonferroni test/ANOVA). Cited from Ref. [29]. Reprinted with*

*-PLGA/–Alexa 488 labeling siRNA-PLGA hybrid mixed micelles*

#### **Figure 4.**

*Western blot analysis of GPC3levels in HM-1 cells treated with siRNA-PLGA hybrid micelles* in vitro*. Data represent the mean SD (n = 3). \*\*p < 0.01 versus the control group (Bonferroni test/ANOVA). Cited from Ref. [29]. Reprinted with permission from Elsevier.*

#### **Figure 5.**

*Anti-metastasis effects of siRNA-PLGA micelles in a mouse peritoneal dissemination model. Representative images of the mesentery after laparotomy. Cited from Ref. [29]. Reprinted with permission from Elsevier.*

*Therapeutic Effect of Glypican-3 Gene Silencing Using siRNA for Ovarian Cancer in a Murine… DOI: http://dx.doi.org/10.5772/intechopen.90311*

#### **2.3 Recognition of cancer cell using Fab**<sup>0</sup> **-PLGA/siRNA-PLGA hybrid mixed micelle** *in vitro*

In previous study, we reported that Gpc3 knocking down using siRNA-PLGA hybrid micelle by intraperitoneal injection was effective to suppress the metastasis in peritoneal dissemination of ovarian cancer mice model [29]. However, it is

#### **Figure 6.**

**Figure 4.**

**Figure 5.**

**72**

*Ref. [29]. Reprinted with permission from Elsevier.*

*Gynaecological Malignancies - Updates and Advances*

*Western blot analysis of GPC3levels in HM-1 cells treated with siRNA-PLGA hybrid micelles* in vitro*. Data represent the mean SD (n = 3). \*\*p < 0.01 versus the control group (Bonferroni test/ANOVA). Cited from*

*Anti-metastasis effects of siRNA-PLGA micelles in a mouse peritoneal dissemination model. Representative images of the mesentery after laparotomy. Cited from Ref. [29]. Reprinted with permission from Elsevier.*

*Effect of GPC3 knockdown caused by treatment with siRNA-PLGA micelles on the secretion of IFN-γ, IL-6, TNF-α in the peritoneal fluid in a mouse peritoneal dissemination model. Data represent the mean* � *SD (n = 5). \*\*p < 0.01 versus the control group (Bonferroni test/ANOVA). Cited from Ref. [29]. Reprinted with permission from Elsevier.*

#### **Figure 7.**

*Efficiency of intracellular uptake of Fab*<sup>0</sup> *-PLGA/–Alexa 488 labeling siRNA-PLGA hybrid mixed micelles in vitro by flow cytometry analysis.*

necessary to develop a carrier which is "targeting" and "systemically administable". That is why, we prepared Fab0 -PLGA/siRNA-PLGA mixed micelle to recognize the target cell. Fab0 -PLGA hybrid was synthesized in a same method as siRNA-PLGA hybrid was synthesized. The drug design was described in **Figure 1B** and **E**.

**3.3 Limitation of assessment using animal**

*DOI: http://dx.doi.org/10.5772/intechopen.90311*

target cancer cell by i.v. in the future.

**Acknowledgements**

**Conflict of interest**

**75**

**4. Conclusion**

In the future as a next step, immunodeficient mice would be indispensable when we establish human model such as patient-derived xenograft (PDX) model. However, there is possibility that we cannot comprehend whether the micelle has medical potential when immunodeficient mice are used because GPC3 might be a molecule that is strongly associated with the immune system. That is why, we considered that we should further examine the usefulness of this therapy using micelles for human cancer cells based on our data using murine cell because there

*Therapeutic Effect of Glypican-3 Gene Silencing Using siRNA for Ovarian Cancer in a Murine…*

In conclusion, our results could indicate that Gpc3 gene silencing using siRNA has a possibility as an effective new therapeutic approach without side effects in ovarian cancer, especially CCC with GPC3 expression. Furthermore, this GPC3 targeting gene therapy is also useful for high GPC3 expression cancer such as HCC, melanoma and lung cancer if appropriate carrier is developed to deliver siRNA to

In addition, this finding is the first study to show that siRNA-PLGA hybrid micelles can effectively deliver siRNA to cancer cells *in vivo* at a low dose with significant anti-metastatic effect on murine ovarian cancer. We expect that novel formulation with more specific effects like siRNA including drug delivery system

This work was supported by JSPS KAKENHI Grant Number JP17K08477, Fukuoka Foundation for Sound Health Cancer Research Fund, and funds (No. 181045)

would be developed for malignant ovarian cancer therapy in the future.

from the Central Research Institute of Fukuoka University.

The authors declare no conflict of interest.

are different characteristics between murine and human cancer cells.

