E2 Transcription Research Articles

Adenovirus E1A Activation Domain Regulates H3 Acetylation Affecting Varied Steps in Transcription at Different Viral Promoters.

How histone acetylation promotes transcription is not clearly understood. Here, we confirm an interaction between p300 and the adenovirus 2 large E1A activation domain (AD) and map the interacting regions in E1A by observing colocalization at an integrated lacO array of fusions of LacI-mCherry to E1A fragments with YFP-p300. Viruses with mutations in E1A subdomains were constructed and analyzed for kinetics of early viral RNA expression and association of acetylated H3K9, K18, K27, TBP, and RNA polymerase II (Pol II) across the viral genome. The results indicate that this E1A interaction with p300 is required for H3K18 and H3K27 acetylation at the E2early, E3, and E4 promoters and is required for TBP and Pol II association with the E2early promoter. In contrast, H3K18/27 acetylation was not required for TBP and Pol II association with the E3 and E4 promoters but was required for E4 transcription at a step subsequent to Pol II preinitiation complex assembly.IMPORTANCE Despite a wealth of data associating promoter and enhancer region histone N-terminal tail lysine acetylation with transcriptional activity, there are relatively few examples of studies that establish causation between these histone posttranslational modifications and transcription. While hypoacetylation of histone H3 lysines 18 and 27 is associated with repression, the step(s) in the overall process of transcription that is blocked at a hypoacetylated promoter is not clearly established in most instances. Studies presented here confirm that the adenovirus 2 large E1A protein activation domain interacts with p300, as reported previously (P. Pelka, J. N. G. Ablack, J. Torchia, A. S. Turnell, R. J. A. Grand, J. S. Mymryk, Nucleic Acids Res 37:1095-1106, 2009, https://doi.org/10.1093/nar/gkn1057), and that the resulting acetylation of H3K18/27 affects varied steps in transcription at different viral promoters.

Abstract 5879: Effect of Minnelide, a prodrug, on cervical cancer growth

Abstract Human Papilloma Virus (HPV) infection is associated with the development of Cervical cancer. Therapeutic resistance and recurrence remain the major obstacle to successful treatment of cervical cancer patients. Recently, a novel pro-drug Minnelide, derived from the diterpenoid triepoxide, triptolide, was found to be very effective against several human cancers in experimental animal models. The goal of the present study is to determine the effect of triptolide (in vitro) or Minnelide (in vivo) on HPV positive cervical cancer cells. Triptolide inhibited the proliferation of human cervical cancer cell lines, Ca Ski, SiHa, Me-180, and C-33a with an IC50 of 50 - 100 nM. Furthermore, triptolide inhibited the expression of viral oncoproteins E6 and E7 transcripts significantly. Consequently, p53, tumor suppressor protein levels were increased in the treated cells which led to increased apoptosis and reduced proliferation. Subsequently, we determined the effect of Minnelide on cervical cancer growth in athymic, nude mice. Tumor (SiHa and CaSki) bearing mice were treated with either 0.2 or 0.4 ug/kg doses of Minnelide. These studies showed that Minnelide inhibited tumor growth by about 70 % when compared to control group of mice and improved survival significantly. In a parallel study, combination treatment with carboplatin and Minnelide showed more than additive inhibition of tumor growth. In summary, Minnelide treatment reduced the transcript levels of HPV oncoproteins and inhibited cellular proliferation by upregulating p53. Our studies further demonstrate that Minnelide can be used either as a monotherapy or in combination with platinum drugs to inhibit the growth of cervical cancers. Citation Format: Vivek Ramakrishnan, Bhuwan Giri, Urvashi Hooda, Peter Wilkinson, Christopher deHaydu, Janneth Oleas, Sabita Roy, Sundaram Ramakrishnan, Ashok Saluja. Effect of Minnelide, a prodrug, on cervical cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5879.

