Expression Of E7 Oncoprotein Research Articles

In vitro study of HPV18-positive cervical cancer HeLa cells based on CRISPR/Cas13a system

The E6 protein is a known oncogene in cervical cancer and plays a key role in the development and progression of cervical cancer by reducing the expression level of the tumor suppressor protein P53 and ultimately leading to enhanced cell proliferation and reduced apoptosis. Therefore, antiviral agents that inhibit the expression of E6 oncoprotein are expected to be potential therapies for human cervical cancer. Here we developed CRISPR/Cas13a: crRNA dual plasmid system and demonstrated that CRISPR/Cas13a could effectively and specifically knock down human papillomavirus 18 E6 mRNA, downregulate the expression level of E6 protein, and restore the expression of the tumor suppressor gene P53 protein, thereby inhibiting the growth of cervical cancer cells and increasing their apoptosis, the E6-2, E6-3, and E6-5 groups resulted in apoptosis rates of 25.4%, 22.4%, and 22.2% in HeLa cells. Moreover, CRISPR/Cas13a enhances the proliferation inhibition and apoptosis induction of cisplatin in cervical cancer HeLa cells. The CRISPR/Cas13a system targeting HPV E6 mRNA may be a promising therapeutic approach for the treatment of human papillomavirus-associated cervical cancer.

Low-Cost Molecular Biomarker HPV-16/18 E6 Oncoprotein Expression in Cervical Intraepithelial Neoplasia (CIN) and Cervical Cancer with Its Relation with Severity of Neoplastic State

This research was carried out with the intention to see the detection rate of HPV-16/18 E6 oncoprotein expression in different categories of cervical neoplasia and to find out its association with increasing neoplastic state, using lateral flow test “Onco E6 Cervical Test” designed for low-resource settings. A cross-sectional study was conducted at Gynecologic Oncology Outpatient Department (OPD) of Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, Bangladesh, from May 2018 to April 2019. Histopathologically diagnosed CIN I, CIN II, CIN III, and cervical cancer cases of age 21–70 years who were compliant to allow cervical swab collection for E6 oncoprotein test were selected by purposive sampling. E6 oncoprotein expression was 4.44% (2/45), 28.57% (4/14), 50% (2/4), and 77.78% (28/36) for CIN I, CIN II, CIN III, and cervical cancer cases, respectively. Thirty-six women of total 99 (N = 99) study participants were found E6 positive (detection rate 36.36%). Type distribution showed 83.33% (30/36) type 16, 13.89% (5/36) type 18, and 2.78% (1/36) co-infection with types 16 and 18. Considering CIN I the reference category, E6 oncoprotein expression was found associated with CIN II (OR = 6.95, p = 0.052), significantly associated with CIN III (OR = 18.10, p = 0.026), and highly significantly associated with cervical cancer (OR = 43.57, p < 0.001). Association of E6 oncoprotein expression is significant for CIN III and highly significant for cervical cancer. The presence of E6 oncoprotein expression in CIN lesions can identify true cancer precursors with risk of cancer development in the future and can be utilized in cancer screening program.

HPV16 E7-impaired keratinocyte differentiation leads to tumorigenesis via cell cycle/pRb/involucrin/spectrin/adducin cascade.

Here, we used codon usage technology to generate two codon-modified human papillomavirus (HPV)16 E7 genes and, together with wild-type E7, to construct three HPV16 E7 gene plasmids: Wt-E7, HB1-E7, and HB2-E7. The three HPV 16 E7 plasmids were used to investigate how HPV16 E7 protein was expressed in different cells and how this oncoprotein deregulated cellular and molecular events in human keratinocytes to induce carcinogenesis. We discovered that codon usage of HPV16 E7 gene played a key role in determining expression of E7 oncoprotein in all tested cells. HPV16 E7 inhibited significantly expression of pRb to impair keratinocyte differentiation and disrupted development of skin epidermis in mice. HPV16 E7 increased substantially the number of G0/G1 cells associated with upregulation of cyclin D2 and downregulation of cyclin B1 in keratinocytes. HPV16 E7 not only inhibited expression of involucrin and α-spectrin but also disrupted the organization of involucrin filaments and spectrin cytoskeleton. Furthermore, HPV16 E7 inhibited expression of β-adducin, destroyed its cytoskeletal structure and induced phosphorylation of β-adducin(Ser662) in keratinocytes. Importantly, HPV16 E7 induced carcinogenesis in mice associated with expression of phosphorylated β-adducin(Ser662) and its nucleus-translocation. In conclusion, we provided evidence that HPV16 E7 oncoprotein inhibited keratinocyte differentiation in vitro and in vivo leading to carcinogenesis through cell cycle arrest and disruption of pRb/involucrin/spectrin/adducin cascade.

