Prevention and Control

#### **Chapter 5**

## Preventing Human Papilloma Virus through Community Education and Vaccination

*Celeste Mulry Baldwin and Lisa Rinke*

#### **Abstract**

Human Papilloma Virus (HPV) affects many members of the community. To better educate the community in a participatory manner, engaging those outside of the health care arena is necessary. To prevent the spread of the disease in the United States, reaching the parents of children at the vulnerable age of 9–11 years of age is critical. The barriers to education of parents and children around the spread of a sexually transmitted disease are vast and difficult to overcome. However, the use of proven vaccinations give healthcare providers and community advocates the main tool for prevention of the spread of the disease. It is often taboo to discuss anything related to sexual promiscuity or sexual activity in the United States in the public schools. The biggest myth includes the fear parents and grandparents have is that if HPV is talked about, then the child may become sexually active sooner. This myth needs to be challenged with science and reality including taking on the those vehemently opposed to vaccines, known as "Anti-Vaxers" that obstruct vaccine education. The strategies utilized in public health outreach to the community should be reviewed and uniquely developed for each diverse community to overcome the challenges in the prevention of HPV.

**Keywords:** Human Papilloma Virus, HPV Vaccines, Preventable Cancers, Vaccine Hesitancy, Community Education, HPV Vaccine Rates in the United States

#### **1. Introduction**

Human Papilloma Virus (HPV) is a sexually transmitted disease that is found in multiple organs in both male and female patients. The role of the provider in this case is extremely vital in reducing the spread of the disease and encouraging vaccines for prevention of cancers. HPV is the one virus that has over 200 variants, which has an effective vaccine regimen available and provides coverage to eliminate the risk for fatal cancers [1]. Thus, it would seem to be an obvious step for children ages 9–11 years of age and those 11–45 years of age to receive the vaccine, yet that is not the case [2]. Less than 30% of children actually receive the vaccine and often, the second dose is avoided.

This vaccine hesitancy is a global issue in that some children live in poverty and do not have access to this preventable cancer vaccine and in addition, those children with high socio-economic statues refusing to be vaccinated at the advice of their parents. With regard to those 11–45 years of age, often this age group has not been

fully educated about the concern over HPV infection and have concerns that they may get the disease.

Lastly, parental and grandparent vaccine hesitancy is due in part to lack of knowledge and concerns that the child may become promiscuous when vaccinated. Dispelling all of the myths surrounding HPV and the vaccine makes it difficult at best for providers to do due diligence in educating and preventing HPV. Which is quite distressing when over 13,00 cases of cervical cancers are diagnosed yearly in the United States (U.S.) and thousands of fatal cancers could be prevented [3].

This chapter outlines and provides the background including the incidence, prevalence, etiology, pathology, and health promotion measures of HPV. To appreciate HPV an understanding of regular screening and clinical practice guidelines are presented. The importance of health promotion and prevention is outlined. The goal of this chapter is to review the management and care to prevent HPV and subsequent complications, as well as present HPV vaccine rates, HPV vaccine hesitancy, and strategies to provide prevention at the community level.

#### **1.1 Prevalence and incidence of HPV**

As the fourth most common cancer, cervical cancer (CC) is a global health issue [4]. It is estimated that 75% of women will contract a HPV infection during their lifetime [5]. The annual incidence rate in the United States is 14 million [6]. A notable variability in the incidence rates of HPV exist worldwide. So, to are the disparities in detection and death of HPV. Tanzania reports 10,000 cases with 7000 death per year [4]. In Korean women, CC ranks as the seventh most common cancer [7]. As a result, the pathological manifestations of HPV may occur in the genital region and oral cavity [5]. Uterine cervical cancer incidence is approximately 600,000 cases per year. In oral pathogenesis, HPV 16 is likely to the primary cause accounting for 90% of malignant neoplasms [5].

According to the Centers for Disease Control (CDC), 40 out of the over 200 types of HPV can infect the genital region [8]. Despite being self-limited, asymptomatic, or unrecognized, sexual activity persons are likely to have become infected at least once. The most common types of oncogenic HPV, specifically 16 and 18, are responsible for the development of cervical, vulvar, vaginal, penile, anal, and oral pharyngeal cancer. Lower risk HPV type 6 and 11 are associated with respiratory papillomatosis and genital warts [8].

The prevalence of oral HPV in the United States between 2011 and 2014 was 7.3% in adults aged 18–69. During the same time frame, non-Hispanics saw a 2.9% rate and non- Hispanic black adults was 9.7% in comparison to 7.3% in non-Hispanic whites and 7% in Hispanic adults. Low prevalence rates occurred among non- Hispanic Asian women with no significance differences noted in non-Hispanic white, non-Hispanic black and Hispanic women. Overall, oral HPV was highest in men within each race and Hispanic group [9].

#### **1.2 Most vulnerable populations that acquire HPV**

According to the World Health Organization, greater than 85% of the 300,000 reported deaths from CC occur in countries with low to middle income. These rates are largely due to vaccination programs and screening practice in countries with higher income earnings [10]. Efforts to reach vulnerable populations are now distempered by the ongoing global pandemic due to the novel coronavirus (COVID-19), delaying and disrupting routine immunizations impinging accessibility, furthering inequalities of health care potentiating healthcare consequences globally [11].

*Preventing Human Papilloma Virus through Community Education and Vaccination DOI: http://dx.doi.org/10.5772/intechopen.98350*

#### **2. The historical perspective of HPV**

Nearly 528,000 women are diagnosed with cervical cancer each year with nearly half of them deceased [12]. The authors suggest that nearly 80% of the cases are found in third world countries are lacking the resources to battle this number of deaths. The evolution of (HPV) in the world is multifactorial leaving women in vulnerable populations more likely to contract one of the over 200 types most often through sexual transmission. The higher risk for women is due in part to the factors that lead to spread of HPV infection. Namely, early age at marriage, intercourse, pregnancy, and use of hormonal contraceptives [12]. Nearly 75% of adults that are sexually active have the disease without symptoms.

#### **2.1 The evolution of HPV and types**

There are over 200 types of HPV with 14 strains considered to be associated with cervical cancer [13]. Nearly 80 million people in the United States have been infected with most clearing the infection without incident [14]. In addition, these authors suggest that there has been a surge in oropharyngeal cancers related to HPV infection with the more notorious strains as the culprit [14].

#### **2.2 Sites of origin of HPV**

HPV is drawn to squamous epithelial cells and these are often found on mucosa such as skin and moist areas. HPV is most commonly found in the female cervix, however other sites of infection include those organs with similar tissue qualities such as the oropharynx, tonsillar tissue, soft palate, penis, and anus as examples. HPV tends to also live in the vagina, nose, nasopharynx, trachea, bronchi, and inner eyelid [15]. Frequently, HPV lives on the skin in the form of warts, however these growths can appear inside the organs described. Prevention of spread of anogenital warts is critical to decreasing the progression of cancer in males and females alike [16].

#### **2.3 The diagnosis of HPV**

The diagnosis of HPV is completed by performing a Papanicolaou (Pap) screening to identify infected cervical tissue [17]. This screening method first utilized in 1950's remains a gold standard for diagnosis. Cervical cancer remains the 4th most common cause of cancer in women globally [18]. The American Cancer Society (ACS) recommends that cervical cancer screening should begin at age 25 for women with an HPV test every five years using the U.S. Food and Drug Administration (FDA) tests only [15]. Those with higher risks such as immunocompromise include patients with HIV infection, organ transplant, or long term use of steroids [15]. If the patient has had a total hysterectomy with cervix removal and are cancer free are exempt from this testing.

#### **2.4 Risks for preventable cancers**

Cervical cancer evolves in four major steps, which include infection, persistence, progression, and invasion [18]. When the patient presents to clinic with oropharyngeal growths on the tongue or soft palate, a biopsy of the site should be performed and a follow up appointment should be scheduled once the diagnosis is confirmed. Most of the 230 genotypes of HPV cause no harm and resolve asymptomatically without the patient noticing with 40 genotypes known to be high risk [19]. Genital

warts are tumors found caused by HPV, are the most common sexually transmitted disease, and generally are benign [19]. Risk factors for genital warts include: number of partners, barrier contraception use, young age at first encounter, circumcision, and male sexual behavior [19]. This high rate of transmission results in infection of multiple partners including females.

However, the clinician should be aware that correlating lymph node enlargement may require further biopsy to assure that squamous cell carcinoma is not in the tissue or lymph nodes. While most head and neck cancers are caused by squamous cell carcinoma and found historically in smoker, drinker, males >50 years of age, more recently it has been found in women in the oropharynx. This is occurring more often in the last decade as teens and young women engage in oral sex as a means to prevent pregnancy. In addition, males having sex with males are in a high risk category for this type of cancer due to multiple partners, as well as high risk for genital warts and anogenital cancer [19]. Recently, an anal Pap smear was created to help diagnose HPV in males having sex with multiple male partners. Recently, HPV is a culprit linked to urothelial bladder cancer as well [20].

#### **3. Current treatments for HPV**

A quadrivalent vaccine for HPV was first recommended by a sub-committee of the (CDC) in the United States known as the Advisory Committee on Immunization Practices (ACIP) in 2006 [21]. In 2009, a bivalent was available and in 2015, nonvalent HPV vaccine was created and is the mainstay in HPV vaccination today. To date, the vaccine rates among girls ages 13–17 years of age remains quite low at nearly 42%, while boys in the same age group are worse at a rate of 28% [21]. Pediatricians and mid-level providers spend an inordinate amount of time working to educate families regarding this cancer prevention vaccine, yet compliance continues to be dismal. Adults should be vaccinated up until 45 years of age.

#### **3.1 The target age group**

The WHO Director in 2018, committed to eliminating cervical cancer with a significant goal of a 90% immunization rate in girls 15 years of age by 2030 [10]. Additionally, the Healthy People 2025 national goals in the U.S. continue to advocate for improved vaccine completion numbers. Approximately 70% of the global target population includes adolescent girls, ages 9–14, living in geographic regions without an immunization program for HPV prevention [21]. Adults less than 45 years of age are encouraged to take the vaccine.

#### **3.2 Vaccine hesitancy**

According to the WHO, if 70% vaccination coverage is achieved in low and middle income countries, approximate 4 million deaths could be prevented [10]. However, despite national recommendations, vaccine hesitancy often ensues [21]. In 2010 an estimated 14% of teenage girls in the USA completed and received all 3 doses, noting parental hesitancy as the primary reason for lack of follow through. One concern being the public policy mandate resulting in suspicion on the part of the parents. Parental opposition stems primarily from compulsory vaccination of their children citing trust and safety for the reluctance to pursue or complete the protocol. Several states in the U.S. require the HPV series to attend school, however, religious exemptions abound and this has not significantly increased the vaccination rate [22].

*Preventing Human Papilloma Virus through Community Education and Vaccination DOI: http://dx.doi.org/10.5772/intechopen.98350*

Knowledge served as a basis for hesitancy to vaccinate. However, parents with the higher levels of education tend to research the topic and decided against vaccination. Those with lower education levels tended to base decisions to vaccinate due to provide recommendation or encouragement opposed pursing knowledge or information regarding disease prevention with vaccination adherence [22]. Patient navigators to increase the vaccination rates and found that white patients are less likely to initiate the HPV vaccine than other ethnicities, however once initiated they were more likely to finish the series [22]. Rural parents were much less likely to encourage HPV vaccines as compared to their urban counterparts [13]. Women in the U.S. in general are basically poorly informed about HPV overall and found therefore, that provider education should target Non-Hispanic Blacks, lower level educated women, and those younger than 65 years of age. Beliefs about the efficacy of the vaccine to prevent cervical cancer remain a barrier to increased vaccine rates overall [22].

Parental attitudes toward vaccination of an STI factors into the decision-making process regarding vaccination prior to FDA approval. Several studies conducted demonstrated favorable attitudes in the USA and UK and accepting vaccination of their children. Specifically noting a mother's sexual values were secondary compared to overall vaccination attitude. In addition, social aversion to vaccination was not seen across various religions groups [23].

#### **4. Community outreach**

Cancer screening efforts are an ongoing effort to take the screening tools out to the community [3]. Educational outreach to vulnerable communities is an incredibly important method of reaching underserved groups. Often, vulnerable and underserved people are fearful of government institutions such as schools, social security, hospitals, and the police. The marginalized people in society have frequently been unsuccessful in navigating the system to obtain access to critical resources. In the case of Immigrants, the concern surrounds deportation or criminal charges. To overcome these barriers to educating and serving the public, healthcare providers are placed in the position of being more creative in how outreach is managed.

#### **4.1 American Cancer Society (ACS) efforts**

The American Cancer Society (ACS) has for decades provided funding, outreach, and resources to all patients in the U.S., as well as their regions and individual chapters. In the last decade, ACS has worked specifically to increase the knowledge base of parents, grandparents, and youth regarding the importance of cancer prevention, specifically for HPV. U.S. national immunization coalitions have devoted large amounts of time, effort, and funding to provide outreach for the public in an effort to prevent HPV. In 2014, a documentary about HPV and cervical cancer was produced in Hollywood called "Someone You Love: The HPV Epidemic." This documentary was shared at a conference in 2016 in Indianapolis, Indiana in the U.S. for a national meeting of all U.S. immunization coalitions. Soon, it spread throughout the U.S. as a tool for HPV prevention for youth and parents. It's real life powerful true stories of women that suffered and some died from cervical cancer caused by HPV. Other efforts in Maui, Hawai'i include having young cancer survivors assist in Relay for Life and ACS outreach events to speak candidly to youth about what HPV is and how disenchanting cancer treatment is along with the burden of fearing that the cancer may return.

Once the youth are aware of what the road for a cancer patient is like, they may not realize that there is one cancer that is preventable, and that is by vaccination against HPV.

#### **5. Conclusion**

Human Papilloma Virus (HPV) is the most common sexually transmitted disease that is found in in both male and female patients. The role of the provider in this case is extremely vital in reducing the spread of the disease and encouraging vaccines for prevention of cancers. HPV is the one virus that has over 200 variants, which has an effective vaccine regimen available and provides coverage to eliminate the risk for fatal cancers [1]. Thus, it would seem to be an obvious step for children ages 9–11 years of age and those 11–45 years of age to receive the vaccine, yet that is not the case [2]. Less than 30% of children actually receive the vaccine and often, the second dose is avoided.

Strategic elements to assist in global vaccination efforts include financial investment on a global level, enhancement of supply, single dose schedules, and effective social marketing [24]. Use of social media platforms to increase awareness of the notion that a vaccine preventable cancer such as cervical cancer may be the wave of the future. Without global concerted efforts to increase the vaccination rate to achieve herd immunity, the fight against HPV infection and the subsequent needless suffering and death will continue to occur.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Celeste Mulry Baldwin\* and Lisa Rinke DNP Online Graduate Program, Young School of Nursing, Regis College, Weston, MA, USA

\*Address all correspondence to: celeste.baldwin@regiscollege.edu

© 2021 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.

*Preventing Human Papilloma Virus through Community Education and Vaccination DOI: http://dx.doi.org/10.5772/intechopen.98350*

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[12] Degarege A, Krupp K, Fennie K, Li T, Stephens DP, Marlow LAV, Srinivas V, Arun A, Madhivanan P. Urban-Rural Inequities in the Parental Attitudes and Beliefs Towards Human Papillomavirus Infection, Cervical Cancer, and Human Papillomavirus Vaccine in Mysore, India. J Pediatr Adolesc Gynecol. 2018 Oct;31(5):494- 502. doi: 10.1016/j.jpag.2018.03.008. Epub 2018 Mar 26. PMID: 29596907; PMCID: PMC6119521.

[13] Chrysostomou, A. C., & Kostrikis, L. G. (2020). Methodologies of Primary HPV Testing Currently Applied for Cervical Cancer Screening. *Life (Basel, Switzerland)*, *10*(11), 290. https://doi. org/10.3390/life10110290

[14] Boakye, E., Tobo, B. B., Rojek, R., Mohammed, K.A., Geneus, C.J., & Osazuwa-Peters, N. (2017) Approaching a decade since HPV vaccine licensure: Racial and gender disparities in knowledge and awareness of HPV and HPV vaccine, Human Vaccines & Immunotherapeutics, 13:11, 2713-2722, DOI: 10.1080/21645515. 2017.1363133

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[16] Skoulakis A, Fountas S, Mantzana-Peteinelli M, Pantelidi K, Petinaki E. Prevalence of human papillomavirus and subtype distribution in male partners of women with cervical intraepithelial neoplasia (CIN): a systematic review. BMC Infect Dis. 2019 Feb 26;19(1):192. doi: 10.1186/s12879- 019-3805-x. PMID: 30808285; PMCID: PMC6390310.

[17] Hirth, J. (2019) Disparities in HPV vaccination rates and HPV prevalence in the United States: a review of the literature, Human Vaccines & Immunotherapeutics, 15:1, 146-155, DOI: 10.1080/21645515.2018.1512453

[18] Yamaguchi, Sekine, Hanley, Kudo, Hara, Adachi, Ueda, Miyagi, & Enomoto. Risk factors for HPV infection and high-grade cervical disease in sexually active Japanese women.

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#### **Chapter 6**

## Cervical Cancer Prevention and Control

*Tariku Laelago Ersado*

#### **Abstract**

Cervical cancer is caused by HPV (human papilloma virus). It is the second most common cancer in women living low developed countries. The components of cervical cancer prevention and control comprises primary prevention, secondary prevention and tertiary prevention. Primary prevention of cervical cancer encompasses prevention of infection with HPV. Giving HPV vaccine for girls aged 9–14 years before they initiate sexual activity is one of the interventions of primary prevention of cervical cancer. Screening and treatment is needed in secondary prevention of cervical cancer. Screening of cervical cancer encompasses testing a target group (women) who are at risk for a cervical pre-cancer. Tertiary prevention of cervical cancer comprises treatment of cervical cancer and palliative care. The components of tertiary care comprise surgery, radiotherapy, chemotherapy and palliative care. Community mobilization, health education and counseling on cervical cancer prevention and control is vital to make ownership on cervical prevention. Monitoring and evaluation of cervical cancer prevention and control on key program indicators should be done regularly.

