Anal Squamous Cell Carcinoma

#### **Chapter 4**

## Evolving Concepts toward Individualized Treatment of Squamous Cell Carcinoma of the Anus

*Luc Dewit, Annemieke Cats and Geerard Beets*

*"If you do not change direction, you may end up where you are heading", Lao Tsu, Chinese philosopher.*

#### **Abstract**

Treatment of squamous cell carcinoma of the anus has evolved over the last 5 decades from radical surgery to combined chemoradiation therapy. Radiation treatment techniques have dramatically improved with the development of more powerful computers, algorithms and treatment machines. The clinical impact of the modern radiation treatment techniques, such as intensity-modulated radiotherapy and volumetric modulated arc therapy, is discussed. The standard-of-care regimen still is concurrent Mitomycin C, 5-fluorouracil and high-dose radiation, as was conceived 45 years ago. Variants of this schedule are discussed in this chapter. International guidelines have been generated and implemented. Whereas concurrent chemoradiation therapy is the treatment of choice for locally advanced tumors, early tumors are probably adequately controlled with either reduced dose chemoradiation therapy or radiation therapy alone. Prognostic factors, such as high-risk human papillomavirus, epidermal growth factor receptor and immune response, will be highlighted. The role of surgery in primary care is limited to local excision of T1N0 tumors ≤ 1 cm of the anal margin. Salvage radical surgery is limited to locoregional recurrent, non-metastasized and resectable tumors after chemoradiation therapy. In addition, new treatment modalities, such as targeted therapy and immunotherapy, will be discussed. Current research aims at refining prognostic subgroups to further individualize treatment strategy, implementing quality assurance protocols in international trials and investigating the molecular profile of squamous cell carcinoma of the anus, in order to identify new treatment avenues. This will hopefully change the landscape of anal cancer treatment in the future.

**Keywords:** anal carcinoma, radiotherapy, chemoradiation therapy, prognostic factors, surgery, biological agents

#### **1. Introduction**

Squamous cell carcinoma of the anus (SCCA) is a rare tumor with an increasing incidence over the last decades [1]. It originates from the basal cells of the epithelial layer of the anal canal, which extends from the anorectal junction to the anal orifice, or anal margin, which extends from the anal orifice to a radius of 5 cm laterally [2]. Tumors arising from the anal margin have a different biological behavior, and this will be briefly discussed later in this chapter. Most, but not all, SCCA are causally related with high-risk human papillomavirus (HPV-HR), mainly subtypes 16 and 18 [3, 4]. These tumors develop from high grade anal intraepithelial neoplasia (AIN3) through a number of consecutive oncogenic steps, which are only partially understood [5]. Radical surgery, which usually implies an abdominoperineal resection with a permanent end colostomy, has been shown to yield 5-year survival rates of only 20–70%, depending on stage and resection margins [6]. Radiation therapy has demonstrated superior survival rates with a high probability of organ preservation. The seminal papers of Nigro and colleagues have shown that the combination of radiation and chemotherapy resulted in even better survival rates, at least for locally advanced cases [7, 8]. This has been confirmed in two landmark randomized phase III trials [9, 10]. Hence, chemoradiation therapy (CRT) has largely replaced radical surgery in the treatment of SCCA.

The focus of this chapter is to highlight the evolving concepts toward individualized treatment of patients with SCCA, based upon prognostic parameters. Emphasis will be given to improved radiation treatment techniques, concurrent and (neo) adjuvant chemotherapy regimens, the role of HPV status, molecular markers and immune response. In addition, the role of surgery will be addressed.

#### **2. Improved treatment of SCCA**

#### **2.1 Technical improvement of radiation treatment of SCCA**

#### *2.1.1 Radiation dose and target volume*

The efficacy of (chemo)radiation treatment for SCCA has been known for several decades. The acute and late toxicity, however, was considerable with the large, non-conformal treatment fields, which often resulted in moderate functional outcome and quality of life [11]. With the development of more powerful computers, algorithms and treatment machines, more sophisticated treatment techniques became available. This has resulted in a shift from standard opposed anteriorposterior fields (AP-PA) or a four-field technique in the fifties through eighties of the previous century to 3D-conformal radiotherapy (3D-CRT) in the nineties and intensity-modulated radiotherapy (IMRT) in the early years of this century and volumetric modulated arc therapy (VMAT) in the last decade.

The difference in toxicity between 3D-CRT and IMRT or VMAT has never been compared in a prospective randomized trial, but several retrospective studies and one recent prospective study have reported an improved toxicity profile with the newer techniques [12–17]. A recent national audit in the UK comparing these techniques confirmed the reduced toxicity with IMRT (**Table 1**) [18]. A few studies also claim a better disease-free survival (DFS) and locoregional control (LRC) with IMRT [12, 14, 19].

Toxicity is largely related to the radiation dose and the volume of normal tissues exposed to radiation, which in turn is related to the gross tumor volume (GTV) and clinical and planning target volume (CTV and PTV). The GTV is determined by the macroscopic local tumor extent and documented macroscopically involved regional lymph nodes, whereas the CTV is dependent on the site of regional lymph nodes that are considered to be at risk for microscopic metastatic disease. In addition, the PTV is determined by the set-up error of patient positioning. With the advent of magnetic resonance imaging (MRI) and fluor-18-deoxyglucose positron

#### *Evolving Concepts toward Individualized Treatment of Squamous Cell Carcinoma of the Anus DOI: http://dx.doi.org/10.5772/intechopen.85545*


#### **Table 1.**

*UK National Audit of anal cancer radiotherapy 2015 [18]. Reproduced with permission of Elsevier.*

emission tomography (18F FDG-PET), much improvement is made over the years in visualizing the primary tumor and involved regional lymph nodes and, hence, delineating GTV. In contrast, the estimation of microscopic metastatic disease remains poor and is largely based upon a few studies with documented locoregional recurrence in relation to tumor size and irradiated volumes [20–22]. The CTV for SCCA is notoriously complex, given the potential involvement of inguinal, iliac, mesorectal and presacral lymph nodes. Consensus contouring guidelines have been developed to assist radiation oncologists in setting up a treatment plan [23, 24]. With respect to the radiation dose, a two or three dose level for microscopic and macroscopic disease has emerged form clinical trials. For instance, in the Radiation Therapy Oncology Group (RTOG) 87-11 trial, a radiation dose of 30.6 Gy was given to the common iliac lymph nodes whereas a dose of 45 Gy was delivered to the lower iliac lymph nodes and 50.4 Gy to the primary tumor [25]. In contrast, in the United Kingdom Coordinating Committee on Cancer Research Anal Cancer Trial (UKCCCR-ACT) I and the European Organization For Research and Treatment of Cancer Radiotherapy (EORTC) 22861 trial the common iliac lymph nodes were not included in the elective radiation field, whereas a dose of 45 Gy was given to the lower iliac and inguinal lymph nodes with a boost to 60–65 Gy to the primary tumor [9, 10]. In the subsequent UKCCCR-ACT II the dose to the iliac and inguinal lymph nodes was limited to 30.6 Gy and the boost to the primary tumor to 50.4 Gy [26]. Despite these differences in radiation dose and volume, no striking difference in LRC was observed between these trials [9, 10, 25]. A number of retrospective studies have reported a better LRC with a higher radiation dose, at least in the locally advanced tumors [27–30]. This was confirmed in a systematic literature review [31] and a recent retrospective study from a large Scandinavian database [32]. However, in the French prospective randomized ACCORD-03 trial, which included only locally advanced cases, a marginal, non-significant increase in colostomy-free survival (CFS), a surrogate endpoint for LRC, was observed after 70 Gy, as compared with 60 Gy [33]. Consequently, in the absence of definitive evidence, current clinical guidelines do not advocate a higher radiation dose for larger tumors [34, 35].

#### *2.1.2 The treatment gap*

In the initial trials, a treatment gap of 6 weeks was included at an intermediate radiation dose [9, 10, 25]. This was done to allow for recovery from acute radiation toxicity, but also to give the tumor time to regress and to assess whether a radiation boost should be given with external beam irradiation or with brachytherapy. As results matured and further insight in tumor radiobiology was gained, this long treatment gap was considered to be potentially hazardous, due to the likelihood of tumor repopulation during the treatment gap. In the subsequent studies, the treatment gap was shortened to 2 weeks, which not only seemed to be feasible, but also resulted in better LRC in some studies [36–40] but not in others [41, 42]. With the advent of IMRT and VMAT, the entire radiation course could be administered without a treatment break. Today, most modern radiotherapy centers have implemented IMRT or VMAT for SSCA.

#### **2.2 Chemotherapy and radiation for SCCA**

#### *2.2.1 Landmark studies*

In June 1973, Dr. Nigro presented 3 cases with SCCA at a meeting of the American Proctologic Society in Detroit, that were treated with radiation therapy (RT) and concurrent Mitomycin C (MMC) and 5-fluorouracyl (5-FU) in a preoperative setting [7]. The rationale for this approach was to improve the LRC and overall survival (OS) of SSCA, since the results with radical surgery alone were modest, at best. Dr. Nigro realized that, in contrast with rectal cancer, SCCA originates from an organ which has an abundant lymphatic vessel supply, that allows rapid lymphatic tumor spread. In addition, there is limited space in the lower pelvis for radical surgery. The radiation dose was 30 Gy in 3–5 weeks via two large anterior-posterior opposed fields, and 30 mg of MMC was given on day 1 in a single bolus infusion and 1500 mg per day of 5-FU on days 2–6 in a continuous infusion. Six to 8 weeks later, two of them underwent an abdominoperineal resection, as planned. No tumor was found on microscopic examination of the operation specimen in these two cases. The third patient refused surgery and remained free of disease 1 year later [7]. This treatment regimen was expanded in a larger series, which confirmed the excellent results [43]. This pioneering work formed the basis for definitive CRT with higher, therapeutic radiation doses.

The superiority of this regimen compared with RT alone was established in two randomized phase III trials, the UKCCCR-ACT I and the EORTC 22861 [9, 10]. These trials were executed almost parallel in time and their design was strikingly similar, except for the eligibility criteria: in the EORTC trial only locally advanced patients were eligible, whereas in the ACT I all stages were accepted for inclusion. Despite this imbalance in patient selection, no major difference in the treatment outcome was observed between these two trials. Both studies showed a significant improvement in LRC control with CRT as compared with RT alone [9, 10]. In the ACT I, 3-year LRC increased from 47% after RT alone to 70% after CRT with concurrent 5-FU and MMC [9]. The corresponding figures in the EORTC 22861 trial were 55 and 68%, respectively [10]. The difference in LRC and progression-free survival (PFS) in the ACT I remained up to 12 years after treatment [44]. However, no difference in OS was found in either of these trials [10, 44].

The value of MMC, in addition to 5-FU, was established in the phase III RTOG 87-04 study [25]. In this trial, however, MMC was given twice in the first and fifth week of the radiation treatment, as opposed to only once in the ACT I and EORTC 22861 trial. It resulted in considerably more grade 4-5 hematological toxicity than was seen in the European trials.

