Introduction

Cervical cancer, a significant global health concern, remains a leading cause of cancer-related mortality among women worldwide. While progress in screening and vaccination programs has demonstrably reduced the incidence of cervical cancer caused by Human Papillomavirus (HPV), a substantial burden persists, particularly in resource-limited settings. Understanding the intricate interplay of molecular alterations driving cervical carcinogenesis is paramount for developing more effective prevention, diagnosis, and therapeutic strategies (1). This review delves into the critical relationship between the loss of retinoblastoma protein (p16) and its downstream target, the retinoblastoma protein (Rb), and the integration of HPV DNA within precursor lesions of the cervix. We will explore the molecular mechanisms underlying this complex interaction, its role in promoting cellular transformation, and its implications for risk stratification and personalized treatment approaches (2).The pathogenesis of cervical cancer is a multistep process, typically initiated by persistent infection with high-risk HPV types, primarily HPV16 and HPV18. These oncogenic HPV types encode proteins, notably E6 and E7, which disrupt crucial cellular regulatory pathways, driving uncontrolled cell proliferation and ultimately leading to malignant transformation (3). The E6 protein binds to and ubiquitinates p53, a key tumor suppressor protein known as the “guardian of the genome.” This ubiquitination targets p53 for proteasomal degradation, effectively silencing its tumor suppressor functions, including cell cycle arrest, apoptosis, and DNA repair (4). Concurrently, the E7 protein binds to and inactivates Rb, a critical regulator of the G1/S cell cycle transition. Rb normally binds to transcription factors like E2F, preventing their activation and thereby halting cell cycle progression (5). When Rb is phosphorylated by cyclin-dependent kinases (CDKs) upon E7 inactivation, E2F is released, enabling the transcription of genes required for S-phase entry and promoting uncontrolled proliferation (6).

The inactivation of both p53 and Rb represents a pivotal event in cervical carcinogenesis. Loss of p53 function leads to genomic instability, increased mutation rates, and impaired DNA repair mechanisms, further accelerating the accumulation of oncogenic alterations. Rb inactivation removes a critical brake on the cell cycle, allowing cells with accumulated DNA damage to proliferate unchecked (7). The synergistic effects of p53 and Rb inactivation create a permissive cellular environment for further genomic alterations, ultimately driving the progression from precancerous lesions to invasive cancer (8).

While HPV E6 and E7 proteins exert a dominant influence on cervical carcinogenesis, the cellular context plays a significant role in shaping the disease trajectory. A particularly important aspect of this context involves the expression and function of p16INK4a, a cyclin-dependent kinase inhibitor that acts as a critical gatekeeper at the G1/S checkpoint (9). p16INK4a binds to and inhibits CDK4 and CDK6, preventing the phosphorylation of Rb and thus maintaining Rb’s tumor suppressor function. Loss of p16INK4a expression, often due to epigenetic silencing through promoter hyper methylation, results in Rb hyper phosphorylation and subsequent E2F activation, contributing significantly to uncontrolled cell proliferation in cervical precursor lesions (10). The loss of p16, therefore, creates a “perfect storm” for malignant transformation, amplifying the effects of E6 and E7(11).

The interplay between p16/Rb loss and HPV integration is complex and bidirectional. The integration of HPV DNA into the host genome can directly disrupt the expression of tumor suppressor genes, including p16. Integration at specific genomic loci can lead to the physical disruption of the p16 gene, preventing its transcription (12). Furthermore, HPV integration can activate oncogenes or disrupt the function of other tumor suppressor pathways, indirectly contributing to p16 loss. Conversely, loss of p16 can create a cellular environment more susceptible to HPV integration (13). The disruption of the G1/S checkpoint by p16 loss increases genomic instability and allows for more efficient integration of HPV DNA, further accelerating the carcinogenic process (14).

The significance of p16/Rb loss and HPV integration extends beyond the development of precancerous lesions. These alterations are also predictive of disease progression and response to therapy. Patients with cervical cancer exhibiting loss of p16 expression or high levels of HPV integration often have a poorer prognosis (15). Moreover, the presence of p16 loss can influence the response to certain chemotherapeutic agents. Understanding these complex interactions is critical for developing targeted therapies that can effectively address the molecular drivers of cervical cancer (16).

