Degradation Of Tumor Suppressor Proteins Research Articles

A Phase II Trial of Pevonedistat and Docetaxel in Patients With Previously Treated Advanced Non–Small-Cell Lung Cancer

BackgroundPostimmunotherapy (IO) treatment options for stage IV non–small-cell lung cancer (NSCLC) remain limited. Docetaxel alone or in combination with ramucirumab remains a standard of care, but response rates and survival benefit are suboptimal. Cullin-RING ligases (CRL) catalyze degradation of tumor suppressor proteins and are overactivated in NSCLC. Neddylation, which is catalyzed by the NEDD8 activating enzyme (NAE), is required for the activation of CRLs. Pevonedistat, a first-in-class small molecule NAE inhibitor, exerted antitumor activity when combined with docetaxel in preclinical studies. MethodsWe conducted a phase II, single-arm, investigator-initiated study evaluating the efficacy of pevonedistat plus docetaxel in patients with relapsed/refractory stage IV NSCLC. Patients received docetaxel 75 mg/m2 on day 1 and pevonedistat 25 mg/m2 on days 1, 3 and 5 of a 21-day cycle. The primary endpoint was objective response rate (ORR). ResultsFrom March 5, 2018 to January 26, 2021, we enrolled 31 patients. The ORR was 22% (1 CR, 5 PR), median PFS was 4.1 months, and median OS was 13.2 months. The incidence of Grade ≥3 adverse events (AE) was 53% in patients (n = 30) who received at least 1 dose of both drugs, with the most frequent being neutropenia and AST/ALT elevation. One patient was taken off study for a Grade 4 transaminase elevation. There were no Grade 5 toxicities. ConclusionOur data suggest that the combination of docetaxel and pevonedistat is safe and exerts activity in patients with relapsed NSCLC. These encouraging results suggest that the neddylation pathway is an antitumor pathway that should be further studied.

A phase II trial of pevonedistat plus docetaxel in patients with previously treated advanced non–small-cell lung cancer (NSCLC).

e21062 Background: For patients with stage IV NSCLC, treatment options are limited after progression on immunotherapy (IO) +/- platinum-based chemotherapy. Docetaxel alone or in combination with ramucirumab remains a standard of care, but response rates and survival benefit are suboptimal. Cullin-RING ligases (CRLs) catalyze the ubiquitylation and degradation of tumor suppressor proteins and are overactivated in NSCLC. Therefore, inhibition of CRLs has potential therapeutic value. Neddylation is required to activate CRLs; NAE (NEDD8 activating enzyme) catalyzes neddylation. In pre-clinical studies, pevonedistat, a first-in-class small molecule NAE inhibitor, exerts anti-tumor effects when combined with docetaxel. Methods: We conducted a phase II, single-arm, investigator-initiated study evaluating the efficacy of pevonedistat plus docetaxel in patients with stage IV NSCLC with progression on or after platinum-based chemotherapy +/- IO. Patients with tumors that had driver mutations in EGFR, ALK, ROS1, and BRAF must have also had progression on targeted inhibitors. A Simon Optimal two-stage design was utilized with 90% power and 15% type I error to detect the drug as promising when the true response rate is 25% and the historical control rate is 10%. A maximum of 37 response-evaluable (R-E) patients could be enrolled. Patients received docetaxel 75 mg/m2 on day 1 and pevonedistat 25 mg/m2 on days 1, 3 & 5 of a 21-day cycle. Response was assessed every 2 cycles based on RECIST v1.1 criteria. The primary objective was overall response rate (ORR). Those who received 2 cycles or had progression prior to the end of cycle 2 were considered R-E. Patients who received any treatment on protocol were evaluable for safety (S-E). Results: From 3/5/2018 to 1/26/2021, we enrolled 31 patients and 27 were R-E with a median follow-up of 38.7 months. The trial was terminated early due to the sponsor’s decision to discontinue the development of pevonedistat. In the R-E population, 52% were female with a median age of 63 and 85% had adenocarcinoma. The median number of prior regimens was 2, and 92% of patients had previously received IO. The ORR was 22% (95%CI 8.6-42.3%) and the disease control rate (SD+CR+PR) was 66.7% (95%IC 46.0- 83.5%). The median PFS was 4.1 (2.8-5.8) months and median OS was 13.1 (6.7-24.8) months. The median duration of response was 14.5 months. The incidence of Grade 3-4 adverse events (AE) was 53% in the 30 S-E patients. The most common grade 3-4 AEs were decrease in blood counts (anemia and neutropenia) and elevation in liver transaminases. There were no Grade 5 toxicities. Conclusions: Our data suggest that the combination of docetaxel and pevonedistat is safe and may have improved anti-tumor activity compared to historical data with docetaxel alone. These results suggest that the neddylation pathway is a targetable anti-tumor pathway that should be further studied in patients with NSCLC. Clinical trial information: NCT03228186 .