*Evolving Concepts toward Individualized Treatment of Squamous Cell Carcinoma of the Anus DOI: http://dx.doi.org/10.5772/intechopen.85545*

#### *2.2.2 Subsequent pivotal studies*

In the subsequent phase III RTOG 98-11 trial, the role of neo-adjuvant and concurrent cisplatin and 5-FU was addressed by comparing it with concurrent MMC and 5-FU [45]. While the combination of cisplatin and 5-FU was less toxic than MMC and 5-FU, the disease-free survival (DFS) and OS was significantly worse with the new regimen [46]. In the UKCCCR-ACT II, concurrent cisplatin, 5-FU and RT was compared with concurrent MMC, 5-FU and RT, with or without adjuvant cisplatin and 5-FU, in a 2 × 2 factorial design [26]. In this trial, which is the largest phase III trial carried out to date for anal cancer, no difference in PFS (**Figure 1**) and toxicity was observed between the four treatment arms [26]. The French phase III ACCORD 03 trial investigated the value of neo-adjuvant and concurrent cisplatin, 5-FU and RT, and radiation dose intensification, also in a 2 × 2 factorial design [33]. Whereas a marginal, non-significant increase in CFS was observed in the group that received the higher radiation dose, no difference in CFS was found between the patients with and without neo-adjuvant chemotherapy. Acute and late toxicity were similar between the four groups [33]. The EORTC 22011-40014 randomized phase II trial compared concurrent MMC, cisplatin and RT with MMC, 5-FU and RT [47]. The new combination proved to be highly effective, but more toxic, with a compliance of only 49% as opposed to 79% for the standard arm [47].

#### *2.2.3 Variant schedules*

In the UKCCCR-ACT I, EORTC 22861 and RTOG 87-04 trials, MMC was given once on day 1 [9, 10] or twice on day 1 and 29 of the radiation treatment [25],

#### **Figure 1.**

*MMC or cisplatin+5FU and radiation + or—adjuvant cisplatin/5-FU for SSCAC [26]. Reproduced with permission of Elsevier.*

whereas 5-FU was administered in a continuous infusion day 1–4 or 5 and day 29–32 or 33. Variants of this treatment schedule have been explored with 5-FU given continuously in lower daily doses over the entire split-course radiation treatment [37], or by replacing 5-FU with capecitabine, an oral prodrug of 5-FU, given twice daily during the radiation treatment [48–51]. These schedules seemed feasible and equally effective as the standard schedule. In addition, capecitabine has the advantage of being able to be given on an outpatient basis.

Taken together, the original regimen of MMC and 5-FU remains the standard of care in CRT for SCCA, 45 years after its inception. There is a trend of using capecitabine instead of 5-FU because it is more patient friendly and equally effective. Arguably, MMC is more toxic than cisplatin in combination with 5-FU or capecitabine and RT [37], but this is dose dependent and seems to be equally effective in a single bolus of 10 mg/m2 as 12 or 15 mg/m2 or twice 10 mg/m2 [9, 10, 25]. Furthermore, the combination of cisplatin and 5-FU is not more effective than MMC and 5-FU, but requires hospitalization for hydration procedures to prevent renal toxicity [26].

#### **3. Prognostic factors in anal carcinoma**

Well-known clinical prognostic factors in SCCA are age (>55 years better than ≤55 years), sex (female better than male), tobacco smoking (worse), primary tumor size and site (anal margin better than anal canal), T- and N-stage, tumor ulceration (worse if present) and histological differentiation grade [32, 52, 53]. Other prognostic factors include HPV-HR and certain genetic alterations.

#### **3.1 Human papillomavirus**

HPV-HR is causally related with the onset and progression of SCCA [5]. Once integrated into the host DNA, the main viral oncoproteins E6 and E7 interact with the tumor suppressor proteins p53 and retinoblastoma protein (pRb), respectively. P53 has a key role in maintaining DNA integrity, whereas pRb is a negative regulator of the cyclin-dependent kinase inhibitor p16. Upon persistent HPV-HR infection, p53 becomes permanently inactivated, disrupting DNA repair processes, and pRb inactivation induces upregulation of p16. As such, p16 is sometimes used as a surrogate marker of HPV-HR infection. These and other oncogenic processes lead to genomic instability, carcinogenesis and tumor progression. As a result, HPV-HR+ SCCA have a number of unique features, some of which have a prognostic or even a predictive value (**Figure 2**) [5].

Patients with HPV-HR+ SCCA have a significantly better outcome after CRT than HPV-HR-tumors [54–56]. Absolute difference in LRC/PFS varies from 32 to 67%, whereas the difference in OS varies from 22 to 52%. Interestingly, within the HPV-HR+ tumors, LRC and OS after CRT are significantly better in patients with tumors carrying a high HPV-HR DNA load than in those with a low HPV-HR DNA load [57]. Intratumoral p16 expression is also correlated with LRC and PFS after CRT for SCCA [58]. An even stronger discriminating effect on LRC and PFS is observed by combining p16 expression and HPV DNA tumor load [57].

P53 and p16 expression/HPV-HR+ are inversely correlated in SCCA [56, 58]. In addition, p53 expression and disruptive *TP53* mutations are associated with a significantly worse outcome after CRT [56, 58].

*Evolving Concepts toward Individualized Treatment of Squamous Cell Carcinoma of the Anus DOI: http://dx.doi.org/10.5772/intechopen.85545*

**Figure 2.**

*Molecular features in HPV positive tumors [5]. Reproduced with permission of Elsevier.*

#### **3.2 Epidermal growth factor receptor**

The epidermal growth factor receptor (EGFR) is frequently overexpressed in SCCA and this may confer a growth and survival advantage. In a subgroup analysis of the RTOG 98-11 trial, overexpression of EGFR and a downstream proliferation marker Ki67 was associated with a significantly worse DFS and OS [59]. In a recent small series of recurrent SCCA, high levels of alterations in the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway, which is a growth and survival promoting pathway downstream of EGFR, were associated with poor OS [60].

#### **3.3 Immune response**

Persistent intratumoral HPV-HR infection can elicit a host immune response, which is mediated by immune checkpoint proteins such as cytotoxic T lymphocyteassociated protein 4 (CTLA-4) and programmed cell death 1 (PD-1), expressed on activated T-cells and programmed cell death ligand 1 (PD-L1), expressed on tumors and various host cells [5, 61]. This can attract CD8+ T-lymphocytes into the tumor,

#### **Figure 3.**

*Prognostic impact of CD8+/PD1 and DC8+/PD-L1 expression on LRC and DFS after CRT in SSCAC [62]. Reproduced with permission of Taylor and Francis.*

so-called tumor-infiltrating lymphocytes (TILs). HPV-mediated intratumoral immune response has a significant influence on LRC and DFS, as illustrated by the amount of CD8+ TILs and PD-1 and PD-L1 expression levels after CRT in SCCA (**Figure 3**) [62].

#### **4. Biological agents**

Although the standard regimen of CRT with MMC and 5-FU is effective in SCCA, there is still room for improvement, in particular in the locally advanced cases and tumors that carry poor prognostic factors. Attempts have been made to investigate newer, promising agents. Here we focus on two avenues that have been explored.

Cetuximab is a chimeric IgG1 monoclonal antibody with a high affinity for EGFR. It has been tested in a few phase II trials in combination with concurrent CRT in SSCA, and turned out to be very toxic and probably also less effective than the standard regimen [63–67].

Two phase II trials have been published on the use of anti-PD-1 monoclonal antibodies in recurrent and/or metastatic SCCA, that is nivolumab [68] and pembrolizumab [69]. Objective responses were observed in 24 and 17%, respectively, and stable disease in 42% of the latter [68, 69]. Adverse events were acceptable.

#### **5. The role of surgery in anal carcinoma**

#### **5.1 Salvage abdominoperineal resection**

Radical surgery for SCCA is restricted to locoregional recurrent, non-metastasized and resectable tumors after CRT. The standard operation procedure is an abdominoperineal resection (APR), sometimes extended with resection of parts of the vagina or prostate, if involved, in order to obtain clear surgical margins [6]. This leaves a large pelvic floor defect, which preferably should be closed with a vertical

*Evolving Concepts toward Individualized Treatment of Squamous Cell Carcinoma of the Anus DOI: http://dx.doi.org/10.5772/intechopen.85545*

rectus abdominis myocutaneous flap (VRAM). Patients are left with a permanent colostomy. After APR, 5-year OS varies between 30 and 75%, depending upon whether or not clear resection margins have been obtained [6, 70]. Morbidity can be substantial, such as wound infections and poor healing of previously heavily irradiated organs and tissues. Wide resections into non-irradiated tissues and reconstructions with plastic flap techniques reduce these serious complications [6].

#### **5.2 Curative local excision**

A particular role for curative surgery in first line treatment of SCCA is reserved for small, T1N0 tumors of the anal margin, suitable for local excision (LE). This is not a trivial decision to make and these patients deserve to be seen by an experienced multidisciplinary team. Based on a recent pattern of care study in Australia, there is a wide variety in management of these small T1 tumors, depending upon the findings after a (non)excisional biopsy (**Figure 4**) [71]. In accordance with the guidelines and expert opinion, it is safe to say that T1N0 tumors < 1 cm, located in the anal margin, are good candidates for LE [34, 35]. This will probably account for only 4% of all anal cancers [72]. If pathological examination of the surgical specimen reveals that the resection is not radical, some form of additional treatment is warranted and should be discussed in a multidisciplinary team. If located in the anal canal, LE carries a risk of sphincter damage and is therefore relatively contraindicated. Nevertheless, a recent retrospective cohort study of

**Figure 4.**

*Reported management of T1N0 anal cancer [71]. Reproduced with permission of Springer.*

the US National Cancer Database on 2243 cases with T1 N0 SCCA has shown that over the period 2004–2012 LE was increasingly used in the more recent years, also for tumors of the anal canal [73]. Although criticized for its lack of information on the exact tumor location, LRC and DFS [74, 75], this study and the Australian survey [71] illustrate that clinicians are reluctant to treat these small tumors with standard CRT.

#### **6. Treatment strategy**

Today's clinical research on SCCA is focused on individualizing treatment as a function of estimated prognosis. A good example, for instance, is the UK trial "PersonaLising rAdioTherapy dOse in anal cancer" (PLATO), which offers a platform of 3 trials, ACT3, 4 and 5, for 3 different risk groups of SCCA [76].

ACT3 is a non-randomized trial for patients with low-risk T1N0 tumors of the anal margin, that undergo LE, followed by active surveillance if the resection margin is >1 mm. If the margin is ≤1 mm, postoperative reduced dose CRT is given locally (41.4 Gy in 23 fractions). In the Netherlands Cancer Institute, we use a somewhat different treatment policy for these tumors, taking a relatively new entity for SCCA into account, known as superficially invasive squamous cell carcinoma (SISCCA). SISCCA is defined as an invasive squamous cell carcinoma with an invasive depth of ≤3 mm and a horizontal spread of ≤7 mm that has been completely excised [77]. In the cervix, SISCCA is known to bear a minimal risk of microscopic lymph node metastasis and it is assumed to be similar for SISCCA of the anus, although the data supporting this are scarce [77]. We therefore have adopted a close surveillance policy for SISCCA of the anal margin. If the resection margin is too close or involved, a wider excision is performed, if possible. If not, postoperative reduced dose RT alone is given to the anus (45 Gy in 25 fractions). For T1N0 tumors that are microscopically >3 mm in invasive depth or >7 mm in horizontal spread, we also irradiate the inguinal lymph nodes to 45 Gy in 25 fractions. We do not advocate CRT in these cases, because the results with RT alone are excellent [35, 78, 79]. Furthermore, CRT is associated with an absolute increase of 9% of non-cancer related deaths compared with RT alone, mainly from cardiovascular cause and secondary tumors [44].

