Targeting HPV Oncogenes (E6/E7) For Cervical Cancer Therapy: Advances in Molecular and Natural Strategies

The broad family of non-enveloped, double-stranded circular DNA viruses known as  papillomaviruses (PVs) infects the mucosa and skin.  Despite the fact that there are over 200 different types of PV, the mechanisms of PV infection are essentially the same: Cellular binding, internalization, and virus trafficking are mediated by two viral capsid proteins, the major capsid protein (L1) and the minor capsid protein (L2). More than 150 different types of viruses make up the large family known as human papillomaviruses, or HPVs.  Nearly all cases of cervical cancer and most cases of anal, head, and neck cancer are caused by HPVs with transforming potential. HPV16 is the most researched HPV type because it causes the majority of cervical cancer cases. The process of HPV16 infection involves several steps. The virus uses an epithelium injury to get to the basal keratinocytes, which are the only cells in the epithelium that undergo mitosis ^[1].

Fig1.1 Structure of HPV

Cervical cancer is the fourth most common cancer in women globally, causing hundreds of thousands of deaths annually, predominantly in low- and middle-income countries. Decades of research have unequivocally established that persistent infection with high-risk Human Papillomavirus (HR-HPV), particularly types 16 and 18, is the necessary causative agent for virtually all cases. The transition from transient infection to malignant neoplasia hinges entirely on the integration of the HR-HPV genome into the host cell's DNA and the subsequent, uncontrolled expression of the viral E6 and E7 oncogenes. These two proteins act as molecular master switches, dismantling the host cell’s critical tumor suppression pathways ^[2].

Given that E6 and E7 are solely responsible for initiating and maintaining the cancerous state, and are not expressed in normal, healthy cells, they present an exceptionally specific and high-value target for therapeutic intervention. Modern oncology is shifting towards precision medicine, making the targeted elimination or inactivation of E6/E7 an active and urgent area of research for developing curative strategies that minimize systemic toxicity ^[3].

Fig. 1.2 Carcinoma of Cervix Stock Vector

Human papillomaviruses (HPVs) are the cause of RRP. The Papillomaviridae family includes the non-enveloped, double-stranded circular DNA virus known as HPV. Together with the early and late harbor regions, which code for proteins involved in transcription, replication, and immunological resistance, the HPV genome also includes the non-coding upstream regulatory region (URR) . The structural proteins L1–L2 are encoded by the late (L) region, which also contributes to virus assembly. Conversely, the early proteins E5, E6, and E7 change the molecular mechanisms of HPV-infected cells and function as oncogenes. To date, over 200 distinct HPV types have been identified based on their genomes, and they are classified as low- and high-risk viruses. According to reports, at least 17 high-risk types—including HPVs 16, 18, 31, 33, 35, 39, 45, 51, 52, 55, 56, 58, 59, 68, 73, 82, and 83—co-occur with other oncogenes to contribute to the onset and progression of cancers ^[4] 

The E6/E7 proteins of high-risk HPVs have been found to enhance DNA integration into the host genome, plausibly due to enhanced chromosome rearrangements in high-risk HPV-infected cells, leading to mutation and/or deletion of both viral and host genes . Furthermore, high-risk HPVs’ E6/E7 proteins have the ability to modify DNA repair pathways. Indeed, high-risk HPVs, especially HPV16, have been implicated in vulvar cancer [14], uterine cervical cancer and its precancerous (CIN) lesions [15], anal [16], and oropharyngeal cancers [17]. Low-risk HPV infections (6 and 11), however, are rarely associated with cancer and can result in benign anogenital papillomas and skin warts. Although several types of HPV may contribute to RRP, current research suggests that the low-risk HPV types (HPV6 and 11) are the most common causative factors for the condition. However, RRP has been linked to high-risk HPV infection. Low-risk HPV11 E6/E7 proteins are just as important for the stable maintenance of episomes as high-risk E6 and E7 proteins, per a study by Stephen et al. In RRP-infected individuals, HPV DNA is usually episomal (HPV infects the basal cells and develops a prolonged infection) rather than fused or integrated with host DNA. Numerous microbial agents, such as G. vaginalis and Sneathia spp., have been shown in studies to potentially aid HPV-induced infection, invasion, and multiplication ^[5].

1.1 HPV Classification and High-Risk Types

HPVs are non-enveloped, double-stranded DNA viruses with a circular genome. They are broadly classified based on their risk for oncogenesis.

Table no. 1

HR-HPV types, particularly HPV-16 and HPV-18, account for over 70% of all cervical cancer cases.