Second Generation Small Molecule Inhibitors of Gankyrin for the Treatment of Pediatric Liver Cancer.

Simple SummaryNew therapy options are needed for children with liver cancer. The goal of this study was to evaluate the role of three new compounds in the treatment of liver cancer cells. These compounds all inhibit a protein called Gankyrin, which is known to promote liver cancer by destroying tumor suppressor proteins. We demonstrated that liver cancer cells have significantly reduced proliferation when treated with these compounds by preventing the degradation of tumor suppressor proteins. We also discovered that these compounds enhance the effects of doxorubicin, which is a chemotherapy drug commonly used in liver cancer. These results support continued efforts in Gankyrin-based therapy for the treatment of pediatric liver cancer.Background: Gankyrin, a member of the 26S proteasome, is an overexpressed oncoprotein in hepatoblastoma (HBL) and hepatocellular carcinoma (HCC). Cjoc42 was the first small molecule inhibitor of Gankyrin developed; however, the IC50 values of >50 μM made them unattractive for clinical use. Second-generation inhibitors demonstrate a stronger affinity toward Gankyrin and increased cytotoxicity. The aim of this study was to characterize the in vitro effects of three cjoc42 derivatives. Methods: Experiments were performed on the HepG2 (HBL) and Hep3B (pediatric HCC) cell lines. We evaluated the expression of TSPs, cell cycle markers, and stem cell markers by Western blotting and/or real-time quantitative reverse transcription PCR. We also performed apoptotic, synergy, and methylation assays. Results: The treatment with cjoc42 derivatives led to an increase in TSPs and a dose-dependent decrease in the stem cell phenotype in both cell lines. An increase in apoptosis was only seen with AFM-1 and -2 in Hep3B cells. Drug synergy was seen with doxorubicin, and antagonism was seen with cisplatin. In the presence of cjoc42 derivatives, the 20S subunit of the 26S proteasome was more available to transport doxorubicin to the nucleus, leading to synergy. Conclusion: Small-molecule inhibitors for Gankyrin are a promising therapeutic strategy, especially in combination with doxorubicin.

From the Evasion of Degradation to Ubiquitin-Dependent Protein Stabilization.

A hallmark of cancer is dysregulated protein turnover (proteostasis), which involves pathologic ubiquitin-dependent degradation of tumor suppressor proteins, as well as increased oncoprotein stabilization. The latter is due, in part, to mutation within sequences, termed degrons, which are required for oncoprotein recognition by the substrate-recognition enzyme, E3 ubiquitin ligase. Stabilization may also result from the inactivation of the enzymatic machinery that mediates the degradation of oncoproteins. Importantly, inactivation in cancer of E3 enzymes that regulates the physiological degradation of oncoproteins, results in tumor cells that accumulate multiple active oncoproteins with prolonged half-lives, leading to the development of “degradation-resistant” cancer cells. In addition, specific sequences may enable ubiquitinated proteins to evade degradation at the 26S proteasome. While the ubiquitin-proteasome pathway was originally discovered as central for protein degradation, in cancer cells a ubiquitin-dependent protein stabilization pathway actively translates transient mitogenic signals into long-lasting protein stabilization and enhances the activity of key oncoproteins. A central enzyme in this pathway is the ubiquitin ligase RNF4. An intimate link connects protein stabilization with tumorigenesis in experimental models as well as in the clinic, suggesting that pharmacological inhibition of protein stabilization has potential for personalized medicine in cancer. In this review, we highlight old observations and recent advances in our knowledge regarding protein stabilization.

Ubiquitination Regulators Discovered by Virtual Screening for the Treatment of Cancer.

The ubiquitin-proteasome system oversees cellular protein degradation in order to regulate various critical processes, such as cell cycle control and DNA repair. Ubiquitination can serve as a marker for mutation, chemical damage, transcriptional or translational errors, and heat-induced denaturation. However, aberrant ubiquitination and degradation of tumor suppressor proteins may result in the growth and metastasis of cancer. Hence, targeting the ubiquitination cascade reaction has become a potential strategy for treating malignant diseases. Meanwhile, computer-aided methods have become widely accepted as fast and efficient techniques for early stage drug discovery. This review summarizes ubiquitination regulators that have been discovered via virtual screening and their applications for cancer treatment.