Deregulated TNF-Alpha Levels Along with HPV Genotype 16 Infection Are Associated with Pathogenesis of Cervical Neoplasia in Northeast Indian Patients.

Multiple factors are associated with human papillomavirus (HPV) infection related cervical anomalies and its progression to cervical carcinoma (CaCx), but data vary with respect to the underlying HPV genotype and with population being studied. No data are available regarding the role of immunological imbalance in HPV infected CaCx pathogenesis from Northeast India, which has an ethnically distinct population, and was aimed to be addressed through this study. The study included 76 CaCx cases, 25 cervical intraepithelial neoplasia (CIN) cases, and 50 healthy female controls. HPV screening and genotyping were performed by PCR. Differential expression of tumor necrosis factor alpha (TNF-α) was studied at serum level by enzyme-linked immunosorbent assay and tissue level by immunohistochemistry and messenger RNA (mRNA) level by real-time PCR. The data were correlated with interferon gamma (IFN-γ) and NF-κβp65 levels at protein level, as well as HPV16 E6 and E7 expression at transcript level statistically. HPV infection and HPV16 genotype were predominant in the studied cohort. TNF-α was found to be downregulated at both mRNA and protein levels in CaCx cases compared to controls; and the gradient downregulation correlated with progression of the disease from normal→CIN→CaCx. TNF-α expression correlated with insufficient modulation of both IFN-γ and NF-κβp65. The HPV16 E6 and E7 transcripts were found to be sharply upregulated in CaCx cases strongly inversely correlated with the TNF-α expression. Significant role of TNF-α downregulation associated with insufficient IFN-γ and total NF-κβp65 modulation and the resulting significant upregulation of viral transcripts E6 and E7 are key to the HPV16 infection mediated CaCx pathogenesis in northeast Indian patients.

HPV genotype determination and E6/E7 mRNA detection for management of HPV positive women

BackgroundClinical management of HPV positive women is difficult since many of the infections, including high-risk oncogene genotypes (hr-HPV), are transient. Therefore only a limited number of patients have a high-grade lesion and sending all HPV positive women for colposcopy would only increase costs and unnecessary treatment, with serious psychological consequences for patients. The need has emerged to identify other HPV related markers able to correctly detect women with a high-risk of developing high-grade lesions. Genotyping and the search for E6/E7 mRNA are among the possible candidates.MethodsThe study was carried out by means of an observational analysis of the data relative to 674 HR-HPV positive women who we had observed from January 2013 to June 2015; the data had been gathered in a database at the HPV Center of the University Hospital of Catania, Italy.Women were considered eligible for this study if the following data was present in the database: Pap TEST, histologic evaluation, HPV TEST and E6/E7 mRNA detection.We calculated the Odds Ratio (OR) of woman who were mRNA positive, with CIN2+ lesions, and Odds Ratio of HPV16 positive women.ResultsTranscripts were detected in 23.6% (69/292) of the women with CIN1 and in 97.2% (210/220) of those with CIN2 + .Regarding genotyping, the 81,8% (180/220) of the women with CIN2+ had genotype 16, while only 18.1% (40/220) had genotype 18, 31, 33, 45.We calculated the OR in the group of HPV16 women with CIN2+ (OR = 4.62; 95% CI = 3.13 to 6.82), this value increased (OR = 106.12; 95% CI = 53.71 to 209.69) in women with CIN2+ and positive mRNA.DiscussionThe presence of the HPV16 genotype in our study was associated with a risk 5 times greater of developing a high-grade lesion (CIN2+) (OR = 4.62 95% CI:3.13–6.82); this supports the hypothesis that it would be opportune to have targeted protocols for the management of HPV 16 positive women. The results showed that there was an association between E6/E7 mRNA expression and histology (OR = 106.12; 95% CI = 53.71 to 209.69). The E6/E7 mRNA test showed a higher prevalence of E6 and E7 transcripts in patients with higher-grade lesions.ConclusionThe results of this study suggest that the HPV genotype determination and E6/E7 mRNA detection would find an important application for management of HPV positive women.