Flax seed oil reduced tumor growth, modulated immune responses and decreased HPV E6 and E7 oncoprotein expression in a murine model of ectopic cervical cancer

Cervical cancer is the second leading cause of cancer death in women in India. Previously, we have reported that alpha linolenic acid (ALA), induced apoptosis in cervical cancer cell lines and reduced expression of E6 and E7 oncoproteins with simultaneous decrease in Cox2/VEGF/MAP kinase proteins. Here, we investigated the tumor retardation potential of flax oil (FO), rich in ALA, in mouse papilloma model. Flax oil significantly reduced tumor volume and weight in mice compared to the Tumor control (TC) group. Interestingly, compared to cisplatin (Cis) alone, there was slightly enhanced decrease in tumor weight when FO was given together with Cis (Cis + FO). A marked increase in plasma antioxidant levels in mice, and increase in lipid peroxidation in tumors with simultaneous decrease in liver tissues was observed in Cis + FO group compared to either TC or Cis groups. FO and Cis + FO significantly modulated immune response in mice by increasing CD8α and IFNγ and decreasing IL-4 expression. Interestingly, when given together with cisplatin, flax oil reduced HPV E6 and E7 oncoprotein expression with concomitant increase in the relative mRNA expression of tumor suppressor genes p53 and Rb. Thus, flax oil could be explored for its therapeutic potential in cervical cancer.

Human Papillomavirus (HPV) 16/18 E6 Oncoprotein Expression in Infections with Single and Multiple Genotypes.

Factors that differentiate risk of cervical cancer associated with infection with single versus multiple HPV types are yet undefined. We hypothesize that E6 oncoprotein is one determining factor. This cross-sectional, multicenter study was performed between 2013 and 2017. A total of 1,781 women were recruited from six hospitals. Samples were tested for presence of 14 types of high-risk HPV DNA. HPV16/18-positive samples were also tested for HPV16/18-E6 oncoprotein. Of 1,781 subjects, 687 (38.6%) tested positive for HPV16/18. HPV16/18 single infections were associated with higher E6 positivity rates compared with multiple infections only for cancer cases (HPV16: 92.2% vs. 76.5%; HPV18: 93.9% vs. 62.1%) but not for normal histopathology or cervical intraepithelial neoplasia. In HPV16/18 coinfection subjects, the positivity rate was 42.9% for HPV16-E6 and 42.9% for HPV18-E6. The combined positivity rate of either HPV16-E6 or HPV18-E6 among HPV16/18 coinfection subjects was 78.6%, similar with HPV16 (74.8%) and HPV18 (79.5%) single-infection subjects. The positivity rates of HPV16/18 E6 oncoprotein varied depending on the HPV-type composition in multiple infection ("clusters") including HPV types other than 16 and 18. Multiple infection clusters most likely to express HPV16-E6 and HPV18-E6 were HPV16/52 (61.5%) and HPV18/52 (66.7%), and the less were HPV16/45 (10.0%) and HPV18/51 (16.7%), respectively. Patterns of E6 oncoprotein expression varied depending on clustering types. However, expression was greatest in women with single HPV-type infections compared with those with multiple HPV types regardless of histopathology. Our findings provided new insight of natural history of cervical cancer.

Cigarette Smoking Promotes Infection of Cervical Cells by High-Risk Human Papillomaviruses, but not Subsequent E7 Oncoprotein Expression.