ACT4 is a randomized phase II trial for intermediate-risk tumors, T1–2 (≤4 cm) N0 or Nx, comparing LRC at 3 year after standard-dose CRT (50.4 Gy in 28 fractions) *versus* a reduced-dose CRT (41.4 Gy in 23 fractions). In the French guidelines, the advice for T1 and small T2 tumors is to treat them with RT alone [35]. In the Netherlands Cancer Institute, we follow the Dutch National guidelines, which advocate RT alone for T1N0 tumors and CRT for all other stages [80].

ACT5 is a randomized seamless pilot/phase II/phase III trial for high-risk SCCA, T1-2N1-3 or T3-4Nany, comparing 3-years' LRC after standard-dose CRT (53.2 Gy in 28 fractions) with that after 2 higher dose levels (58.8 and 61.2 Gy in 28 fractions) [76]. In the Netherlands Cancer Institute, we use CRT for these tumors with a relatively high radiation dose of 59.4 Gy in 30 fractions. We do not consider a lower radiation dose, because with VMAT the toxicity profile is acceptable [79].

#### **7. Conclusions and future prospects**

The treatment of SCCA has evolved over the last 5 decades from a mutilating radical surgical treatment with a modest survival probability to an individualized *Evolving Concepts toward Individualized Treatment of Squamous Cell Carcinoma of the Anus DOI: http://dx.doi.org/10.5772/intechopen.85545*

radiation treatment with or without concurrent chemotherapy with good survival outcome and acceptable morbidity. Important improvements in radiation treatment techniques have been made, modern guidelines have been implemented and quality assurance is provided. However, there is still room for improvement. Quality of life analyses have infrequently been performed and are rarely taken into account in treatment decision making (e.g. [11, 81–83]). A good step forward in this respect is the development of a core outcome set of data, which should be the minimal information required in future clinical trials for anal cancer [84]. Radiation dose de-escalation and omitting concurrent chemotherapy for early tumors with good prognosis are important avenues to explore. On the other hand, new treatment modalities are needed for poor prognostic cases, such as HPV-HR negative SCCA. Immunotherapy seems to be a promising modality, either alone [68, 69] or in combination with chemotherapy [85]. Exploring the molecular profile of SCCA may reveal new potentially therapeutic targets and prognostic and predictive markers [60, 86, 87]. Circulating tumor DNA at baseline and in follow-up may become an important tool in treatment decision making [88]. These new insights and therapeutic avenues may eventually change the landscape of anal cancer treatment in the near future.

### **Conflict of interest**

The authors have declared no potential conflict of interest.

#### **Nomenclature**



*Evolving Concepts toward Individualized Treatment of Squamous Cell Carcinoma of the Anus DOI: http://dx.doi.org/10.5772/intechopen.85545*

#### **Author details**

Luc Dewit1 \*, Annemieke Cats2 and Geerard Beets3

1 Department of Radiation Oncology, The Netherlands Cancer Institute (Antoni van Leeuwenhoek), Amsterdam, The Netherlands

2 Department of Medical Oncology, The Netherlands Cancer Institute (Antoni van Leeuwenhoek), Amsterdam, The Netherlands

3 Department of Surgical Oncology, The Netherlands Cancer Institute (Antoni van Leeuwenhoek), Amsterdam, The Netherlands

\*Address all correspondence to: l.dewit@nki.nl

© 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.

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Section 5

## Cutaneous Squamous Cell Carcinoma

### **Chapter 5**

## Mechanical Force and Actin Dynamics during Cutaneous Squamous Cell Carcinoma (cSCC) Progression: Opportunities for Novel Treatment Modalities

*Sarah Boyle and Zlatko Kopecki*

### **Abstract**

Cutaneous squamous cell carcinoma (cSCC) accounts for 25% of cutaneous malignancies diagnosed in the Caucasian population. Surgical removal in combination with radio- and chemotherapy is an effective treatment; however, prognosis for patients suffering from aggressive cSCC is still relatively poor. Increasing prevalence coupled with high mortality and morbidity in aggressive metastatic forms of cSCC highlights the need for development of novel targeted therapeutics. Metastasis is a complex process requiring dramatic reorganization of the cell cytoskeleton. Recent studies have highlighted the importance of mechanical forces and actin dynamics in cancer cells' intrinsic ability to invade adjacent tissues, intravasate into vasculature, and ultimately metastasize. Tight regulation of the biochemical and mechanical properties of the actin cytoskeleton drives cellular processes involved in cSCC progression including polarity establishment, morphogenesis, and motility. Here we will provide a short introduction to disease pathogenesis, give an overview of the role of key regulatory proteins governing the mechanical forces and actin dynamics critical to cSCC progression, and describe the contribution of actin remodeling and actomyosin signaling to cSCC progression. We will also discuss how targeting protein regulating mechanical force and actin dynamics may have clinical utility in development of novel treatment modalities for patients suffering from aggressive cSCC.

**Keywords:** cutaneous squamous cell carcinoma, actin cytoskeleton remodeling, mechanical force, contraction, systemic therapy

### **1. Introduction**

Cutaneous squamous cell carcinoma (cSCC) most commonly arises in actinically damaged skin and accounts for 25% of cutaneous malignancies diagnosed in the Caucasian population [1]. The incidence of cSCC continues to rise annually, with an estimated 50–200% increase in incidence in the last three decades in USA alone, and is predicted to increase in future years due to an aging global population [2]. Solar ultraviolet radiation is the primary environmental extrinsic cause of cSCC. Intrinsic

immunosuppression, the second most common cause, leads to the formation of aggressive cSCC in organ transplant patients, patients on immunomodulatory therapies, and those suffering from recessive dystrophic epidermolysis bullosa, a genetic skin blistering disease [3–5]. The incidence of cSCC is higher in individuals who are fair-skinned and have a sun-sensitive phenotype; however, the aggressive forms of cSCC are more common in men and the elderly [3]. Despite its prevalence, the relatively low fatality rate of cSCC means that its health and economic burden is often substantially underestimated [3], albeit latest data showing that in addition to significant morbidity cSCC accounts for up to 8000 deaths per year and costs approximately \$4.8 billion annually in USA alone [6].

cSCC generally presents as a scaly, red or bleeding abnormal lesion on sunexposed areas, and is associated with relatively benign outcomes and a low risk of metastasis. However, cSCC can demonstrate dramatic histopathological heterogeneity, resulting in a wide range of clinical outcomes [7]. Histopathologic subtypes of cSCC are broadly divided into low-grade SCC (**Figure 1A**) that are well-differentiated but have low metastatic potential (keratoacanthomas, SCC *in situ* and verrucous carcinoma), or high-grade SCC (**Figure 1B**) that are poorly differentiated, have high potential of metastasis and recurrence, and are associated with a poor prognosis for patients (desmoplastic cSCC, adenosquamous cSCC and cSCC associated with non-healing ulcers or scarring processes arising from chronic wounds) [2, 7]. Once developed, the natural history of untreated SCC is one of local invasion followed by metastasis via the lymphatic system, blood or perineural invasion, which can lead to death [7]. In most cases, cSCCs are detected early and can be successfully eradicated by surgical excision. However, if not detected and/or left untreated, disease progression to high-grade cSCC will often lead to mortality. Clinically, the most powerful predictor of disease pathogenesis is nodal metastasis and size, followed by invasion beyond fat, location, and lastly perineural invasion. These parameters are used in clinical staging systems for cSCC [3]. Management of cSCC is primarily surgical, with adjuvant chemoradiation approaches based on risk factors, patient and tumor features, as well as care features including access to treatment and associated costs [2].

With increasingly longer life expectancy, the health and economic burden of cSCC is likely to continue to increase significantly. Hence, a better understanding of factors contributing to cSCC progression and metastasis is necessary to aid development of novel therapies, aimed at combatting cSCC in the community. Despite

#### **Figure 1.**

*Representative histopathological features of low-grade and high-grade cSCC. (A) Low-grade cSCC in situ with prominent dyskeratosis and aberrant mitosis at all levels of the epidermis, with marked parakeratosis and intact basement membrane. (B) High-grade poorly-differentiated cSCC lesions showing prominent keratinization and the formation of "pearl like" structures where dermal nests of keratinocytes attempt to mature. Adapted from Yanofsky et al. [7] and modified with approval.*

#### *Mechanical Force and Actin Dynamics during Cutaneous Squamous Cell Carcinoma (cSCC)… DOI: http://dx.doi.org/10.5772/intechopen.86041*

recent advances in gene expression screening technologies that have begun to identify candidate genes commonly mutated in patients with cSCC (including TP53, CDKN2A, Ras and NOTCH1), which may be responsible for regulating motility and invasion in cSCC, a comprehensive understanding of the factors contributing to cSCC invasion including mechanical tension and actin dynamics is still emerging [8]. One thing that is clear is that patient outcome directly correlates with the degree of local and regional invasion, and coordinated regulation of the actin cytoskeleton is critical to cell motility, invasion and metastasis [9]. Consequently, the signaling pathways involved in mediating chemotactic cues from the extracellular environment that regulate the actin cytoskeleton and mechanical forces, guiding cancer cell invasion and metastasis, have been and continue to be an area of intense study.

Recent studies have revealed a number of proteins and molecules that are aberrantly expressed in cSCC. These proteins link cell migratory signals to the actin cytoskeleton, thereby playing an instrumental role in the ability of cancer cells to resist chemotherapy and/or metastasize [10]. In this chapter we will describe the actin dynamics and mechanical force governing tumor cell migration, invasion and metastasis. We will outline the main signaling pathways governing the formation of invasive protrusions by cancer cells with regard to the function of key regulatory proteins involved in actin cytoskeleton remodeling in cSCC.

