Cervical cancer and HPV infection: ongoing therapeutic research to counteract the action of E6 and E7 oncoproteins

Highlights

• Human papillomavirus (HPV) has a primordial role in the development of cervical cancer.

• Therapies currently available for cervical cancer treatment are reviewed.

• The relevance of nucleic-acid-based therapies targeting HPV functions is discussed.

• HPV oncoprotein silencing is a promising path to explore in cervical cancer treatment.

Cervical cancer is the fourth most common cancer among women worldwide and its development is mainly associated with human papillomavirus infection, a highly sexually transmissible virus. The expression of E6 and E7 viral oncoproteins deregulates cell repairing mechanisms through impairment of tumor suppressor protein functions, such as p53 or retinoblastoma protein. Although the implementation of new preventive vaccines has decreased the infection rate and cervical cancer progression, there are still many women who are affected by this pathology. Nowadays, the main treatment often requires the use of invasive techniques. From well-established strategies, like DNA vaccines and gene therapy, to innovative gene silencing technologies; different methodologies are currently under scrutiny that target the E6 and E7 oncoproteins and/or their modes of action.

Introduction

The connection between human papillomavirus (HPV) and cancer development, more specifically cervical cancer, was first established in 1983 by Harald zur Hausen and co-workers [1]. At the time, it had already been possible to correlate the development of certain carcinomas in animals with the infection of specific viruses – leading scientists to pursue the same line of thought and study HPV in cancer biopsies, given that HPV DNA had been reported in genital warts [2]. Hence, for the first time, zur Hausen was able to identify HPV type 16 and demonstrated its presence in several malignant tumor biopsies [1]. Later on, zur Hausen’s research group found the presence of HPV-16 and HPV-18 DNA in cervical cancer cell lines, as well as in cervical carcinoma biopsies [3]. The first steps to understand the development of cervical cancer were taken, and the groundbreaking work performed by the German scientist was later recognized in the form of a Nobel prize. Ever since such scientific discoveries were performed, many efforts have been made toward the establishment of new detection, screening, preventive and therapeutic methodologies against cervical cancer, mainly by exploring the crucial role of HPV in the development of this pathology.

Although the incidence of cervical cancer has been slowly decreasing with the advances in this field, it still represents one of the most common cancers among women nowadays and it is mandatory to search for new methodologies that will help to overcome this problem. To fully understand the ultimate treatment for this disease, it is first necessary to unveil the background of cervical cancer, to decode the mechanisms responsible for the development and progression of HPV infection and to explore different and promising therapeutic approaches.

Cancers are some of the deadliest pathological conditions globally. In particular, cervical cancer is considered the fourth most common cancer in women, accounting for 266 000 deaths in 2012 [4]. When consulting data collected and provided by GLOBOCAN 2018, it is possible to infer that the cervical cancer burden is higher in less developed countries [5]. Actually, in Eastern and Central Africa, this cancer is known as the primary cancer found in women. This might be strongly associated with the fact that less developed countries present very poor health resources in comparison to developed countries, perhaps leading to a late diagnosis [6]. In addition, the recent distribution of several HPV vaccines worldwide probably contributed to a decrease in HPV infection, and a consequent decrease in the number of the cervical cancer cases in countries with easy access to vaccination, such as developed countries. Given its severe implication in the development of cervical cancer, it is therefore very important to understand the biological mechanisms by which HPV operates within the cervical epithelium and further develop suitable strategies that might prevent or overturn its infection and tumorigenicity.

According to World Cancer Reports, ‘persistent epithelial infection with one or more oncogenic types of HPV may lead to the development of precancerous lesions…which may progress to invasive cervical cancer’ [6]. As a matter of fact, between 79% and 100% of invasive cervical cancer cases worldwide account with the presence of DNA belonging to high-risk HPV types, from which ˜70% are related to HPV-16 and HPV-18 7, 8. Even though HPV is directly associated with cervical cancer, other cancers have also been found to be correlated with HPV infection such as anal, vaginal, vulvar, penile and head and neck cancers [9]. Indeed, there are about 200 HPV genotypes currently documented; however, only some present carcinogenic potential and are known to have the ability of inducing cancer [10]. The fact that different HPV types are related to different clinical symptoms has led scientists to classify each HPV type according to the severity of the clinical outcomes, namely by low, intermediate and high risk. The high-risk types encompass HPV that can induce the development of tumor cells [11]. The severity of the symptoms is usually associated with the affinity presented by E6 and E7 oncoproteins within each HPV type toward the target proteins: tumor suppressors p53 and retinoblastoma protein (pRB), respectively.