Persistent cervical infection with high-risk human papillomaviruses (hrHPVs) is a necessary, but not sufficient, condition for the development of cervical cancer. Therefore, there are other co-factors facilitating the hrHPV carcinogenic process, one of which is smoking. To assess the effect of smoking on high-risk (hr) HPV DNA positivity and on the expression of HPV E7 oncoprotein, as a surrogate of persistent hrHPV infection, we used data from women recruited for the PIPAVIR project, which examined the role of E7 protein detection in cervical cancer screening. Women were tested for hrHPV DNA, using Multiplex Genotyping (MPG), and E7 protein, using a novel sandwich ELISA method, and gave information on their smoking habits. Among 1473 women, hrHPV prevalence was 19.1%. The odds ratio (OR) for hrHPV positivity of smokers compared to non-smokers was 1.785 (95% confidence intervals (CI): 1.365–2.332, p < 0.001). The ORs for E7 positivity, concerning hrHPV positive women, ranged from 0.720 to 1.360 depending on the E7 detection assay used, but this was not statistically significant. Smoking increases the probability of hrHPV infection, and smoking intensity is positively associated to this increase. Smoking is not related to an increased probability of E7 protein positivity for hrHPV positive women.

Human papillomavirus E7 oncoprotein expression by keratinocytes alters the cytotoxic mechanisms used by CD8 T cells.

Cervical cancer is a malignant transformation of keratinocytes initiated by the E7 oncoprotein of human papillomavirus (HPV). These tumors are characterized by keratinocyte hyperproliferation and are often infiltrated with activated CD8 T cells. HPV infection confers changes to gain immunological advantage to promote chronic infection, and these persist with malignant transformation. We investigated the relative importance of the many redundant mechanisms of cytotoxicity used by CD8 T cells to kill keratinocytes expressing HPV E7 oncoprotein using extended-duration time-lapse microscopy that allows examination of cell-to-cell interactions during killing. E7 expression by keratinocytes increased susceptibility to cell-mediated killing. However, while killing of non-transgenic keratinocytes was traditional, perforin-mediated, and caspase-dependent, E7-expression favored killing by perforin-independent, caspase-independent mechanisms. The roles of perforin, TNFα, IFNγ, Fas/FasL and PD1/PD-L1 were graded according to target cell survival to produce a hierarchy of killing mechanisms utilized in killing E7-expressing cells. TNFα was essential for perforin-mediated killing of E7-expressing cells, but not perforin-independent killing. IFNγ facilitated killing by Fas/FasL interaction, especially in the absence of perforin. Additionally, expression of E7 offered protection from killing by up regulation of PD-L1, Fas and FasL expression on keratinocytes promoting fight-back by target cells, resulting in effector cell death. This study shows that keratinocytes expressing E7 are highly susceptible to killing by CD8 T cells, but utilizing different armamentarium. Down-regulation of CD8 T cell cytotoxicity in HPV-related tumors may be due to suppression by E7-expressing keratinocytes. Immunotherapy for HPV-related cancers may be improved by suppression of PD-L1, or by suppression of FasL.

Risk stratification and long-term risk prediction of E6 oncoprotein in a prospective screening cohort in China.

E6 oncoprotein is a necessary agent of HPV driven oncogenic transformation. This study is aimed at evaluating the risk stratification potency of HPV 16/18 E6 oncoprotein (E6) as a triage method for HPV positivity. Moreover, it also acts as a predictor of cervical intraepithelial neoplasia grade 3 or worse (CIN3+). The screening cohort of 1,997 women was followed for a 15 year period in approximate five-year intervals. Participants were concurrently screened by HPV DNA testing (HC2), liquid based cytology (LBC), visual inspection with acetic acid (VIA) and were referred to colposcopy and biopsy if any tests reflected positive. E6 was performed on cervical samples collected from this cohort in 2005 and 2014. The ability of E6 to predict CIN3+ risk after the five- and ten-year interval was evaluated. Among HPV positive women in 2005, E6 indicated the lowest positive rate (9.9%) compared to LBC (48.4%) and VIA (28.0%), however, a higher prevalence rate (10.3%) and 10-year cumulative incidence rate (53.0%) of CIN3+ were detected among women who were E6 positive. Meanwhile, only 4.2% and 2.9% of women with abnormal LBC and positive VIA were diagnosed as prevalent CIN3+ in 2005, 23.0% and 16.5% developed to CIN3+ after year 10, respectively. Strong associations were found between precedent and subsequent HPV persistence and E6 oncoprotein expression (ORadjusted = 40.0 and 21.2, respectively). E6 oncoprotein could serve as a low-cost, highly specific, strongly indicative point-of-care method in the triage and treatment of HPV positive women.