The metastatic spread of aggressive cancers, including cSCC, is a highly selective process involving a series of sequential and orchestrated steps in the so-called "metastatic cascade": detachment from the primary tumor site, cell migration and invasion of the surrounding extracellular matrix (ECM), intravasation into vasculature, extravasation at a secondary site, and interaction with the extracellular environment to form metastatic tumors [11]. Each of these steps offers the potential for design of different therapeutic approaches to combat aggressive cSCC. Indeed, a number of recent studies have identified novel therapeutic approaches including both adjuvant and neoadjuvant treatments, with clinical trials utilizing epidermal growth factor receptor inhibitors and immune checkpoint blockers (nivolumab, pembrolizumab, and ipilimumab) showing promising early results as potential treatments of cSCC [12]. Recent trials using cytotoxic chemotherapy have, however, shown limited advances for the treatment of cSCC, and trials investigating combined immune checkpoint inhibitor and radiation therapies, which may have synergistic effects in treatment of cSCC, are still pending [13]. This highlights the need for increased research to close the gaps in our knowledge of cSCC biology, including better understanding of the factors that lead to aggressive cSCC, the role of microbiomes and HPV infection, the role that mechanical force and actin dynamics plays in this process, prediction of clinical response to therapies including immune checkpoint blockade, and how to tailor better prevention and treatment strategies to individual risk factors and needs [6]. Emerging evidence on the crosstalk between different components of the cytoskeleton in metastatic progression combined with clinical data illustrating strong relationships between cytoskeletal alterations and metastasis in various cancers pinpoints important opportunities for potential therapeutic targets [11]. Later in this chapter we will describe current research that has attempted to identify the steps of the metastatic cascade suitable and most amenable for therapeutic intervention, with a focus on harnessing our knowledge of actin cytoskeleton remodeling and mechanical forces to postulate therapeutic strategies targeting cytoskeletal and cytoskeletal-associated proteins critical in cSCC.

#### **2. Cytoskeletal dynamics and regulation during cSCC progression**

The skin is exposed to and responds to a wide range of mechanical signals throughout homeostasis and through to malignancy. Mechanical forces have been shown to regulate these normal cellular processes including stem cell renewal, lineage differentiation and proliferation, wound healing, as well as transformation through changes in the actin cytoskeleton—the ability to protrude, adhere to the ECM, migrate through tissue and invade into the underlying basement membrane.

The types of mechanical forces exerted upon the skin can vary depending on the context. In homeostasis, tensile/stretch forces and compressive forces arise as a result of muscle and joint movements, and physical location—skin stretched over bone is under significantly more stress than when over fat or muscle [14]. Tensile forces cause cells to elongate and expand, and therefore are generated at sites of wounding as epithelial cells migrate in and contract to close the wound. This can generate scarring and fibrosis, which can lead to skin cancer including cSCC [15]. Compressive forces generate different biomechanics in skin cells compared to tensile force [16]. Compressive forces are able to activate Rho-ROCK signaling (described below) in the skin, which has been shown to play a role in tumor progression [17]. In melanoma, it was found that stress-bearing areas of the foot were more conducive to cancer development due to increased mechanical compressive stress [18]. Changes in substrate stiffness underlie these mechanical signals (**Figure 2**), and it has been shown that stiff stroma can lead to an activation of integrin signaling and subsequent cSCC development [19].

Cells have the ability to sense these changes in their environment (process referred to as "mechanosensing"). The mechanical signal is then converted to a biochemical signal in a process called mechanotransduction, and the biochemical signals initiate a cascade of changes within the cell at the transcriptional, translational and post-translational levels that result in a cell that can appropriately and reciprocally respond to the extracellular signals (process referred to as "mechanoreciprocity") [20]. In disease states including cSCC, the heightened and/ or constitutive extracellular signals generate a detrimental loop of ever-increasing mechanoreciprocal signaling, hence leading to enhanced tumor progression, invasion and eventually metastasis [20].

Triggered by the changes in the cell microenvironment, the actin cytoskeleton undergoes a number of changes that allows a cell to become more motile and/or invasive. The ability of a cell to undergo directed migration is essential to its ability to metastasize, and is characterized by an ordered process (**Figure 3**) of membrane protrusion at the leading edge (filopodia and lamellipodia) and sides (invadopodia)

#### **Figure 2.**

*Mechanical forces acting upon skin cells. The major types of mechanical forces experienced by skin cells are compressive (inward pushing) and tensile (stretching) forces, which in cSCC progression are generated by an increase in extracellular matrix stiffening. These are sensed by the cell, which then is able to respond accordingly.*

#### *Mechanical Force and Actin Dynamics during Cutaneous Squamous Cell Carcinoma (cSCC)… DOI: http://dx.doi.org/10.5772/intechopen.86041*

of the cell, contact and adhesion between the protrusion and the matrix, movement of the main cell body, and retraction of the trailing edge [21, 22]. Lamellipodia are flat membrane protrusions containing dendritic arrays of actin filaments that branch out like a sheet from the leading edge of a cell. This particular form of protrusion is thought to have a major role in cell migration as their morphology allows the cell to make multiple contacts with the underlying substrate and pull the cell forward. Filopodia are narrow protrusions made up of bundled and cross-linked actin filaments that also stretch out from the leading edge. Invadopodia, unlike lamelli- and filopodia, are protrusions branching out from the sides of a cell, which have increased membrane remodeling and matrix degradation proteins [21]. These particular membrane protrusions are often seen in cancer cells including during cSCC progression [23].

Adhesions between the cancer cells and matrix are necessary for invasion and are largely mediated by integrins (discussed in detail below) [22]. During cSCC progression, there is an increase in cellular attachment to the ECM and, concurrently, a decrease in attachment to neighboring epithelial cells signified by a reduction in levels of E-cadherin expression. Following matrix adhesion, the trailing edge of the cell contracts, allowing the cell to move forward. Myosin II is required for this actin filament contraction, and is largely regulated by signaling through the small G-protein Rho. Once the cell contracts, its tail detaches as focal adhesion complex components are cleaved [22]. Actin remodeling proteins are essential in these processes, and often regulate cell-cell and cell-stroma attachment and turnover of focal adhesions, allowing cell traction and movement to take place. The coordination of actin polymerization and contraction allows the cSCC cell the ability to migrate through dense, stiff ECM and stroma to metastasize to lymph node or surrounding organs.

A number of different pathways are activated downstream of mechanical signals, causing changes in the actin cytoskeleton. Signaling pathways involved in cytoskeletal dynamics during cSCC progression are also activated as a result of constitutive or heightened growth factor signaling [24]. Together, the combination of mechanical and biochemical signals can trigger a multitude of intracellular signaling cascades that ultimately affect cell morphology. In this section, we will discuss the broad families of proteins regulating actin dynamics and mechanical forces in cSCC, an overview of which is illustrated in **Figure 4**. The overview provided in this chapter will not cover all the pathways or actin remodeling proteins involved but focus on those most relevant to cSCC progression, invasion and metastasis.

#### **Figure 3.**

*Cell motility as controlled by the actin cytoskeleton. Upon sensing of extracellular cues, intracellular signaling cascades generate cytoskeletal protrusions (lamellipodia and filopodia) at the leading edge containing actin filaments, as well as invasive protrusions (invadopodia) at the sides of the cell, necessary for cSCC invasion. The cell adheres to the matrix, forming integrin-mediated focal adhesions. The nucleus and cell body are then pushed forward as the trailing edge contracts, via stress fibers. The rear of the cell then detaches, allowing the cell to migrate forward.*

#### **2.1 Mechanosensing: integrins**

Integrins are well-characterized as the first point of contact for mechanical signal transduction. Inactive heterodimers on the cell surface, they are partially activated by intracellular proteins ("inside-out" signaling) before full activation upon binding to extracellular ligands ("outside-in" signaling) [25]. Binding to the extracellular ligand results in full activation of the integrin receptor and leads to the formation of either an intracellular focal adhesion complex to link the ECM to the actin cytoskeleton, or hemidesmosomes, linking the ECM to intermediate filaments. The integrin that is activated is context-dependent in regards to the particular focal adhesion complexes that are formed, broadly encompassing a range of adaptor proteins including: talin, vinculin, paxillin, Flightless I (Flii), focal

#### **Figure 4.**

*Major pathways regulating cytoskeletal dynamics downstream of mechanical signals in cSCC. Graphical representation of signaling pathways which respond to mechanical force and govern downstream actin remodeling during cSCC progression.*

#### *Mechanical Force and Actin Dynamics during Cutaneous Squamous Cell Carcinoma (cSCC)… DOI: http://dx.doi.org/10.5772/intechopen.86041*

adhesion kinase (FAK) and integrin-linked kinase (ILK). FAK is rapidly recruited to focal adhesions upon integrin activation and is auto-phosphorylated, driving downstream signaling. FAK phosphorylation, stimulated by integrin activation, allows binding of Src-family kinases that are then able to trans-phosphorylate FAK. This leads to activation of ERK/MAPK signaling, and this complex is therefore able to control cell shape and regulate focal adhesions [26]. In cSCC, a step-wise increase in activation of FAK from unaffected margin skin to hyperproliferative skin and invasive cSCC [27] results in elevated integrin-FAK-Src signaling that stimulates keratinocyte migration [28] and drives progression of benign papillomas to aggressive cSCC [29, 30]. In cSCC, it has been demonstrated that FAK function is required for cancer stem cell maintenance, regulating cSCC initiation, growth, regression, and progression [31]. Actin remodeling protein Flii, which will be discussed in detail in Section 2.4, has been shown not to directly bind integrin receptors but form focal adhesion complexes with adaptor proteins and regulate integrin activation, downstream Src/paxillin signaling and focal adhesion turnover in a Rac1 dependent manner [25, 32]. Additionally, latest research has shown that actin remodeling proteins Flii and gelsolin, which have always been thought to be intracellular, have also both been shown to be secreted where they function to sequester extracellular actin post tissue injury, modulate inflammation and affect collagen VII anchoring fibril formation. Flii is able to modulate inflammation via toll-like receptor 4 (TLR-4) signaling, as Flii leucine-rich repeat (LRR) domains have 50% similarity to LRR domains of TLR-4, by which the immune system is able to detect infection or injury. The binding of LRRs to PAMP and DAMP molecules activates intracellular TLR signaling and ultimately results in the release of proinflammatory cytokine secretion [33]. The extracellular roles of Flii and gelsolin in respect to mechanosensing and cSCC progression are still to be examined. Nevertheless, it is clear that the coordinated activation of integrin receptors, focal adhesion complex formation and downstream signaling stimulation is essential for cytoskeletal changes that are necessary for cell migratory and invasive capability during cSCC progression.

#### **2.2 Mechanotransduction: Rho GTPases**

Downstream of integrin activation are the Rho small GTPases, which are part of the Ras superfamily and key regulators of cell cytoskeletal dynamics through both actin polymerization and organization, hence driving cancer cell motility [34]. Of this subfamily, RhoA, Rac1 and Cdc42 are the best-characterized.