HPV is a small double-stranded DNA virus, belonging to the family Papillomaviridae, and one of the most common sexually transmissible viruses worldwide. It presents a nonenveloped icosahedral structure and its genome contains eight open reading frames (ORFs) [12]. These ORFs are responsible for coding eight proteins, which can be divided into early stage (E1, E2, E4-E7) and late stage (L1 and L2). As the name implies, early proteins are the proteins to be first expressed upon virus infection of the host cell and are associated with the infection itself and the possible transformation of infected cells. By contrast, late proteins constitute the viral capsid and contribute to the spreading of the infection through the host system, via the release of virus particles within the superficial epithelial cells [12].

Figure 1a illustrates the normal cell repairing pathway. Viral infection with HPV begins when the virus can penetrate the cervical epithelium through micro-abrasions. Then, the E1 and E2 protein expression leads to regulation of the viral replication within infected cells, resulting on the expression of other early-stage proteins. At this point, E5, E6 and E7 oncoproteins begin to be expressed, contributing to cell survival and uncontrolled proliferation, as presented in Fig. 1b 13, 14, 15. Such a carcinogenic mechanism is strongly dependent on the HPV ability in mainly expressing E6 and E7 viral oncoproteins. Both proteins are known to interfere with cell-cycle regulation, affecting the signaling pathways for cell repair and apoptosis 13, 14.

The E6 protein can induce tumor suppressor p53 degradation, hindering its function as an apoptosis signaling cascade regulator, through E6AP protein binding. The latter is unable to bind itself to p53, in normal circumstances. However, in the presence of E6 protein, E6AP and E6 form a protein complex that can recognize and bind to p53. Such attachment can impair transcriptional activation or repression of p53-responsive promoters by preventing its binding to specific DNA motifs and ultimately lead p53 to degradation through ubiquitination, resulting in a decrease in p53 levels 13, 16. The inactivation of p53 function favors the continuous replication of damaged DNA and abnormal cell survival, which would otherwise be repaired or eliminated by apoptosis induction if p53 was expressed in normal levels (Fig. 1a) [13]. Moreover, E6 has been shown to be able to interact with other proteins that also regulate cell signaling pathways. For instance, E6 can also interact with p300, a promoter that activates p53 through acetylation in an E6AP-independent manner [17]. Also, E6 can target for degradation of other proteins involved in the signaling cascade for apoptosis, namely Bak, the adaptor molecule Fas-associated death domain (FADD) and procaspase 8 [17]. Taking these interactions altogether, the disruptive effect that E6 can exert on the regulation of cell-cycle proliferation and repair is notorious. By contrast, E7 protein is involved in the impairment of tumor suppressor pRB function. As portrayed in Fig. 1a, whereas p53 activates apoptosis, pRB functions mainly rely on the inactivation of transcription factors, such as E2F. This molecule, when freely available, stimulates cellular cycle transition to S phase. Thus, upon viral infection, E7 is expressed and binds to pRB, disrupting its complex with E2F and contributing to continuous cell proliferation (Fig. 1b) 14, 18. Moreover, E7 oncoprotein is also involved with the interference of p107 and p130 functions, two proteins that also regulate cell-cycle proliferation through E2F transcription factor binding. The ultimate inhibition of pRB, p107 and p130 by E7 viral protein contributes to the uncontrolled cell proliferation and progression to malignant transformation seen in HPV-infected cervical epithelium 14, 19. Although E6 and E7 proteins are considered the main causative agents for HPV-infected cell transformation, E5 also presents carcinogenic activity. This protein has the ability to enhance cell proliferation through interaction with epidermal growth factor (EFG), a known stimulator of cell growth, therefore contributing to tumor progression (Fig. 1b) [15].

With the evolution of HPV infection, normal cervical tissue begins to lose its normal features and cells become more undifferentiated. The progression of the disease leads to the establishment of cervical intraepithelial neoplasia (CIN), which can be mild (CIN1) or moderate (CIN2) and characterized by cytologic abnormalities in the tissue. Without suitable treatment and as a result of persistent infections, CIN2 can evolve into high-grade lesions such as severe CIN (CIN3) or invasive carcinoma [20]. Therefore, given the timeline of HPV infection and possible progression into cervical cancer, it is crucial to detect and treat the HPV infection before its evolution into more serious conditions.