Human papillomavirus 16 E6 and E7 oncoprotein expression alters microRNA expression in extracellular vesicles

Extracellular vesicles released by cancer cells are mediators of intercellular communication that have been reported to contribute to carcinogenesis. Since they are readily detected in bodily fluids, they may also be used as cancer biomarkers. The E6/E7 oncoproteins drive human papillomavirus (HPV)-associated cancers, which account for approximately 5% of all human cancers worldwide. Here, we investigate how HPV16 E6/E7 oncogene expression in primary human epithelial cells alters miR expression in extracellular vesicles and compare these to changes in intracellular miR expression. Examining a panel of 68 cancer related miRs revealed that many miRs had similar expression patterns in cells and in extracellular vesicles, whereas some other miRs had different expression patterns and may be selectively packaged into extracellular vesicles. Interestingly, the set of miRs that may be selectively packaged in HPV16 E6/E7 extracellular vesicles is predicted to inhibit necrosis and apoptosis.

High stromal Foxp3-positive T cell number combined to tumor stage improved prognosis in head and neck squamous cell carcinoma

ObjectivesHead and neck squamous cell carcinomas (HNSCC), one of the most frequent cancers in the world, are largely infiltrated by inflammatory immune cells. Our aim was to evaluate the number of Foxp3+ T cells in HNSCC, reporting its prognostic power in comparison to other risk factors. Material and methodsOur clinical series was composed of 21 tumor-free peri-tumoral epithelia, 49 low grade dysplasia, 43 high grade dysplasia and 110 carcinoma samples including some cases with HPV infection. In vivo experiments were conducted on 80 C3H/HeN mice which were orthotopically injected with SCCVII CT, E7, E6 and E6/E7 cell lines. ResultsFoxp3+ T cell infiltration increased with tumor progression from normal epithelia, dysplasia to carcinoma and the increase is more important in HPV+ patients than in negative ones. Animal experiments revealed that E7 oncoprotein expression was significantly associated with an increase in Foxp3+ T cell recruitment in tumor, a delay in tumor onset and improved animal survival. Univariate Cox regression analyses demonstrated that high Foxp3+ T cell number in stromal compartment is associated with longer patient recurrence-free and overall survivals. Foxp3+ T cell number improved the prognostic value of tumor stage. Multivariate analyses reported that stromal Foxp3+ T cell number is a strong prognostic factor independent of classical risk factors such as tobacco, alcohol, and HPV status. ConclusionFoxp3+ T cell number is a significant prognostic factor for HNSCC, improving the tumor stage, and that viral E7 may play a role in the Foxp3+ T cell infiltration to the tumor.

Elevated Expression of Human Papillomavirus-16/18 E6 Oncoprotein Associates with Persistence of Viral Infection: A 3-Year Prospective Study in China

An association between high-risk human papillomavirus (HR-HPV) oncoprotein expression and viral persistence has been suggested by the outcome of etiology studies, but there are no epidemiologic studies evaluating that link. We performed a 3-year prospective study in which 2,498 Chinese women ages 25 to 65 years were screened by six screening tests, including the Onco E6: Cervical Test (Arbor Vita Corporation) in 2011 (baseline). Six-hundred and ninety women who were positive for any of the tests and a random sample of 164 women with all negative results received colposcopy, and cervical specimens for the cobas 4800 HPV test ("cobas," Roche Molecular Systems) were collected before colposcopy; of this group, 737 cervical specimens were collected to perform cobas and Onco E6: Cervical Test in 2014 (follow-up). Twenty-four cases of HPV16/18 E6 positives and 204 selected controls at baseline, 13 cases of HPV16/18 E6 positive and another 204 selected controls at follow-up were analyzed separately using unconditional logistical regression models to estimate ORs and 95% confidence intervals (CI). Compared with women who were HPV16 E6 oncoprotein negative at baseline, women in the E6-positive group had a much higher risk of HPV persistence (adjusted OR, 54.64; 95% CI, 7.19-415.09) at 3-year follow-up; a statistically strong association was also found between HPV16/18 HPV persistence and E6 oncoprotein expression detected at follow-up (adjusted OR, 360.57; 95% CI, 28.30-4,593.55). A single detection of HPV16/18 E6 oncoprotein expression was strongly associated with viral persistence. HPV16/18 E6 oncoprotein constitutes a marker for risk of HPV persistence. Cancer Epidemiol Biomarkers Prev; 25(7); 1167-74. ©2016 AACR.