Regulation of p14ARF by Siva1 in H. pylori-infected gastric epithelial cells.

243 Background: Helicobacter pylori ( H. pylori) is the strongest known risk factor for gastric cancer. Bacterial degradation of tumor suppressor proteins affect the host microbe’s interactions and host cellular response, which contribute to tumorigenesis. p14ARF, a crucial tumor suppressor protein that activates p53 protein under oncogenic stress plays a major role in oncogenic stress response (OSR) regulation. However, little is known about the mechanism of ARF and OSR regulation in H. pylori-infected gastric epithelial cells. Methods: The expression of p14ARF and cytotoxin-associated gene A (CagA) were analyzed in gastric cells co-cultured with H. pylori strains isolated from high-gastric risk and low-gastric risk areas by immunoblotting. To investigate the potential role of CagA in regulation of p14ARF, we employed isogenic cagA− and cagE− H. pylori mutants in gastric epithelial cells, and C57BL/6 mice (n = 10). We also analyzed the expression of Siva1 in human individual infected with cagA-positive (n = 13) and cagA-negative (n = 13) bacteria as well as uninfected human subjects (n = 6). siRNA was used to inhibit activity of Siva1 protein. Results: In this study, H. pylori strains expressing high levels of CagA virulence factor and associated with a higher gastric cancer risk more strongly suppress p14ARF compared with low-risk strains in vivo and in vitro. We found that degradation of p14ARF induced by CagA is mediated by E3 ubiquitin ligase Siva1, which works in concert with another E3 ubiquitin ligase TRIP12. Decreased expression of Siva1 protein and consequent up-regulation of p14ARF was also found in gastric mucosa of H. pylori-infected mice and human individuals. Tumorigenic strain 7.13 was more potent in upregulation of Siva1 and downregulation of p14ARF than non-tumorigenic strain B128. Inhibition of p14ARF protein by H. pylori causes inhibition of autophagy in infected cells. Conclusions: Our results provide first evidence that carcinogenic H. pylori strains significantly alter the host tumor suppressor protein p14ARF, leading to suppression of host OSR and autophagy, which may affect host-bacteria interactions and tumorigenic alteration in the stomach.

A novel therapeutic opportunity for the most aggressive sub‐type of pancreatic cancer: Targeting keratin 17 dependency on nuclear export

Introduction and hypothesesThe purpose of this study was to uncover a novel targeted therapeutic approach for pancreatic ductal adenocarcinoma (PDAC). We previously discovered that keratin 17 (K17) is an independent negative prognostic biomarker for PDAC, where high K17 expression is associated with decreased patient survival. In our pre‐clinical mouse model, PDACs that overexpress K17 were four times more likely than controls to result in fatality within 7 weeks, suggesting that K17 drives tumor aggression. We determined that K17 functions as an oncoprotein by translocating into the nucleus of PDAC cells and enhances XPO‐1‐ mediated nuclear export and degradation of tumor suppressor proteins (TSPs).Here, we hypothesized that 1) K17 depends on nuclear export to drive tumor aggression and is highly sensitive to nuclear inhibition, compared to K17‐negative PDACs, and 2) blocking K17 enhanced XPO‐1‐mediated nuclear export causes enhanced apoptosis and cell cycle arrest.Methods and resultsTo test the hypothesis that K17 depends on nuclear export to drive tumor aggression and is more sensitive to nuclear inhibition than K17‐negative PDACs, we performed pre‐clinical studies in mouse models and orthotopically implanted isogenic murine PDAC cells (LSL‐KrasG12D, LSL‐Trp53R172HPdx‐1‐Cre, KPC) that expressed human K17 (KPC‐K17) or not (KPC‐EV) in immunocompetent animals. After animal randomization, mice were treated three times weekly via oral gavage with selinexor, an inhibitor of the nuclear exporter, XPO‐1, that is currently in phase 2 clinical trials. We found that selinexor inhibited the growth of K17‐positive PDACs in mice, compared to K17‐negative PDACs, suggesting that K17sensitizes PDACs to selinexor treatment. To determine if this decreased tumor growth was due to a tumor‐cell nuclear export blockage, we performed in vitro studies and found that KPC‐K17 cells elicited a four‐fold increase in cell death and a two‐fold increase in G1/S cell cycle arrest compared to KPC‐EV cells. In addition, selinexor treatment led to nuclear retention of K17. To test the dependency of KPC‐K17 cells to K17‐mediated nuclear export, we challenged the cells to chemotherapeutic and targeted therapeutic agents that induce nuclear import inhibition (ivermectin), G1/S transition inhibition (palbociclib), and DNA‐damage (oxaliplatin). In contrast to selinexor treatment, there were no differences in cell death or cell cycle arrest between KPC‐K17 and KPC‐EV cells upon treatment with these compounds.ConclusionsOur results suggest that K17‐positive tumors are specifically vulnerable to inhibition of nuclear export. Given that K17 expression is a biomarker that drives tumor aggression in PDACs, our results suggest that treatment with selinexor is a novel therapeutic opportunity for the most aggressive sub‐type of pancreatic cancer.Support or Funding InformationPancreatic Cancer Action Network