**Keywords:** cervical cancer, primary prevention, secondary prevention, tertiary prevention, control, vaccination

#### **1. Introduction**

Cervical cancer is caused by sexually acquired infection with certain types of HPV (human papilloma virus). HPV is a group of viruses that are extremely common worldwide. There are more than 100 types of HPV, of which at least 14 are cancer-causing [1]. Worldwide, cervical cancer is the fourth most frequent cancer in women. There were 570 000 new cases of cervical in 2018. More than 311 000 deaths from cervical cancer occur every year. More than 85% of these deaths occur in low and middle income countries. Seventy-percent of cervical cancers worldwide are caused by only two HPV types (16 and 18) [1, 2].

Abnormal vaginal bleeding is the common symptom of cervical cancer. The bleeding can occur after sexual intercourse. Bleeding after menopause or increased vaginal discharge may also be symptoms [3].

There are numerous risk factors the can cause cervical cancer. Educational status, place of residence, using old sanitary napkins, younger age at marriage, sexual transmitted infections, number of partners and health service utilization are associated with cervical cancer. Bathing daily and during menstruations is found to be preventive factors for cervical cancer [4, 5]. Women who have HIV infection

have an increased risk for cervical cancer than women who have no HIV infection [3, 6]. Non access to cervical cancer screening, commence sexual intercourse at early age, cigarette smoking and long term use of oral contraceptives are also related with higher risk of cervical cancer [3, 5]. History of genital warts, immunosuppression, multiparty, diet low in folates, carotene and vitamin C are also included in risk factors of cervical cancer [7].

The component of cervical cancer prevention and control comprise primary, secondary and tertiary prevention. Cervical cancer can often be prevented by having regular screenings with pap tests and HPV tests to find any pre-cancers and treat them. It can also be prevented by receiving the HPV vaccine [8]. World health organization (WHO) recommended vaccine that can protect HPV 16 and 18 and the vaccine is approved for use in many countries [9]. Avoiding exposure to risk factors is additional actions to prevent cervical cancer [8].

WHO put new cervical elimination targets of 90% HPV vaccination coverage, 70% screening coverage, 90% access to treatment for cervical pre-cancer and cancer and access to palliative care by 2030. Attaining these targets can decrease more than 40% of new cervical cancer cases and 5 million associated mortality by 2050. To achieve this targets efforts should be increased [6]. Availing updated evidence based information on cervical cancer prevention and control is important to increase the information coverage and to develop best strategies that focus on cervical cancer prevention and control. The aim of this chapter is providing the best available information on cervical cancer prevention and control. The chapter described three components of cervical cancer prevention, community mobilization, education and counseling on cervical cancer prevention and monitoring and evaluating cervical cancer prevention and control.

#### **2. Prevention and control of cervical cancer**

The goal of any comprehensive cervical cancer prevention and control programme is to decrease the burden of cervical cancer. This can be done by reducing HPV infections, detecting and treating cervical, pre-cancer lesions, and providing timely treatment and palliative care for invasive cancer [9].

The key components of comprehensive cervical cancer prevention and control contains three interdependent components: primary, secondary and tertiary prevention (**Figure 1**). In **Figure 1**, programmatic interventions to prevent HPV infections and cervical cancer is also illustrated.

Even though, effective cervical cancer methods such as HPV vaccination, screening and safe sex practice exists, affordability and putting into practice remain challenge for most countries [5].

Unless cervical cancer prevention and control measures are effectively executed, it is estimated that by 2030, nearly 800,000 new cases of cervical cancer will be annually diagnosed. The huge majority of these cases will be in developing countries [10]. To reduce this burden, community mobilization, education and counseling on cervical cancer prevention should be implemented at all levels. Monitoring and evaluation of cervical cancer prevention and control on key program indicators should also be done on a regular basis.

#### **2.1 Primary prevention of cervical cancer**

Prevention of HPV infection is included in primary cervical prevention and control. There are different subtypes of HPV that can cause cervical cancer but, the major subtypes are 16 and 18 [9].

#### **Figure 1.**

*Programmatic interventions to prevent HPV infections and cervical cancer.*

The public health goal of primary prevention of cervical cancer is to reduce HPV infections. Primary prevention can be realized through behavioral change approaches and the use of biological mechanisms, including HPV vaccination. The interventions for primary prevention of cervical cancer include: providing immunization for girls aged 9–14 years before the start sexual intercourse, health education on healthy sexuality for both boys and girls and promotion of condom use. HPV vaccines are not intended to treat women with past or present HPV infection [9, 11].

The target age group for HPV vaccination is 9–14 years earlier to becoming sexual active. Two doses of HPV vaccine with six month interval is required. There is no maximum interval between the two doses. But, the interval of not greater than 12–15 months is suggested to allow girls to complete the schedule on time prior to becoming sexually active. If the interval between doses is shorter than 5 months, then a third dose should be offered at least six months after the first dose. A three dose schedule (at 0, 1–2, and 6 months) is recommended for females 15 years and older and for those known to be immunocompromised and/or HIV-infected [10, 12, 13].

It is not essential to screen for HPV infection or HIV infection prior to HPV immunization. Pre-immunization assessments (e.g., HPV testing of any kind, cervical cancer screening or Pap testing, pregnancy testing, or "virginity testing") are not mandatory [10, 13].

If girls are age ≥ 15 years and received their first dose before age 15 years, they may complete the three doses. If no doses were taken before age 15 years, three doses should be administered. In both scenarios, immunization can be given up to 26 years. If adequate resources remain after immunizing girl's age 9 to 14 years, girls who received one dose may take extra doses between age 15 and 26 years. If there is ≥50% coverage in the priority female target population, sufficient resources and cost effectiveness, boys may be immunized to prevent other non-cervical human papillomavirus related cancers and diseases [12].

The HPV vaccines prevent over 95% of HPV infections caused by HPV types 16 and 18. It may have some cross-protection against other less common HPV types which cause cervical cancer [14]. There are three various vaccines, which vary in the number of HPV types they comprise and target. However, not all are obtainable in everywhere.


Cervarix is the best cost effective vaccine with proved efficacy in one dose. The WHO commends two doses for either Gardasil 9 or Cervarix for those up to 15 years of age and three doses for women 15 years or older. The WHO commends are grounded on induced antibody titers at month 7 for Gardasil and Gardasil 9 as there are at present no efficacy data for these vaccines in fewer than three doses. The WHO recommendations for Cervarix are built on efficacy information in addition to immunogenicity information. Three dose efficacy prevents cervical intraepithelial neoplasia (CIN) 2 or worse by any HPV type is around 62% for both Cervarix and Gardsail9. The three dose efficacy prevents CIN 3 or worse by any HPV type is 93% for Cervarix and 43% for Gardasil, with no information for ardasil9 [10, 15] (**Table 1**).

There are numerous HPV vaccination distribution approaches. The followings are commonly used distribution approaches:


Educational interventions announcing the risk of HPV and the benefits of vaccines are important, especially in low and middle income countries [17]. Education and effective communication is vital in attaining successful immunization programme [18].

#### **2.2 Secondary prevention of cervical cancer**

In secondary prevention of cervical cancer, screening and treatment as desired is included. Screening comprises testing women who are at risk for a cervical precancer. The aim of screening is to detect and treat those people identified as having early signs of the illness, usually by means of inexpensive, precise, and reliable test that can be practical widely. The other aim of screening is to decrease the death related with cervical cancer through identifying the illness when still at an early treatable stage or through detecting precursor lesions. The systematic removal of CIN lesion during screening also leads to reductions of the incidence of invasive cervical cancers of all stages.

There are numerous cervical cancer screening tests in use or being studied around the world. Cervical cytology has been in use for the past 50 years. Newer screening tests are HPV DNA testing and visual screening tests [19]. Increasing the acceptance of screening has many sigfinaces in preventing cervical cancer through early detection and treatment of pre-cancerous changes before malignancy grows. Approaches of inspiring women to start cervical cancer screening include inviting, reminding, teaching, communication framing, counseling, risk factor identification and financial interventions. Use of invitations and to a lesser degree educational resource are supported by evidence as a good methods of encouraging women to undertaken cervical cancer screening [20].


#### **Table 1.**

*Characteristics of HPV vaccines.*

Screening of cervical cancer is identifying for pre-cancer. Cervical cancer screening is recommended for woman aged 30 up to 49 years at least one in life time. Early detection and treatment of precancerous lesions can prevent the majority of cervical cancers.

HPV vaccination does not substitute cervical cancer screening. In countries where HPV vaccine is introduced, screening programs may need to be developed or strengthened [21]. Visual inspection of the cervix without magnification was the first technique of screening of the cervix. Nowadays, three types of tests are encouraged:


The randomized trial studies done in different places on cervical cancer screening have shown the efficacy of visual inspection, cytology screening and HPV screening [22–24]. Many studies have acknowledged that in countries where the resources exist to confirm high value and good coverage of the people, cytology screening provides to decreasing the incidence of advanced stage cancers and death related with cervical cancer [25–27].

The treatment methods mostly used are cryotherapy, loop electrosurgical excision procedure or cold-knife conisation [5].

There are two kinds of HPV tests:


HIV infected women should undergo cervical cancer screening twice in the first year after diagnosis of HIV infection and then annually. For women with two successive normal cytological examinations, the recommendation is that annual follow up includes a detailed visual inspection of the anus, vulva, and vagina, as well as the cervix [5].

Cervical screening based on HPV testing can prevents more invasive cervical cancer and precancerous lesions. It can offers innovative options such as selfcollection of specimens to improve screening uptake broadly [28].

#### **2.3 Tertiary prevention of cervical cancer**

Tertiary prevention of cervical cancer comprises treatment of cervical cancer and palliative care. Surgical treatment, chemotherapy, radiotherapy and palliative are included in tertiary cervical cancer prevention [16]. The public health goal of tertiary prevention of cervical cancer is to reduce the number of mortality due to cervical cancer.

The interventions for tertiary prevention of cervical cancer comprise:


#### **2.4 Community mobilization, education and counseling on cervical cancer prevention**

Community mobilization is a process of engaging communities and generating support for all those in need of health services, resulting in sustainable community ownership and involvement. Effective communication can increase rates of vaccination

#### *Cervical Cancer Prevention and Control DOI: http://dx.doi.org/10.5772/intechopen.99620*

and screening and save women's lives. Health care workers and others involved in cervical cancer control at all levels should be trained in basic counseling skills, so that they can communicate effectively with clients. The content of the counseling encounter will vary according to the client's problems or concerns and her individual situations. It can address prevention, screening, follow-up, referral, diagnosis, treatment of precancerous lesions, treatment of invasive cancer and/or palliative care [9].

#### **2.5 Monitoring and evaluating (M & E) cervical cancer prevention and control**

Monitoring and assessing the improvement of objectives and targets at country level is crucial. The followings are crucial indicators of cervical cancer preventions and control:


Essential impact indicators of cervical cancer are incidence and death. Establishing cancer register is important to monitor the incidence and death rate of cervical cancer. The register will help to assess long term impacts of cervical cancer screening, treatment and vaccination [21]. The main recording and reporting tools that are used for immunization should be adapted to include HPV vaccine. The recording and reporting tools comprises: immunization register, tally sheet, immunization card, defaulter tracking system, stock record and integrated monthly report [10].

M & E helps the management team to determine the extent to which the program is meeting the stated goals, objectives, targets and make corrections accordingly [16].

An effective program of prevention and control of cervical cancer must address several issues, including the coverage and quality of screening services, availability of diagnosis, treatment and monitoring [29].

Depending on the country setting and resources available, M & E of cervical cancer prevention can be done by using different approaches.

The approaches includes:


#### **3. Conclusion**

Cervical cancer prevention and control components are primary prevention, secondary prevention and tertiary prevention. Primary prevention comprise HPV vaccination of girls 9–14 years old. Secondary prevention include screening and treatment with low technology VIA followed by cryotherapy. Tertiary prevention of cervical cancer incorporates treatment of invasive cancer and providing palliative care. Mobilizing community, giving health education and counseling is very important in prevention and control of cervical cancer. M & E of cervical cancer prevention and control on key program indicators should also be done regularly.

#### **4. Terminology**

**Bivalent:** a vaccine that works by stimulating an immune response against two different antigens; e.g. Cervarix is a bivalent vaccine that helps protect the body against infection with HPV types 16 and 18.

**Chemotherapy**: The term that usually describes the use of drugs to treat cancer but which may also describe the use of antibiotics to treat infectious diseases.

**Cervical intraepithelial neoplasia (CIN**): abnormalities in the cells of the cervix which may become cancerous.

**Cryotherapy:** the use of cold or freezing in treatment.

**Cytology**: the study of individual cells. Cytology's main use in medicine is to detect abnormal cells. It is widely used to screen for cancer (as in the cervical smear test) or to confirm a diagnosis of cancer.

**DNA** (**deoxyribonucleic acid**): the principal molecule carrying genetic information in almost all organisms.

**Immunogenicity**: the property of eliciting an immune response.

**Neoplasia**: the pathological process that results in the formation and growth of a tumor.

**Palliative treatment**: treatment that relieves the symptoms of a disorder but does not cure it.

**Opioid**: a type of drug used to relieve strong pain, e.g. morphine.

**Quadrivalent**: a vaccine that works by stimulating an immune response against four different antigens; e.g. Gardasil is a quadrivalent vaccine that helps protect the body against infection with HPV types 6, 11, 16 and 18.

**Screening**: The application of a test to people who are as yet asymptomatic for the purpose of classifying them with respect to their likelihood of having a particular disease. It is not undertaken to diagnose a disease, but to identify individuals with increased probability of having either the disease itself or a precursor of the disease.

**Prognosis:** An assessment of the probable course and outcome of a disease.

*Cervical Cancer Prevention and Control DOI: http://dx.doi.org/10.5772/intechopen.99620*

### **Author details**

Tariku Laelago Ersado Department of Nursing, Wachemo University Durame Campus, Durame, Ethiopia

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

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

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[2] WHO. human papillomavirus and cervical cance. Available from: https:// www.who.int/news-rooms/fact-sheets/ detail/human-papillomavirus-(hpv) and-cervical-cancer WHO; 2019.

[3] American cancer society. Cancer Facts & Figures. American cancer society; 2015.

[4] Kashyap N, Krishnan N, Kaur S, Ghai S. Risk factors of cervical cancer: a case control study. Asia-Pacifc Journal of Oncology Nursing. 2019;6(3):7.

[5] Ngoma M, Autier P. Cancer prevention: cervical cancer. ecancer. 2019;13(952):6.

[6] WHO. WHO releases new estimates of the global burden of cervical cancer associated with HIV. 2020.

[7] Aggarwal P. Cervical cancer: Can it be prevented? World Journal of Clinical Oncology. 2014;5(4):7.

[8] cervical cancer: screening and prevention. http://www.cancer.net. types/cervical-cancer/screening-andprevention: Cancer. Net; 2020.

[9] WHO. Comprehensive Cervical Cancer Control: A guide to essential practice. Geneva, Switzerland: WHO; 2014.

[10] WHO. Guide to INTRODUCING HPV VACCINE INTO NATIONAL IMMUNIZATION PROGRAMMES. In: Immunization VaB, editor. Geneva: Expanded Programme on Immunization (EPI) 2016. p. 104.

[11] P.Reddi Rani, Reddy KS. Primary Prevention of cerivcal Cancer JPOG MAR/APR. 2016:6.

[12] Silvina Arrossi, Temin S, Garland S, Eckert LON, Bhatla N, † XC, et al. Primary Prevention of Cervical Cancer: American Society of Clinical Oncology Resource-Stratified Guideline. JGO – Journal of Global Oncology. 2017;3(5):24.

[13] Meites E, Gee J, Unger E, Markowitz L. Human Papillomaviruses.

[14] Arbyn M, Xu L, Simoens C, PPL M-H. Prophylactic vaccination against human papillomaviruses to prevent cervical cancer and its precursors Cochrane Database Syst Rev 2018.

[15] Habadi MI, Aljohani HHN, Altayeb MA, Alwosibai HA, Allehyani MAS, Almozain MAM, et al. HUMAN PAPILLOMA VACCINE OVERVIEW. INDO AMERICAN JOURNAL OF PHARMACEUTICAL SCIENCES. 2019;6(2):7.

[16] FMOH. Guideline for Cervical Cancer Prevention and Control in Ethiopia. Adis Ababa: FMOH; 2015. p. 62.

[17] Cheng L, YanWang, Du J. Human Papillomavirus Vaccines: An Updated Review. vaccines. 2020;8(391):15.

[18] Bello F, Enabor O, Adewole I. Human Papilloma Virus Vaccination for Control of Cervical Cancer: A Challenge for Developing Countries. African Journal of Reproductive Health. 2011;15(1):6.

[19] Alliance for Cervical Cancer Prevention. Planning and Implementing Cervical Cancer Prevention and Control Programs: A MANUAL FOR MANAGERS. Alliance for Cervical Cancer Prevention; 2004.

[20] Everett T, Bryant A, Griffin MF, Martin-Hirsch PPL, Forbes CA, RG J. Interventions targeted at women to

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encourage the uptake of cervical screening (Review). The Cochrane Collaboration. 2011(5):98.

[21] WHO. WHO GUIDANCE NOTE: Comprehensive cervical cancer prevention and control: a healthier future for girls and women. Switzerland: WHO; 2013.