The main risk factor for adults is increased sexual activity with multiple partners. Approximately 0.7% of mothers who have HPV genital warts infect their babies. Up to 80% of newborns born to mothers with genital HPV have HPV DNA. There are three types of vertical HPV transmission: perinatal (during or immediately following birth), prenatal (during pregnancy), and periconceptional (around the time of conception). In adults, the main risk factor is increased sexual activity with multiple partners. About 0.7% of mothers with HPV genital warts pass the disease to their infants. HPV DNA is detected in up to 80% of the neonates born to mothers with genital HPV . The vertical transmission of HPV can be categorized into periconceptional (around the time of conception), prenatal (pregnancy), and perinatal (birth or immediately after that). It is known that juvenile-onset RRP is acquired during delivery by vertical transmission, either through the birth canal or through infected placenta and amniotic fluid, from the mother's anogenital site to the child's respiratory tract . Children born to mothers with active HPV infection had a significant risk (231 times higher) of juvenile RRP than children born to mothers without the virus, according to a study by Venkatesan and colleagues [6].

Fig. 1.3 Sexually transmitted infection -MAPEH G8

1.2 The E6/E7 Oncogenic Mechanism

The malignant transition occurs when the HR-HPV genome integrates into the host cell DNA, leading to constitutive, high-level transcription of the early genes E6 and E7.

5.  E6 Function: Targeting p53

The E6 oncoprotein primarily targets the host cell's major guardian of the genome, the p53 tumor suppressor protein.

• Mechanism: E6 interacts with an E3 ubiquitin ligase called E6-AP (UBE3A). The E6/E6-AP complex then specifically targets p53 for ubiquitination and rapid degradation by the proteasome.
• Outcome: The loss of functional p53 abrogates the cell’s ability to initiate apoptosis (programmed cell death) or cell cycle arrest in response to DNA damage or stress. This allows genetically damaged cells to survive and proliferate.

5. E7 Function: Targeting pRb

The E7 oncoprotein disrupts the crucial G1-to-S phase cell cycle checkpoint maintained by the retinoblastoma protein (pRb).

• Mechanism: E7 binds directly to the active, hypophosphorylated form of pRb. This binding causes the dissociation of pRb from the E2F transcription factors.
• Outcome: Free E2F factors promote the transcription of genes necessary for DNA synthesis (S-phase entry), effectively overriding the normal cellular restriction point and forcing the cell into uncontrolled division ^[7].

5. Synergy in Transformation

The concurrent inactivation of both p53 and pRb by E6 and E7, respectively, provides the optimal environment for malignant transformation: the cell gains unlimited proliferative capacity (via E7) while simultaneously losing the ability to initiate cell death or repair DNA damage (via E6) ^[8] .

Fig. 1.4 HPV and its Effects

1.3 History:

The Foundational Science (1980s–1990s)

1.3.1  The Rationale: E6/E7 as the Achilles' Heel

The entire field of therapeutic targeting of cervical cancer began with a crucial discovery in the 1980s by Harald zur Hausen: High-risk Human Papillomavirus (HPV), specifically types HPV-16 and HPV-18, is the necessary cause of nearly all cervical cancers.

• 1.1. Constitutive Expression: Unlike the transient expression of most viral genes during initial infection, the two small oncoproteins, E6 and E7, are continuously and constitutively expressed in all HPV-driven premalignant and malignant lesions. This makes them the ultimate tumor-specific antigens.
• 1.2. Oncogenic Dependency: Experiments in the late 1980s and early 1990s demonstrated that if E6 and E7 expression is silenced in cervical cancer cell lines, the cells stop proliferating and undergo senescence (growth arrest) or apoptosis (programmed cell death). This confirmed the cancer's absolute dependence on these viral proteins for survival ^{[9] .}

1.3.2. The Primary Molecular Mechanisms of E6/E7 Action

Targeting strategies are built upon understanding how E6 and E7 drive cancer:

Table no.2

5. Early Molecular Strategies and the Dawn of Immunotherapy (1990-Early 2000s)

1.3.3. First Generation Molecular Targeting: Gene Therapy

The initial attempts focused on genetically disrupting the E6/E7 mRNA or DNA, aiming to     revert the malignant phenotype.

• E2 Protein Reintroduction: In persistent HPV infection, the viral DNA often integrates into the host genome, disrupting the E2 gene. The E2 protein normally acts as a negative regulator, suppressing the expression of E6/E7. Early gene therapy trials (mid-1990s) sought to reintroduce a functional E2 gene into cancer cells to "turn off" the oncogenes. While conceptually sound, challenges included inefficient gene delivery and E2's own potential to modulate cell motility.
• RNA Interference (RNAi) Pioneers: The discovery of RNAi was a major revolution. By the late 1990s and early 2000s, researchers were using short interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) specifically designed to cleave and destroy the E6/E7 mRNA transcripts .
  • Historical Significance: RNAi demonstrated powerful in vitro and in vivo efficacy in animal models, proving that E6/E7 knockdown was a viable therapeutic approach ^[10] .