RhoA is involved in actomyosin contractility, formation of actin stress fibers and assembly of focal adhesion complexes. The main regulator of cytoskeletal dynamics leading to formation of stress fibers is myosin II, and its regulatory subunit myosin regulatory light chain-2 (MLC2) can be activated by RhoA signaling leading to contraction of actin fibers. Rho-associated kinases ROCK1 and ROCK2 are serine/ threonine kinases that contain a Rho-binding domain and are activated by RhoA in its active GTP-bound form, directly activate MLC2 via phosphorylation. Due to its roles in cell contractility and movement, Rho-ROCK signaling has been implicated as a driver for invasiveness during cSCC progression but also plays a positive role in physiological normal wound healing processes [35]. In human cSCCs, ROCK is not only highly expressed but also activated in the hyperproliferative skin and invasive regions of the tumor, as shown by phosphorylation of the ROCK substrate myosin phosphatase (MYPT1) [27]. In the skin, this ROCK-mediated actomyosin contractility is required for proliferation of the epidermis, as ROCK activation stabilizes β-catenin through phosphoinositide 3-kinase (PI3K), Akt, and inhibition of its phosphorylating kinase GSK3β [36]. During cancer progression from normal skin through to hyperproliferative and invasive cSCC, nuclear localization of active

β-catenin and inactivation of GSK3β is increased, accompanied also by a progressive increase in FAK activation [27, 36]. It has further been demonstrated that a negative regulator of ROCK signaling, 14-3-3ζ, is significantly down-regulated in human cSCCs. As genetic deletion of 14-3-3ζ results in significantly larger papillomas in the two-stage chemical carcinogenesis (DMBA-TPA) mouse model of SCC, this suggests that uncontrolled ROCK signaling can drive cSCC tumor growth [35].

Cdc42 is involved in formation of F-actin microspikes and filopodia in both normal and cSCC cells, by via actin polymerization at the leading edge and at the sides of the cells, contributing to cSCC invasion. Traf6 has been demonstrated to regulate Cdc42 to induce these F-actin microspikes in SCC cells [37]. When Cdc42 is absent in keratinocytes, cells are no longer able to properly process and deposit ECM components or integrin receptors, hence halting cellular migration [38]. Taken together, these studies demonstrate roles for Cdc42 in both skin cell migration and invasion, necessary cellular processes for progression of cSCC. Cdc42 is necessary for proper cellular polarity in normal and migratory cells [34], and this in turn activates G-proteins of the Rac subfamily of Rho small GTPases [39].

Rac1 is hyperactivated in cSCC via integrins including α3β1, and is important for keratinocyte cell proliferation [40, 41]. Rac1 stimulates polymerization of actin via multiple kinase signaling cascades including that of MAP kinase (elevating the transcription factors AP-1, NFκB and CRE). This therefore allows the cell to form a branched actin network, necessary for leading-edge lamellipodia formation and membrane ruffling [42]. Indeed, actin remodeling protein, Flightless I (Flii), has been shown to regulate focal adhesion by inhibition of paxillin phosphorylation via a Rac1 dependent pathway [32]. Rac1 can be activated by Tiam1, and it was shown in the twostage chemical carcinogenesis cSCC model that genetic deletion of Tiam1 significantly reduced tumor incidence, burden and growth. However, SCC tumors that did arise in Tiam-null mice were significantly more invasive and malignant, potentially due to a loss of cell-matrix adhesion [43]. This highlights the dual homeostatic and tumorpromoting roles that actomyosin regulatory pathways can play during cSCC progression.

Rho-associated kinase ROCK can also phosphorylate and activate LIM kinases 1 and 2, which are then able to phosphorylate and inactivate cofilin, an F-actin severing protein, resulting in F-actin filament stabilization [44]. Accordingly, it has been shown that LIMK1 levels are increased in cSCC tumor tissue compared to normal skin, and that LIMK1 silencing can suppress cell growth and invasion in cSCC cell lines [45]. In addition, it has been suggested that LIMK is required in the microenvironment in leading fibroblasts, to allow for efficient remodeling of the ECM and subsequent cSCC invasion [46]. It has been demonstrated that cofilin phosphorylation can be abolished by treating cSCC cells with LIMK inhibitors. This reduces β-catenin accumulation and epidermal proliferation via reversing actomyosin contractility [36], however clinical trials using these inhibitors are still pending. The involvement of Rho GTPases in cellular migration and invasion in cSCC due to cytoskeletal rearrangement implicates this family of proteins as drivers of cancer initiation, progression and metastasis. Hence, targeting Rho pathway signaling, in particular that of RhoA-ROCK signaling, is an attractive therapeutic option that will be explored later in this chapter.

#### **2.3 Actin polymerization: WASP, cortactin, and Arp 2/3**

Actin nucleation promoting molecules are activated downstream of Rho GTPases and growth factor receptors. Wiskott-Aldrich syndrome protein (WASP) family members, via Erk, paxillin, and Src signaling together with cortactin, act to stimulate the actin-related protein (Arp) 2/3 complex which in turn mediates actin polymerization [47].

*Mechanical Force and Actin Dynamics during Cutaneous Squamous Cell Carcinoma (cSCC)… DOI: http://dx.doi.org/10.5772/intechopen.86041*

The WASP family consists of WASP proteins and WASP-family verprolinhomologous (WAVE) proteins. WASP proteins interact with Rho GTPases in order to form cellular protrusions and allow the cell to migrate, for example, N-WASP is involved in formation of filopodia and invadopodia upon its activation by Cdc42. WASP proteins bind to G-actin and Arp2/3 resulting in their activation and hence triggering actin filament production. WASP and WAVE proteins also bind profilin, which transports actin monomers onto the growing ends of actin filaments, and is therefore also an important factor in cell motility [47]. As a loss of N-WASP in keratinocytes causes epidermal hyperplasia and a reduction in epithelial cell tight junctions [48], this highlights the need for proper control of these processes. The formation of cellular protrusions also relies on the Src protein cortactin, which binds to and activates the Arp2/3 complex independently of WASP, thereby regulating actin filament nucleation. Cortactin binds to F-actin, stabilizing actin filaments and allowing it to properly activate Arp2/3 [49, 50]. In head and neck SCC, overexpression of cortactin increases cancer cell proliferation and increases cell survival in anchorage-independent conditions [51], while in oral SCC, silencing of cortactin was shown to significantly impair invasiveness and downregulate the levels of epithelial markers, indicating an epithelial to mesenchymal transition (EMT) [52], a process by which epithelial cells lose their adhesion to one another and acquire a migratory and invasive mesenchymal phenotype (discussed in further detail below). Indeed, Arp2/3 complex proteins are required for cell proliferation and migration in other forms of SCC [53, 54], and based on the Arp2/3 role in actin filament polymerization, it is clear that Arp2/3 is also critical for actin cytoskeletal remodeling leading to cancer cell motility and invasion.

#### **2.4 Actin remodeling: tropomyosin, Flightless I, and podoplanin**

The actin cytoskeleton is composed of three distinct elements including microfilaments, microtubules and intermediate filaments. Tight regulation of cytoskeletal elements must be coordinated, and latest research has shown that interplay between actin and microtubules is bidirectional [55]. Actin-based motility is also dependent on the balanced activity of number of specific actin remodeling proteins. In this section we will highlight main actin remodeling proteins that have been shown to have specific functions in cSCC progression, including members of the tropomyosin family of actin-associated proteins, the gelsolin family of actin remodeling proteins, and Podoplanin, a simple glycoprotein with important roles in cSCC progression.

Members of the tropomyosin family of actin remodeling proteins display a tissue- and time-specific expression, while their association with actin filaments impairs isoform-specific regulation of actin filament dynamics [56]. Tropomyosin proteins assemble as polymers in the major groove of the polymerized actin filament and their association drives actin filament turnover, hence playing an important part in a number of cellular functions including motility and metastasis [57]. There are over 40 different isoforms of tropomyosin and few have been described as having an important role in cSCC progression. High expression of Tm5NM1, a specific cytoskeletal tropomyosin isoform, has been shown to inhibit cell migration and invasion as well as impair normal wound healing via its effects on Src activation, focal adhesion stabilization, increased actin filament tension, and paxillin phosphorylation [58]. Current research is examining the effect of Tm5NM1 inhibitor TR100 on cSCC progression (see Section 3). On the other hand, downregulation of tropomyosin-1 and complete loss of β-tropomyosin has been identified in human esophageal SCC, while α-tropomyosin has been shown to be preferentially

expressed in keratinocytes of the multistage model of murine cSCC, collectively suggesting isoform specific functions [59–61].

The dynamic remodeling of the actin cytoskeleton is also tightly regulated by the gelsolin family of actin remodeling proteins, which includes: gelsolin, villin, adseverin, capG, advillin, supervillin, and Flightless I (Flii) [62]. These actin binding proteins function in the cytoplasm of the cells where they control actin organization by severing pre-existing filaments, capping the fast growing filament ends and bundling filaments, enabling filament reassembly into new cytoskeletal structures that are required for cell motility, invasion and metastasis [63]. Studies have shown that downregulation of gelsolin proteins counteracts cancer cell invasion *in vitro* [64], however in cSCC, gelsolin and Flii have been the most studied to-date. Gelsolin over-expression has been shown to promote cell growth and motility in oral SCC [64, 65], while Flii, through its effects on apoptosis, has been linked to promotion of breast cancer progression and invasion and progression of cSCC [23, 66]. Flii is an important regulator of cell adhesion, migration and proliferation and a number of previous studies have described the role of Flii protein in wound healing and demonstrated the therapeutic effect of Flii neutralizing antibodies (FnAb) in acute and chronic wounds, skin blistering diseases and inflammatory skin conditions [25, 32, 67–72]. Flii modulates cell adhesion and paxillin signaling, and regulates actin polymerization, tight junction formation and ECM production during wound repair suggesting that similar roles may govern Flii activity in cSCC progression [23, 25, 32, 72]. Indeed, altering Flii levels both genetically and using Flii neutralizing antibodies significantly augments cSCC progression [23]. Therapeutic approaches targeting Flii in cSCC are described in Section 4.

The expression of Podoplanin, a small mucin-like protein, has also been linked to remodeling of the actin cytoskeleton in cSCC. Podoplanin is upregulated in the invasive front of a number of human carcinomas including cSCC and has been shown to induce collective cell migration by filopodia formation, via downregulating the function of Rho small GTPases [73, 74]. Podoplanin has also been linked to an increase in the migration of cancer-associated fibroblasts as well as endothelial network formation [75]. Collectively these findings suggest that Podoplanin is able to induce an alternative pathway of tumor cell invasion in the absence of traditional epithelial-mesenchymal transition.

Taken together, these studies highlight the important role of actin remodeling in cSCC progression and outline the importance of bidirectional stimulation of actin remodeling by both intrinsic factors and the microenvironment, critical to tumor invasion/metastasis. These findings provide a rationale for development of novel therapeutic strategies that target tumor invasion and metastasis.

#### **3. Physiological effects of actin remodeling**

Changes in the actin cytoskeletal structure result in changes to cell morphology, creating a cell more conducive to invasion. One of the commonly recognized requirements of metastasis is a cellular transition from epithelial to mesenchymal phenotype (EMT). This transition is characterized by upregulation of genes including vimentin, SNAI1 (snail), SNAI2 (slug) and Zeb1, and a downregulation of epithelial genes including cadherins, as well as concurrent loss of cell-cell junctions [76]. For example, in head and neck SCC an increase in matrix stiffness and hence increased mechanical signaling caused an increase in EMT markers in tumor-initiating cells [71]. Cells undergoing EMT develop an elongated spindle-like morphology, due to the enhanced membrane protrusion formation [77]. It has been shown in A431 cells, a human epidermal SCC cell line, that loss of T-cadherin induces elongation of cells and formation

*Mechanical Force and Actin Dynamics during Cutaneous Squamous Cell Carcinoma (cSCC)… DOI: http://dx.doi.org/10.5772/intechopen.86041*

of lamellipodia and multiple leading edges via changes in EGF-stimulated motility and invasion, as T-cadherin influences EGFR localization and responsiveness [78]. Of note, RhoA activation was also increased upon the loss of T-cadherin [79]. Likewise, Podoplanin is also capable of transforming cells to an invasive state without having to undergo EMT, due to rearrangements of the actin cytoskeleton [74].