[22] Van der Aa MA, Pukkala E, Coebergh JW, Anttila A, S S. Mass screening programmes and trends in cervical cancer in Finland and the Netherlands Int J Cancer. 2008;122(8):5.

[23] Sigurdsson K, Sigvaldason H. Longitudinal trends in cervical histological lesions (CIN 2-3+): a 25-year overview Acta Obstet Gynecol Scand. 2006;85(3):6.

[24] Tan N, Sharma M, Winer R, Galloway D, Rees H, RV B. Modelestimated effectiveness of single dose 9-valent HPV vaccination for HIVpositive and HIV-negative females in South Africa. Vaccines. 2018;36:7.

[25] Li X, Stander MP, Van Kriekinge G, N D. Cost-effectiveness analysis of human papillomavirus vaccination in South Africa accounting for human immunodeficiency virus prevalence BMC Infect Dis. 2015;15(566).

[26] Sinanovic E, Moodley J, Barone MA, Mall S, Cleary S, J H. The potential cost-effectiveness of adding a human papillomavirus vaccine to the cervical cancer screening programme in South Africa Vaccine. vaccines. 2009;27(44):7.

[27] Tracy JK, Schluterman NH, Greene C, Sow SO, Gaff HD. Planning for human papillomavirus (HPV) vaccination in sub-Saharan Africa: a modeling-based approach vaccines. 2014;32(26):7.

[28] Ogilvie G, Nakisige C, Huh WK, Mehrotra R, Jeronimo ELF. Optimizing secondary prevention of cervical cancer: Recent advances and future challenges. Int J Gynecol Obstet. 2017;138(1):5.

[29] Manuel V-HV. Screening and Prevention of Cervical Cancer in the World. J Gynecol Res Obstet. 2017; 3(3):7.

[30] WHO, Pan African Health Organization. Monitoring national cervical cancer prevention and control programmes: quality control and quality assurance for visual inspection with acetic acid (VIA)-based programmes. Swizerland WHO, Pan African Health Organization; 2013. p. 41.

Section 4

## Treatment and Prognosis

#### **Chapter 7**

## Insights of Cisplatin Resistance in Cervical Cancer: A Decision Making for Cellular Survival

*Elizabeth Mahapatra, Salini Das, Souvick Biswas, Archismaan Ghosh, Debomita Sengupta, Madhumita Roy and Sutapa Mukherjee*

#### **Abstract**

The clinical scenario of acquired cisplatin resistance is considered as a major impediment in cervical cancer treatment. Bulky drug-DNA adducts formed by cisplatin elicits *DNA damage response (DDR)* which either subsequently induces apoptosis in the cervical cancer cells or enables them to adapt with drug assault by invigorating pro-survival molecular cascades. When HPV infected cervical cancer cells encounter cisplatin, a complex molecular interaction between *deregulated tumor suppressors*, *DNA damage-repair enzymes*, and *prosurvival molecules* get initiated. Ambiguous molecular triggers allow cancer cells to cull apoptosis by opting for a survival fate. Overriding of the apoptotic cues by the pro-survival cues renders a *cisplatin resistant phenotype* in the tumor microenvironment. The present review undrapes the impact of deregulated signaling nexus formed due to crosstalk of the key molecules related to cell survival and apoptosis in orchestrating platinum resistance in cervical cancer.

**Keywords:** HPV, Cervical cancer, Cisplatin resistance, tumor suppressors, DNA-damage repair, prosurvival signaling

#### **1. Introduction**

Cervical cancer, one of the widespread gynecological cancers, accounts for the maximum deaths amongst women across the globe. As per GLOBOCAN 2018, cervical cancer is helmed as the fourth leading cause of mortality and morbidity in women after breast and ovarian cancers [1]. As revealed from the data collated by World Health Organization (WHO) in 2013, over 85% of the cervical cancer cases had surfaced mostly from developing countries with a poor socio-economic backdrop [2]. Women, owing to lack of awareness, often arrive for seeking medical help when the malignant growth of cervix has attained advancement [3].

Infections with a special class of oncogenic DNA viruses called *Human Papilloma Viruses (HPVs)*, hailing from the viral family *Papillomaviridae,* are highly accredited for the malignant transformation of cervix. Principally, HPVs are sexually transmitted [4]. On the basis of its carcinogenic potentials, HPVs can be categorized as *–(i) low-risk HPVs(lr-HPVs)* like *HPV 6, 11, 42, 43* and *44*, and *(ii) high-risk* 

*HPVs(hr-HPVs)*like *HPV 16, 18, 31, 33, 34, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68* and *70* [4, 5]. Persistent and prevalent infections with hr-HPVs contribute in development of cervical cancer alongside other cancers such as the cancer of vagina, vulva, penis, anus, head and neck. HPV infections may also give rise to anogenital warts and recurrent respiratory papillomatosis [5, 6]. Besides HPVs, several other risk factors have been implicated in the etiology of cervical cancer. These mainly include long term use of oral contraceptive [7], smoking [8] and infections with *Chlamydia trachomatis* [9].

Cervical cancer progresses through stages of *mild dysplasia***,** *moderate dysplasia* and *severe dysplasia* to finally aggravate into *carcinoma in situ* and *invasive cancer* **stages** [10]. The International Federation of Gynecology and Obstetrics (FIGO) classify these developmental grades of *cervical intraepithelial neoplasia (CIN)* into various stages [11, 12]. Rise in the global disease burden is majorly due to *treatment failure* and *disease recurrence* [13]. The advent of vaccination has allowed for preinfection protection [14]. However, lack of cost-effectiveness has limited its use to only a certain section of the society, particularly in low income countries like India. The therapeutic modality therefore, is skewed to *chemotherapy* and *radiotherapy***.** Stage specific treatment regime is followed for treating cervical cancer [15]. As per FIGO conventions, *Stage IIB-IVA* denotes invasive stages where treatment is ensued in forms of conventional modes of chemotherapy and radiotherapy [11]. Traditionally, chemotherapy involves use of *platinum ligated drugs* like *cisplatin (cisdiamminedichloroplatinum; CDDP)* [16, 17]. Cisplatin in combination with other chemotherapeutics is often employed for treating invasive stages [16]. Patients are often subjected to treatment with cisplatin as a **'***radiosensitizer'* prior to radiotherapeutic intervention in the *Concurrent Chemoradiotherapy (CCRT)* regime [16]. Accordingly, cisplatin is the *'drug of choice'* to oncologists for treating cervical cancer irrespective of its different stages.

In the process of HPV mediated cervical carcinogenesis several molecular changes are incited which remodels the metabolic profiles of the cervix [18]. HPV induced metabolic paradigm shift bestows the cells with therapy evasive properties. Consequentially, neoplastic cells emerge as highly dynamic and evolving entities [19]. On encountering drugs, the rewired signaling cascades of the tumor cells residing in the cervix get triggered. These eventuate in increased metabolism of chemotherapeutics like cisplatin, finally catering in reduced intracellular drug accumulation [20], paving a way for acquired cisplatin resistance. This chapter majorly discusses the mechanisms underlying the acquirement of resistance towards cisplatin as a result of deregulated activities of tumor suppressors, DNA damage repair enzymes and prosurvival molecules, mediated due to HPV infections.

#### **2. HPVs: Integral to etiology of cervical cancer**

HPVs are relatively small non-enveloped viruses with a diameter of 55 nm. It has a double stranded circular DNA genome which is 8 kb long and is enclosed within an *icosahedral capsid* composed of *72 capsomers* [21, 22]. Functionally, the HPV genome is regionalized into-i) a non-coding regulatory region called the *long control region (LCR)* or the *upper regulatory region (URR)*, ii) an early region which houses *E1, E2, E4, E5, E6* and *E7 genes*, and (iii) a late region which is made up of late expressing genes such as *L1* and *L2* [23]. LCR regulates the process of viral DNA replication via controlling the transcription of Open Reading Frames (ORFs). The lately transcribed proteins L1 and L2 are the structural proteins of the viral capsid. The early genes are dictators of viral replication, transcription, assembly and

#### *Insights of Cisplatin Resistance in Cervical Cancer: A Decision Making for Cellular Survival DOI: http://dx.doi.org/10.5772/intechopen.98489*

oncogenesis. Particularly, E6 and E7 are oncogenic and they degrade the cell cycle regulators like p53 and pRb to eventuate in cervical carcinoma [23].

These miniscule infectious agents access the cervical epithelial layer through crevices or microabrasions that generally forms due to mechanical shock or injury. Following entry, HPVs integrate their genome with that of the host to initiate the process of malignant transformation of the cervix (**Figure 1**). The carcinogenesis of cervical epithelium begins with the onset of viral lifecycle which initiates with viral entry into basal cell layer of the epithelium [24]. The basal cell layer of the cervical epidermis enables multiplication and replication of the virus by providing them with a suitable microenvironment. Molecules expressed by the basal cells such as *integrins (*α*6*β*1,* α*6*β*4), heparan sulphate,* and *proteoglycans* are chemoattractants for HPVs [25, 26]. No sooner does the virus enter the basal cells the viral replication starts but owing to poor copy number the duplication of viral DNA becomes non-reproductive. However, as the infection load spreads into the parabasal and intermediate layers, which are majorly comprised of semi-differentiated cells or terminally differentiated keratinocytes, DNA copy number increases and productive viral replication commences [27]. Meanwhile, the process of cervical carcinogenesis gets driven as the virus multiplies and sustains itself in the host system.

#### **Figure 1.**

*Host cell hijacking by HPVs. HPVs enter the cervical epithelium through micro abrasions to finally integrate its genome into the DNA of the basal cells to promote loss of genomic integrity. Carcinogenesis is accompanied by viral multiplication in the cervical epithelium.*

#### **3. Concomitant molecular changes during cervical carcinogenesis upon HPV infection: an escape route to cisplatin therapy**

#### **3.1 Onslaught of HPVs deregulates tumor suppressors**

HPV mediated neoplastic transformation of cervix kick starts with the abridgement of tumor suppressor functions. An array of experimentations conducted in *in vitro* and *in vivo* models have successfully established the immortalizing capacities of E6 and E7 viral gene products; ensued via degradation of cell cycle controllers like p53 and pRb [28, 29]. E6 promotes ubiquitin mediated degradation of p53 in assistance with *E6-associated protein (E6AP),* a homolog for *MDM-2* expressed in cells infected with hr-HPVs [30, 31]. As per reports, E6AP very efficaciously reduces the half-life of p53 in HPV infected cervical carcinoma cells, precisely as *MDM-2,* the conventional p53 inhibitor [32]. The guardian of the genome, p53, controls and coordinates the major genetic players involved in *cell cycle arrest* [33]. On top of this, p53 choreographs *DNA damage repair*, and *apoptotic events* [34]. As the episome formation is successfully accomplished by the virus, *DNA damage response (DDR)* is triggered. Absence of functional p53 allows the cervical cells to skip *G1-arrest* [35]. These functions which are central to cell survival and death get violated in the HPV immortalized cervical cells owing to reduced p53 levels. A higher E6 level is inversely proportional to cellular p53 levels [36]. Contrarily, the oncoprotein E7 binds with hypophosphorylated retinoblastoma protein (pRb); releasing the growth promoter E2F from the Rb-E2F complex. E2F translocate to the nucleus to enable expression of genes that drive the infected cells through S-phase of the cell cycle [37]. Cumulative loss of function of both of these tumor suppressors enables the infected cells to progress through G1 and S phases even with genetic errors. Shortfall of repair processes ultimately paves a way for genomic disintegrity to prevail; mediating neoplastic growth. Recent reports suggest that E6 and E7 intervene into the tumor suppressor activity by recruiting methyl groups on their promoter region [38, 39]. These oncogenic viral proteins also methylate *cyclinA1* promoter and deregulate cell cycle progression to mediate tumorigenesis [40].

#### **3.2 Cisplatin insensitivity: a consequence of HPV driven deregulation of tumor suppressors**

HPV immortalized cervical cancer cells, especially those at the invasive stages, are subjected to treatment with platinum-ligated drugs like cisplatin. Following its cellular entry, cisplatin transforms into a very strong electrophilic species by hydrolytic activation. Such an activated drug generates an electrophilic attack on cellular nucleophiles like DNA and results in formation of bulky drug-DNA adducts which are beyond repair [41]. Inevitably, cancer cells harboring complex cisplatin modified DNA will be arrested in the G1 phase of the cell cycle particularly; due to generation of DDR response and subsequent activation of p53. Generation of cisplatin-DNA adducts activates *Ataxia Telangiectasia Mutated (ATM) vis a vis ATM- and Rad3-related (ATR)* proteins; culminating into phosphorylation at serine 15 residue and stabilization of p53 [42]. ATR along with various other proteins form an axis of ATR/CHK2/p53/p21; which ultimately mediates apoptosis [43–46]. Therefore, p53 functional status is central to mediation of cisplatin cytotoxicity. This was first demonstrated in a study conducted with small-cell lung cancer cells where adenoviral delivery of p53 sensitized them to cisplatin and resulted in apoptosis [47]. A similar study carried out with ovarian cancer cells, reflected cisplatin induced apoptotic death upon adenovirus mediated delivery of p53 [48]. This tumor suppressor takes up multi-modal routes to facilitate cisplatin-induced cell death. Specifically, p53 increases the susceptibility of cancer cells towards cisplatin by degrading *FLIP* 

*Insights of Cisplatin Resistance in Cervical Cancer: A Decision Making for Cellular Survival DOI: http://dx.doi.org/10.5772/intechopen.98489*

*(FLICE-like inhibitory protein)* and by binding with the anti-apoptotic mediator *Bcl-xl* to inactivate its function [49, 50]. It further activates other tumor suppressors like *phosphatase and tensin homolog (PTEN)* to shut down PI3K/Akt pathway [51]. Sometimes, hyperinduction of p53 can disable *AMP-kinase (AMPK)* [52]**;** thereby forcing the cancer cells to succumb to cisplatin cytotoxicity**.**

In HPV infected cells, this entire p53 dictated cell-death inducing pathway is compromised owing to functional absence of the tumor suppressors. E6 mediated prior degradation of p53 in cervical cancer cells, unprecedentedly makes them tolerant to the drug. It has been experimentally demonstrated that p53-Bax signaling axis elicited cisplatin induced apoptosis in cervical cancer cells [53]. Even in multiple clinical studies, patients retaining wild-type p53 have been found to respond better to platinum based chemotherapy [54]. Expression patterns of p53 are predictive of success rate of cisplatin treatment in adeno-carcinoma of the uterine cervix [55]. A very recent report has envisaged the contribution of p53 in restoring cisplatin sensitivity in CDDP resistant cervical cancer cells, particularly during combination treatment with doxorubicin [56].

#### **3.3 HPV mediated impairment of DNA repair machinery: an auto-corrector of cisplatin-DNA adducts in cervical cancer**

Early genes E1 and E2 drive the process of viral replication in the host by acting as an *origin recognition factor (ORF)* and by imparting helicase [57]. Mostly, the viral replication is dependent upon host cell factors, especially those which are involved in DNA damage repair pathways [58]. Not only HPVs, but other viruses like hepatitis C virus (HCV), Epstein–Barr virus (EBV) and human cytomegalovirus (HCMV) concocts the components of DNA damage repair pathway to survive in the host cells [59, 60]. HPV, while attempting to integrate its genome into the host cell's DNA, incurs DNA damage that eventually evokes DDR.

The host cell has various repair pathways working in a well-knitted fashion to clear off irrelevant mistakes that may arise during the process of DNA replication. Some of these include- *base excision repair (BER), nucleotide excision repair (NER)*, *mismatch repair (MMR)*, *homologous recombination (HR)* and *non-homologous end joining (NHEJ)*. This machinery actively functions to correct errors incorporated in DNA during replication while the cell is gradually traversing through different stages of the cell cycle. In instances of assault to DNA architecture hurled as single strands or double strand breaks, recruitment of ATM or ATR proteins occur at the site of damage. Protein complex comprised of *MRN (MRE11-RAD50-NBS1)* and *Tip60* recognizes and recruits these proteins to the site of damage. ATM phosphorylates a series of downstream effectors which includes *CHK2* and the *histone H2A (H2AX)* to begin with the repair process. For correcting double strand breaks, ATM switches over to HR pathway wherein the process of repair is executed by *breast cancer 1/2 (BRCA1, BRCA2), RAD51* and *Partner and Localizer of BRCA2 (PALB2)* molecules [61–63]. In all cases, p53 is found to be the sole dictator of the process as it allows repair to occur by arresting cells with erroneous DNA.

Cervical cancer cells infected with hr-HPVs exhibit an upregulation of *ATM pathway*. Throughout the viral lifecycle, ATM response is constitutively kept activated owing to phosphorylation of its downstream effectors namely CHK2, NBS1 and BRCA1 [62, 63]. Oncogenic early protein E7 along with higher levels of E1 keeps ATM activity always at a hike. E1, the ORF while imparting helicase action, forms pseudoviral replication origins which initiate the process of DDR by stalling replication forks [64, 65]. In high-risk HPV infected cervical cells, the candidates of ATM pathway accumulates in the nucleus [66]. The differentiated and undifferentiated cells of the cervical epithelium packed with viral genomes also exhibit an upregulation of homologous recombination factors [67, 68]. Studies with pharmacological inhibitors

have further delineated that ATM remains activated all throughout the viral life-cycle. It aids in amplification of viral genome within the differentiated squamous cells [69].

In addition to ATM, HPVs also activate ATR pathway. In HPV infected cells considerably higher levels of *ATR-interacting protein* or *ATRIP* and *DNA topoisomerase 2-binding protein 1* or *TopBP1* were noted [70]. Multiple studies have shown that in cells infected with HPVs has remarkably higher levels of total and phosphorylated forms of ATR as well as its downstream effectors, CHK1 [70]. ATM and ATR pathways provide the virus with an access to the host replicative machinery. E7 destroys Rb along with other related tumor suppressors such as p107 and p130, comprising the family of pocket proteins to control the transit of the cells from G1 phase to S-phase. Moreover, the released E2F translocates to the nucleus and lead to translational activation of the responsive gene, some of which are candidates of DNA damage repair pathways [70].