Separate analysis of human papillomavirus E6 and E7 messenger RNAs to predict cervical neoplasia progression.

A few studies previously suggested that human papillomavirus (HPV) E6 messenger RNA (mRNA) may exist uniformly in all grades of cervical intraepithelial neoplasia (CIN), whereas the detection rate of E7 mRNA may increase with disease progression from low-grade CIN to invasive carcinoma. The aim of this study was to clarify the different roles of E6 and E7 mRNAs in cervical carcinogenesis. The presence of each E6 and E7 mRNA was analyzed in 171 patients with pathologically-diagnosed CIN or cervical carcinoma. We utilized a RT-PCR assay based on consensus primers which could detect E6 mRNA (full-length E6/E7 transcript) and E7 mRNAs (spliced E6*/E7 transcripts) separately for various HPV types. E7 mRNAs were detected in 6% of CIN1, 12% of CIN2, 24% of CIN3, and 54% of cervical carcinoma. The presence of E7 mRNAs was significantly associated with progression from low-grade CIN to invasive carcinoma in contrast with E6 mRNA or high-risk HPV (HR-HPV) DNA (p = 0.00011, 0.80 and 0.54). The presence of both E6 and E7 mRNAs was significantly associated with HPV16/18 DNA but not with HR-HPV DNA (p = 0.0079 and 0.21), while the presence of E6 mRNA was significantly associated with HR-HPV DNA but not with HPV16/18 DNA (p = 0.036 and 0.089). The presence of both E6 and E7 mRNAs showed high specificity and low sensitivity (100% and 19%) for detecting CIN2+ by contrast with the positivity for HR-HPV DNA showing low specificity and high sensitivity (19% and 89%). The positive predictive value for detecting CIN2+ was even higher by the presence of both E6 and E7 mRNAs than by the positivity for HR-HPV DNA (100% vs. 91%). In 31 patients followed up for CIN1-2, the presence of both E6 and E7 mRNAs showed significant association with the occurrence of upgraded abnormal cytology in contrast with E6 mRNA, HR-HPV DNA, or HPV16/18 DNA (p = 0.034, 0.73, 0.53, and 0.72). Our findings support previous studies according to which E7 mRNA is more closely involved in cervical carcinogenesis than E6 mRNA. Moreover, the separate analysis of E6 and E7 mRNAs may be more useful than HR-HPV DNA test for detecting CIN2+ precisely and predicting disease progression. Further accumulation of evidence is warranted to validate our findings.

Association of an intact E2 gene with higher HPV viral load, higher viral oncogene expression, and improved clinical outcome in HPV16 positive head and neck squamous cell carcinoma.

To assess the relationship of E2 gene disruption with viral gene expression and clinical outcome in human papillomavirus (HPV) positive head and neck squamous cell carcinoma, we evaluated 31 oropharyngeal and 17 non-oropharyngeal HPV16 positive carcinomas using two PCR-based methods to test for disruption of E2, followed by Sanger sequencing. Expression of HPV16 E6, E7 and E2 transcripts, along with cellular ARF and INK4A, were also assessed by RT-qPCR. Associations between E2 disruption, E2/E6/E7 expression, and clinical outcome were evaluated by Kaplan-Meier analysis for loco-regional recurrence and disease-specific survival. The majority (n = 21, 68%) of HPV16 positive oropharyngeal carcinomas had an intact E2 gene, whereas the majority of HPV16 positive non-oropharyngeal carcinomas (n = 10, 59%) had a disrupted E2 gene. Three of the oropharyngeal tumors and two of the non-oropharyngeal tumors had deletions within E2. Detection of an intact E2 gene was associated with a higher DNA viral load and increased E2/E6/E7, ARF and INK4A expression in oropharyngeal tumors. Oropharyngeal carcinomas with an intact E2 had a lower risk of loco-regional recurrence (log-rank p = 0.04) and improved disease-specific survival (p = 0.03) compared to tumors with disrupted E2. In addition, high E7 expression was associated with lower risk of loco-regional recurrence (p = 0.004) as was high E6 expression (p = 0.006). In summary, an intact E2 gene is more common in HPV16 positive oropharyngeal than non-oropharyngeal carcinomas; the presence of an intact E2 gene is associated with higher HPV viral load, higher viral oncogene expression, and improved clinical outcome compared to patients with a disrupted E2 gene in oropharyngeal cancer.