This review will delve deeper into the molecular mechanisms linking p16/Rb loss and HPV integration, exploring the epigenetic and genetic factors that contribute to these alterations. We will discuss the role of various signaling pathways, including those involving cyclin-dependent kinases, and the impact of these alterations on cell cycle regulation, DNA repair (17), and apoptosis. Furthermore, we will examine the clinical implications of p16/Rb loss and HPV integration, focusing on their prognostic value, their role in treatment response, and their potential as therapeutic targets (18).

Material and methods

This narrative review systematically gathered relevant literature by querying major electronic bibliographic databases, including PubMed/MEDLINE, Scopus, Web of Science, and Google Scholar. The search strategy employed a combination of medical subject headings (MeSH terms) and free-text keywords to maximize capture of relevant studies pertaining to cervical carcinogenesis, HPV oncogenes (E6/E7), p16, and the Rb pathway. Specific search strings included combinations such as: “cervical cancer” AND “HPV” AND “p16 expression,” “HPV E7” AND “Rb degradation,” and “CIN” AND “p16/Rb axis.” Only articles published in the English language, with full-text availability, and indexed before January 1, 2026, were considered for inclusion. Exclusion criteria primarily involved case reports, editorials, non-peer-reviewed abstracts lacking full methodology, and studies focusing exclusively on non-human models or vaccines without detailing molecular pathogenesis.

Results

p16/Rb Pathway Dysregulation in Cervical Precursor Lesions

Cervical cancer remains a significant global health disparity, persisting as a leading cause of cancer-related mortality, particularly in low- and middle-income countries, despite the existence of effective prophylactic vaccines. The overwhelming causality in virtually all cervical cancer cases is directly attributable to persistent infection by high-risk human papillomavirus types, most notably HPV-16 and HPV-18(19). The progression from initial hrHPV infection to the development of high-grade squamous intraepithelial lesions, or cervical intraepithelial neoplasia grade 3, and ultimately invasive carcinoma, constitutes a well-defined, multi-step carcinogenic cascade. This process is fundamentally driven by the integration of the viral genome into the host cell’s DNA, leading to the constitutive expression of two primary oncogenes: E6 and E7(20). These viral proteins act as master disruptors of essential host cell cycle and apoptotic machinery. E6 targets the tumor suppressor p53 for proteasomal degradation, thereby abrogating cell cycle arrest and apoptosis in response to DNA damage. Concurrently, E7 targets the retinoblastoma protein Rb, the gatekeeper of the G1 to S phase transition. By binding Rb and promoting its degradation, E7 effectively releases the E2F transcription factors, forcing the cell into uncontrolled proliferation a state termed oncogene-induced replication stress (21). The critical diagnostic and prognostic hallmark reflecting this disruption in the Rb pathway is the dramatic and sustained upregulation of the CDKN2A-encoded protein p16INK4a. In an effort to compensate for E7-mediated Rb inactivation, the cell overproduces p16, which serves as a robust surrogate biomarker for active high-risk HPV oncogene expression and definitive histologic transformation, making p16 immunohistochemistry a cornerstone in assessing the biological significance of underlying HPV infection in precursor lesions (22) (figure 1).

Figure 1. cervical carcinogenesis pathway

 The central premise of current research in cervical carcinogenesis pivots on the precise molecular dialogue between these viral drivers and the hosts genetic response, specifically focusing on the p16/Rb axis within the context of viral integration. While p16 overexpression serves as an excellent marker for established hrHPV activity, the mechanistic depth required for precision diagnostics necessitates a deeper interrogation of the host-pathogen interface (23). Therefore, the objective of this investigation is to meticulously examine the reciprocal relationship between the functional status of the p16/Rb pathway and the molecular events associated with HPV genome integration in CIN lesions. Specifically, we aim to correlate patterns of p16 expression, which indicate the degree of Rb pathway perturbation, with the physical state of the HPV DNA namely, whether it remains episomal or becomes integrated across the spectrum of pre-invasive disease (24). Understanding how the intensity of E6/E7 activity translates into the magnitude of p16 induction and whether p16 integrity can predict the transition toward stable, high-risk integration will provide refined criteria for triaging patients with persistent HPV positivity (25).