The remodeling of the actin cytoskeleton also creates intracellular reciprocal forces that balance out the forces received by the cell from the extracellular microenvironment. Actin polymerization extends the filament network, and as filaments in the leading edge are compressed between transient associations with the cell membrane and the bulk of the actin cytoskeletal network behind them, intracellular force is generated. As protrusions are extended and retracted, actin filaments experience tension from transient bonds with the membrane, becoming bent or compressed [80]. Activation of the mechanotransduction pathways described above, downstream of ECM stiffness in cSCC, can also increase the propensity for augmented interactions with the stroma, and generate a tumor-promoting environment that enhances mechanoreciprocal signaling [20, 36].

#### **4. Therapeutic approaches targeting actin cytoskeletal regulatory pathways**

Metastasis is a complex process requiring significant reorganization of the actin cytoskeleton and coordinated involvement of number of key proteins. These proteins interact directly and indirectly with both actin and microtubule networks, hence significantly influencing migratory and metastatic cell phenotypes. Strong clinical relationships between actin cytoskeletal alterations and cutaneous cancer metastasis have been previously described [11], offering potential opportunities for therapeutic intervention. For example, up-regulation of cortactin, an actin-binding adaptor protein in melanoma, has been directly linked to increased distal metastasis and reduced disease-free survival, while up-regulation of Ras mRNA has been directly linked to Stage III and Stage IV disease in head and neck SCC [81, 82]. The complex nature of cellular migration and invasion presents challenges in developing therapeutic approaches, as compensatory pathways may overcome the effects of specific inhibitors. This highlights the need for development of combinational and adjuvant therapies targeting multiple pathways that are involved in actin dynamics to treat aggressive cSCC. Pharmacological inhibitors of actin have failed clinical development due to non-specific effects on normal actin function in tissue, resulting in high levels of cardiotoxicity. Hence, research efforts have centered on therapeutic approaches that can modify signaling pathways regulating the actomyosin cytoskeleton and/or target cytoskeletal and cytoskeletal-associated proteins [11].

Increasingly it has been recognized that microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) are involved in regulating cytoskeletal dynamics through regulation of gene expression. lncRNAs have been shown to regulate lamellipodia formation by downregulating integrin expression in cSCC [83]. A number of miR-NAs have also been shown to play a role in regulating cell cytoskeletal dynamics and interactions with stroma in cSCC, including: miR-340 [84], miR-20a [45], miR-31 [85] and miR-125b [86]. These miRNAs act via inhibiting RhoA, LIMK1, WAVE3 and matrix metalloproteinase (MMP)-13 respectively. The use of miRNAs in the clinic has clear potential, however clinical trials are yet to be undertaken.

One of the signaling pathways participating in regulation of cancer cell motility, invasion and metastasis is the ROCK signaling pathway, described in detail above. Hyperactivation of this pathway promotes cancer cell invasion in many solid tumors and studies have shown that Rho signaling through ROCK promotes the rounded

bleb-associated mode of amoeboid motility, thereby promoting tumor cell metastasis [87]. Earlier reports have shown that treatment with the selective ROCK inhibitor Y-27632 increases SCC cell adhesion, upregulates expression of E-cadherin, and decreases the phosphorylation of cofilin (thereby activating it), resulting in altered actin cytoskeleton rearrangement [88]. In the two-stage chemical carcinogenesis model, Y27632 treatment resulted in significantly smaller and fewer papillomas, with a reduced rate of cSCC conversion. This was associated with reduced collagen deposition in the ECM, which would indicate a decrease in mechanical signaling due to ECM stiffness [36]. This illustrates that inhibition of ROCK is a potential strategy for treatment of solid cancers including cSCC.

A potential upstream regulator of ROCK-mediated cell migration is gammaactin. Modulation of gamma-actin changes the directional cell migration via effects on microtubule dynamics and cell polarity, hence highlighting the crosstalk between actin cytoskeleton and microtubule signaling as a potential modality for targeting specific components of the network [89]. More recent studies, using newer generations of ROCK inhibitors and pharmacological small molecule inhibitors of the downstream effectors of ROCK that have micro-tubule stabilizing effects, are also showing some promise in regulating tumor metastasis, however no compounds are yet clinically approved [90]. Another potential therapeutic strategy is harnessing the ability of 14-3-3ζ, a negative regulator of ROCK signaling, to moderate mechanoreciprocity in cSCC [35]. These approaches are particularly enticing due to the negative effects that clinical targeting of ROCK itself can potentially have [91].

Interestingly, studies investigating the interactions between SCC cells and cancer-associated fibroblasts have shown that ROCK activity is also an important requirement for adjacent stromal fibroblasts. ROCK activity positively influences the JAK1-STAT3 signaling pathway resulting in increased actomyosin contractility and proinflammatory cytokine secretion, favoring cSCC cancer cell invasion [92]. Consequently, these studies suggest that approaches aimed at inhibiting ROCK signaling have the potential to interrupt both intrinsic and microenvironment-derived signals during cSCC progression.

Actin remodeling proteins have long been implicated in cSCC, as a dysregulated actin cytoskeleton and an aberrant tumor microenvironment is a hallmark of aggressive cSCC [11, 93]. One particular actin remodeling protein, Flightless I (Flii), has been identified as a tumor promoter with transcriptional activity in colorectal, breast and hepatocellular carcinoma cell lines [66]. However, recent studies have also shown that Flii is significantly increased in human and mouse cSCC tissue samples, while secreted Flii is elevated in the sera of patients with cSCC and is increased in different cSCC cell lines established from human primary, recurring and metastatic cSCC as well as immortalized keratinocytes [23]. Human cSCC samples show positive staining for Flii in invading keratinocytes, surrounding tumor stroma and the outer hyperkeratotic layer of cSCC nodules present in the deep dermis [23]. Together, these data suggest that Flii is not only an important regulator of the actin cytoskeleton involved in cSCC progression but also a potential therapeutic target and diagnostic marker of cSCC severity. Indeed, overexpression of Flii resulted in severe cSCC development via evasion of apoptosis, while reducing Flii expression using intradermal injections of FnAb during cSCC initiation and progression significantly reduced Flii expression in both the tumor microenvironment and in the serum, and led to significantly smaller tumor size (**Figure 5**) and decreased cellular sphere formation and invasion *in vitro* [23].

Remodeling and polymerization of actin filaments is critical during cSCC invasion and formation of invadopodia. Increased Flii levels have shown to weaken cellstroma and cell-cell adhesions via alteration of GTPase and Src/paxillin signaling pathway activity [32] and augmented integrin-facilitated cell migration [25]. This

*Mechanical Force and Actin Dynamics during Cutaneous Squamous Cell Carcinoma (cSCC)… DOI: http://dx.doi.org/10.5772/intechopen.86041*

#### **Figure 5.**

*Reducing Flii expression prior to initiation and development of cSCC using FnAb results in decreased cSCC progression. As Flii is increased in human SCC samples, the effect of preventative FnAb treatment prior to SCC induction was investigated in FliiTg/Tg mice. Mice were treated with FnAb 2 weeks prior to cSCC induction and every second week throughout the trial and SCC development. (A and B) Reducing Flii levels in mice skin using preventative FnAb treatment prior to cSCC induction and during development resulted in decreased tumor progression and size relative to IgG control mice that have significantly larger and more developed necrotic and ulcerated tumors. (n = 12/treatment) Mean ± SD \* p < 0.05. (C) Representative images of H&E stained tumors treated with FnAb or IgG control show more severe ulcerated tumor pathology in the IgG control group (black arrows). Scale Bars = 500 μm and 100 μm.*

promotes tumor progression and facilitates invadopodia formation and subsequent tumor invasion into surrounding tissue [23]. Indeed, Flii is significantly increased in invading cSCC and has been demonstrated to associate with cortactin at leading edges of invadopodia and to regulate the invasive properties of cSCC keratinocytes [23]. Systemic and topical therapeutic approaches using FnAb are currently in development with FnAb as a therapy for wound healing now entering the final preclinical validation stage [68, 94]. Flii has been shown to colocalize with structural (Claudin-1, -4 and -6) and adaptor (ZO-1 and -2) tight junction proteins and its overexpression in keratinocytes results in an altered F-actin/G-actin ratio, which can be restored using FnAb [72]. Therefore, taken together, these studies suggest that therapies targeting Flii may be a potential strategy for reducing the severity of cSCC in the community, however clinical trials using FnAb are still pending.

Pharmacological inhibition of actin-associated proteins aimed at compromising the survival and invasion of tumor cells may also have clinical benefit. One example of this strategy is harnessing TR100 inhibition of the tropomyosin isoform Tm5NM1. Tm5NM1 belongs to a family of actin-associated proteins that regulate the activity of several effectors of actin filament dynamics [95], as described above. The TR100 inhibitor has been shown to preferentially disrupt the actin cytoskeleton of tumor cells, impairing tumor cell motility and viability, and reducing melanoma growth both *in vitro* and *in vivo*. This therefore provides a pathway for development of a novel class of anti-actin compounds for the potential treatment of wide variety of cancers including cSCC [96].

Microtubule targeting agents of both synthetic and natural design, and microtubule stabilizing and destabilizing agents, have been the focus of anti-cancer therapy in the last decade and remain one of the most successful group of agents in the clinic [97, 98]. Their ability to regulate the tubulin-microtubule equilibrium disrupts the mitotic spindle, halting the cell cycle and resulting in cell death. They have been shown to be effective in combination with anti-angiogenic and anti-vascular properties and in some cases have demonstrated the ability to overcome multi-drug resistance, supporting their utilization as a chemotherapy [99]. Epothilones are a new class of anti-microtubule agents currently in clinical trials. Epothilones have shown activity in cSCC cell lines and in melanoma clinically, however clinical trials on cSCC patients are still pending [100, 101]. Other examples of microtubule-targeting agents, which have shown clinical promise in different subtypes of SCC including metastatic and recurrent disease, include semisynthetic compounds docetaxel and eribulin and a natural compound called rhizoxin [102–104]. While microtubule-targeting compounds are widely used as chemotherapeutic agents, they do have variability in different cancers, cancer cells frequently develop resistance to them, and they can be toxic to normal tissue, highlighting the need for better research and refinement of these compounds as well as a need to further understand their interactions with microtubule-associated proteins [105]. It is possible that microtubule-targeting agents also exert broader effects on tumor cell migration, invasion and metastasis and future studies should explore their effects on cSCC in combination with actin pathway inhibitors. Gaining a better understanding on the interplay of regulatory proteins governing the mechanotransduction and actin cytoskeletal remodeling involved in tumor cell migration, invasion and metastasis will lead to increased efforts to exploit therapeutic avenues targeting the actin cytoskeleton to treat aggressive cSCC.