Synthetic siRNA targeting human papillomavirus 16 E6: a perspective on in vitro nanotherapeutic approaches.

High-risk human papillomaviruses infect skin and mucosa, causing approximately 5% of cancers worldwide. In the search for targeted nanotherapeutic approaches, siRNAs against the viral E6 transcript have been molecules of interest but have not yet seen successful translation into the clinic. By reviewing the past approximately 15years of in vitro literature, we identify the need for siRNA validation protocols which concurrently evaluate ranges of key treatment parameters as well as characterize downstream process restoration in a methodical, quantitative manner and demonstrate their implementation using our own data. We also reflect on the future need for more appropriate cell culture models to represent patient lesions as well as the application of personalized approaches to identify optimal treatment strategies.

Localization of Felis catus Papillomavirus Type 2 E6 and E7 RNA in Feline Cutaneous Squamous Cell Carcinoma.

Findings from polymerase chain reaction-based methods have suggested a role of Felis catus papillomavirus 2 (FcaPV-2) in the development of feline cutaneous squamous cell carcinoma (SCC). However, because polymerase chain reaction cannot localize deoxyribonucleic acid or ribonucleic acid within the lesion, it is difficult to differentiate a coincidental FcaPV-2 infection and a causative association. Given that a key event in the pathogenesis of human papillomavirus-induced cancer is the expression of viral E6 and E7 oncogenes, localization of FcaPV-2 E6 and E7 transcription within neoplastic cells in feline SCCs would support a causative role for this papillomavirus. Therefore, RNAscope in situ hybridization was used to localize FcaPV-2 E6 and E7 transcripts in 18 formalin-fixed paraffin-embedded samples of cutaneous SCC. Positive signals were present within 5 of 9 samples (56%) from ultraviolet-protected sites and 0 of 9 samples from ultraviolet-exposed sites. In the 4 in situ hybridization-positive samples that contained adjacent hyperplastic skin, hybridization patterns in these regions were characterized by intense nuclear signals within the superficial epidermis and punctate signals within the basal epithelial layers. However, within the 5 SCCs, punctate signals were present within all layers of the epidermis, with progressive loss of intense nuclear signals within the superficial epidermis. This hybridization pattern is consistent with unregulated E6 and E7 transcription and decreased viral replication and is similar to the pattern observed in human papillomavirus-induced cancers as they progress from hyperplastic lesions containing productive infections to nonproductive neoplasms. These findings support a causative role for FcaPV-2 in the pathogenesis of feline SCC.

HPV RNA CISH score identifies two prognostic groups in a p16 positive oropharyngeal squamous cell carcinoma population

HPV-related and HPV-unrelated oropharyngeal squamous cell carcinomas are two distinct entities according to the Union for International Cancer Control, with a better prognosis conferred to HPV-related oropharyngeal squamous cell carcinomas. However, variable clinical outcomes are observed among patients with p16 positive oropharyngeal squamous cell carcinoma, which is a surrogate marker of HPV infection. We aimed to investigate the prognostic value of RNA CISH against E6 and E7 transcripts (HPV RNA CISH) to predict such variability. We retrospectively included 50 histologically confirmed p16 positive oropharyngeal squamous cell carcinomas (p16 positive immunostaining was defined by a strong staining in 70% or more of tumor cells). HPV RNA CISH staining was assessed semi-quantitatively to define two scores: RNA CISH “low” and RNA CISH “high”. Negative HPV RNA CISH cases were scored as RNA CISH “low”. This series contained 29 RNA CISH low cases (58%) and 21 RNA CISH high cases (42%). Clinical and pathologic baseline characteristics were similar between the two groups. RNA CISH high staining was associated with a better overall survival in both univariate and multivariate analyses (p = 0.033 and p = 0.042, respectively). Other recorded parameters had no prognostic value. In conclusion, HPV RNA CISH might be an independent prognostic marker in p16 positive oropharyngeal squamous cell carcinomas and might help guide therapeutics.