 The Viral Oncogenic Mechanism: HPV E6 and E7 Functions

The oncogenic potential of high-risk Human Papillomaviruses (HPV), particularly types 16 and 18, is fundamentally mediated by the sustained expression of two key viral proteins: E6 and E7, which act in concert to subvert critical host cell cycle checkpoints and apoptotic pathways. The E6 protein executes its primary transforming function through the targeted degradation of the tumor suppressor protein p53, often referred to as the guardian of the genome. E6 achieves this by binding directly to the cellular ubiquitin ligase E6AP (UBE3A), forming a complex that recognizes p53 as a substrate for proteasomal destruction (26). This binding event bypasses the normal regulatory mechanisms that stabilize p53 in response to cellular stress, DNA damage, or oncogenic signaling. The resulting dramatic decrease in functional p53 levels abolishes the cell’s ability to enforce cell cycle arrest at the G1/S or G2/M checkpoints, prevents DNA repair processes dependent on p53 transcriptional activity, and crucially, inhibits p53-mediated apoptosis in the presence of accumulating genomic aberrations (27). The loss of p53 function licenses the cell to continue proliferation despite significant genotoxic stress, establishing the first critical component of cellular immortalization and genomic instability necessary for malignant progression. Furthermore, E6 has been shown to interact with other cellular targets, including PDZ domain-containing proteins, contributing to disruption of cell polarity and enhanced telomerase activity, though its central role remains the unchecked elimination of p53 (28).

The second, equally vital component of the HPV oncogenic mechanism is executed by the E7 protein, which specifically targets the Retinoblastoma protein (Rb), the master regulator of the G1 to S phase transition. E7 binds to the pocket domain of the Rb protein with high affinity. This binding event mimics the action of host-cell cyclin-dependent kinases (CDKs) that normally phosphorylate Rb in late G1. Phosphorylation of Rb causes its conformational change, leading to its dissociation from the E2F family of transcription factors (29). In the context of E7-mediated inactivation, the binding of E7 directly sequesters Rb or promotes its degradation, effectively liberating the bound E2F transcription factors. Free E2F then translocates to the nucleus to drive the transcription of genes essential for DNA synthesis and cell cycle progression, such as cyclins E and A, DHFR, and CDC2. By constitutively activating the E2F pathway, E7 forces the cell into the S phase, independent of external growth signals, thereby overriding the primary brake on cellular division. The synergistic effect of E6 neutralizing p53 preventing apoptosis in response to hyper proliferation and E7 liberating E2F driving hyper proliferation constitutes the prototypical “Double Hit” hypothesis against major tumor suppressor pathways (figure 2) (30). This dual disruption of the p53 and Rb axes provides the fundamental molecular underpinning for the uncontrolled cellular growth and accumulation of damage characteristic of HPV-driven carcinogenesis, particularly in precursor lesions like Cervical Intraepithelial Neoplasia (CIN) (31).

Figure 2. HPV E6/E7 double-hit mechanism

he Host Cell Cycle Regulator: p16INK4a and Rb Pathway

The p16INK4a protein serves as an indispensable guardian of the host cell cycle, operating upstream of the Retinoblastoma (Rb) protein within the critical G1 restriction point mechanism. Functionally, p16INK4a is a potent cyclin-dependent kinase inhibitor (CKI) belonging to the INK4 family, specifically designed to inhibit the activity of CDK4 and CDK6 complexes. These kinases, when active and bound to Cyclin D, are responsible for initiating the hyper phosphorylation cascade targeting the Rb protein (32). In its normal, unperturbed state, the cell maintains sufficient p16INK4a expression, which sequesters and inactivates CDK4/6. This sustained inhibition prevents the phosphorylation of Rb, thereby maintaining Rb in its active, hypo phosphorylated state. In this active configuration, Rb tightly binds to and sequesters the E2F family of transcription factors, effectively halting the transcription of genes required for DNA replication and entry into the S phase (33). This checkpoint acts as a crucial fail-safe, ensuring that the cell only proceeds into proliferation when appropriate growth signals are present and the genome is intact. The preservation of Rb in its active, growth-repressive conformation, mandated by functional p16INK4a, is therefore paramount for preventing premature cell cycle entry and the propagation of genomic errors. The pathway’s integrity is constantly challenged by oncogenic stressors, such as uncontrolled expression of cyclins or viral oncoproteins, making the status of p16INK4a expression a critical determinant of cellular fate; its functional loss removes the primary inhibitory constraint on CDK4/6 and thus unleashes E2F-driven transcription, overriding the G1 safeguard (34).