#### **3.4 HPV seized DNA repair machineries of cervical cancer cells encourages acquired Cisplatin resistance**

Upregulated activities of DNA damage repair enzymes empower cervical cancer cells to quickly repair the cisplatin-DNA adducts. Cisplatin generates intrastrand crosslinks in the DNA to primarily activate *nucleotide excision repair (NER) system* [71]. It has been proposed that NER prevents apoptosis in cisplatin treated cells via activation of the members of the ATM pathway followed by its recruitment to the site of damage in the DNA. In cervical cancer cells, already activated ATM, immediately starts chewing away drug-DNA adducts; leading to resistance. Over 20 proteins hailing from the *excision repair cross-complementation group 1 (ERCC1)* partake in this process of clearing away the cisplatin-DNA conjugates [72]. At the 5′ site of the bulky cisplatin-DNA lesions, ERCC1 gets co-recruited with ERCC4 for excising away DNA-adducts [73]. In HCA-1R, a cisplatin resistant cervical cancer cell line, an upregulation of ERCC1 expression is recognized. Poor cisplatin responders with locally advanced cervical squamous cell carcinoma exhibit elevated levels of ERCC1 [74]. ERCC1, therefore, is considered as a prognostic biomarker for assessing the survival rate of patients receiving chemotherapy or CCRT [75, 76]. Another evolutionarily conserved DNA repair pathway is *Mismatch Repair (MMR)* pathway which is highly implicated in cisplatin resistance of cervical cancer cells [77]. Amongst the MMR proteins, *MutS homolog 2 (MSH2)* has been identified as a contributor of cisplatin resistance in cervical cancer cells [78]. *Post-meiotic segregation 2 (PMS2),* another key member of the MMR system is found to be negatively correlated with cervical cisplatin resistance [79–82].

#### **3.5 HPV mediated upregulation of prosurvival signaling cascades: Another contributor of cisplatin resistance in cervical cancer**

*'Abortive infection'* often referred to active HPV infection, induces the genesis of both benign and malignant neoplasms of the cervix [83]. The oncogenic viral early gene products initiates cervical carcinogenesis by interacting with the crucial prosurvival signaling cascades of the host cell [84]. Besides abrogating p53 and pRb functions, HPVs opportunistically modulate four important cellular survival pathways by interacting with their upstream effectors such as *growth factor receptor, notch receptor, Ras* along with *phosphatidylinositol 3-kinase subunit C (PI3KCA)* gene which is second messenger activating Akt kinases [85, 86].

#### *3.5.1 Activation of PI3K/Akt signaling*

PI3K, particularly was found to be amplified and overtly activated in HPVinduced cervical cancers [87, 88]. The activation of MAPK/ERK in turn alters

*Insights of Cisplatin Resistance in Cervical Cancer: A Decision Making for Cellular Survival DOI: http://dx.doi.org/10.5772/intechopen.98489*

transcription of multiple genes that are important for regulation of cell-cycle progression and cell proliferation. Thus, activation of PI3K begets in Akt activation via phosphorylation of the protein in most of the HPV infected cancers. HPV16 E6 activates receptor protein tyrosine kinases (RTKs) viz. epidermal growth factor receptor (EGFR), insulin receptor beta and insulin-like growth factor receptor beta; lying upstream of the PI3K/Akt pathway [89]. Activation of Akt results into a series of changes in downstream targets. Akt, furthermore can phosphorylate E6 to promote its ability to interact with 14–3-3σ, an important protein required for carcinogenic progression [90]. A strong association between HPV and surged c-myc expression has been evidenced [91–93]. Reportedly, interaction between E6 and c-myc activates the enzymatic function of *telomerase* [94, 95]. In a clinical study, thirty nine out of 46 cervical cancer specimens evinced phosphorylation of Akt at serine 473 [96]. Akt activation was obtained in about, forty-eight percent of stage Ib2-IIb cervical cancer patients. HPV infection destabilizes the host genome for which mutations may be incurred in PIK3CA gene. Some mutations may be activator mutations accounting in Akt hyperactivation in cervical as well as many other HPV-induced cancers [95]. Oncogenic mutations and translational amplification of PIK3CA gene, switch on PI3K/Akt signaling invigoratingly; driving HPV mediated tumorigenesis.

#### *3.5.2 Activation of mTOR signaling*

mTOR kinase functions as a cellular rheostat that amalgamates cellular signaling pathways after sensing growth factor, starvation and energy status. Recently, it has been reported that Akt/mTOR activation occurs immediately after exposure to HPV16 pseudovirions [96]. mTOR activation is frequently observed in cervical squamous cell carcinoma, as well as in most HPV positive head and neck squamous cell carcinomas (HNSCC), and oropharyngeal cancers (OPSCC) [93, 97]. HPV oncoproteins E7 and E6 can chronologically activate AKT through pRb binding and subsequently stimulate mTOR in its complex 1 (mTORC1). These upregulated prosurvival signaling molecules lead to a shift in metabolic paradigm of the cancer cells. When subjected to cisplatin treatment, the HPV infected cervical cancer cells start to metabolize the drug faster than usual. This result in rapid drug efflux and eventually lessens intracellular cisplatin levels to orchestrate cisplatin resistance. Therefore, most cisplatin resistant cervical cancer cells are often characterized by the presence of greater levels of cisplatin efflux pumps [98, 99]. Of late, Li et al. showed in their study that in cisplatin resistant cervical cancer cells with upregulated PI3K/Akt pathway, espouses surged levels of *Lysosome-associated protein transmembrane 4*β*-35 (LAPTM4B-35)* which is another cisplatin exporter [100, 101].

#### *3.5.3 Activation of the Wnt pathway*

Nuclear accumulation of β-catenin due to activation of the canonical Wnt/β-Catenin pathway leads to transcriptional activation of a plethora of proliferative genes. This is highly characteristic to HPV16-positive invasive cancers as well as early dysplastic lesions [102, 103]. This phenomenon of nuclear accumulation of β-catenin positively correlates with progression of cervical cancer [104]. Accordingly, β-catenin was found in higher frequencies within the nucleus of cervical cancer cell line SiHa (bearing integrated HPV16) and HeLa (bearing integrated HPV18) [105]. Lichtig et al. proposed that HPV16 E6 could mechanistically activate Wnt/β-catenin pathway in a p53 independent fashion [106]. β-catenin signaling pathway exhibited a regulatory activity over acquired resistance to cisplatin via upregulation of Bcl-xl [107]. Cisplatin resistance got promoted in neoplasia due to shut down of GSK-3β owing to activation of Wnt/β-catenin signaling [108].

#### *3.5.4 Activation of the Notch pathway*

Cellular prosurvival juxtracrine signaling axis involving TGFβ/Notch1 is found to be exhilarated in invasive cervical cancer [109]. As the cervical lesions progressed from intraepithelial lesions III to microinvasive carcinoma, Notch1 translocated from the cytoplasm to the nucleus for ease of function [110]. HPV E6 has been identified as an activator of Notch protein in multiple cervical cancer cell lines [111]. Upregulated activities of notch protein induce stemness in cervical cancer cells, thereby enabling them to evade cisplatin driven cytotoxicity. Inhibition of Notch1was found to revert epithelial to mesenchymal transition (EMT); restoring cisplatin sensitivity [112].

#### *3.5.5 Telomerase activation*

*Viral oncoprotein* E6 escalates human telomerase activity by upregulating its catalytic subunit hTERT or telomerase reverse transcriptase [113, 114]. E6 on being

#### **Figure 2.**

*Concomitant molecular changes induced by HPV contribute in cisplatin resistance in cervical cancer. The viral oncoproteins, particularly E6 and E7 deregulate the crucial molecules involved in cellular metabolism, cell cycle progression, DNA damage repair differentiation, survival, and apoptotic death. These changes provide the cervical cancer cells to evade cytotoxic cell death upon treatment with cisplatin; leading to acquirement of resistance.*

*Insights of Cisplatin Resistance in Cervical Cancer: A Decision Making for Cellular Survival DOI: http://dx.doi.org/10.5772/intechopen.98489*

aided by E6AP binds to the hTERT promoter region to increase its transcriptional activity [112]. NFX1–123, an mRNA interacting protein gets positively regulated by E6, to maintain higher telomerase expression [115]. E6 upon binding to hTERT protein increases telomerase activity via posttranscriptional modifications [116]. Telomerase reverse transcriptase was reported to promote cisplatin resistance by suppressing apoptosis.

Orchestration of HPV induced signaling nexus in promoting cisplatin resistance in cervical cancer is well depicted in **Figure 2**.

#### **4. Perspective insights**

As the virus hijacks the host system, it flips the molecular dynamics according to its own survival benefit. As discussed in this review, loss of function of tumor suppressors, magnified activities of DNA repair enzymes and constitutional activation prosurvival signaling cascades in the HPV infected cervix, make the situation precarious. The conundrum of drug resistance that arises as a result of these existent changes, stymies therapy. Tracking these prior change can aid in planning conventional therapeutic regimes. Thus, these molecules can act as valuable prognostic biomarker before administration of cisplatin based chemotherapy to cervical cancer patients.

#### **Acknowledgements**

The authors are indebted to the Director, Chittaranjan National Cancer Institute, Kolkata for encouraging the idea of the manuscript. The authors are grateful to Ministry of Health & Family Welfare, Govt of India for financial support to undertake the research.

#### **Conflict of interest**

Authors declare no conflict of interest.

#### **Author details**

Elizabeth Mahapatra, Salini Das, Souvick Biswas, Archismaan Ghosh, Debomita Sengupta, Madhumita Roy and Sutapa Mukherjee\* Department of Environmental Carcinogenesis and Toxicology, Chittaranjan National Cancer Institute, Kolkata, India

\*Address all correspondence to: sutapa\_c\_in@yahoo.com

© 2021 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

## HPV and Carcinogenesis

#### **Chapter 8**

## Human Papillomavirus and Cervical Cancer

*Saliha Sağnıç*

#### **Abstract**

Cervical cancer is one of the leading female cancers especially in developing countries and a common cause of death among middle-aged women. The main role of Human Papillomavirus (HPV) in both cervical cancer and pre-invasive lesions of the cervix has been proven in studies. Reducing the incidence of the disease can be achieved by the regular cervical screening of women and vaccination of appropriate age groups. The disease can be better controlled by better elucidating the details of HPV carcinogenesis, the interaction between the host and the virus, and determinants of the systemic and cellular immune response to the viral infection. HPV causes oropharyngeal and anogenital diseases in both men and women and is usually sexually transmitted. Most infections are transient and could be cleared spontaneously by the host immune system. After the first encounter with HPV infection, it takes years to progress to cervical cancer, which gives clinicians a long period to follow these patients in terms of precancerous lesions and to investigate the pathogenesis of the disease. HPV plays a major role in the development of cervical cancer, but histological types have different relationships with HPV genotypes. HPV can remain latent for a long time and the most important thing determining the persistence is the type of HPV. HPV vaccination provides a direct benefit to both men and women by providing safe protection against cancers that may result from persistent HPV infection.

**Keywords:** cervical cancer, human papillomavirus, casualty, etiology, screening, vaccine

#### **1. Introduction**

Worldwide, cervical cancer is the fourth most common cancer among females and the third most common female genital tract cancer. 570,000 cases of invasive cervical carcinoma were diagnosed and 311,000 cervical cancer deaths occurred in 2018 [1, 2]. In low-income countries that do not have access to cervical cancer screening and prevention programs, cervical cancer continues to be a major cause of cancer diseases and deaths. The prevalence of cervical cancer and precancerous lesions depends on how effectively cancer screening programs and HPV vaccines are used in populations. The causal relationship between HPV and cervical cancer has been well documented [3**–**6] and HPV can be detected in 99.7 percent of cervical cancers [7]. Studies have consistently shown strong geographical correlations between HPV-DNA prevalence and cervical cancer incidence [6]. The worldwide spot prevalence of HPV is about 10 percent detected in a meta-analysis of studies involving more than 150,000 images with normal cervical cytology [8].

Africa is the most prevalent place of HPV infection in the world, where HPV is detected in 22% of African women. The most common types worldwide are HPV types 16 and 18, however, there appears to be geographic variation in the distribution of HPV genotypes.

HPV causes oropharyngeal and anogenital diseases in both men and women and is usually sexually transmitted. HPV infections are considered the most common sexually transmitted disease in sexually active individuals. It is estimated that at least 80% of sexually active individuals have been exposed to HPV once in their lifetime [9].

The most common histological type of cervical cancer is squamous cell carcinoma (SCC) (70%). Although the incidence of invasive cervical adenocarcinoma and its variants has gradually increased in the last few years; this type of neoplasia accounts for approximately 25% of all invasive cervical cancers diagnosed today. Other rare variants also constitute 3–5% of cervical cancer. HPV plays a major role in the development of cervical cancer, but histological types have different relationships with HPV genotypes. Available data state that HPV 18 accounts for 15% of SCCs and about 50% of adenocarcinomas [10]. The highest prevalence of HPV infection typically occurs within the first decade after sexual intercourse, typically between the ages of 15 and 25 in most western countries. Many sexually active young women have sequential infections with different types of oncogenic HPV. These infections are usually detected temporarily, but often reversible cytological changes occur. HPV spreads from the skin to the skin surface, and cutaneous HPV infections are common in the general population. Person-to-person transitions are typically asymptomatic [11]. Therefore, unprotected penetrative sexual intercourse (both vaginal and anal) or close physical contact from skin to skin is the most important factor for HPV infection [12]. Other risk factors are the number of partners [13, 14], new sexual partners [14], high-risk sexual partner, previous sexually transmitted disease, young age, not being married, non-Hispanic black, being the highest school graduate, poverty, low-income, first coitus at younger than 18 years old [8], primary or secondary immunodeficiency conditions. Spread from other HPV-infected genital organs, such as post-toilet wiping from front to back, may also play a role in the transmission of other types of contact [14, 15]. Penetrating vaginal and anal intercourse is not required for passage, but the prevalence of infection is much lower in virgins. Female-to-male transmission may occur at a higher rate than male-to-female transmission [16]. Regular condom use reduces the risk of HPV infection [17]. However, condoms do not completely prevent the transmission of HPV because the virus is spread through skin-to-skin contact.

HPV usually makes its first peak at an early age in unvaccinated sexually active women. Humoral and cellular immunity is provided with natural immunity partially [18, 19]. The presence of anti-HPV antibodies in patients with previous HPV type 16 infection has been associated with a lower risk of infection later, and it is thought that protective immunity is formed [18, 20**–**22]. However, it is not known how long and how much this protection lasts. It has been documented that some individuals with HPV infection did not develop antibodies [22, 23]. The second peak is in the postmenopausal period [24, 25]. This may be due to weakened cellular immunity or persistence or reactivation of a previously acquired infection. Reactivation may be the main source of newly detected HPV infection in HIVpositive women [26]. The oncogenic activity of HPV increases in the postmenopausal period. New HPV infection in older women does not usually progress to preinvasive disease or cancer.

HPV can remain latent for a long time and the most important thing determining the persistence is the type of HPV. HPV infection is best documented by molecular tests. PCR and in situ hybridization are mostly used in HPV typing.

#### *Human Papillomavirus and Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.98490*

With cytological examinations, only 30% of the patients represent the presence of a cytological disorder caused by HPV. A high-risk HPV type is responsible for 95% of pre-invasive lesions of the cervix and cervical cancer.

HPV has more than 200 types and can be divided into subgroups as mucous or cutaneous types according to their tissue tropism. Typing depends on DNA sequence and homology. Each type was separately identified as having less than 90% DNA base pair homology with another HPV strain. In addition to HPV genotypes, HPV intratypic variants also have epidemiological and oncogenic value in cervical cancer [27]. Different HPV types tend to infect different parts of the human body and are therefore associated with different diseases. The most common types of HPV associated with certain lesions vary according to the geography and demography of the population studied, but generally HPV types 6 and 11 cause condyloma acuminata, while type 16,18,31,52 cause intraepithelial neoplasms of the cervix [26, 28]. Over 40 HPV types showing tropism to the anogenital epithelium enter the epithelium of the penis, scrotum, perineum, anal canal, perianal area, vaginal introitus, vulva, and cervix. Approximately 15 HPV types are known as high risk, carcinogenic, or cancer-related [8]. High risk HPV types; 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 low-risk HPV types; 6, 11, 40, 42, 43, 44, 53, 54, 61, 72, 73, and 8. Among these, the most common HPV type in cervical cancer is type 16, and the intraepithelial neoplasia it causes is the type most likely to progress to cancer [3]. The reason of low-risk HPV types not causing cancer is that they cannot integrate into the host cell's chromosome. The E6 and E7 genes of such HPV viruses bind weakly to p53 and pRb. The presence of a cervical transformation site is not necessary for oncogenic HPV to infect the female genital tract. Because HPV can also cause cancer in the vulva, vagina, and anal region, the epithelium of which is mentioned before, squamous keratinized epithelium. HPV 18 causes disease more frequently in younger women and recurrence is more than that of HPV 16. In cervical cancer, the specificity of HPV 18 is higher than that of HPV 16. HPV 16 and 18 involve the cervix more than other HPV types with low oncogenic potential.

#### **2. Basic virology**

The link between genital HPV infections and cervical cancer was first demonstrated in the early1980s by Harold Zur Hausen, since then, the biology of HPV viruses has been extensively studied and has proven well connected with neoplasia.