The full transcription map of mouse papillomavirus type 1 (MmuPV1) in mouse wart tissues.

Mouse papillomavirus type 1 (MmuPV1) provides, for the first time, the opportunity to study infection and pathogenesis of papillomaviruses in the context of laboratory mice. In this report, we define the transcriptome of MmuPV1 genome present in papillomas arising in experimentally infected mice using a combination of RNA-seq, PacBio Iso-seq, 5’ RACE, 3’ RACE, primer-walking RT-PCR, RNase protection, Northern blot and in situ hybridization analyses. We demonstrate that the MmuPV1 genome is transcribed unidirectionally from five major promoters (P) or transcription start sites (TSS) and polyadenylates its transcripts at two major polyadenylation (pA) sites. We designate the P7503, P360 and P859 as “early” promoters because they give rise to transcripts mostly utilizing the polyadenylation signal at nt 3844 and therefore can only encode early genes, and P7107 and P533 as “late” promoters because they give rise to transcripts utilizing polyadenylation signals at either nt 3844 or nt 7047, the latter being able to encode late, capsid proteins. MmuPV1 genome contains five splice donor sites and three acceptor sites that produce thirty-six RNA isoforms deduced to express seven predicted early gene products (E6, E7, E1, E1^M1, E1^M2, E2 and E8^E2) and three predicted late gene products (E1^E4, L2 and L1). The majority of the viral early transcripts are spliced once from nt 757 to 3139, while viral late transcripts, which are predicted to encode L1, are spliced twice, first from nt 7243 to either nt 3139 (P7107) or nt 757 to 3139 (P533) and second from nt 3431 to nt 5372. Thirteen of these viral transcripts were detectable by Northern blot analysis, with the P533-derived late E1^E4 transcripts being the most abundant. The late transcripts could be detected in highly differentiated keratinocytes of MmuPV1-infected tissues as early as ten days after MmuPV1 inoculation and correlated with detection of L1 protein and viral DNA amplification. In mature warts, detection of L1 was also found in more poorly differentiated cells, as previously reported. Subclinical infections were also observed. The comprehensive transcription map of MmuPV1 generated in this study provides further evidence that MmuPV1 is similar to high-risk cutaneous beta human papillomaviruses. The knowledge revealed will facilitate the use of MmuPV1 as an animal virus model for understanding of human papillomavirus gene expression, pathogenesis and immunology.

UVC radiation as an effective disinfectant method to inactivate human papillomaviruses.

Endocavitary ultrasound probes are part of a commonly used procedure in the clinical arena. The cavities examined, vaginal canal and cervix, anal canal, and oral cavity are all areas commonly infected with the human papillomavirus (HPV), thus making them susceptible to contamination by HPV. It has been demonstrated that these probes can remain contaminated with high-risk HPV even when approved disinfection protocols have been performed. we have previously shown that HPV is resistant to some high-level disinfectant (HLD). In our present study we analyzed efficacy of using high-level ultra-violet C (UVC) radiation against HPV16 and HPV18 using a hard-surface carrier test. Stocks of infectious authentic HPV16 and HPV18 virions were dried onto carriers with a 5% (v/v) protein soil or 4ppm hard water. Efficacy testing were performed with the automated device, Antigermix S1 device (UVC radiation at 253.7nm) and 0.55% OPA in quadruplicate with matched input, neutralization, and cytotoxicity controls. Hypochlorite was included as a positive control for viral deactivation. Infectivity was determined by the abundance (qRT-PCR) of the spliced E1^E4 transcript in infected recipient cells. The automated Antigermix S1 device showed excellent efficacy against HPV16 and HPV18 whereas OPA showed minimal efficacy. While HPV is highly resistant to OPA, high-level UVC radiation offers an effective disinfection practice for ultrasound probes. Our results suggest that healthcare facilities using endocavitary ultrasound probes need to strongly consider disinfection methods that are effective against HPV.