As shown in **Figure 7**, *in vitro* experiment, intracellular uptake of siRNA using Fab0 -PLGA/siRNA-PLGA mixed micelle was significantly increased compared with control. In particular, cytotoxicity was accelerated caused by treatment with Fab0 - PLGA/siRNA-PLGA mixed micelle compared with siRNA-PLGA hybrid micelle. This result suggests that the characteristics of the targeting used by antibody may be expected to have an additive effect of the function of Fab<sup>0</sup> itself in addition to the increase in the intracellular uptake efficiency by cell recognition. In some antibodies, the target protein knockdown effect is dramatically obtained using Fab0 - PLGA/siRNA-PLGA mixed micelle (data not shown). From these results, Fab0 - PLGA/siRNA-PLGA mixed micelles are believed to be useful as one of the targeting formulations to recognize the target cell.

#### **3. Expected side effect caused by gene therapy and limitation of assessment using animal**

#### **3.1 Off-target effects caused by RNAi**

The technique of RNAi in the medical field is expected to have not only therapeutic effects for human induced by knock-down specific genes but also suffers from off-target effects. Previous study reported that algorithm or open-source desktop software was developed to design RNAi sequences to exert strong and selective suppression of target genes and predict off-target [45, 46]. However, it is difficult to predict specific side effects that appear due to off-target effects in human. Furthermore, we suggested that the details of the off-target effect are often unclear due to the fact that commercial nucleic acid medications have a short period of use. In some cases, mouse results may not be compatible with humans because off-target effects vary by its sequences though there were no noticeable side effects in our experiment *in vivo*.

#### **3.2 Cytotoxicity of exogenous siRNA or polymer in development of formulation**

Until now, some polyplex or lipoplex with high membrane permeability formulations have been used for siRNA delivery system [47, 48]. A number of polymers have been popularly utilized to form stable and nanocomplexes with its cytotoxicity problem [27, 49–53]. PEI is also probably the most frequently used polycation in gene delivery, our LPEI-coated micelles did not exhibit cytotoxic effects. The fact that no toxicity was found in our experiments at the concentrations we used was consist with previous reports [54]. The greatest feature of this micelle is that it consists of a safe polymer, PLGA. PLGA is known as one of the biodegradable polymers used in marketed medication [55]. In some cases, siRNA can be immunogenic such as virus vectors induce multiple component of the immune response, cytotoxic T-lymphocyte (CTL) response can be elicited against viral gene products of exogenous transgene products [25]. Regarding the immunogenicity of this micelle, it is unlikely that immunogenicity was shown due to the fact that cytokines in the peritoneal fluid were suppressed.

*Therapeutic Effect of Glypican-3 Gene Silencing Using siRNA for Ovarian Cancer in a Murine… DOI: http://dx.doi.org/10.5772/intechopen.90311*

#### **3.3 Limitation of assessment using animal**

In the future as a next step, immunodeficient mice would be indispensable when we establish human model such as patient-derived xenograft (PDX) model. However, there is possibility that we cannot comprehend whether the micelle has medical potential when immunodeficient mice are used because GPC3 might be a molecule that is strongly associated with the immune system. That is why, we considered that we should further examine the usefulness of this therapy using micelles for human cancer cells based on our data using murine cell because there are different characteristics between murine and human cancer cells.

#### **4. Conclusion**

necessary to develop a carrier which is "targeting" and "systemically administable".

As shown in **Figure 7**, *in vitro* experiment, intracellular uptake of siRNA using

PLGA/siRNA-PLGA mixed micelle compared with siRNA-PLGA hybrid micelle. This result suggests that the characteristics of the targeting used by antibody may be expected to have an additive effect of the function of Fab<sup>0</sup> itself in addition to the increase in the intracellular uptake efficiency by cell recognition. In some antibodies, the target protein knockdown effect is dramatically obtained using Fab0

PLGA/siRNA-PLGA mixed micelle (data not shown). From these results, Fab0

**3. Expected side effect caused by gene therapy and limitation of**

**3.2 Cytotoxicity of exogenous siRNA or polymer in development of**

cytokines in the peritoneal fluid were suppressed.

Until now, some polyplex or lipoplex with high membrane permeability formulations have been used for siRNA delivery system [47, 48]. A number of polymers have been popularly utilized to form stable and nanocomplexes with its cytotoxicity problem [27, 49–53]. PEI is also probably the most frequently used polycation in gene delivery, our LPEI-coated micelles did not exhibit cytotoxic effects. The fact that no toxicity was found in our experiments at the concentrations we used was consist with previous reports [54]. The greatest feature of this micelle is that it consists of a safe polymer, PLGA. PLGA is known as one of the biodegradable polymers used in marketed medication [55]. In some cases, siRNA can be immunogenic such as virus vectors induce multiple component of the immune response, cytotoxic T-lymphocyte (CTL) response can be elicited against viral gene products of exogenous transgene products [25]. Regarding the immunogenicity of this micelle, it is unlikely that immunogenicity was shown due to the fact that