HPV is an epithelotrophic virus from the Papillomaviridae family, small, non-enveloped, encapsulated, 55 nm in diameter, containing double helix 8 kilobase circular DNA. Human papillomaviruses are a big family with the systematic classification of five genera (α, β, γ, μ, and ν), 48 species, and 206 types [27]. The genetic map of HPV-16 is illustrated in **Figure 1** [29]. It is coated with 72-surface icosahedral protein capsid which contains at least two capsid proteins, L1 and L2. HPV genome contains 7900 base pairs. It encodes 8 genes that can encode early and late proteins and URR, which control the transcription of late genes without coding [30]. Early proteins are associated with viral gene regulation and cell transformation, while late proteins form the coat of the virus [31, 32]. Specific gene products are duplicated at each differentiation level of squamous keratinocyte [33]. At the most superficial level, the L1, L2, and E4 genes are duplicated for assembly of the viral capsid in which the HPV genome is packaged. After the short-lived superficial cells desquamate, infectious HPV virions are released for the next round of infection. E1,2,5,6 and 7 are expressed in the early period of differentiation of HPV in the epithelium, L1 and L2 are expressed in the late period, and E4 is expressed during differentiation. E4 is the gene most associated with viral release. E1 and E2 play

**Figure 1.** *Genome organization of human papillomavirus. The genetic map of HPV-16.*

a critical role in participating in the structure of host DNA. E1 enables the regulation of DNA replication and keeping the virus in episomal form. E2 cooperates with E1, ensures viral DNA replication, downregulates E6 and E7 expression [34]. The HPV genome remains a stable viral episome in the nucleus of the cell, independent of the host cell nucleus. When it causes cervical cancer or pre-invasive diseases of the cervix, the HPV genome in the host nucleus integrates into the host cell's DNA. Viral integration into cellular DNA was proposed as a marker of progression to cervical cancer. Integration is rarely seen in the pre-invasive disease. Whether integration in HSIL stages progresses to cervical cancer is unknown [6]. When E2 is added to the DNA structure, it degrades and as a result, E6 and E7 expression is increased [35]. In other words, the production of E6 and E7 is mainly under the control of E2. E6 and E7 are the major HPV oncoproteins and they work together to immortalize epithelial cells [36]. Both E6 and E7 proteins are consistently expressed in cancerous tissues. E6 can lead to persistent infections and invasive cancer development with its telomerase activity. E6 activates c-myc and increases telomerase activity of the catalytic subunit gene (by increasing hTERT transcription) [37]. It has also been shown that E6 and E7 antagonize the inhibition of hTERT via BRCA [38].

E6 and E7 proteins of HPV 16 bind more tightly to their targets than other HPV types, so HPV 16 becomes more persistent. E6 binds and suppresses p53, which blocks the G1 → S step in the cell cycle [39]. Following E6 binding of p53, p53 is disrupted in the presence of E6-associated protein [40]. If p53 does not participate, DNA damage cannot be repaired. The result is that the global cycle cannot be controlled, there is no apoptosis, and chromosomal mutations accumulate because there is no DNA repair [41, 42]. E7 binds to the retinoblastoma that regulates apoptosis and forces the cell to enter the synthesis step [33]. Retinablostome inactivates the E2F transcription factor that controls DNA synthesis, cyclin function

*Human Papillomavirus and Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.98490*

and promotes the S phase of the cell cycle. When E7 binds to the Rb protein, E2F is released, allowing cyclin A to promote cell turnover [43]. Thus, a cell with unstable chromosomes and high-risk HPV can turn into a malignant cell. E6 and E7 are essential proteins in immortality transformation. But these two genes are not the only ones responsible for cancer development [44]. Progression to neoplasia possibly involves a genetic change in the pathways that control intracellular or intercellular signaling [45].

E5, on the other hand, disrupts the antigen presentation of MHC-I and MHC-II. E5 also activates the EGF pathway [34]. The L1 protein self-assembles in the absence of the viral genome to form a virus-like particle (VLP). L1 VLP is the immunogen used in HPV vaccines. L2 is the minor capsid protein that mediates HPV infection with L1 [46, 47].

#### **3. HPV infection causality in cervical cancer**

The role of the HPV virus in cervical cancer development is proven by the demonstration of HPV DNA and viral oncogenes E6 and E7 in cancerous tissues, that E6 and E7 gene products have host cell transforming properties, and HPV has been shown as a major factor in cervical cancer development in epidemiological studies. There are four main steps in the development of cervical cancer [48];


Initial infection of the basal cell of cervical epithelium occurs as a result of microscopic breaks in the epithelium [33]. HPV targets and binds to the heparin sulfate proteoglycan receptor located in the basement membrane [49] indicating that HPV infection starts from the basement membrane. The replication cycle of the virus depends on the maturation of the keratinocyte. Since HPV does not enter the bloodstream, it does not cause viremia and inflammation. Therefore, the antigen is not formed against HPV and HPV cannot be detected by blood tests. There are also no FDA-approved serological or blood tests to detect HPV infection.

Although HPV infection is common in the population, cervical cancer develops in a minority of these infected patients. Because most HPV infections are temporary and additional factors are required for cancer development. The period from the first infection with HPV to the development of cancer is approximately 15 years. Different subtypes of HPV are detected at different rates in histological types of cervical cancer. In squamous cell carcinoma, HPV 16,18,58,33and 45 is found in 59%, 13%, 5%,5% and 4% of cases,respectively. In adenocarcinoma, HPV 16,18,45,31 and 33 is found in 36%, 37%, 5% percent, 2%,2% of cases, respectively [2].

The most expected and most likely outcome in women infected with HPV is complete resolution of the infection within 2 years [50, 51]. The least expected result is the development of neoplasia [52] and it occurs as a result of persistent infection. Currently, there is no effective treatment for HPV persistence [53].

Spontaneous recovery of HPV infection is more likely in young women [6]. Lowgrade lesions caused by HPV can be detected clinically when smears are used for screening, but they are usually temporary, however, HPV can become latent [54]. It may be reactivated in immunocompromised patients however, it is not known which HPV infections become latent and whether recurrent HPV infections carry a significant cancer risk. The possibility of pre-cancerous or cancerous lesions increases with persistent HPV infections. More than one HPV type can be positive in a woman. When women with multiple types of infection were compared with women who were positive for only one HPV type, no increased risk was identified. This suggests that each HPV type causes disease independently from the other [55].

Although HPV is the most powerful cause for cervical cancer development, the presence of HPV alone is not sufficient for cervical cancer, and additional factors are required. These factors can be causes such as smoking, endogenous and exogenous hormones. HPV is positive in 99% of patients diagnosed with cervical cancer. It is thought that an HPV virus type that causes cervical cancer is encountered around the age of 21 on average [56]. While HPV 16 is responsible for 50% of cervical cancer, HPV 18 is responsible for 20% [57]. The remaining cases (19%) are caused by HPV 31,33,45,52,58 [58]. While persistent HPV infection progressing to CIN3 in 5 years [59], CIN3 progresses to invasive cancer in 30% of patients after 30 years [60]. Factors such as smoking, multiparity, age at first birth, and the use of oral contraceptives facilitate the progression of HPV-infected epithelial cells to cancer [61].

Excessive viral load in the lesion does not mean that the lesion will progress to cancer, except for HPV 16 [62]. Very high dose viral load may be detected in some low-grade lesions, but these lesions may regress over time [8]. Therefore, viral load measurement is not useful in the clinic and does not provide any additional benefit [63].

#### **4. HPV vaccination**

Routine HPV vaccination is recommended for adolescents and young adults in many countries. HPV vaccination provides a direct benefit to both men and women by providing safe protection against cancers that may result from persistent HPV infection. This protective effect has been best demonstrated in cervical cancer, one of the most common women's cancers worldwide. Inactive HPV vaccination can prevent HPV infection and its sequelae. Vaccination status does not change recommendations for screening. HPV vaccine provides protection not only from cervical caser but also from vulvar, vaginal, oropharyngeal, anal cancers, and anogenital warts. There is also evidence of decreased genital warts among men of similar age in areas with a high proportion of vaccinated women [64].

The vaccine does not protect against 100 percent of the types known to cause cervical cancer since the most extensive vaccine protects against only 9 types of HPV. Therefore, women should continue to have cervical screening regardless of whether they are vaccinated or not. However, in societies that cannot include the HPV vaccine in the routine vaccination program due to economic concerns, it is recommended that public health efforts focus primarily on the vaccination of young women, the group with the absolute benefit and cost-effectiveness of HPV vaccination. However, none of the existing HPV vaccines will cure pre-existing vaccine-type HPV infections or related diseases [27], as the vaccine is effective if used in primary prevention. Sexually active individuals should be vaccinated consistently with recommendations specific to their age. Abnormal Papanicolaou test, genital warts, or a history of HPV infection is not a contraindication to HPV

#### *Human Papillomavirus and Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.98490*

vaccination [65]. The HPV vaccine is recommended for these patients as it can still protect against infection with HPV vaccine types that have not been encountered yet [66, 67]. However, if the individual is previously infected with any HPV type in the vaccine, it reduces the protection of the vaccine.

These vaccines contain virus-like particles, but without producing the effect of the virus, only activate the body's immune system, in other words, by initiating the production of HPV-type antibodies, enable the woman to become resistant to HPV for a long time. Many studies have reported that the prevalence and incidence of HPV infection and HPV-related disease decreased following the initiation of HPV vaccination [68**–**71].

The vaccine does not contain virus DNA, it contains capsid particles of the virus. The vaccine is produced against the L1 and L2 capsid proteins. L1 VLP vaccines strongly stimulate cellular and humoral immunity. There are three types of HPV vaccine, bivalent, quadrivalent, and 9-in-1 vaccine, although not all of them are available everywhere. Bivalent vaccine is protective against the HPV types 16 and 18, quadrivalent vaccine and 9-shot to vaccine to,HPV types 16,18,6,11, and HPV types 16,18,6,11,31,33,45,52,58 respectively. Although it is not available everywhere, it is more advantageous to be vaccinated with a 9-shot vaccine since it contains more HPV types that cause cervical cancer. Characteristics of the three human papillomaviruses (HPV) vaccines licensed for use in the United States are demonstrated in **Table 1** [72]. Therapeutic vaccines are under development but not clinically available [73].

The best time for HPV vaccination is before an individual has sexual intercourse. Clinical trial data on vaccine efficacy in men and women show that vaccination with the HPV vaccine is most effective among people not infected with HPV. Although it can be applied to individuals of all age groups, routine HPV vaccination is recommended between 11 and 12 years of age [66]. In this age group, the protection of the vaccine is almost 100% [74**–**77]. The resulting titers are generally higher in younger people than in older individuals [72]. There is no minimum threshold titer defined for protection. The natural history and the determinants of the immune response to HPV are still poorly understood [6]. The World Health Organization (WHO)


#### **Table 1.**

*Characteristics of the three human papillomavirus (HPV) vaccines licensed for use in the United States.*

recommends that the primary target of HPV vaccination programs is women between the ages of 9 and 14 and that local public health programs only recommend that older women be vaccinated if it is cost-effective and does not divert resources from vaccinating the primary target population or cervical cancer screening [66]. It is recommended to vaccinate men between the ages of 16–26 [72]. The vaccine can be administered from the age of 9 years [72], but it is not recommended before the age of 9. Compensatory vaccination is recommended for adolescents and adults aged 13–26 who have not been vaccinated before or have not completed the vaccine series [78] and for males aged 13 through 21 years who have not been vaccinated previously or who have not completed the 3-dose series. Males aged 22 through 26 years may be vaccinated. ACIP recommends vaccination of men who have sex with men and immunocompromised persons through age 26 years if not vaccinated previously [72]. The decision to vaccinate individuals over the age of 26 should be made on an individual basis. Because in this age group, the protection of the vaccine decreases [79]. While the protection of the vaccine is 81% in individuals between the ages of 26–35, it decreases to 75% in individuals between the ages of 35–46. The rate of protection is 44% in individuals who have been previously infected with HPV [80]. In these cases, the vaccine protects against other types of HPV. Persons who are virgin under the age of 26 can have the 9-inoculation vaccine if they have had a bivalent or quadrivalent vaccine before. The HPV vaccine is not recommended during pregnancy due to limited safety information [72]. Those who were vaccinated by mistake during pregnancy do not need termination because no relationship has been shown between the HPV vaccine and abortion or poor fetal outcomes [81]. Women of reproductive age do not need a pregnancy test before vaccination [72]. If conception is achieved between doses, the remaining doses are postponed, the remaining doses are completed after pregnancy, and do not start over [82]. Vaccine is safe for nursing mothers because inactive vaccines do not affect the safety of breastfeeding. There is no need to screen the individual for HPV before HPV vaccination [67]. Measurement of post-vaccination antibody titers has no clinical use [6] since the protective titer is unknown.

In addition, studies are showing that HPV is present in the smoke that occurs during the surgical removal or ablation of HPV-infected tissues and that nasal or oropharyngeal HPV infection may develop if this smoke is inhaled by healthcare workers [83]. Therefore, it would be beneficial to vaccinate healthcare workers who are at risk of such exposure [84]. Studies have shown that the HPV vaccine protects women against high-grade lesions of the cervix, vulva, and vagina for 10 years, and persistent antibody levels have been found [85**–**87].

The vaccine is administered in 3 doses [72]. The peak immunity achieved with 3 doses of vaccination is greater than that of native HPV infection. After 2 years, the antibody level drops but is still higher than that of innate immunity [78]. There is no evidence of the additional benefit of a booster dose. For individuals under 15 years of age, 2 doses are sufficient (between 0 and 6–12 months). In this age group, if the second dose is administered less than five months after the first dose, the dose should be repeated at least 12 weeks after the second dose and at least five months after the first dose. It can be applied in 0,1 and 6 months or 0, 2, and 6 months. The interval between the first and second doses should be a minimum of 4 weeks. The interval between the second and third doses should be a minimum of 12 weeks. The minimum interval between the first and third doses should be 5 months. If a dose has been administered at a shorter interval, it should be repeated at the minimum recommended interval after the last dose has passed [66, 67, 88]. If the dosing schedule is got out of order the remaining doses are made quickly, not starting over [67, 72]. Vaccines that can provide the same protection after 1 dose are in the development phase. Such a vaccine could be an important breakthrough

#### *Human Papillomavirus and Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.98490*

for low-income countries to prevent disease. If possible, the vaccination should be continued with whatever type of vaccine it started. However, if there is a problem in accessing the vaccine or if the first vaccine is not known, it can be continued with other types of vaccines [72]. Although direct efficacy data on HPV vaccination in immunocompromised hosts are lacking, immunocompromised individuals can also be vaccinated in the same manner [66]. The HPV vaccine can be safely administered in a different anatomical region at the same time with other age-appropriate vaccines. Coadministration of HPV vaccine with other vaccines does not affect the immune response [83, 89].

Side effects of HPV vaccination are generally limited to mild local reactions (regional reaction, systemic malaise, fever) [72] and syncope. Mild injection site reactions were the most common side effects in studies [90]. Syncope is not specific to the HPV vaccine [90, 91]. None of the side effects already seen are characteristic of the HPV vaccine. Following HPV vaccination, a routine waiting period of 15 minutes in a sitting or supine position is recommended [67]. Other reported side effects include headache, nausea, vomiting, tiredness, dizziness, and weakness [43]. The adjuvant aluminum hydroxyphosphate found in the quadrivalent and 9-vaccine and the adjuvant aluminum hydroxide + monophosphoryl lipid found in the bivalent vaccine is used to increase the immunological response of the vaccine. The higher the amount of adjuvant in the vaccine, the more side effects it has. For this reason, since the 9-vaccine contains more adjuvant substances, its side effects occur more [72]. However, the protection of the 9-in-1 vaccine against HPV 16 and 18 is approximately as much as the quadrivalent vaccine [72].

#### **5. Conclusions**

Cervical cancer is preventable cancer worldwide with the organized and strict compliance of early screening methods, vaccination programs, and changing sexual behavior. Aggressive treatments of early or ambiguous cytologic lesions related to HPV may result in a decrease in rates of more advanced disease although increases in the incidence of cervical adenocarcinomas have been reported in several populations. Because cervical screening tests are insufficient in detecting adenocarcinoma. Since the HPV vaccine is protective against high-risk types, it is beneficial to apply it to the recommended age groups.

#### **Acknowledgements**

Thank you very much to Ömer Harun Sağnıç,M.D. and Özer Birge,M.D. for their contributions and supports**.**

No financial support has been received in writing this article.

#### **Conflict of interest**

The author declare no conflict of interest.

#### **Notes/thanks/other declarations**

None.

*Cervical Cancer - A Global Public Health Treatise*

### **Author details**

Saliha Sağnıç Division of Gynecologic Oncology, Department of Gynecology Obstetrics, Akdeniz University, Antalya, Turkey

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

© 2021 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.

*Human Papillomavirus and Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.98490*

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#### **Chapter 9**

## The Importance of the Extracellular Matrix in HPV-Associated Diseases

*Joana Sampaio, Joana Ferreira, Ana Carolina Santos, Manuel Bicho and Maria Clara Bicho*

#### **Abstract**

The extracellular matrix (ECM) is the non-cellular component of the tissues of our organism. It is the dynamic element that maintains a biochemical structure capable of supporting the organization and architecture of the tissue constituents. The diversity of ECM's constituents gives it the biochemical and biophysical properties necessary to regulate its behavior and differentiation. ECM has an important role in the biology of cancer cell development and progression. Human papillomavirus infection (HPV) is the principal etiological agent of the most common sexually transmitted diseases. It is a virus that can cause lesions precursors of epithelial squamous and glandular tumors. Type 16 (HPV16) is the leading cause of pre-malignant lesions and invasive cancers in these tissues. This work will focus on HPV infection to understand the role of ECM in the invasion, spread, and pathogenesis of the lesions caused by this virus. Cancer is no longer considered a pathology explained only by uncontrolled proliferation and apoptosis but also by the deregulation of the microenvironment.