Analysis of p53 codon 72 polymorphism and HPV 5,8 E6 oncoprotein expression in Basal cell carcinoma in Basrah

Analysis of p53 codon 72 polymorphism and HPV 5,8 E6 oncoprotein expression in Basal cell carcinoma in Basrah

Human papillomavirus E6 and E7 oncoproteins affect the expression of cancer-related microRNAs: additional evidence in HPV-induced tumorigenesis.

Human papillomaviruses (HPVs) are the causative agents of cervical cancer and are also associated with other types of cancers. HPVs can modulate microRNAs (miRNAs) expressed by infected cells. The production of extracellular vesicles is deregulated in cancer, and their cargo delivered to the microenvironment can promote tumorigenesis. The involvement of HPV oncoproteins on miRNA expression in cells and exosomes was analyzed in keratinocytes transduced with E6 and E7 from mucosal HPV-16 or cutaneous HPV-38 (K16 and K38). MiRNAs were investigated through the TaqMan Array Human MicroRNA Cards, followed by real-time RT-PCR assay for specific miRNAs. Selected miRNA targets were analyzed by Western blot and correlated to the HPV oncoproteins by specifically silencing E6 and E7 expression. Exosomes, isolated from K16 and K38 supernatants by differential centrifugations, were quantified through the vesicle-associated acetylcholinesterase activity. MiRNAs deregulated in K16 and K38 cells were identified. HPV-16 and/or HPV-38 E6 and E7 single proteins can modify the expression of selected miRNAs involved in the tumorigenesis, in particular miR-18a, -19a, -34a and -590-5p. The analysis of the content of exosomes isolated from HPV-positive cells revealed the presence of E6 and E7 mRNAs and few miRNAs. MiR-222, a key miRNA deregulated in many cancers, was identified in exosomes from K16 cells. HPV E6 and/or E7 oncoprotein expression can induce the deregulation of some miRNAs. Through the production and function of exosomes, HPV oncogenes as well as HPV-deregulated miRNAs can potentiate the virus oncogenic effects in the tumor cell microenvironment.

1020 The Importance of E7 Oncoprotein Expression in a Mouse Model of HPV Carcinogenesis

1020 The Importance of E7 Oncoprotein Expression in a Mouse Model of HPV Carcinogenesis

Environmental exposure and HPV infection may act synergistically to induce lung tumorigenesis in nonsmokers.

Most studies of lung tumorigenesis have focused on smokers rather than nonsmokers. In this study, we used human papillomavirus (HPV)-positive and HPV-negative lung cancer cells to test the hypothesis that HPV infection synergistically increases DNA damage induced by exposure to the carcinogen benzo[a]pyrene (B[a]P), and contributes to lung tumorigenesis in nonsmokers. DNA adduct levels induced by B[a]P in HPV-positive cells were significantly higher than in HPV-negative cells. The DNA adduct formation was dependent on HPV E6 oncoprotein expression. Gene and protein expression of two DNA repair genes, XRCC3 and XRCC5, were lower in B[a]P-treated E6-positive cells than in E6-negative lung cancer cells. The reduced expression was also detected immunohistochemically and was caused by increased promoter hypermethylation. Moreover, mutations of p53 and epidermal growth factor receptor (EGFR) genes in lung cancer patients were associated with XRCC5 inactivation. In sum, our study indicates that HPV E6-induced promoter hypermethylation of the XRCC3 and XRCC5 DNA repair genes and the resultant decrease in their expression increases B[a]P-induced DNA adducts and contributes to lung tumorigenesis in nonsmokers.

A novel dithiocarbamate derivative induces cell apoptosis through p53-dependent intrinsic pathway and suppresses the expression of the E6 oncogene of human papillomavirus 18 in HeLa cells.