PLGA/siRNA-PLGA mixed micelles are believed to be useful as one of the targeting

The technique of RNAi in the medical field is expected to have not only therapeutic effects for human induced by knock-down specific genes but also suffers from off-target effects. Previous study reported that algorithm or open-source desktop software was developed to design RNAi sequences to exert strong and selective suppression of target genes and predict off-target [45, 46]. However, it is difficult to predict specific side effects that appear due to off-target effects in human. Furthermore, we suggested that the details of the off-target effect are often unclear due to the fact that commercial nucleic acid medications have a short period of use. In some cases, mouse results may not be compatible with humans because off-target effects vary by its sequences though there were no noticeable side effects


hybrid was synthesized. The drug design was described in **Figure 1B** and **E**.






That is why, we prepared Fab0

*Gynaecological Malignancies - Updates and Advances*

formulations to recognize the target cell.

**assessment using animal**

in our experiment *in vivo*.

**formulation**

**74**

**3.1 Off-target effects caused by RNAi**

target cell. Fab0

Fab0

In conclusion, our results could indicate that Gpc3 gene silencing using siRNA has a possibility as an effective new therapeutic approach without side effects in ovarian cancer, especially CCC with GPC3 expression. Furthermore, this GPC3 targeting gene therapy is also useful for high GPC3 expression cancer such as HCC, melanoma and lung cancer if appropriate carrier is developed to deliver siRNA to target cancer cell by i.v. in the future.

In addition, this finding is the first study to show that siRNA-PLGA hybrid micelles can effectively deliver siRNA to cancer cells *in vivo* at a low dose with significant anti-metastatic effect on murine ovarian cancer. We expect that novel formulation with more specific effects like siRNA including drug delivery system would be developed for malignant ovarian cancer therapy in the future.

#### **Acknowledgements**

This work was supported by JSPS KAKENHI Grant Number JP17K08477, Fukuoka Foundation for Sound Health Cancer Research Fund, and funds (No. 181045) from the Central Research Institute of Fukuoka University.

#### **Conflict of interest**

The authors declare no conflict of interest.

*Gynaecological Malignancies - Updates and Advances*

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s12885-019-6084-4

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*DOI: http://dx.doi.org/10.5772/intechopen.90311*

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[8] Lin H, Huber R, Schlessinger D, Morin PJ. Frequent silencing of the GPC3 gene in ovarian cancer cell lines. Cancer Research. 1999;**59**(4):807-810

[9] Nakatsura T, Nishimura Y. Usefulness of the novel oncofetal antigen glypican-3 for diagnosis of hepatocellular carcinoma and

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*Therapeutic Effect of Glypican-3 Gene Silencing Using siRNA for Ovarian Cancer in a Murine…*

melanoma. BioDrugs. 2005;**19**(2):71-77. DOI: 10.2165/00063030-200519020-

[10] Nakatsura T, Yoshitake Y, Senju S, Monji M, Komori H, Motomura Y, et al. Glypican-3, overexpressed specifically in human hepatocellular carcinoma, is a novel tumor marker. Biochemical and Biophysical Research Communications. 2003;**306**(1):16-25. DOI: 10.1016/

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[12] Shirakawa H, Suzuki H,

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Cancer Science. 2009;**100**(8): 1403-1407. DOI: 10.1111/ j.1349-7006.2009.01206.x.

et al. Release of GPI-anchored

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[7] Gutiérrez J, Brandan E. A novel mechanism of sequestering fibroblast growth factor 2 by glypican in lipid

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[4] Lopes-Coelho F, Gouveia-Fernandes S, Gonçalves LG, Nunes C, Faustino I, Silva F, et al. HNF1β drives glutathione (GSH) synthesis underlying intrinsic carboplatin resistance of ovarian clear cell carcinoma (OCCC). Tumour Biology. 2016;**37**(4):4813-4829. DOI:

### **Author details**

Mai Hazekawa\*, Takuya Nishinakagawa, Tomoyo Kawakubo-Yasukochi and Manabu Nakashima Department of Immunological and Molecular Pharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan

\*Address all correspondence to: mhaze@fukuoka-u.ac.jp

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Therapeutic Effect of Glypican-3 Gene Silencing Using siRNA for Ovarian Cancer in a Murine… DOI: http://dx.doi.org/10.5772/intechopen.90311*

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and Manabu Nakashima

Mai Hazekawa\*, Takuya Nishinakagawa, Tomoyo Kawakubo-Yasukochi

Department of Immunological and Molecular Pharmacology, Faculty of

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan

\*Address all correspondence to: mhaze@fukuoka-u.ac.jp

*Gynaecological Malignancies - Updates and Advances*

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### Section 2