The in-depth knowledge of ECM dynamics and its complexity is central and promising, specifically in developing new targeted therapies.

**Keywords:** Extracellular matrix, human papillomavirus, heparan sulfate proteoglycans, metalloproteinases, heparanase

#### **1. Introduction**

The extracellular matrix (ECM) can be defined as a three-dimensional, noncellular macromolecular network made of collagen, proteoglycans, elastin, fibronectin, laminins, and other glycoproteins structural support for the organization of cellular constituents. It is known to be a physiologically active component of living tissue. The various parts of the matrix bind to each other and cell adhesion receptors, forming a complex network on which cells rely in all tissues and organs. Through transduction of extracellular signals originating from the ECM, cell surface receptors regulate diverse cellular functions such as survival, growth, migration, and differentiation and are vital for homeostasis maintenance [1, 2].

Each organ has a unique combination of elements in its constitution so that the specific function of the tissue itself can be fulfilled. This unique composition arises from biophysical and biochemical feedback between cellular components and the microenvironment, where they are inserted during the genesis of the tissue. The ECM continuously undergoes remodeling mediated by different decomposition enzymes, the proteinases being a highly dynamic structure. The balance between ECM degradation and secretion, orchestrated by ECM-modifying cells, is responsible for tensional homeostasis and organ-specific properties such as elasticity and compressibility [2–6].

In vitro, most animal cells have been shown to remain viable only when adherent to a substrate. Thus, the cell relies heavily on its sense of touch to survive by adhering and spatially interacting with the surrounding ECM- the concept of anoikis. The ability to attach and communicate with its environment is responsible for several growth factor receptors, and adhesion molecules arranged along the cell membrane, namely integrins. Indeed, cells have been shown to translate signals from the ECM to coordinate crucial morphological organization and signal events by regulating gene transcription. A cell converts external mechanical stimuli into a downstream intracellular chemical signal. This process is known as mechanotransduction. The sensitivity with which cells respond to biophysical and biochemical signals from the ECM demonstrates the importance of tissue homeostasis in maintaining healthy host cells.

Consequently, dysregulation of ECM remodeling has been shown to contribute significantly to cellular evolution through various fibrotic conditions, characterized by excessive ECM deposition and increased ECM stiffness. Due to increased interstitial pressure, irreversible loss of tissue homeostasis has been linked to an increased risk for various pathological conditions such as osteoarthritis, cardiovascular disease, and cancer [3, 4, 6–10].

#### **2. ECM constitution**

Various proteins that constitute the ECM result in different structures and properties. The main components of ECM include collagen, proteoglycans, laminin, and fibronectin. As each structure has a function, different subtypes and combinations of ECM molecules confer different functions essential to the correct functioning of the whole organism, **Figure 1** [11, 12].

#### **Figure 1.**

*ECM components and their organization. Organization of the different collagens, proteoglycans (HPSG), laminins, and fibronectin in the basement membrane and the extracellular matrix. In the basement membrane, laminin is attached to the cell, forming a fibrillar network. It is then linked to the type IV collagen network through nidogen and proteoglycans such as perlecan and agrinin. The different proteoglycans hold the fibrils together to form a collagen fiber. Fibronectin is bound to the cell by integrins and syndecans.*

*The Importance of the Extracellular Matrix in HPV-Associated Diseases DOI: http://dx.doi.org/10.5772/intechopen.99907*

#### **2.1 Collagen**

Collagen is the basis of ECM architecture, is the most significant functional component, and the most abundant protein in human tissue. It can be classified into fibrillar (I-III, V, and XI) and non-fibrillar. Collagen fibers give ECM its tensile strength by limiting tissue distensibility. Collagens are trimeric molecules composed of three α-polypeptide chains that contain the sequence repeat (G-X-Y). This repeat allows the formation of a triple helix that gives the characteristic structure of this superfamily, **Figure 1**. Currently, there are 28 unique subtypes of collagen discovered. Each member of the collagen family has at least one triple helix domain. Most collagens bind to and interact with several ECM proteins forming supramolecular aggregates [3, 6, 8, 11].

Fibrillar collagens form fibrous structures often found in tendons, cartilage, skin, and cornea. Each collagen fiber is made up of several collagen subtypes in response to their tissue location.

Fundamentally we can define four classes of collagens:


Types I, III, and V, predominantly produced by fibroblasts, are essential for the structure of the interstitial matrix. Their function as a pericellular "glue and structure" is necessary in tissue repair. Type IV is primarily located in the basement membrane and underlying epithelial or endothelial cells, ensuring their specialized polarization and function. Type IV collagen is the main constituent of basement membranes in tissues such as the lung, kidney, skin, intestine, and liver. It is mainly produced by endothelial and epithelial cells, and seen as intelligent collagen necessary for tissue repair processes that allow polarized cells (endothelial and epithelial) to survive and function, enable regular tissue function [6, 13].

#### **2.2 Proteoglycans**

Proteoglycans are the functional modifiers of ECM and provide additional properties. They are proteins characterized by being covalently linked to glycosaminoglycans (GAGs). These GAGs are long, negatively charged chains conferred by sulfate and carboxyl groups (heparan sulfate (HS), dermatan sulfate, and keratan sulfate (KS). The addition of sulfate and carboxyl groups to GAGs gives them this negative charge, enabling them to sequester water and cations (sodium and calcium) and have cellular lubrication and filling functions, **Figure 1** [6, 12, 14].

There are about three dozen extracellular matrix proteoglycans encoded in mammalian genomes, divided into several families. The two largest families include the LRR (leucine-rich repeat) proteoglycans and the hyalectans (such as versican). In addition to those mentioned, perhaps the most significant of all is perlecan (HSPG2), a multidomain protein that is part of all basement membranes. There are also two small families of transmembrane proteoglycans: glypicans and syndecans, both of which have heparan sulfate side chains, as does CD44 [12, 14].

Syndecans are encoded by four different genes and represent the most abundant transmembrane heparan sulfate proteoglycans (HSPGs). Their core protein comprises an extracellular domain, a single transmembrane domain, and a short cytoplasmic domain that interacts with the cell cytoskeleton. Glypicans are encoded by six different genes and are anchored to the cell membrane via glycosylphosphatidylinositol (GPI). The multiple organs of the human body contain different isoforms of HSPG with various polysaccharide compositions and sulfation patterns [12, 15].

The extracellular domain of syndecans is intrinsically disorganized, a feature that allows it to interact with a huge variety of molecules and perform a wide variety of biological functions. Some of these functions involve acting as coreceptors for tyrosine kinase receptors linked to growth factors. The HS chains of these proteoglycans share various ligands, such as matrix proteins, growth factors, cytokines, and chemokines, which are presented to high-affinity receptors present on the cell surface [3, 4, 6, 10].

In conclusion, proteoglycans are highly variable in their shape and structure to exert different functions in the ECM, i.e., and they are highly pleiotropic. This characteristic makes them essential in maintaining a healthy ECM, and without them, its entire structure would collapse.

#### **2.3 Laminins**

Laminins are trimeric glycoproteins formed by α, β, and γ chains often found in the basal lamina and mesenchymal compartments. The three chains form a coiled α-helical structure that builds the long arm, while the three short arms are each composed of one chain. At the end of the long arm are five laminated G-type (LG) domains of the α-chain that serve as binding points for the cell. Integrins, dystroglycan, lutheran glycoprotein, or sulfated glycolipids bind to these LG domains. At the end of each short arm are the N-terminal laminin (LN) domains that are important for laminin polymerization and the formation of the basement membrane [5, 6, 11].

Laminins have cell-specific functions such as adhesion, differentiation, migration, maintenance of phenotype, and resistance to apoptosis (anoikis). By binding to integrins, laminins can create a dynamic link between the cell and the ECM. The unique heterotrimeric laminins have the integrins as anchored partners allowing the induction of signaling pathways and the organization of the intracellular cytoskeleton. It has been observed that heparan sulfates directly mediate the interaction between laminins and collagen IV. Laminins play crucial roles in both basal membrane formation and interactions between cells and the ECM. While collagen, proteoglycans, and hyaluronic acid constitute the main structural component of the MEC, laminins are one of the molecules that bridge the cell-ECM interaction gap [6, 11, 12, 14].

#### **2.4 Fibronectin**

Fibronectin is a high molecular weight protein composed of two subunits linked together by two cysteine persulfide bonds. It is secreted in a soluble form by hepatocytes into the bloodstream or expressed in tissues by fibroblasts, forming a fibrillar network. The structure of fibronectin and its multiple post-translational modifications result in an immense variety of interactions with various ECM components (growth factors and GAGs) that mediate cell attachment and motility, ECM remodeling, host-pathogen interactions, among others. A single gene encodes this glycoprotein with 20 human isoforms resulting from alternative mRNA excisions

#### *The Importance of the Extracellular Matrix in HPV-Associated Diseases DOI: http://dx.doi.org/10.5772/intechopen.99907*

(primary transcript). Similar to collagen, fibronectin forms a fibrillar network in the ECM. The structure of the fibronectin matrix is mediated by selective binding to α5β1 integrins. Compact, soluble fibronectin is secreted and unfolded through these integrins, revealing specific binding sites for other fibronectin molecules to form its fibrillar network. Binding to fibronectin induces integrin aggregation, which provides high local concentrations of fibronectin on the cell surface. This phenomenon promotes fibronectin–integrin interactions through the N assembly domains of each molecule [5, 6, 16].

Once fibronectin is attached to the cell surface by integrins, the actin cytoskeleton can drag molecules to the fibronectin to change its conformation. This will affect the C-terminal regions of fibronectin, revealing binding sites for fibronectin, heparan sulfates, heparin, collagen, and other ECM molecules. Through strong non-covalent protein–protein interactions, the fibronectin network matures and becomes insoluble, although other ECM proteins can mediate mature lateral interactions between fibrils. These interactions stabilize the relatively weaker binding sites [5, 6, 16].

In conclusion, fibronectin works as a skeleton upon which the bioavailability and activity of various growth factors are orchestrated. The interaction of fibronectin with growth factors (e.g., TGF-β, PDGF, HGF, VEGF, FGF) can impact cell migration, cell proliferation, survival signals, and angiogenesis as downstream outcomes of their activation through mechanical or enzymatic activation [5, 6, 14, 16].

#### **3. Functions of the extra cellular matrix**

The countless unique molecules that are part of the constitution of ECM give it various functions that simultaneously influence biochemical and biophysical processes in the cell. Although ECM was for many years considered an inert component that only provided structure to cells in tissue formation, its role in determining cellular functions and phenotypes has been clarified in the last two decades [2, 6, 14, 17].

The many proteolytic processes that modify ECM, by the action of proteolytic enzymes, play roles in ECM remodeling and are thought to release ECM-binding growth factors and expose cryptic activities in ECM, including the release of antiangiogenic factors. Similarly, enzymes that degrade GAGs, such as heparanases and sulfatases, can also change the properties of proteoglycans in the ECM. Remodeling of the ECM by these various processes has important effects on development and associated pathologies [2, 14, 17].

Finally, ECM is known to transmit mechanical signals to cells and activate intracellular signaling mechanisms and the cytoskeleton machinery. The importance of ECM and its varied functions in the development and maintenance of cellular balance or homeostasis is indisputable [2, 14, 17].

#### **3.1 Migration and proliferation**

Cell migration is essential for tissue development, a fact demonstrated by neural crest cells, which migrate from the periphery of the neural tube to different parts of the embryo to form the heart, nerves, skin, and skull [2, 6].

The ECM influences the path and speed of migrating cells through its topography, composition, and physical properties. Cells migrate from regions with low ECM concentration to high ECM concentrations due to an adhesion gradient called haptotaxis. Proteases that degrade ECM also play a facilitating role in cell

migration through a process involving matrix metalloproteinases (MMPs), their inhibitors, among other enzymes. It is essential to realize that constant remodeling of the ECM occurs during development [2, 4, 6].

#### **3.2 Development**

The topographic variation in the structure and elasticity of ECM provides cells with the ability to adapt and form complex morphological systems that are essential for different organs [2, 4, 6].

ECM modulates tissue growth to form complex structures that are necessary for these organs to function. In addition, it provides structural organization not only through its action as a physical barrier to cell growth and by activating intracellular signaling in a time- and context-dependent manner. ECM exerts this effect by modulating the distribution of growth factors, physical anisotropy, and anchoring [2, 4, 6, 11].

ECM also has an essential role in highlighting the influence on cell fate. This role is shown in mammary gland differentiation. Even with hormonal stimulation in vitro, mammary gland cells do not secrete milk proteins. However, after exposure of these cells to laminin-1, they begin to secrete these proteins. This phenomenon indicates to us that an appropriate ECM microenvironment is indispensable for the cell to be able to fulfill its functions [2, 4, 6].

To conclude, cells can sense the physical properties of the adjacent matrix and activate the appropriate intracellular mechanisms for their differentiation. Therefore, the physical properties of the ECM have cell differentiation capabilities.

#### **3.3 Tissue homeostasis**

ECM is a highly dynamic structure. Even after development, ECM is constantly being deposited, degraded, and modified to maintain tissue homeostasis. This is especially important for preserving cell phenotype and physiological processes such as wound healing, angiogenesis, and bone remodeling [2, 4, 6].

To maintain tissue homeostasis, cells in contact with the ECM perceive ECM properties through receptors and adhesion complexes. In turn, the cell regulates the expression of ECM components and enzymes based on the signals it receives. This leads to a feedback mechanism in which cell also influences ECM, resulting in a balance of deposition and degradation of ECM components [2, 3, 6].

The response of cells to other stimuli is ultimately influenced by the ECM components. The complexity and importance of the feedback mechanism between the ECM and the cell is essential to maintain tissue homeostasis [2, 3, 6].

The imbalance in ECM deposition and degradation leads to disease and is a hallmark of cancer and other conditions that course with fibrosis. Overall, the role of ECM in tissue homeostasis is to direct the appropriate cellular response and phenotype to maintain mechanical integrity and tissue function [6].

#### **4. ECM disruption in cancer progression**

Traditional views of cancer have been changing, and the significant role of ECM in the regulation of cell proliferation, migration, and apoptosis have been highlighted. At the microscopic level, the organization of ECM constituents forms a specific microenvironment that plays a critical role in tumor progression. ECM is constantly remodeling and actively influences cell adhesion and migration. Thus,

#### *The Importance of the Extracellular Matrix in HPV-Associated Diseases DOI: http://dx.doi.org/10.5772/intechopen.99907*

small changes in the homeostasis of the microenvironment can result in significant effects on cancer cell proliferation. As the main component of ECM, collagen, dictates the main properties of the matrix, changes in its deposition or degradation can lead to loss of ECM homeostasis [2–4, 6, 11].

As cancer cells proliferate, the surrounding matrix changes a dynamic interaction between cells and the microenvironment. Changes such as increased fibronectin secretion, collagen type I, II and IV, indicate that tumor progression requires continuous interaction between the ECM and tumor cells. Increased deposition of matrix proteins promotes tumor progression as it interferes with cell–cell adhesion, cell polarity, and the amplification of growth factor signaling. High collagen deposition has been shown to result in tumor progression through increased integrin signaling [6].

The increased stiffness of the matrix activates integrins as well as cytoskeletal tension, promoting cell adhesion and motility [2–4, 6, 11]. It has been observed that local cell invasion of tumors is directed along collagen fibers aligned, suggesting that this linearization of the fibers facilitates invasion. These densely aligned collagen fibers are believed to act as cues for proliferating neoplastic cells to migrate outward from the tumor [2–4, 6, 11].

Tumor tissue hydration also has some impact on ECM dysregulation. Since it is strongly influenced by specific tissue GAGs due to their anionic structure and their ability to attract water, it is known that as hydration increases, the increase in intra-tumor hydrostatic pressure also increases, altering the biomechanical properties of the tissue that are known to be crucial for invasion [2–4, 6, 11].

Elevated levels of hyaluronic acid (carboxylated and free glycosaminoglycan) in ECM correlate with an increased likelihood of malignancy and poor prognosis. As a ubiquitous linear polysaccharide, hyaluronic acid (HA) is fundamental in determining the compressive properties of most biological tissues. Combining tensile strength due to collagen conformation and compressive strength due to hyaluronic acid creates the ideal biophysical properties for tissue homeostasis. Furthermore, hyaluronic acid is an induction signal for epithelial-mesenchymal transition (EM) and a migration substrate. It is also important in regulating vascular endothelial barrier permeability by stabilizing cell–cell junctions [2–4, 6, 11, 18].

Although increased levels of collagen directly promote ECM stiffness and mechanistically drive cell motility and proliferation, the exact role of hyaluronic acid in cancer metastasis remains unclear. However, its downregulation may serve as a key biomarker for invasion and metastization [2–4, 6, 11, 18].

#### **5. Human papillomavirus (HPV)**

#### **5.1 The virus**

The human papillomavirus (Human papillomavirus-HPV) is a small (55 nm) non-enveloped virus that belongs to the Papillomaviridae family. It can be classified according to its tropism (cutaneous, mucocutaneous, and mucosal). Since the availability of cellular factors expressed in different layers of the epithelium plays a role in viral gene expression and genome amplification, the viral cycle is strictly dependent on the epithelial differentiation program HPV infections are associated with some hyperproliferative pathologies of epithelia and mucosa, and most cervical cancer and warts cases. It has been described more than 200 types of HPV. Almost 40 types exhibit a particular tropism for the anogenital region's cellular floor epithelium and mucous membranes. HPV types in this subgroup are classified as being of high or low oncogenic risk, depending on the clinical lesions they cause.