1.3.4. The Rise of Therapeutic Cancer Vaccines

The fact that E6 and E7 are viral proteins, foreign to the host, makes them perfect targets for the immune system. The goal of a therapeutic vaccine is to stimulate a robust, tumor-killing cytotoxic T-lymphocyte (CTL) response against E6 and E7.

• Peptide/Protein Vaccines: These earliest vaccine concepts delivered synthetic E6/E7 peptides or recombinant proteins to prime T-cells. They showed limited clinical success due to poor immunogenicity (the ability to trigger an immune response).
• Early Ad/VV: To enhance T-cell priming, researchers began using live, attenuated vectors (like Vaccinia virus or Adenovirus) engineered to express E6/E7. These vectors infect host cells, mimic a natural infection, and efficiently deliver the antigens to the immune system. These led to the first meaningful clinical trials showing E6/E7-specific T-cell responses in patients with high-grade pre-cancers (CIN 2/3) ^[10] .

5. Natural Strategies: Phytotherapy and Nutraceuticals (1990s–Present)

1.3.5. The Rationale for Natural Strategies (Phytotherapy)

As an alternative to complex molecular and immunological approaches, significant research has focused on natural compounds (phytochemicals) that can disrupt the E6/E7 pathway.

• History in Traditional Medicine: Many compounds come from plants used in traditional medicine for centuries, now being investigated with a molecular focus on E6/E7 disruption.
• Key Mechanisms of Action:
  • Transcriptional Repression: Directly inhibiting the promoter that drives E6/E7 expression.
  • Protein-Protein Interaction Blockade: Preventing E6 from binding to p53-mediating proteins (like E6-AP) or E7 from binding to pRb.
  • Induction of Apoptosis/Senescence: The ultimate goal of E6/E7 targeting ^[11].

1.3.6. Major Historical and Current Natural Agents

Table no.3

Clinical Relevance: While in vitro data is strong, clinical translation is challenging due to low bioavailability, making these compounds better suited for high-grade pre-cancers (CIN 2/3) or as adjunct therapies [12] .

1.3.7 The Global Burden of Cervical Cancer and the HPV Link

Cervical cancer remains a significant global health challenge, particularly in developing countries. It is the fourth most common cancer in women worldwide, with an estimated 604,000 new cases and 342,000 deaths annually. The vast majority of cervical cancer cases (over 99%) are caused by persistent infection with high-risk human papillomaviruses (HPVs). Among these, HPV types 16 and 

18 are responsible for approximately 70% of all cervical cancers.The journey from HPV infection to cervical cancer is a multi-step process. Initial HPV infection often clears spontaneously. However, in a subset of women, the infection persists, leading to precancerous lesions and, if left untreated, invasive cervical cancer. Understanding this progression has been crucial in developing preventive and therapeutic strategies.

Fig.1.5 Global Burden of Cervical Cancer

1.3.8 The Role of HPV Oncogenes E6 and E7 in Carcinogenesis

The high-risk HPV types encode two key oncoproteins, E6 and E7, which are central to the development and progression of cervical cancer. These proteins manipulate cellular pathways to promote uncontrolled cell growth and evade immune surveillance.

The E6 oncoprotein primarily targets and degrades the tumor suppressor protein p53. p53 is a critical guardian of the genome, initiating cell cycle arrest or apoptosis in response to DNA damage. By degrading p53, E6 allows cells with damaged DNA to continue proliferating, accumulating mutations that can drive cancerous transformation.

The E7 oncoprotein primarily binds to and inactivates the retinoblastoma tumor suppressor protein (pRb). pRb is a crucial regulator of the cell cycle, preventing cells from entering the S-phase (DNA replication) until conditions are favorable. By inactivating pRb, E7 disrupts cell cycle control, leading to uncontrolled cell division. Additionally, E7 can interfere with other cellular pathways involved in DNA repair and immune responses.

The persistent expression of both E6 and E7 is essential for maintaining the malignant phenotype of HPV-positive cervical cancer cells. This makes these oncogenes attractive targets for therapeutic intervention [13].

Fig.1.6 the Role of Crideus Cancer Strategies

1.3.8 Natural Strategies and Immunotherapy

Beyond direct molecular interventions, natural compounds and immunotherapeutic approaches are gaining traction in the fight against HPV-driven cervical cancer.