The Myb-related protein MYPOP is a novel intrinsic host restriction factor of oncogenic human papillomaviruses

The skin represents a physical and chemical barrier against invading pathogens, which is additionally supported by restriction factors that provide intrinsic cellular immunity. These factors detect viruses to block their replication cycle. Here, we uncover the Myb-related transcription factor, partner of profilin (MYPOP) as a novel antiviral protein. It is highly expressed in the epithelium and binds to the minor capsid protein L2 and the DNA of human papillomaviruses (HPV), which are the primary causative agents of cervical cancer and other tumors. The early promoter activity and early gene expression of the oncogenic HPV types 16 and 18 is potently silenced by MYPOP. Cellular MYPOP-depletion relieves the restriction of HPV16 infection, demonstrating that MYPOP acts as a restriction factor. Interestingly, we found that MYPOP protein levels are significantly reduced in diverse HPV-transformed cell lines and in HPV-induced cervical cancer. Decades ago it became clear that the early oncoproteins E6 and E7 cooperate to immortalize keratinocytes by promoting degradation of tumor suppressor proteins. Our findings suggest that E7 stimulates MYPOP degradation. Moreover, overexpression of MYPOP blocks colony formation of HPV and non-virally transformed keratinocytes, suggesting that MYPOP exhibits tumor suppressor properties.

Gankyrin Promotes Tumor-Suppressor Protein Degradation to Drive Hepatocyte Proliferation.

Background & AimsUncontrolled liver proliferation is a key characteristic of liver cancer; however, the mechanisms by which this occurs are not well understood. Elucidation of these mechanisms is necessary for the development of better therapy. The oncogene Gankyrin (Gank) is overexpressed in both hepatocellular carcinoma and hepatoblastoma. The aim of this work was to determine the role of Gank in liver proliferation and elucidate the mechanism by which Gank promotes liver proliferation.MethodsWe generated Gank liver-specific knock-out (GLKO) mice and examined liver biology and proliferation after surgical resection and liver injury.ResultsGlobal profiling of gene expression in GLKO mice showed significant changes in pathways involved in liver cancer and proliferation. Investigations of liver proliferation after partial hepatectomy and CCl4 treatment showed that GLKO mice have dramatically inhibited proliferation of hepatocytes at early stages after surgery and injury. In control LoxP mice, liver proliferation was characterized by Gank-mediated reduction of tumor-suppressor proteins (TSPs). The failure of GLKO hepatocytes to proliferate is associated with a lack of down-regulation of these proteins. Surprisingly, we found that hepatic progenitor cells of GLKO mice start proliferation at later stages and restore the original size of the liver at 14 days after partial hepatectomy. To examine the proliferative activities of Gank in cancer cells, we used a small molecule, cjoc42, to inhibit interactions of Gank with the 26S proteasome. These studies showed that Gank triggers degradation of TSPs and that cjoc42-mediated inhibition of Gank increases levels of TSPs and inhibits proliferation of cancer cells.ConclusionsThese studies show that Gank promotes hepatocyte proliferation by elimination of TSPs. This work provides background for the development of Gank-mediated therapy for the treatment of liver cancer. RNA sequencing data can be accessed in the NCBI Gene Expression Omnibus: GSE104395.

FXR-Gankyrin axis is involved in development of pediatric liver cancer.