#### **5. Conclusions**

The contribution of actin cytoskeletal remodeling and actomyosin signaling during SCC progression is significant and cannot be undervalued in the search for new treatment modalities. Recent research has identified a number of potential novel therapeutic targets within regulatory actin and microtubule signaling pathways that should be explored as potential therapeutic adjuvants to immunomodulatory therapies currently in clinical trials. A comprehensive understanding of the regulatory network of cutaneous mechanotransduction, mechanical forces and actin dynamics in cSCC, as discussed in this chapter, will facilitate the development of novel approaches to curb the incidence and progression of aggressive cSCC in the community, generating new inroads toward development of novel, individually personalized and efficient therapeutic approaches.

#### **Acknowledgements**

Dr. Zlatko Kopecki is supported by a Future Industries Institute Foundation Fellowship from University of South Australia. Dr. Sarah Boyle is supported by a Royal Adelaide Hospital Research Fund Early Career Fellowship.

#### **Conflict of interest**

The authors declare no conflict of interest.

*Mechanical Force and Actin Dynamics during Cutaneous Squamous Cell Carcinoma (cSCC)… DOI: http://dx.doi.org/10.5772/intechopen.86041*

### **Nomenclature**


#### *Squamous Cell Carcinoma - Hallmark and Treatment Modalities*


#### **Author details**

Sarah Boyle1 and Zlatko Kopecki2,3\*

1 Centre for Cancer Biology, University of South Australia and SA Pathology, Adelaide, South Australia

2 Future Industries Institute, University of South Australia, Adelaide, South Australia

3 School of Pharmacy and Medical Sciences, University of South Australia Cancer Research Institute, Adelaide, South Australia

\*Address all correspondence to: zlatko.kopecki@unisa.edu.au

© 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.

*Mechanical Force and Actin Dynamics during Cutaneous Squamous Cell Carcinoma (cSCC)… DOI: http://dx.doi.org/10.5772/intechopen.86041*

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### Section 6

## Molecular Aberrations in Squamous Cell Carcinoma

#### **Chapter 6**

## Comprehensive Molecular Characterization of Squamous Cell Carcinomas

*Corina Lorz, Carmen Segrelles, Ricardo Errazquin and Ramon Garcia-Escudero*

#### **Abstract**

Over the last two decades, a number of high-throughput technologies (genomeand proteome-based) have been developed and applied on different cancer types such as squamous cell carcinomas (SCCs) arising from aerodigestive and genitourinary tracts. These analyses, when comprehensively utilized, have clearly contributed to a better understanding of the molecular hallmarks, oncogenic pathways and immunological features of SCCs. This chapter aims to describe the SCCs most important molecular aberrations as well as their molecular classification, highlighting the commonalities and differences among them, independent of their body site origin. The most frequently altered oncogene is PIK3CA, involved in the PI3K/ AKT/mTOR pathway and frequently activated in many human cancers. However, alterations in the cell-cycle control TP53 gene occur in the vast majority of SCCs. New possible molecular therapies, common to all SCCs, are discussed in light of a comprehensive, panSCC analysis.

**Keywords:** squamous cell carcinoma, human papillomavirus, genomics, Fanconi anemia, TCGA, mutation, copy number alteration, cancer treatment, biomarker

#### **1. Introduction**

Squamous cell carcinomas (SCCs) represent highly common solid cancers that arise from stratified and pseudo-stratified epithelia of the skin, and aerodigestive and genitourinary tracts. Although SCCs from different body sites share histological characteristics, they are molecularly and clinically heterogeneous, and a major cause of cancer mortality [1]. Reported risk factors for SCCs, depending on the body site, include alcohol intake (head and neck, and esophagus), cigarette smoking (bladder, lung, head and neck, and esophagus), UV light exposure (skin) and infection with human papillomavirus (HPV) (skin, head and neck, and cervix uteri). HPV infects epithelial cells and transforms them through the oncogene action of viral genes. E6 and E7 genes from some HPVs infecting head and neck and cervix uteri inhibit the function of the important tumor suppressors p53 and pRb, respectively [2, 3]. The initiation of SCCs is due to genomic perturbations, genetic mutations, and/or altered expression of key molecules mainly involved in cellcycle control, signaling and cell adhesion pathways, squamous differentiation and chromatin regulation [1, 4]. A number of reports show that SCCs from different

anatomical locations have common features despite the fact that they are clinically treated as separate entities. These findings suggest an integrated view of the disease and possible new methods for prevention and treatment.

Here we review reports in which hundreds of SCCs have been comprehensively characterized at the molecular level using different high-throughput technologies. Such analyses highlighted commonalities and differences between SCCs, independent of body site origin, and allow their classification based on molecular aberrations. New possible molecular therapies, common to all SCCs, are discussed in light of the comprehensive, panSCC analysis.

### **2. Molecular features of SCCs**

SCCs from different anatomical sites have been molecularly characterized using various genome-wide technologies (**Table 1**). Despite early reports describing most frequent mutations using next-generation sequencing (NGS) such as whole-exome sequencing (WES) in many cancer types [5], most of the comprehensive analyses have been done within the context of The Cancer Genome Atlas (TCGA) consortium. TCGA is an USA project which has generated comprehensive, multi-dimensional maps of the key genomic changes in the main types of cancer (http://cancergenome.nih. gov/). Microarray- and/or NGS-based technologies have been used in order to determine mutations in protein coding genes, expression levels of messenger RNA (mRNA) and micro RNA (miRNA) molecules, DNA-methylation and genome copy-number variation (CNA) (**Table 1**). Moreover, an important subset of cancers has been characterized at the protein level, using Reverse Phase Protein Array (RPPA) (**Table 1**). Recently, the TCGA launched a set of publications reporting pancancer analyses of more than 11,000 tumors from 33 types of cancers [4] (https://www.cell.com/ pb-assets/consortium/pancanceratlas/pancani3/index.html), including SCCs from 5 individual body sites: lung (LUSC), head and neck (HNSC), esophageal (ESCA), cervical (CESC), and bladder (BLCA) cancers. Most of the molecular features described here are based on the TCGA panSCC analysis, in which around 1400 SCCs from those body sites were analyzed simultaneously [6]. Although skin SCC is the second most frequent cancer in Caucasians [7], no comprehensive, genome-wide analysis has been reported. Interestingly, most frequent mutations using NGS-based technologies in skin SCC showed many similarities with SCC from other body sites [8, 9].


*messenger RNA; miRNA: micro RNA; proteome: reverse phase protein assay (RPPA).*

#### **Table 1.**

*List of publications with genome-wide analysis of SCC.*

#### **2.1 Mutations in cancer genes**

The most frequent mutated gene found in SCCs is *TP53* (64% in panSCCs) [4, 6, 16], (**Figure 1**) a tumor suppressor gene whose main function is to prevent genome mutations [17]. Missense "hot spots" mutations are very common, which result in dominant-negative and/or gain-of-function properties [18]. Although *Tp53* was found to be highly altered in many other cancer types [19], frequencies depends on the type, stage, body site, and other factors. Mutations in *TP53* are infrequent in HPV(+) SCC cancers, possibly because p53 functions are compromised as the protein is degraded by the activity of the viral E6 oncogene. Individually, frequent *TP53* mutations are found in SCCs within BLCA [15], ESCA [12], HNSC [10] and LUSC [11], and to a lesser extent in CESC whereby the majority of tumors are HPV(+) [13]. Other mutated genes involved in cell-cycle control include CDK inhibitor *CDKN2A* and the *RB1* gene, although less frequently. The incidence of *CDKN2A*/*RB1* mutations is much reduced in HPV(+) HNSC and CESC, as the E7 viral oncogene can bind and inactivate pRb protein, coded by *RB1*, thus rendering direct genetic mutation dispensable [10, 13]. Another important group of mutated genes include regulators of squamous differentiation, such as *NOTCH1*, *AJUBA* or *ZNF750* (**Figure 1**) [4, 6, 16].

#### **Figure 1.**

*Relevant mutations and CNA alterations in SCCs from BLCA, CESC, LUSC, HNSC and ESCA. Tumors are grouped into iC10, iC25 and iC27 clusters. Genes are grouped into functions. Frequency of alterations per gene is shown. HPV infected samples are indicated. Tumors having WES and CNA data, and belonging to iC10, iC25 and iC27 clusters are shown (n = 1098). Data are from the cBioportal for Cancer Genomics (http://www. cbioportal.org/) [20].*

Other mutated genes include *KMT2D*, *NSD1*, *EP300*, or *KDM6A*, all of them involved in chromatin regulation through histone post-translational modifications. *PIK3CA*, *PTEN*, *FAT1*, *EPHA2* or *RASA1* genes, also mutated, are involved in important signaling and cell adhesion pathways of epithelial cells. There are also mutations in genes important in cell survival, like *TGFBR2* or *CASP8*. Mutations of *HLA-A* and *HLA-B* and deletions of *B2M*, implicated in immune escape, also exist (**Figure 1**) [4, 6, 16].

#### **2.2 panSCC molecular clustering**

High-throughput technologies have allowed the identification of tumor subgroups within specific cancer types, like the 'intrinsic subtypes' of breast cancer [21], occasionally having important clinical differences and outcomes [22]. Tumor subgroups based on genome-wide molecular analyses have been reported also for HNSC [10], LUSC [11], BLCA [14, 15], CESC [13] and ESCA [12]. Such classifications are based on molecular features like mutations, CNA, DNA-methylation, or expression of mRNAs, miRNAs, proteins [10–15] and long non-coding RNAs [15, 23, 24].

The existence of hundreds of primary tumors from different cancer types within TCGA having multiplatform molecular data have allowed the integrated identification of their differences and commonalities, regardless of body site [4, 6, 16]. One of such analyses, performed over 1400 SCCs from five different locations (LUSC, HNSC, CESC, ESCA and BLCA), discovered the existence of different SCC tumor clusters based on CNA (six clusters), DNA methylation (five clusters), mRNA expression (six clusters), miRNA expression (five clusters), and RPPA-based protein expression (eight clusters) [6]. These clusters highlight significant molecular features in SCC versus non-SCC, and between SCCs. Moreover, the iClustering method [25], which performs clustering from multi-type genomic data, showed the presence of 3 main iClusters: iC10, iC25 and iC27 [6] (**Figure 1**). Most HPV(−) tumors grouped in iC10 and iC25, associated with smoking history, organ site and molecular aberrations (**Figure 2A**), while most HPV+ CESC and HNSC samples mapped within iC27 having non-smoking individuals (**Figure 2A**). All tree SCC-clusters displayed significant chromosome 5q and 3p copy gains, concomitant with overexpression in 3q genes SOX2, TP63, and TP73, implicated in squamous differentiation and stemness (**Figure 1**) [6]. iC25 cluster bear 11q gains, and iC10/iC25 included 9p losses. Most iC10/25 HPV(−) SCC tumors displayed genome-wide hypomethylation with high DNA CNA, and associated augmented mRNA and miRNA levels. Some HPV(−) SCCs and most iC27 HPV(+) HNSCs and CESCs, showed wider hypermethylation and reduced CNAs, correlated with reduced mRNA and miRNA expression [6].