Natural Strategies: Several natural compounds have demonstrated anti-HPV activity by modulating E6/E7 expression or function. For example, compounds like curcumin (from turmeric), resveratrol (from grapes), and epigallocatechin gallate (EGCG) from green tea have been shown to downregulate E6/E7 expression, restore p53 and pRb function, and induce apoptosis in HPV-positive cancer cells. These compounds often work through various mechanisms, including epigenetic modulation and interference with signaling pathways critical for viral gene expression. While promising, further research and clinical trials are needed to establish their efficacy and safety as therapeutic agents.

Immunotherapy: The immune system plays a crucial role in controlling HPV infection and eliminating HPV-infected cells. However, E6 and E7 can also promote immune evasion. Immunotherapeutic strategies aim to re-engage the host immune system to recognize and destroy HPV-positive cancer cells. These include:

• Therapeutic HPV vaccines: These vaccines are designed to elicit a strong cytotoxic T-lymphocyte (CTL) response against E6 and E7 proteins, thereby clearing existing HPV infections and precancerous lesions.

• Checkpoint inhibitors: Drugs that block immune checkpoints (e.g., PD-1/PD-L1) can unleash anti-tumor immune responses that were previously suppressed by the cancer cells.
• Adoptive cell therapy: Involves isolating and expanding HPV-specific T cells from the patient, which are then re-infused to target and destroy cancer cells

These natural and immunotherapeutic approaches offer diverse avenues for combating cervical cancer, often with fewer side effects than conventional treatments [14] .

Fig.1.7 Natural Strategies and Inntural Strategies

1.4 Cervical Cancer

Cervical cancer (CC) is the fourth most common cancer in women globally, according to Bray et al. (2024). The International Agency for Research on Cancer (IARC) reports that the incidence of CC rose from 13.3 cases per 100,000 in 2020 to 14.1 cases per 100,000 in 2022. During that period, the mortality rate remained high at roughly 7 per 100,000 people (Sung et al., 2021; Bray et al., 2024). The outlook for CC is worrisome due to the increasing incidence rates and the consistently high mortality rates. Even more concerning is the disproportionate burden of CC, which is concentrated in low-income areas like Southeast Asia, South America, Melanesia, and sub-Saharan Africa (Bray et al., 2018, 2024; Sung et al., 2021). and vaccines. A thorough understanding of the natural history of HPV infection and the progression of precancerous lesions is essential for developing effective screening, prevention, and treatment strategies for CC. The underlying causes of this imbalance are probably disparities in screening accessibility, treatment uptake, health awareness, healthcare resource allocation (Qju et al., 2021). and vaccines. Creating efficient screening, prevention, and treatment plans for CC requires a deep comprehension of the natural history of HPV infection and the development of precancerous lesions. Particularly noteworthy is the link between cervical carcinogenesis and human papillomavirus (HPV) infection. The development of CC has been linked to persistent HPV infection (Arbyn et al., 2015; Wright et al., 2015), which calls for improvements in HPV testing methods and vaccinations. Creating efficient screening, prevention, and treatment plans for CC requires a deep comprehension of the natural history of HPV infection and the development of precancerous lesions .

Fig. 1.8 HPV Lifecycle and CC Progression

CC progression and the HPV life cycle. The HPV life cycle starts when the virus infects basal epithelial cells, where it replicates at a low level. The viral oncogenes are progressively expressed as the cells differentiate, which causes the host cells to proliferate uncontrollably. Following this, the virus collects and emerges from differentiated epithelial cells. CIN, which can range from mild abnormalities (CIN1) to severe abnormalities (CIN3), can be caused by a chronic HR-HPV infection. This may eventually progress to invasive CC if treatment or reversal is delayed [15]

2. METHODOLOGY

5. HPV Detection Methods:

2.1: Introduction and The Role of Cytology

Human Papillomavirus (HPV) infection is the primary cause of cervical cancer and is implicated in other anogenital and oropharyngeal cancers. The clinical utility of HPV detection methods lies in primary screening, triage of women with abnormal cytology, and monitoring after treatment. Given that persistent infection with high-risk (HR) HPV types (e.g., HPV 16, 18, 31, 33, 45, etc.) is the prerequisite for cancer development, most current assays are focused on detecting the nucleic acid (DNA or mRNA) of these carcinogenic types.

1. 1. 1. The Historical Standard: Cytology (Papanicolaou Smear)

The Papanicolaou (Pap) smear, or cytology, is not a direct HPV detection method but is the traditional cornerstone of cervical cancer screening. It identifies the cytopathic effects of the virus on cervical epithelial cells.