The development of hepatoblastoma (HBL) is associated with failure of hepatic stem cells (HSC) to differentiate into hepatocytes. Despite intensive investigations, mechanisms of the failure of HSC to differentiate are not known. We found that oncogene Gankyrin (Gank) is involved in the inhibition of differentiation of HSC via triggering degradation of tumor suppressor proteins (TSPs) Rb, p53, C/EBPα and HNF4α. Our data show that the activation of a repressor of Gank, farnesoid X receptor, FXR, after initiation of liver cancer by Diethylnitrosamine (DEN) prevents the development of liver cancer by inhibiting Gank and rescuing tumor suppressor proteins. We next analyzed FXR-Gank-Tumor suppressor pathways in a large cohort of HBL patients which include 6 controls and 53 HBL samples. Systemic analysis of these samples and RNA-Seq approach revealed that the FXR-Gank axis is activated; markers of hepatic stem cells are dramatically elevated and hepatocyte markers are reduced in HBL samples. In the course of these studies, we found that RNA binding protein CUGBP1 is a new tumor suppressor protein which is reduced in all HBL samples. Therefore, we generated CUGBP1 KO mice and examined HBL signatures in the liver of these mice. Micro-array studies revealed that the HBL-specific molecular signature is developed in livers of CUGBP1 KO mice at very early ages. Thus, we conclude that FXR-Gank-TSPs-Stem cells pathway is a key determinant of liver cancer in animal models and in pediatric liver cancer. Our data provide a strong basis for development of FXR-Gank-based therapy for treatment of patients with hepatoblastoma.

E6AP promotes prostate cancer by reducing p27 expression.

Prostate cancer (PC) is the most common cancer in men. Elevated levels of E3 ligase, E6-Associated Protein (E6AP) were previously linked to PC, consistent with increased protein expression in a subset of PC patients. In cancers, irregular E3 ligase activity drives proteasomal degradation of tumor suppressor proteins. Accordingly, E3 ligase inhibitors define a rational therapy to restore tumor suppression. The relevant tumor suppressors targeted by E6AP in PC are yet to be fully identified. In this study we show that p27, a key cell cycle regulator, is a target of E6AP in PC. Down regulation of E6AP increases p27 expression and enhances its nuclear accumulation in PC. We demonstrate that E6AP regulates p27 expression by inhibiting its transcription in an E2F1-dependent manner. Concomitant knockdown of E6AP and p27 partially restores PC cell growth, supporting the contribution of p27 to the overall effect of E6AP on prostate tumorigenesis. Overall, we unravelled the E6AP-p27 axis as a new promoter of PC, exposing an attractive target for therapy through the restoration of tumor suppression.

Gel-based chemical cross-linking analysis of 20S proteasome subunit-subunit interactions in breast cancer.

The ubiquitin-proteasome system plays a pivotal role in breast tumorigenesis by controlling transcription factors, thus promoting cell cycle growth, and degradation of tumor suppressor proteins. However, breast cancer patients have failed to benefit from proteasome inhibitor treatment partially due to proteasome heterogeneity, which is poorly understood in malignant breast neoplasm. Chemical crosslinking is an increasingly important tool for mapping protein three-dimensional structures and proteinprotein interactions. In the present study, two cross-linkers, bis (sulfosuccinimidyl) suberate (BS(3)) and its water-insoluble analog disuccinimidyl suberate (DSS), were used to map the subunit-subunit interactions in 20S proteasome core particle (CP) from MDA-MB-231 cells. Different types of gel electrophoresis technologies were used. In combination with chemical cross-linking and mass spectrometry, we applied these gel electrophoresis technologies to the study of the noncovalent interactions among 20S proteasome subunits. Firstly, the CP subunit isoforms were profiled. Subsequently, using native/SDSPAGE, it was observed that 0.5 mmol/L BS(3) was a relatively optimal cross-linking concentration for CP subunit-subunit interaction study. 2-DE analysis of the cross-linked CP revealed that α1 might preinteract with α2, and α3 might pre-interact with α4. Moreover, there were different subtypes of α1α2 and α3α4 due to proteasome heterogeneity. There was no significant difference in cross-linking pattern for CP subunits between BS(3) and DSS. Taken together, the gel-based characterization in combination with chemical cross-linking could serve as a tool for the study of subunit interactions within a multi-subunit protein complex. The heterogeneity of 20S proteasome subunit observed in breast cancer cells may provide some key information for proteasome inhibition strategy.