In the context of precancerous lesions, such as those induced by high-risk HPV, the integrity of this regulatory axis is frequently compromised through various mechanisms that lead to the functional inactivation or loss of p16INK4a expression, often preceding or accompanying the direct action of viral proteins like E7. While HPV E7 directly binds and inactivates Rb, rendering p16INK4a somewhat redundant for immediate G1 release, the independent epigenetic silencing of the CDKN2A locus which encodes p16INK4a represents a convergent and highly significant pathway for tumor progression, particularly in later stages or in other cancer types that mimic HPV-driven events (35). The primary mechanism for this functional loss is hyper methylation of the p16INK4a promoter region, an epigenetic modification that leads to transcriptional repression and subsequent absence of the protein. Additionally, homozygous deletions or large-scale genomic loss events encompassing the CDKN2A locus on chromosome 9p21 result in complete gene ablation (36). The loss of p16INK4a function, regardless of the upstream cause (HPV-independent mutation or methylation), results in the constitutive activation of CDK4/6, leading to Rb hyper phosphorylation, E2F release, and uncontrolled cell cycle progression. Immunohistochemically detection of p16 accumulation, paradoxically, is often used as a surrogate marker for high-risk HPV infection because the viral E7 protein inactivates Rb, which in turn triggers a compensatory feedback loop leading to the overexpression of p16 in many precursor lesions, although true loss of function via methylation or deletion represents a more definitive, HPV-independent oncogenic event driving tumor maintenance (37).

 Viral Integration and Genomic Instability

The integration of the high-risk Human Papillomavirus (HPV) DNA into the host cell genome represents a critical, non-reversible transition point in the trajectory from low-grade cervical intraepithelial neoplasia (CIN 1) to high-grade lesions (CIN 2/3) and invasive carcinoma. This event, which occurs in approximately 80% of invasive cervical cancers, fundamentally alters the viral life cycle; the viral genome is fractured, leading to the loss of the upstream regulatory E2 gene, which normally represses the transcription of the oncogenes E6 and E7(38). A particularly consequential effect is the direct interruption or dysregulation of host genes located near the integration site, most notably the CDKN2A locus encoding the tumor suppressor p16INK4a. When HPV integrates near or into this critical gene, the structural damage can lead to its functional inactivation, thereby contributing synergistically to the loss of G1 checkpoint control initiated by viral oncoproteins (39). Furthermore, the insertion often places the remaining E6/E7 expression cassette under the control of strong, constitutively active host cell promoters, leading to massive overexpression of these oncogenes, which surpasses the levels typically seen in episomal infections where viral gene expression is tightly regulated by the viral enhancer/promoter system. This elevation in E6 and E7 activity is the molecular engine driving malignant transformation (40).

The persistent, high-level expression of the HPV oncoproteins E6 and E7 following integration directly dictates the severity and progression potential of the lesion by overwhelming the host cell’s natural defense mechanisms. The E7 oncoprotein’s primary and most potent function is the binding and subsequent proteasomal degradation of the Rb protein, effectively neutralizing its ability to sequester E2F transcription factors. With Rb functionally eliminated, the cell is perpetually driven into S phase, regardless of external signals or the presence of DNA damage, creating a state of unchecked proliferation that defines high-grade dysplasia (41). Concurrently, E6 targets the p53 tumor suppressor for ubiquitin-mediated degradation, abrogating the critical G1/S and G2/M checkpoints that would normally halt cell division in response to DNA damage or cellular stress damage that is exacerbated by the integration process itself and the resulting genomic chaos. Therefore, HPV integration serves as a dual-hit event: it creates new genetic vulnerabilities by disrupting host genes and simultaneously locks in the expression of viral factors (E6/E7) at high levels that guarantee the inactivation of both the Rb and p53 pathways (42). This sustained, aggressive subversion of the two major tumor suppression networks explains why integration marks the transition to irreversible malignancy, as the cells gain the hallmark capabilities of uncontrolled growth and resistance to apoptosis necessary for invasive behavior (43) (figure 3).

Figure 3. HPV integration and the Double Hit oncogenic mechanism

 The Interplay: Synergistic Effects in Precursor Lesions