Dithiocarbamates (DTCs) exhibit a broad spectrum of antitumor activities, however, their molecular mechanisms of antitumor have not yet been elucidated. Previously, we have synthesized a series of novel dithiocarbamate derivatives. These DTCs were examined for cytotoxic activities against five human cancer cell lines. In this study, one of dithiocarbamate (DTC1) with higher potential for HeLa cells was chosen to investigate molecular mechanisms for its anti-tumor activities. DTC1 could inhibit proliferation, and highly induce apoptosis in HeLa cells by activating caspase-3, -6 and -9; moreover, activities of caspase-3, -6 and -9 were inhibited by pan-caspase inhibitor, Z-VAD-FMK. Furthermore, DTC1 decreased the levels of Bcl-2 and Bcl-xL, and increased expression of cytosol cytochrome c, Bak, Bax and p53 in a time-dependent manner but had no effect on the level of Rb. It was shown that DTC1 induced HeLa cells apoptosis through a p53-dependent pathway as tested by the wild type p53 inhibitor, pifithrin-α. Additionally, the relative expression of E6 and E7 were evaluated in HPV18-positive (HeLa cells) by real-time PCR and western blotting. The results firstly demonstrated that DTC1 suppressed both expression of E6 mRNA and E6 oncoprotein, but had no effect on the expression of E7 mRNA and protein in HPV18. Our results suggested that DTC1 may serve as novel chemotherapeutic agents in the treatment of cervical cancer and potential anti-HPV virus candidates that merit further studies.

TLR9 expression in uterine cervical lesions of Uyghur women correlate with cervical cancer progression and selective silencing of human papillomavirus 16 E6 and E7 oncoproteins in vitro.

Cervical cancer is listed as one of high-incidence endemic diseases in Xinjiang. Our study aimed to evaluate the expression of TLR9 in uterine cervical tissues of Uyghur women and examine associations with clinicopathological variables. We further characterized the direct effects of TLR9 upon the selective silencing of human papillomavirus (HPV) E6 and E7 oncoprotein expression in HPV 16-positive human cervical carcinoma cells treated with siRNA in vitro. Immunohistochemistry was applied to evaluate TLR9 expression in 97 formalin-fixed paraffin-embedded cervical samples from Uyghur women; 32 diagnosed with cervical squamous cell carcinomas (CSCC) , 14 with low-grade cervical intraepithelial neoplasias (CINI), 10 medium-grade (CINII), 24 high-grade (CINIII), and 17 chronic cervicitis. BLOCK-iTTM U6 RNAi Entry Vector pENTRTM/U6-E6 and E7 was constructed and transfected the entry clone directly into the mammalian cell line 293FT. Then the HPV 16-positive SiHa human cervical carcinoma cell line was infected with RNAi recombinant lentivirus. RT-PCR and Western blotting were used to determine the expression of TLR9 in both SiHa and HPV 16 E6 and E7 silenced SiHa cells. Immunohistochemical staining showed that TLR9 expression was undetectable (88.2%) or weak (11.8%) in chronic cervicitis tissues. However, variable staining was observed in the basal layer of all normal endocervical glands. TLR9 expression, which was mainly observed as cytoplasmic staining, gradually increased in accordance with the histopathological grade in the following order: chronic cervicitis (2/17, 11.8%) <CINI (4/19, 28.6%) <CINII (3/10, 30.0%) <CINIII (12/24, 50.0%) <CSCC (17/32, 53.1%) (p<0.05), but not with tumor differentiation. RT-PCR and Western blotting showed that TLR9 expression was up-regulated in HPV16 E6 and E7 silenced SiHa cells at both mRNA and protein levels. TLR9 expression increases according to the histopathological grade of cervical pathological process. HPV E6 and E7 oncoprotein have negative effects on the expression and function of TLR9.