Cytokeratin 7 in Oropharyngeal Squamous Cell Carcinoma: A Junctional Biomarker for Human Papillomavirus-Related Tumors.

Background: Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (SCC) represents a distinct subgroup of head and neck tumors. We analyze the expression of cytokeratin 7, a junctional biomarker with a SEQIKA fragment, which stabilizes HPV-16 E7 transcripts, in oropharyngeal SCCs.Methods: Archived tumor specimens and epidemiologic data were collected from patients with oropharyngeal SCCs over 10 years. Briefly, DNA was extracted from tissue blocks, and HPV testing was carried out using SPF10 HPV PCR and INNO-LiPA HPV Genotyping. Immunohistochemical staining for CK7 and p16ink4a was performed on the Ventana BenchMark Ultra Immunostainer. Analysis was by light microscopy using the H-score. CK7 expression was correlated with epidemiologic data, p16ink4a positivity, and HPV status using SPSS.Results: CK7 expression was observed specifically and uniformly in the tonsillar crypt epithelium of normal tonsils and tumor specimens. There were 226 cases of oropharyngeal SCCs, with 70 demonstrating both HPV and p16 positivity. Of 216 cases evaluated for CK7, 106 demonstrated some positivity, whereas H-score > 60 was seen in 55 of these. CK7 H-score > 60 was significantly associated with tonsillar subsite and HPV and p16 positivity.Conclusions: An association between CK7 and HPV has been demonstrated. CK7-expressing tonsillar crypt cells potentially represent an oropharyngeal subsite susceptible to HPV-related SCC.Impact: Along with the cervix and anorectum, specific oropharyngeal expression of CK7 in a site predisposed to HPV-related tumors may suggest a role for CK7 in the pathogenesis of this subgroup of tumors. Further research is warranted to characterize the association between CK7 and HPV-related head and neck SCC. Cancer Epidemiol Biomarkers Prev; 26(5); 702-10. ©2017 AACR.

The 5\u2032UTR in human adenoviruses: leader diversity in late gene expression

Human adenoviruses (HAdVs) shut down host cellular cap-dependent mRNA translation while initiating the translation of viral late mRNAs in a cap-independent manner. HAdV 5′ untranslated regions (5′UTRs) are crucial for cap-independent initiation, and influence mRNA localization and stability. However, HAdV translational regulation remains relatively uncharacterized. The HAdV tripartite leader (TPL), composed of three introns (TPL 1–3), is critical to the translation of HAdV late mRNA. Herein, we annotated and analyzed 72 HAdV genotypes for the HAdV TPL and another previously described leader, the i-leader. Using HAdV species D, type 37 (HAdV-D37), we show by reverse transcription PCR and Sanger sequencing that mRNAs of the HAdV-D37 E3 transcription unit are spliced to the TPL. We also identified a polycistronic mRNA for RID-α and RID-β. Analysis of the i-leader revealed a potential open reading frame within the leader sequence and the termination of this potential protein in TPL3. A potential new leader embedded within the E3 region was also detected and tentatively named the j-leader. These results suggest an underappreciated complexity of post-transcriptional regulation, and the importance of HAdV 5′UTRs for precisely coordinated viral protein expression along the path from genotype to phenotype.