The high-risk HPV types are associated with almost all cases of cervical cancer, and the low-risk HPV types are the cause of nearly all anogenital warts and lowgrade lesions with a slight tendency for malignant progression. The most prevalent high-risk HPVs are HPV16 and HPV18, while the most common low-risk types are HPV6 and HPV11. Infections with specific high-risk HPV types are etiologically related to a significant proportion of vulvar, vaginal, anal, penile, and head and neck carcinomas [19–22].

#### **5.2 Structure of HPV**

HPV is a double-stranded circular DNA virus with 6.8–8.4 kb and can be divided into three functional regions:


The two oncogenes, E6 and E7, play a major role in carcinogenesis, contribute to immortalizing normal human keratinocytes in cell culture systems, and are essential to maintain the transformed phenotype in vivo. The main role of these proteins during the HPV cycle is to generate a permissive cellular microenvironment for viral replication. This includes the induction of DNA replication machinery, immune evasion, and the downregulation of apoptosis. To achieve this, the E6 and E7 proteins give rise to critical cellular regulatory pathways, including those dominated by p53 and pRb. The E6 protein (16–19 kDa) associates with p53 (tumor suppressor protein) and promotes its degradation. E7 (10–14 kDa) inactivates the function of another tumor suppressor protein, the retinoblastoma protein (pRB). Together, these two proteins promote the mechanisms involved in the genesis of tumors caused by these viruses, favoring cell transformation and immortalization [19, 24].

#### **5.3 Viral replication**

As already described, the life cycle of this virus is synchronized with cell differentiation and division.

Whether or not the viral life cycle is complete depends on the nature of the epithelial site where infection occurs and external factors such as hormones and cytokines. It is suggested that infection requires access to the viral particles (composed of viral DNA and the capsid proteins, L1 and L2) to the basal lamina and their interaction with HSPGs laminin [23]. Structural changes in the virion capsid facilitate transfer to a secondary receptor in the basal keratinocyte, necessary for virus internalization and subsequent transfer of the viral genome to the nucleus. Once internalized, virions undergo endosomal transport and pass into the nucleus, where the capsid disassembles and DNA release occurs. The L2-DNA protein

#### *The Importance of the Extracellular Matrix in HPV-Associated Diseases DOI: http://dx.doi.org/10.5772/intechopen.99907*

complex ensures the correct nuclear entry of the viral genomes, while the L1 protein is retained in the endosome and ultimately subject to lysosomal degradation [20, 23, 24].

Infection is thought to require an epithelial wound or micro-wound to allow the virus access to the basal lamina. Indeed, active cell division, as occurs during wound healing, is required to enter the virus genome into the nucleus. It has been proposed that lesion formation requires the initial infection of a mitotically active cell [19, 20].

It is also known that in the basal cells of the epithelium, the expression of viral genes is repressed and expression of early (E) and late (L) genes only happens at the level of keratinocytes or the upper mucosal layers. Viral replication is associated with excessive cell proliferation of all epidermal layers except the basal layer. Since the basal cells of the stratified sidewalk epithelium are the only ones capable of dividing, they are the initial target of HPV infection [19, 20, 23].

Regardless of the nature of the infected basal cell, infection is followed by an initial phase of genome amplification and then the maintenance of the viral episode at a low copy number. The viral replication proteins E1 and E2 are considered essential for this initial amplification phase. The precise role of the HPV E6 and E7 proteins in infected basal cells is uncertain, particularly for low-risk HPV types that are not generally associated with neoplasia and whose viral DNA does not integrate into the chromosomes. They are thought to produce lesions following infection of a basal stem cell at the site of a wound. The role of the curative response in driving the initial proliferation of the infected cell may well be critical, with local microenvironment signaling influencing viral gene expression and/or protein functions. In the case of high-risk types that cause neoplasia, there is the integration of the viral genome into the chromosomal DNA, and the role of the viral E6 and E7 proteins in cell proliferation in the basal and parabasal cell layers is quite clear, especially at cervical sites where neoplasia can occur. It is clear that many functional differences exist between high- and low-risk E6 and E7 proteins and that these contribute, along with differences in promoter activity and gene expression patterns, to the different HPV-associated pathologies seen in vivo. Indeed, recent studies have suggested that a critical event in determining the neoplastic grade is the downregulation of E6/E7 expression, even in the absence of genome integration, which is classified according to the extent to which basal-like cells extend into suprabasal epithelial layers. While such functional differences undoubtedly contribute to the respective abilities of high and low-risk HPV types to cause neoplasia and cancer, it is important to remember that a key function of the E6 and E7 proteins in most HPV types is not to promote basal cell proliferation but rather to stimulate cell cycle re-entry into the middle epithelial layers to allow genome amplification [19, 23, 24].

#### **6. HPV-MEC interaction**

As previously mentioned, the role of HSPGs in HPV infection is quite relevant [15, 25].

The HSPGs typically consist of a core protein and GAG chains. The core protein of syndecans is composed of an extracellular domain, a unique transmembrane domain, and a short cytoplasmic domain that interacts with the cytoskeleton. The glypicans are GPI-labeled HSPGs. The GAG chain comprises unbranched anionic polysaccharides composed of repeated disaccharide units formed by sulfated uronic acid and hexosamine residues [15–18].

As components of the ECM, HSPGs contribute to the organization of the basement membrane and mediate cell adhesion and motility. HSPGs bind to cytokines, chemokines, and growth factors on the cell surface, preventing their degradation, creating temporary storage sites or morphogen gradients important in development. Still, on the cell surface, they also serve as endocytosis receptors, and regulate the lysosomal degradation of extracellular molecules and provide nutrients to the cells. In addition, they are involved in the endocytosis of cell receptors. They mediate the transcellular transport of chemokines through endothelial cells. They also serve as co-receptors for a fibroblast growth factor (FGF) and its receptor. They mediate intracellular signaling or intracellular stress through the proteolytic shedding of syndecans and play an important role in developing and maintaining stem cell niches [15, 18].

The strategic localization of HSPGs in tissues is critical to their functional role. The localization of SDCs and GPCs at the plasma membrane regulates intracellular and cell-to-ECM signaling. The localization of HSPGs in the basement membrane regulates their barrier functions and coordinates cell–cell and cell-MEC interactions [15, 18].

Degradation of heparan sulfate chains by heparanase produces heparin-like fragments that activate FGF-2 mitogenicity. Therefore, the biological role of an HSPG depends on the properties of its core protein, the number of GAG chains attached, its location in cells and tissues, and the biosynthetic modifications that its GAG chains receive in situ. A wide range of biological functions is attributed to GAGs in cancer metastasis and other biological events due to their controlled, highly heterogeneous, and complex structure that allows for the regulation of tissue-specific functions [15, 18].

#### **6.1 HSPGs as viral receptors**

HSPGs are receptors hijacked by numerous viruses to bind to host cells. This typically occurs through electrostatic interactions between the negative charges of HSPGs and the basic amino acid portions of viral surface proteins. A consistent amount of data supports the natural dependence of HSV, DENV, and HPV on HSPGs for their binding to host cells [15, 26].

HSPGs, due to highly sulfated GAG chains, exhibit an overall negative charge that can interact electrostatically with the basic residues of viral surface glycoproteins or viral capsid proteins of non-enveloped viruses. Viruses exploit these weak interactions to increase their concentration on the cell surface and increase their chances of binding to a more specific entry receptor. HSPGs directly serve as entry receptors in rare cases, as described for herpes simplex virus (HSV)-1. Another study showed that HSPGs are crucial for SARS-CoV entry. Prophylactic treatment with bacterial heparinase I or heparin showed a reduction in SARS-CoV infectivity. Thus, either loss of HS or competitive inhibition confers some protection to cells against SARS-CoV. Given the structural similarities between SARS-CoV and the novel SARS-CoV-2, it would be interesting to study the effects of removing HS on SARS-CoV-2 infections. There are multiple ways to reduce viral contact with cell surface HS (HPSE, heparinase, heparin, soluble HS, and MMPs), investigating that this binding may give insight into SARS-CoV-2 entry and possible therapeutics [15, 26, 27].

All papillomaviruses are believed to rely on HSPGs for their initial binding. However, HPV-16 is the serotype whose pathogenesis is most studied due to its oncogenic potential and prevalence [15].

As already mentioned, the HPV infection cycle starts from the basal membrane of the vaginal mucosa, exploring abrasions or lesions in the epithelium (**Figure 2A** and **B**). The entry of HPV particles into host cells is a multistep process that begins with binding to HSPGs expressed on the cell surface of basal *The Importance of the Extracellular Matrix in HPV-Associated Diseases DOI: http://dx.doi.org/10.5772/intechopen.99907*

#### **Figure 2.**

*Schema of the role of HSPGs in HPV16 infection. (A) Bottom is a normal cervix epithelia disrupted. Upper figure represents the details of wounded tissue healing mechanism, (a) MMP/ADAM cleaved GF (growth factor) bound to HSPG of cell membrane and also to the complex with laminin (b) shedd HSPG/GF complex, bounds to (c) adjacent cells GFR/EGFR as a co-receptor. (B) In bottom of figure HPV16 infected wound tissue release, as shown in upper figure, HPSE (heparanase) and HPV16 (a), HPV16 is coated with HSPGs and HS (heparansulfate) released respectively by MMPs/ADAM and HPSE, (b), HPV16 bounds to the complex HSPG-laminin in ECM, (c) adjacent keratinocytes through membrane EGFR-RTK and HS receptor bound as a complex (d) is endocytosed by the cell (e). (C) HPV16 integration by the infected keratinocytes during wound healing, after endocytosed in (B), progresses through CIN I, CIN II and CINIII to invasive cancer, after expression of E6 and E7 HPV oncogenic proteins.*

keratinocytes or in the ECM. The interaction of the HPV16 L1 capsid protein with the HS chains of proteoglycans is well known but generally considered to have a passive role in infection. Binding to HSPGs induces conformational changes in the capsule and facilitates proteolytic cleavage of L1. This cleavage allows interactions between capsid and cyclophilin B, which results in further conformational changes that expose the N-terminus of L2. The exposed N-terminus contains a conserved consensus cleavage site for the extracellular proprotein furin convertase. This interaction is essential for successful HPV infection since cleavage of furin results in the exposure of a binding site on L1 postulated to be recognized by an unknown receptor. The described changes in virion conformation further facilitate the reduction in binding affinity to HSPGs, thus facilitating binding to the unknown receptor(s). These findings underscore the role of initial HSPG attachment to facilitate the critical step of L2 cleavage by furin and association with the putative second receptor for entry. Cleavage of furin has also been implicated in successful endosomal escape before transporting the viral L2/ADN complex into the nucleus, emphasizing the necessity of furin cleavage for successful HPV infection [15, 20].

Syndecan-1, the most abundant HSPG in keratinocytes, plays an important role in this initial binding due to its expression in epithelial cells and its overproduction during wound healing. It has been shown that syndecan-1 when released plays a major role in the infection of keratinocytes by HPV. Instead of separating it from its HS chains, HPV particles are released from the cell surface through the normal process of HSPGs, remaining bound to HS chains (heparan sulfate) and growth

factors. They can bind to the epidermal growth factor receptor (EGFR). The specificity of growth factors is the bridge for the interaction of the virus with cellular receptors, for example, tyrosine kinase, whose signaling promotes infection **Figure 2A** and **B** [15, 28].

Considering the biology of HSPGs, it stands to reason that HPV particles bound to HSPGs would associate with ECM, and indeed, free syndecan forms bind tightly to ECM via their HS chains. Many studies have shown that HPV particles accumulate in the ECM, and LN-332, a component of the ECM, is a proposed attachment factor for HPV. There is already evidence of direct protein–protein interaction between HPV16 and LN-332 **Figure 2A** and **B** [15, 25, 28].

Thus, HS chain cleavage enzymes are known to increase the release and infectivity of HPV particles bound to ECM, indicating that many virus particles bind to ECM via these HS chains. Free syndecan-1 interacts with HPV16 and LN-332, demonstrating it to be an ECM-binding factor for HPV16, in addition to its role in binding HPVs to the plasma membrane. The interaction of snd-1 with LN-332 is expected based on reports demonstrating the concentration in the EMC of free snd-1 with its native HS chains and that LN-332 binds plasma membrane resident snd-1 with high affinity and specificity through HS chains. LN-332 has been identified as a binding factor between ECM and HPV. Since LN- 332 intrinsically lacks HS chains but contains HS-binding domains, it seems more likely that HS chains bridge the gap between HPV and LN-332, and this could account for the co-localization in the ECM of HPV and LN-332 **Figure 2A** and **B** [15, 25, 28].

Models have been developed for explaining the mechanism of HPV release from HSPGs:


None of these models addressed ECM-bound virus release and infectivity. Still, a recent study suggested another model: high-speed processing of normal HSPGs from HPVs to gain infectious entry into keratinocytes. Inhibited viral release from ECM, cellular access, and infectiousness from ECM can be easily explained by this model. Proteases and heparanase play an essential role in HPV release from primary receptors [28].

This model, in which HPV usurps the processing of HSPGs and GFR/RTK signaling to promote infection, reflects the role of epithelial injury in mediating papillomavirus infections in vivo. Consequential breaks in epithelial damage result in an influx of HRs and cytokines involved in syndecan dissemination. Snd-1 expression is enormously increased in keratinocyte migration and proliferation, and free syndecans present in wound fluids regulate the activity of GFs and MMPs. Thus, HPVs appear to have evolved to control the epithelial wound to gain access to mitotically active basal cells and take advantage of the factors and architecture that favor infection. Many intracellular pathogens of the female genital tract (HIV, herpesviruses, chlamydia, Neisseria) interact with cellular HSPGs. Thus, it is tempting to infer that these pathogens also appropriate the biology of HSPGs during infection. In summary, there is new knowledge about the transmission of oncogenic HPVs, and high-speed pathogens usually function during infection of their hosts.

#### *The Importance of the Extracellular Matrix in HPV-Associated Diseases DOI: http://dx.doi.org/10.5772/intechopen.99907*

These findings may point to additional targets for preventing HPV infections and potentially those of similarly acting pathogens **Figure 2A** and **B** [28].

Upon contact with HSPGs, the HPV capsid undergoes conformational changes assisted by extracellular cyclophilin B and cleavage of the capsid protein L2 by furin. This leads to a loss of affinity for HSPGs and binding to different secondary receptors. Identification of the internalization receptor is ongoing, but α6 integrins, EGFR, and the tetraspanin family may be involved. The entry kinetics of HPV appears to be asynchronous and slower than for most other viruses, but the cause is not yet fully known. Some research suggests that it may be linked to the cell cycle phase or the involvement of multiple receptors. Subsequently, the virus is internalized through endocytosis, but there are conflicting reports on different HPV types and cells **Figure 2A** and **B** [15, 20].

The main goal in developing microbicides against HPV (or any viral infection) is to block the interaction between the virion proteins and the cell surface receptors used by the virus to gain entry into cells. As discussed earlier, the initial binding of HSPG is an important step for successful HPV internalization, as its inhibition has been shown to decrease HPV infection in vitro and in vivo. Since several different viruses use HS chains as the initial receptor/corrector to bind to the cell surface, it is considered a viable drug target, particularly about producing a microbicide with broad-spectrum protection against a range of HPVs (as well as other sexually transmitted viruses). Efforts in this direction will aid the development of antiviral drugs that are effective not only on many existing viruses but also on unpredictable emerging viruses [15, 20].

#### **6.2 Pathogenesis and immunity**

Once the basal layer of keratinocytes is reached, the virus can remain latent or take advantage of cell differentiation to replicate and initiate the disease. As for the host immune response, it is known that it can eliminate the infection or silence it (latent). The virus can persist with low infectivity, survive a weak immune response (persistence) and later induce pathology [19, 22, 24].

The mark of HPV infections is the effective evasion of innate immune recognition. The viral productive life cycle is exclusively intraepithelial, there is no viremia, no virus-induced cytolysis or cell death, and viral replication and release are not associated with inflammation. HPV globally decreases innate immune signaling pathways in infected keratinocytes, and pro-inflammatory cytokines are not released, activation signals to Langerhans cells, cell migration from and recruitment of stromal dendritic cells (DCs) and macrophages are absent or inadequate [19, 24].

Despite the high impact of HPV protein expression on cellular homeostasis, these viruses are incomplete carcinogens. Therefore, further changes in the cell and its microenvironment are required for tumor establishment and progression. This process includes dysregulation of the ECM. In some cases, changes in the levels and activity of defined ECM components have been experimentally associated with the expression of HPV-specific proteins, suggesting the direct involvement of the virus in the downregulation of these factors. Other studies, mainly those performed with clinical specimens, have identified changes in the levels of ECM molecules during the progression of HPV-related diseases [21, 29].

#### **7. ECM alterations in HPV-associated diseases**

The natural history of cervical cancer development begins with a precursor lesion called cervical intraepithelial neoplasia (CIN). CIN 1 CIN 2 lesions are

classified as productive lesions, in which the viral cycle is complete. On the other hand, CIN 2 lesions and CIN 3 lesions are potential precursors of cervical cancer. The development of these lesions is mainly caused by persistent infection with oncogenic types of HPV. Intraepithelial lesions show low to moderate histological changes and may regress spontaneously within 1 to 2 years. In persistent high-risk HPV infections, high-grade precancerous lesions (CIN 2 and CIN 3) may develop within 3 to 5 years. Morphologically, CIN 3 (carcinoma in situ-CIS) represents a heterogeneous disease and can be considered a precancerous lesion of a more advanced cervical cancer **Figure 2C** [21, 30].

HPV infection has been associated with several changes in tissue organization and architecture, including downregulation of the expression and activity of MMPs. It has been shown that up-regulation of MMP-2 and MMP-9 expression and activity are associated with high-grade CIN, and their respective inhibitors have reduced expression levels in these lesions

[4, 21]. On the analysis of MMP-11 and MMP-12 expression in high- and low-grade lesions, it was shown that both might be associated with the appearance of cancer precursor lesions and suggested that increased expression of these proteins may be considered an early event during the development of preneoplastic cervical lesions **Figure 2C** [21].