P0166 Human papillomavirus disrupts P130/dream complexes through different mechanisms

Background High-risk HPV E6 and E7 oncoprotein expression is important for malignant transformation of cervical cancer cells. In particular, the E7 protein of high-risk HPV binds to pRB family members (pRB, p107, and p130) for degradation. p107 and p130 pocket proteins are members of mammalian DREAM complexes, which are temporally regulated during cell cycle. E7 targets p130 for HPV16 to promote the host cell to exit from quiescence (G 0 ) state and enter S phase. However, p130 is also degraded in HPV48 even though HPV48 E7 does not binding directly to the pocket protein. Therefore we explored the mechanisms responsible for the activities of HPV16 E7 and HPV48 E7 on disruption of p130/DREAM complex. Methods The experiments were done with a p130 mutant that is defective in binding the E7 LXCXE motif (p130mE7) and another mutant that cannot be phosphorylated by CDK (p130PM22). In addition, a double mutant (p130PM22/mE7) was constructed. The p130 mutants and the wild-type p130 were expressed in HPV-transformed cell lines and FACS analysis was done. Subsequently, the T98G glioblastoma cell line was used to ectopically express both HPV16 and 48 with the p130 mutants. Findings The p130mE7 and p130mE7/PM22 mutants prevented the binding of 16E7 in CaSki cells, whereas p130PM22 and p130mE7/PM22 mutants prevented the binding of 48E7 in T98G cells. Interpretation HPV16 E7 targets p130 predominantly through direct interactions via the LXCXE motif whereby HPV48 E7 disrupts p130/DREAM via CDK2 phosphorylation.

Tumor suppressor p16 INK4A is necessary for survival of cervical carcinoma cell lines

The tumor suppressor p16(INK4A) inhibits formation of enzymatically active complexes of cyclin-dependent kinases 4 and 6 (CDK4/6) with D-type cyclins. Oncogenic stress induces p16(INK4A) expression, which in turn triggers cellular senescence through activation of the retinoblastoma tumor suppressor. Subversion of oncogene-induced senescence is a key step during cancer development, and many tumors have lost p16(INK4A) activity by mutation or epigenetic silencing. Human papillomavirus (HPV)-associated tumors express high levels of p16(INK4A) in response to E7 oncoprotein expression. Induction of p16(INK4A) expression is not a consequence of retinoblastoma tumor suppressor inactivation but is triggered by a cellular senescence response and is mediated by epigenetic derepression through the H3K27-specific demethylase (KDM)6B. HPV E7 expression causes an acute dependence on KDM6B expression for cell survival. The p16(INK4A) tumor suppressor is a critical KDM6B downstream transcriptional target and its expression is critical for cell survival. This oncogenic p16(INK4A) activity depends on inhibition of CDK4/CDK6, suggesting that in cervical cancer cells where retinoblastoma tumor suppressor is inactivated, CDK4/CDK6 activity needs to be inhibited in order for cells to survive. Finally, we note that HPV E7 expression creates a unique cellular vulnerability to small-molecule KDM6A/B inhibitors.

Association of epidermal growth factor receptor mutations with human papillomavirus 16/18 E6 oncoprotein expression in non–small cell lung cancer

Lung cancers in women, in nonsmokers, and in patients with adenocarcinoma from Asia have more prevalent mutations in the epidermal growth factor receptor (EGFR) gene than their counterparts. However, the etiology of EGFR mutations in this population remains unclear. The authors hypothesized that the human papillomavirus (HPV) type 16/18 (HPV16/18) E6 oncoprotein may contribute to EGFR mutations in Taiwanese patients with lung cancer. One hundred fifty-one tumors from patients with lung cancer were enrolled to determine HPV16/18 E6 and EGFR mutations using immunohistochemistry and direct sequencing, respectively. Levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in lung tumors and cells were evaluated using immunohistochemistry and liquid chromatography-mass spectrometry/mass spectrometry. An supF mutagenesis assay was used to determine H2 O2 -induced mutation rates of lung cancer cells with or without E6 expression. Patients with E6-positive tumors had a greater frequency of EGFR mutations than those with E6-negative tumors (41% vs 20%; P = .006). Levels of 8-oxo-dG were correlated with EGFR mutations (36% vs 16%; P = .012). Two stable clones of E6-overexpressing H157 and CL-3 cells were established for the supF mutagenesis assay. The data indicated that the cells with high E6 overexpression had higher H2 O2 -induced SupF gene mutation rates compared with the cells that expressed lower levels of E6 and compared with vector control cells. HPV16/18 E6 may contribute in part to EGFR mutations in lung cancer, at least in the Taiwanese population.