The Significance of Ubiquitin Proteasome Pathway in Cancer Development

The ubiquitin proteasome pathway is the most significant intracellular proteolytic pathway. The target proteins are usually ubiquitinated prior to degradation by the proteasome; however, ubiquitin-independent targeting mechanisms have also been reported (e.g., the antizyme-mediated degradation of ornithine decarboxylase). Aberrations in the components of the ubiquitin proteasome pathway are commonly observed in many cancers, and uncontrolled growth of cancer cells can result either from stabilization of oncoproteins (e.g., c-jun) or increased degradation of tumor suppressor proteins (e.g., p53). In addition, due to the pleiotropic functions of the ubiquitin proteasome pathway in cells, there is great interest in developing inhibitors to specifically block this pathway for cancer treatment. This review summarizes the recent literature and several patented inventions on the ubiquitin proteasome pathway with respect to its role in cancer development and treatment.

TRIAD1 inhibits MDM2-mediated p53 ubiquitination and degradation

Murine double minute (MDM2) is an E3 ligase that promotes ubiquitination and degradation of tumor suppressor protein 53 (p53). MDM2-mediated regulation of p53 has been investigated as a classical tumorigenesis pathway. Here, we describe TRIAD1 as a novel modulator of the p53-MDM2 axis that induces p53 activation by inhibiting its regulation by MDM2. Ablation of TRIAD1 attenuates p53 levels activity upon DNA damage, whereas ectopic expression of TRIAD1 promotes p53 stability by inhibiting MDM2-mediated ubiquitination/degradation. Moreover, TRIAD1 binds to the C-terminus of p53 to promote its dissociation from MDM2. These results implicate TRIAD1 as a novel regulatory factor of p53-MDM2.Structured summary of protein interactions:p53 physically interacts with Mdm2 and Triad1 by anti tag coimmunoprecipitation (View Interaction: 1, 2, 3)Mdm2physically interacts with Triad1 by anti tag coimmunoprecipitation (View interaction)p53physically interacts with Mdm2 by anti tag coimmunoprecipitation (View interaction)Triad1binds to p53 by pull down (View interaction)Mdm2physically interacts with p53 by anti tag coimmunoprecipitation (View interaction)p53physically interacts with Triad1 by anti tag coimmunoprecipitation (View interaction)

Human Papillomavirus 16 E6/E7 Transcript and E2 Gene Status in Patients with Cervical Neoplasia

The viral transforming genes E6 and E7 of human papillomavirus (HPV) 16 cause the degradation of tumor suppressor proteins. Expression of these oncoproteins increases following the integration of viral DNA into the host cell, resulting in the disruption of the E2 open reading frame (ORF). To detect and correlate HPV-16 oncogene transcripts and HPV-16 E2 DNA in cervical biopsies obtained from women (n = 68) with cervical neoplasia. HPV-16 E6/E7 transcript and HPV-16 E2 DNA detection was performed on the cervical biopsies of 42 women positive for HPV-16 (36 with invasive cervical carcinoma and 6 with cervical intraepithelial neoplasia [CIN]). PCR was used to detect HPV DNA in cervical biopsies then restriction fragment length polymorphism (RFLP) was used to type the HPV DNA. Reverse-transcription (RT)-PCR for HPV-16 E6/E7 oncogene mRNA transcripts and a PCR to detect the HPV-16 E2 DNA was performed on HPV-16-positive samples. HPV-16 E6/E7 mRNA transcripts were not detected in any of the CIN I or II biopsies, but were detected in all cases of CIN III and invasive cancer in different combinations (E6 alone, E6*I, E6*I/E6*II, E6/E6*I/E6*II) except for one patient with stage IIB cancer treated with radiotherapy. The incidence of episomal E2 DNA was high in this study with 52.4% of the samples positive for episomal E2. It was even detected in patients with advanced stage cancer with 50%, 42%, and 66.6% of samples positive in stages IIB, IIIB, and IV, respectively. HPV-16 E6/E7 mRNA oncogene transcripts, in various combinations, were uniformly detectable in the majority of the high-grade cervical lesions examined. Intact episomal E2 DNA was seen in a high proportion of samples, even from advanced cervical lesions. Conservation of the E2 gene with concomitant expression of viral oncogenes in advanced cervical lesions may point to alternate mechanisms, other than integration, bringing about the enhanced expression of E6/E7 mRNA. This study suggests that the detection of the HPV-16 oncogene transcripts could serve as an indicator for assessing the prognosis of patients on radiotherapy. The majority of HPV-16-positive cervical neoplastic lesions are transcriptionally active and express the oncogene transcripts. The increased occurrence of intact HPV-16 episomal E2 DNA in advanced lesions further substantiates the fact that the disruption of E2 ORF is not mandatory for increased oncogene expression. Thus, this study underscores the significance of investigating alternative mechanisms of oncogene expression in HPV-16.