Alterations in other components of the ECM have also been explored in the context of cervical tissue transformation. Expression of the 67-kDa laminin receptor (LR67) was found to progressively increase with CIN grade. LR67 is associated with CIN 2 to 3 and can be considered a marker of cell proliferation in cervical tissue. These authors also demonstrated that combined analysis of LR67 and vascular endothelial growth factor-C (VEGF-C) could improve the clinical detection of high-grade CIN **Figure 2C** [21].

HPV infection has also been linked to a percentage of lesions in other epithelia of the anogenital tract, including the vulva, vagina, and anus. Vulvar, vaginal, and anal cancer precursor lesions are called vulvar intraepithelial neoplasia (VIN), vaginal intraepithelial neoplasia, and anal intraepithelial neoplasia (AIN), respectively. As with cervical precursor lesions, VIN and AIN also progress through degrees of epithelial transformation. The analysis of the expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 by IHC in samples of VIN 1, 2, and 3 and invasive vulvar carcinoma suggested that overexpression of MMP-2, MMP-9, and TIMP-2 proteins may be related to the progression of VIN to invasive carcinoma **Figure 2C** [21].

#### **8. ECM composition in HPV-associated cancers**

The loss of regulation of ECM remodeling by unbalanced proteolysis plays a significant role in the loss of tissue homeostasis and pathological processes such as cancer. In cancer, this event may impact tissue tension and release chemotactic fragments of ECM components that influence the local microenvironment. It promotes cell migration and recruitment of stromal, endothelial, and immune cells to the tumor vicinity. The heterogeneous association of cancer cells and other elements observed in the tumor microenvironment, such as inflammatory infiltrate, endothelial cells, and tumor-associated fibroblasts, should also be explored to understand ECM remodeling changes fully [4, 21].

The most investigated proteases present in this process are the MMPs, as described previously. These MMPs play a central role in basement membrane breakdown and cell invasion and neoangiogenesis, and metastasis. Excess MMP activity generates topographical changes in the tumor microenvironment through

#### *The Importance of the Extracellular Matrix in HPV-Associated Diseases DOI: http://dx.doi.org/10.5772/intechopen.99907*

modifications associated with proteolysis of the structural skeleton of the ECM. Indeed, linearization and thickening and/or degradation of specific collagen are common events observed in areas of epithelial tissue adjacent to tumor-associated blood vessels where cancer cells invade. The activity of MMPs also regulates cell migration and release of ECM fragments with biological functions, such as growth factors. The crucial role of specific MMPs in the process of carcinogenesis has set an objective task for researchers to explore the potential of MMPs as therapeutic targets. However, the use of broad-spectrum MMPIs offered no clinical advantages due to dose-limiting side effects [4, 21, 31].

Several authors have studied ECM alterations in both structural and remodeling molecules in invasive cervical cancer. More specifically, changes in the expression/ activity of galectins, collagens, proteoglycans, laminins, fibronectins, integrins, proteases, and regulators have been observed in cervical cancer samples derived cell lines [21].

The claudins (CLDNs) and occludins are families of proteins associated with tight junction establishment, epithelial cell polarity, and intercellular permeability. The expression levels of CLDN type 1, 2, 4, and 7 proteins are increased in HSIL lesions and invasive cervical tumors when compared with normal cervical tissues. On the other hand, occludin is expressed in the basal cell layer of normal cervical tissues. Its protein level is reduced in invasive cervical carcinomas compared with CIN samples. Thus, changes in cell adhesion and ECM structure are a common early feature in cervical cancer progression [21].

Expression of the high-affinity laminin-binding protein 67-kd (67LR) has also been shown to increase both CIN and cervical cancer samples compared to normal cervical tissues.

Versican, an ECM proteoglycan, was evaluated in cervical cancer samples by IHC (Immunohistochemistry) and situ hybridization (ISH). Expression of high levels of versican in tumor stromal myofibroblasts was associated with a lower frequency of CD8-positive T cells, more significant invasion and depth of tumor parametrial infiltration, and no change in cervical cancer survival. Interestingly, the beta-galactoside- galectin-1 binding protein was more expressed in stromal cells adjacent to cancer when compared to normal stroma associated with the cervical tissue. Furthermore, a higher expression level of galectin-1 was associated with increased local tumor recurrence and poor cancer-specific survival in patients with stage I-II cervical tumors after radiation treatment. However, it could not predict distant metastasis [21].

Laminin-1 and smooth muscle actin proteins (SMA) showed increased expression, mainly in the surrounding cervical tumor stroma compared to the normal cervical stroma. In addition, tumor cells especially expressed laminin integrin a6b4 receptors, and tumor-associated fibroblasts showed higher levels of laminin-a1 and laminin-b1 and lower levels of laminin-5, fibronectin, collagen III, TIMP-1, and the hyaluronic acid receptor CD44 when compared to normal fibroblasts. Finally, MMP-7 and MMP-9 expression has been shown to correlate with CD44 expression in skin cancer cells [21].

The data discussed above show that ECM composition and function alterations are common in HPV-associated lesions and cancers. Taken together, these changes highlight the complex molecular pathways that lead from initial infection to disease. For example, analysis of the impact of HPV on components of the MMP family has produced a spectrum of data that could be used for disease diagnosis and identification of targets for therapy. The data summarized here also show that, concerning the mechanisms by which HPV modulates MMP expression and activity, there is still much to learn. Finally, alterations in the ECM may impact the microenvironment of HPV-infected tissue, affecting the

recruitment of inflammatory infiltrates, altering the fate of different cell populations present in the tumor, and ultimately determining disease progression and prognosis [4, 21].

More studies are needed to understand how HPV proteins affect the dynamic balance of the ECM in associated pathologies. This will help us to understand the disease genesis and define more appropriate clinical interventions. Importantly, the vast majority of the ECM changes described have also been observed in HPV-independent tumors. Therefore, understanding the virus-mediated molecular events that lead to ECM disruption may be useful for understanding the basic mechanisms of carcinogenesis and developing more general antitumor approaches [21, 32].

#### **8.1 Heparanase and heparan sulfate/syndecan-1 axis**

As mentioned earlier, syndecans are a family of four HSPGs that can be soluble or membrane-anchored. Syndecan 1 (SDC-1) is the one that has been most studied and is found mainly on the surface of epithelial cells. Loss of syndecan-1 and E-cadherin from the cell surface is known to be a critical step in the transition to epithelial neoplasia [18, 33].

The heparanase/SDC-1 axis is a key point regulating cell signaling when tumor cells are present and in their respective microenvironment. This heparanase/SDC-1 axis modulates cell proliferation as it regulates hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF). HGF is a cytokine that increases the growth, motility, angiogenesis of tumor cells. Free syndecan-1 binds to secreted HGF and ultimately facilitates a paracrine and autocrine signaling cascade through the cellular receptor c-Met. It binds to syndecan-1 in the ECM, stimulating angiogenesis and tumor invasion via the Erk pathway secreted VEGF. To regulate gene expression, heparanase and syndecan-1 can also be transported to the nucleus to regulate gene expression. Overall, nuclear HS chains and syndecan-1 are considered anti-proliferative and decrease gene transcription. Specifically, highly sulfated HS chains are mostly inhibitory, contrasting with free syndecan-1 that promotes angiogenesis, proliferation, and cell invasion. Conversely, heparanase present in the nucleus increases gene expression and promotes growth. Thus, syndecan-1 expression is considered a prognostic tool in solid and hematologic malignancies. A high level of stromal expression of syndecan-1 is a negative prognostic factor, and low levels of epithelial expression are indicators of advanced disease and poor prognosis. Loss of syndecan-1 is believed to represent cancer cells with high malignant and metastatic potential [4, 17, 33].

#### **9. Heparanase**

Heparanase is an endo-β-D-glucuronidase that cleaves the side chains of HS. This results in the release of bioactive HS fragments from the ECM and in structural changes. Over the past two decades, much work has been dedicated to studying the role of heparanase in cancer biology. Various analysis methods have revealed that heparanase expression is increased in numerous cancers, including hematological malignancies, carcinomas, and sarcomas. In addition, elevated heparanase levels are associated with reduced postoperative survival, increased angiogenesis, and metastasis. All of these factors have triggered the development of heparanase inhibitors as novel anti-cancer agents [4, 17, 33].

Mammalian cells express a single functional heparanase enzyme, heparanase-1. Heparanase-2, a homolog of heparanase, has been cloned but cannot perform HS

#### *The Importance of the Extracellular Matrix in HPV-Associated Diseases DOI: http://dx.doi.org/10.5772/intechopen.99907*

degrading activity. It can, however, regulate the activity of heparanase-1. The heparanase structure contains a TIM barrel fold, which incorporates the enzyme's active site and a distinct C-terminus domain with non-catalytic properties and is involved in the non-enzymatic signaling and secretion function of heparanase [17, 33].

The expression of heparanase is under tight regulation. In non-cancer cells, the heparanase promoter is constitutively inhibited, secondary to promoter methylation and p53 activity, which suppresses heparanase gene transcription by binding directly to its promoter. In addition, further regulation occurs during post-translational processing. Cathepsin L is required for post-translational activation of heparanase, and cathepsin L inhibitors prevent the formation of active heparanase. In non-pathological states, heparanase expression is restricted primarily to platelets, activated white blood cells, and the placenta with little or no expression in normal connective tissue or epithelium. In addition, it is most active under acidic conditions (pH 5–6), during inflammation, or within the tumor microenvironment [17, 33].

#### **9.1 Role of heparanase in HPV viral pathogenesis**

As already mentioned, increased expression of heparanase in numerous malignancies is associated with poor prognosis. And the direct role of this enzyme in neoplasms was confirmed when inhibition/silencing of heparanase in cancer cell lines resulted in a significant reduction in the invasive phenotype of the cells [4, 33].

The primary enzymatic activity of heparanase in the cleavage of the HS side chains of HSPGs and consequent release of growth factors and cytokines give rise to cell signaling pathways capable of inducing ECM remodeling. Heparanase can also release proangiogenic growth factors bound to heparan sulfates such as bFGF, HGF, PDGF, and VEGF, from the extracellular matrix to promote endothelial cell migration and proliferation indirectly. Tumors with high levels of heparanase have significantly higher microvascular density than tumors with low levels of this enzyme [4, 33].

Several heparanase capabilities have been demonstrated in the progression of cancers. These include increased cell proliferation via insulin, increased resistance to chemotherapy, expression of mesenchymal markers, increased autophagy, increased cell adhesion, and even a procoagulant function [26, 33].

Focusing on human papillomavirus infection, we remain to understand what role heparanase plays in HPV viral pathogenesis. Recent studies show that HPV16 particles bind to the ECM through HS chains. Reducing the activity of matrix metalloproteinases and heparanase drastically reduces the release of viruses from the ECM, which results in the loss of viral uptake and infection of human keratinocytes. On the other hand, exogenous heparanase promotes viral release and disease. This phenomenon may be necessary for explaining, especially at the wound site, the host healing response and RTK/GFR signaling that increases HS release, allowing an ideal environment for HPV to infect keratinocytes **Figure 2B** [3, 34].

Other research work has shown the significance of the HPV E6 gene in HPVheparanase interaction in head and neck squamous cell carcinoma (HNSCC). The HPV E6 gene interacts with p53, decreasing its activity, leading to increased expression of heparanase since p53 is a potent inhibitor of transcription of this enzyme; expression of p21, a downstream component of the p53 pathway, correlates positively with heparanase expression on tissue section staining, confirming the heparanasep53 signaling event. Polysaccharide segments of the HS chains serve as attachment sites for many growth factors, cytokines, chemokines, and various bioactive ligands. Cleavage of the HS chains by heparanase releases these bioactive factors increasing tumor invasion and malignancy in the case of HPV-induced HNSCC16 [3, 34].

#### **10. The potential of future targeted therapies**

Current statistics on the prevalence of HPV and cervical cancer alone underscore the need for alternative therapies to prevent and treat HPV infections. Existing HPV vaccines, while highly effective, do not offer protection against all high-risk HPV types, let alone low-risk HPV types. Furthermore, these vaccines are purely prophylactic and are not easily accessible to women in low- and middle-income countries (LMIC). For these reasons, alternative therapies that have broad-spectrum protection against HPV types, as well as other sexually transmitted infections, are worth exploring.

Since heparanase is an influential enzyme in tumor progression, it is the ideal therapeutic target. And since it is typically not expressed in healthy tissue, the side effects of its inhibition would be minimal. A series of heparanase inhibitors have been studied and produced, namely heparin, logically because it is a molecule close to HS. However, it is limited as an anti-cancer therapy because of its anticoagulant effects. Similarly, when LMWH (Low molecular weight heparins) was tried as an alternative for the same effect, but the results were controversial [4, 17, 33].

In addition to heparins, strategies have been developed to inhibit heparanase, such as HS mimetic molecules, modified heparins, etc. HS mimicking molecules have lower anticoagulant activity and greater selectivity for heparanase than heparin, allowing a wider therapeutic window. Some inhibitors already investigated are PI-88 (Mupaphosphat), PG545, SST0001 (Roneparstat), M402 (Necumparanib). In addition to HS mimetics, the non-steroidal anti-inflammatory aspirin, widely suggested to have a long-term anti-cancer effect, binds directly to the active site of HPSEs, inhibiting their enzymatic activity and preventing HPSE-dependent cancer cell migration, metastasis, and angiogenesis both in vitro and in vivo. As HPSE inhibition may seem attractive for cancer mitigation, it is important to note the critical role of the enzyme in the infiltration of activated NK cells in primary tumors and metastasis sites. Consequently, potential inhibitors must be highly selective and thoroughly investigated to limit adverse effects [4, 17, 33].

As we see, several heparanase inhibitors have entered clinical trials for various cancers, but none yet for viral diseases. Early results already suggest that using heparanase as a target may have rewarding benefits in controlling many viral infections, and thus the inhibitors listed above may have beneficial effects. Now, the promise that inhibiting this enzyme or the upstream effector, p65, could provide a novel therapeutic intervention to treat the disease. Overall, emerging knowledge about heparanase as an essential regulator of viral infections and associated morbidities could one day make a broad-spectrum antiviral drug a real possibility [34].

#### **11. Conclusion**

In this paper, we have discussed the extracellular matrix's very complex and important role in developing and progressing cancers and, more specifically, in human papillomavirus infection. Indeed, in recent years the ECM has been increasingly considered a crucial component in physiological processes such as cell proliferation, adhesion, and migration. The perspective of cancer and its progression has also changed. We no longer consider a disease caused only by dysregulated cell proliferation. Still, we give importance to the microenvironment and its changes and adaptations to cellular stress. In-depth knowledge of the ECM components, their complex interactions, and their constant and dynamic remodeling during all stages of tumor development brings some hope in developing promising targeted

*The Importance of the Extracellular Matrix in HPV-Associated Diseases DOI: http://dx.doi.org/10.5772/intechopen.99907*

therapies to combat these pathologies. Likewise, knowledge of the components of viral particles and host cell entry factors and their specific interactions may allow the design of efficient antiviral strategies [15, 22].

Indeed, the role of HSPGs in HPV interaction with the ECM is undisputed and developed throughout the work. The enzyme heparanase, which we know has a significant impact on tumor progression and metastasis. Thus, as the influence of the tumor microenvironment on cancer progression becomes more evident, the focus on inhibiting enzymes that degrade HSPGs highlights an approach to maintain normal tissue architecture, inhibit tumor progression, and block metastasis. This review addresses the role of these enzymes, namely heparanase, in the context of the tumor microenvironment and their promise as a therapeutic target for cancer treatment, particularly cervical cancer [15, 17].

#### **Acknowledgements**

The Authors would like to acknowledge the Instituto de Investigação Científica Bento da Rocha Cabral for support.

### **Conflict of interest**

The authors declare that they have no competing interests.

### **Acronyms and abbreviations**


### **Author details**

Joana Sampaio1 , Joana Ferreira1,3,5, Ana Carolina Santos1,3,5, Manuel Bicho1,3,5 and Maria Clara Bicho1,2,3,4\*

1 Faculty of Medicine, University of Lisbon, Lisboa, Portugal

2 Institute of Preventive Medicine and Public Health and ISAMB – Environmmental Health Institute, Faculty of Medicine, University of Lisbon, Lisboa, Portugal

3 Genetics Laboratory and ISAMB – Environmmental Health Institute – Ecogenetics and Human Health Unit, Faculty of Medicine, University of Lisbon, Lisboa, Portugal

4 Dermatology Research Unit, Instituto de Medicina Molecular, University of Lisbon, Lisboa, Portugal

5 Instituto de Investigação Científica Bento da Rocha Cabral, Lisboa, Portugal

\*Address all correspondence to: mcbicho@medicina.ulisboa.pt

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*The Importance of the Extracellular Matrix in HPV-Associated Diseases DOI: http://dx.doi.org/10.5772/intechopen.99907*

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### *Edited by Rajamanickam Rajkumar*

Although it is preventable and curable, cervical cancer is the fourth most common form of cancer among women worldwide. As such, the World Health Organization adopted a Cervical Cancer Elimination Initiative, which aims to eliminate cervical cancer by 2030. This book discusses plans, programs, strategies, solutions, research, and revolutions necessary to achieve this goal. Chapters cover such topics as epidemiology, HPV vaccination, screening and treatment, and prevention and control.

Published in London, UK © 2021 IntechOpen © HeitiPaves / iStock

Cervical Cancer - A Global Public Health Treatise

Cervical Cancer

A Global Public Health Treatise

*Edited by Rajamanickam Rajkumar*