Table no.4

2.1.2. Limitations of Cytology

• High False-Negative Rate: Subjectivity in interpretation and variations in sample collection can lead to significant false-negative results.
• Low Reproducibility: Results are highly dependent on the skill and experience of the cytologist.
• Does not Type HPV: Cytology cannot distinguish between high-risk and low-risk HPV types, nor can it provide specific genotyping (e.g., HPV 16 vs. HPV 18) ^[16] .

2.2 Signal Amplification Assays (Hybrid Capture)

Nucleic acid hybridization methods were among the first molecular tests approved for HPV screening. These methods use probes to bind to target HPV DNA, which is then detected through a signal amplification system.

2.2.1 Hybrid Capture 2 (HC2) Technology

The HC2 test (QIAGEN) is a classic example of a signal-amplification assay and was historically the most widely used.

Table no.5

Providing a comprehensive, five-page, in-depth technical analysis requires a structured approach that details the principles, protocols, advantages, and limitations of the primary HPV detection methods.

Here is a detailed breakdown of HPV detection methods used in clinical and research settings [17]

2.2.2 Signal Amplification Assays (Hybrid Capture)

For HPV screening. These methods use probes to bind to target HPV DNA, which is then detected through a signal amplification system.

• Hybrid Capture 2 (HC2) Technology:

The HC2 test (QIAGEN) is a classic example of a signal-amplification assay and was historically the most widely used.

Table no.6

Advantages and Limitations of HC2

• Advantages: High sensitivity; clinically validated; detects the entire group of HR-HPV types in a single reaction; quantitative (provides a relative measure of viral load).
• Limitations: No specific genotyping (only reports 'HR-HPV positive' or 'negative'); lower specificity compared to {PCR}based tests because it detects transient infections that may not progress to cancer; requires specialized equipment ^[18] .

2.3 Target Amplification: Polymerase Chain Reaction (PCR)

• Polymerase Chain Reaction {PCR} is the most sensitive and versatile method for HPV detection, allowing for massive amplification of the viral DNA sequence, enabling the detection of even very low viral copy numbers.
  1. 1. Polymerase Chain Reaction (PCR)-Based Methods
• PCR and its variants (e.g., Real-Time {PCR}) are the current standard for high-throughput HPV testing and genotyping.

5. Conventional PCR with Consensus Primers

• Principle: Uses consensus primers that target highly conserved regions of the HPV genome, most commonly the {L}1capsid gene, allowing the amplification of a broad range of HPV types in one reaction.
• Consensus Primers: GP5+/ GP6+ or {PGMY}09/{PGMY}11 are the most common primer sets.
• Detection/Typing: After amplification, the {PCR} product must undergo a secondary process for genotyping, such as Reverse Line Blot (RLB) Hybridization (e.g., Linear Array, INNO-LiPA).

• RLB Principle: The amplified DNA is hybridized to a set of type-specific oligonucleotide probes immobilized on a membrane or strip. This allows for simultaneous detection and specific genotyping of dozens of HPV types.

2.3.2 Real-Time Quantitative PCR (qPCR)

• Principle: Combines DNA amplification and detection/quantification in a single step, using fluorescent probes (e.g., TaqMan probes) or dyes (e.g., SYBR Green).
• High-Throughput Assays: Commercial qPCR assays (e.g., cobas {HPV Test) are the preferred methods for primary screening:
  • They typically provide specific genotyping for {HPV} 16 and t{HPV} 18, which are the highest-risk types, and pool the detection of the remaining 12 high-risk types into a single result.
  • Advantage: Faster, closed-tube system minimizes contamination risk, and results are quantitative, allowing for the potential assessment of viral load.
    3. Droplet Digital PCR (ddPCR)

• Principle: Partitions the PCR reaction mixture into thousands of tiny, uniform water-in-oil droplets. PCR amplification occurs independently within each droplet.
• Detection: Droplets are individually analyzed for fluorescence (a positive signal indicates a droplet contained at least one target HPV DNA molecule).
• Advantage: Offers absolute quantification (copy number/sample) without a standard curve and superior sensitivity compared to {qPCR}, making it highly valuable for liquid biopsy applications (e.g., cell-free {HPV}DNA in plasma for cancer recurrence monitoring) ^[19]

2.4 Emerging and Future Technologies

Advancements in molecular biology are yielding even more sensitive and comprehensive HPV detection platforms, particularly for genotyping and cancer monitoring.

2.4.1 Next-Generation Sequencing (NGS)

NGS technology is moving into clinical research for HPV detection due to its ability to comprehensively type the virus and detect novel or rare variants ^[20].

Table no.7

2.4.2 Comparison of Key Clinical HPV Assays

Table no.8