Kaplan-Meier curves demonstrated significant differences in overall survival and progression-free interval between the iClusters, even after adjusting for distinct body sites or disease stages (**Figure 2B**). Patients within iC25 display poorer prognosis, possibly associated to higher CNA aberrations and genome instability (**Figures 1** and **2B**). Therefore, panSCC analysis showed the existence of a prevalent SCC group, having a combination of recurrent CNA and other alterations, and other subtypes whereby HPV infection and other alterations have a greater role.

#### **2.3 Cancer genes in CN alterations**

Oncogenic transformation from normal tissues occurs upon the accumulation of small mutations and also larger alterations, giving rise to deletion (DEL) or amplification (AMP) of regions and altering the normal diploid state of the genome. Negative regulators of cell-cycle control like CDKN2A and RB1 are frequently deleted in SCCs (**Figure 1**). Contrarily, CCND1, MYC, and CCNE1 genes appear frequently amplified, and therefore, their function in cell proliferation. Important

*Comprehensive Molecular Characterization of Squamous Cell Carcinomas DOI: http://dx.doi.org/10.5772/intechopen.85988*

#### **Figure 2.**

*Clinical features of main panSCC iClusters, including body site distribution and patient smoking history frequencies within each iC10, iC25 and iC27 (A), and survival curves with 2 endpoints: overall survival and progression-free interval (B). Clinical data obtained from Liu et al. [26]. P-values were calculated after multivariate Cox regression analysis, using iClusters and body sites or pathologic tumor stage.*

positive mediators of signaling and cell adhesion pathways are frequently amplified (EGFR, ERBB2, FGFR1, PIK3CA, AKT1, AKT3, MAPK1, YAP1), and tumor suppressors like PTEN or FAT1 are deleted. Chromosome 3q genes TP63 and SOX2 are highly frequently co-amplified, and overexpression of their mRNAs is a common SCC feature as mentioned above (**Figure 1**) [6]. Squamous differentiation genes which are deleted also exist, like NOTCH1 and ZNF750. Frequent deletion of chromatin regulation genes occurs, like ARID1A, NSD1, KMT2C or KDM6A. There are also alterations in cell survival genes, like NFE2L2 (AMP), BCL2L1 (AMP), and BCL2L2 (DEL). Importantly, some main immune escape regulators are segregated in CNA regions, like PD-L1 (AMP) or B2M (DEL) (**Figure 1**).

#### **3. SCC and Fanconi DNA repair pathway**

Fanconi anemia (FA) is a rare autosomal recessive genetic disorder in which patients can develop a life-threatening bone marrow failure in the early years after birth [27], which frequently requires allogeneic hematopoietic stem cell transplant [28]. In addition to this blood disorder, FA patients can develop leukemias and solid tumors, mainly SCC in the head and neck, skin, and anogenital regions [29].

Incidence of HNSC in FA is >500 times higher than in the general population, and average age of appearance is significantly earlier. Mutations occur in genes involved in the 'FA pathway' which is activated as a result of DNA replication or DNA damage, especially the damage triggered from DNA crosslinking agents. Some of these FA genes include *BRCA1* and *BRCA2* genes, well known breast cancer-susceptibility genes. Hitherto, there is no explanation for the high incidence of FA-HNSC, but it has been suggested that FA pathway defects might accelerate oncogenic transformation through the accumulation of mutations in a DNA repair-defective context [30]. In this sense, a number of reports showed tumor suppressor functions by FA genes, both in the FA as well as in non-FA human cancer [31].

Campbell et al. reported an unexpectedly high frequency (around 12%) of molecular aberrations involving top 10 FA pathway genes in panSCC from TCGA [6]. An analysis using all 22 FA pathway genes reported so far, demonstrated that almost 30% of SCCs within iC10, iC25 and iC27 clusters from BLCA, CESC, ESCA, HNSC and LUSC (314 out of 1098) display either point mutations or deletions in any FA gene (**Figure 3**). Whether all of these FA gene alterations are associated with defects in DNA-repair is unknown, but clinical implications would be important

#### **Figure 3.**

*Mutations and deep deletion in all 22 FA pathway genes in SCCs from BLCA, CESC, LUSC, HNSC and ESCA. (A) Tumors are grouped into iC10, iC25 and iC27 clusters. Frequency of alterations per gene is shown. HPV infected samples are indicated. (B) Alteration frequency in any FA gene is shown per body site. Tumors having WES and CNA data, belonging to iC10, iC25 and iC27 clusters, and having mutation/deep deletion are shown (n = 314). Data are from the cBioportal for Cancer Genomics (http://www.cbioportal.org/) [20].*

as the FA pathway is a major predictor of cisplatin response in HNSC [32]. These findings suggest that acquired as well as germline alterations in this pathway may contribute to the development of a subset of SCC.

### **4. Molecular therapies against SCC**

Patients suffering squamous cell carcinoma display poor overall survival, and the disease is difficult to treat. Independent of body site, the standard of care is based on surgery, radiotherapy and chemotherapy. Still, few molecular therapies are being used so far, and only in the latest stages of the disease, such as immunotherapies, cetuximab (antibody to EGFR) in HNSC or bevacizumab (antibody to VEGF) in cervical cancer. There is a clear need to develop new targeted therapies accompanied with accurate response biomarkers, so we can give more effective and less aggressive treatments to SCC patients. The profound knowledge about the molecular biology of SCCs that we have acquired over the last recent years, together with comparative efforts of tumors from different body sites, should help to design new clinical trials challenging current treatment modalities.

#### **4.1 Immunotherapies in SCCs**

As understanding of the underlying cancer biology and the complex interactions within the tumor microenvironment improves, there is gathering interest in and evidence for the role of immunomodulating agents in the management of cancer. Immune checkpoint inhibitors, which aim to hinder the inhibitory interaction between programmed cell death protein 1 (PD-1) and its ligand PD-L1, have demonstrated durable improvements in patient outcomes in many cancer types. Thus, pembrolizumab (anti-PD1) has been approved to treat HNSC, CESC, LUSC, and BLCA [33–35]. Clinical trials for pembrolizumab in ESCA are giving good responses [36]. Nivolumab has also reach FDA approval for HNSC, BLCA and LUSC [33–35]. Other existing immunotherapies include avelumab, atezolizumab and durvalumab for BLCA [34]. Although the use of immunomodulating agents in SCC treatment is giving good results, none of them are being used in first line so far and many patients do not respond. Therefore, future analyses and trials should focus on developing accurate response predictors to accelerate their use as first line in therapy.

#### **4.2 Possible new therapies targeting SCCs biomarkers**

Deep molecular analyses of SCCs, as explained above, suggest that certain targeted therapies, at different stages of clinical trial or approval, might be adequate for SCC treatment. These include targeting the following biomarkers:

i.PIK3CA, which encodes p110α, a catalytic subunit of phosphoinositide 3-kinase (PI3K). Activated PI3K can activate PDK1 and AKT, triggering downstream effects on transcription, protein synthesis, metabolism, proliferation and apoptosis. The gene is amplified or mutated in about 37% of SCCs (**Figure 1**), and constitutes the most frequently mutated oncogene in cancers like HNSC, CESC, ESCA and LUSC. A number of clinical trials with p110α inhibitors as possible antitumor therapies are currently running. We have recently identified that HPV(−), HNSC tumors that overexpress PIK3CA display poor outcome and activation of the YAP1-nuclear function, a transcriptional co-factor within the Hippo growth pathway [37]. Therapies targeting nuclear YAP1 might also be effective in a subgroup of SCC patients [38].


#### **5. Conclusions**

Squamous cell carcinomas arising from five different body sites (bladder, cervix uteri, lung, head and neck, and esophagus) share many molecular aberrations, so that the majority of them can be classified in 3 main molecular clusters (iC10, iC25 and iC27). Principal differences between clusters include HPV infection, genome-wide DNA-methylation and CNA, and mutations/CNA in subsets of cancer genes. Amplification in CCND1 is prevalent in iC25 samples, and TP53 and CDKN2A deleterious modifications in HPV(−) tumors. iC25 tumors are HPV(−), display frequent genome alterations and smoking patients, as well as poorer clinical outcome. Importantly, there exist common features between panSCC clusters, such as oncogene PIK3CA mutations/amplifications, amplification in TP63 and SOX2, or mutations in chromatin modifier regulators (like KMT2C and KMT2D). The comprehensive, panSCC molecular analyses suggest that current and future clinical trials targeting aberrations in signaling/ cell adhesion pathways (PIK3CA and EGFR inhibitors) and cell-cycle control (CDK4/CDK6 inhibitors) might have a great impact on SCC treatment and independently of their body site. Future research efforts should focus on developing accurate biomarkers of immunotherapies. Finally, basic and clinical investigators should work together to discover SCC vulnerabilities and derive new treatments, as well as understanding basic mechanisms of oncogenesis, tumor progression and therapy resistance.

*Comprehensive Molecular Characterization of Squamous Cell Carcinomas DOI: http://dx.doi.org/10.5772/intechopen.85988*

### **Acknowledgements**

This work was supported by FEDER cofounded ISCIII [grant numbers PI18/00263, CB16/12/00228] and a grant from Fundación Anemia de Fanconi [grant number 2018/127].

### **Conflict of interest**

No 'conflict of interest' to declare.

### **Author details**

Corina Lorz1,2,3, Carmen Segrelles1,2,3, Ricardo Errazquin1,2 and Ramon Garcia-Escudero1,2,3\*

1 Biomedicine Research Institute, Hospital 12 Octubre, Madrid, Spain

2 Molecular Oncology Unit, CIEMAT, Madrid, Spain

3 CIBERONC, Madrid, Spain

\*Address all correspondence to: ramon.garcia@ciemat.es

© 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.

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### *Edited by Hamid Elia Daaboul*

With the support of multinational specialists, each with different background and separate field of expertise in oncology, this book had the occasion to emerge and to offer physicians, researchers and academics efficient, well-organized and updated scientific information related to the characteristics and treatment modalities of squamous cell carcinoma. It provided in-depth information regarding the comprehension of the molecular interaction of signalling pathways and new phenotypes that might result and lead to further cell proliferation and metastases. It also emphasized on the management and individualization of treatment strategies in different types of SCC, applying molecular profiling and approved protocols in order to identify new treatment opportunities. This book also discussed the potential therapeutic modalities that might arise upon understanding and exploring the role of key regulatory proteins that govern the process of cutaneous SCC progression. And Lastly, it underlined the molecular aberrations present in SCC of different organs and the possibility of emerging new therapeutic drugs by targeting these abnormalities. It is really an innovative record that combined novel information from various medical sources and mixed it to become unified in its scientific profit.

Published in London, UK © 2020 IntechOpen © LCBallard / iStock

Squamous Cell Carcinoma - Hallmark and Treatment Modalities

Squamous Cell Carcinoma

Hallmark and Treatment Modalities

*Edited by Hamid Elia Daaboul*