HPV 16 E5 oncoprotein is expressed in early stage carcinogenesis and can be a target of immunotherapy

ABSTRACTHPV16 persistent infection is a well-known condition that precedes human cancer development. High risk HPV E5 proteins cooperate with E6/E7 oncogenes to promote hyper-proliferation of infected cells leading to possible cancer progression. Thus, presence of E5 viral transcripts could be a key marker of active infection and, in turn, a target of immunotherapy. Purpose of the study is to detect E5 transcripts in clinical samples and to explore the activity of novel anti-HPV16 E5 DNA vaccines. HPV transcripts were detected by PCR with specific primers encompassing the splice-donor sites of E5 transcript. For E5-based immunotherapies, 2 E5-based versions of DNA vaccines carrying whole E5 gene or a synthetic multiepitope gene were improved by fusion to sequence of PVX coat protein. These vaccines were challenged with a new luminescent animal model based on C3-Luc cell line. E5 transcripts were detected in clinical samples of women with HPV positive low-grade SIL, demonstrating the validity of our test. In C3 pre-clinical mouse model, vaccine candidates were able to induce a strong cellular immunity as indicated by ELISPOT assays. In addition, E5-CP vaccines elicited strong anti-tumor effects as showed by decreased tumor growth monitored by animal imaging. The tumor growth inhibition was comparable to those obtained with anti-E7 DNA vaccines. In conclusion, detection of E5 transcripts in clinical samples indicates that E5 is a possible target of immunotherapy. Data from pre-clinical model demonstrate that E5 genetic immunization is feasible, efficacious and could be utilized in clinical trials.

Modified Adenovirus Reduces De Novo Peritoneal Adhesions in Rats and Limits Off-Target Transfection. Role of EZH2 in Adhesion Formation

ABSTRACTAim of the study: Adenovector encoding tissue plasminogen activator (tPA) was shown to reduce experimental peritoneal adhesion. We investigated the targeting potential of our modified adenovector, its ability to reduce adhesions and the epigenetic role of histone methyltransferase EZH2 in adhesion formation. Materials and methods: Control lacZ, nonmodified tPA or modified tPA vectors were instilled in the peritoneal cavity after injury in de novo adhesions or after lysis of adhesions in recurrent adhesions. Adhesion severity was scored and adhesions and liver tissues were examined for adenovirus E4 gene and tPA mRNA expression. Levels of tPA, plasminogen activator inhibitor-1 (PAI-1), transforming growth factor-β1 (TGF-β1), and EZH2 expression were measured. Results: E4 transcripts were detected in adhesions of nonmodified and modified and in livers of nonmodified but not in livers of modified de novo adhesions. Both nonmodified (p = 0.021) and modified vectors (p = 0.036) reduced the severity of de novo adhesions compared to lacZ vector. Levels of tPA in nonmodified (p = 0.021) and modified adhesions (p = 0.001) were elevated while PAI-1 (p = 0.013 and p = 0.001, respectively) and TGF-β1 levels (p = 0.002 and p = 0.016, respectively) were reduced compared with lacZ group. All vectors were not expressed in recurrent adhesions and severity score were not different among groups. EZH2 levels were elevated in de novo nontreated (p = 0.001) and was further increased in recurrent (p = 0.001) nontreated adhesions compared with noninjured peritoneum. Conclusion: Modified adenovirus successfully targeted de novo adhesions but not liver tissues and reduced the severity of de novo adhesions. EZH2 is involved in the development and progression of peritoneal adhesions.

E4F1 controls a transcriptional program essential for pyruvate dehydrogenase activity

The mitochondrial pyruvate dehydrogenase (PDH) complex (PDC) acts as a central metabolic node that mediates pyruvate oxidation and fuels the tricarboxylic acid cycle to meet energy demand. Here, we reveal another level of regulation of the pyruvate oxidation pathway in mammals implicating the E4 transcription factor 1 (E4F1). E4F1 controls a set of four genes [dihydrolipoamide acetlytransferase (Dlat), dihydrolipoyl dehydrogenase (Dld), mitochondrial pyruvate carrier 1 (Mpc1), and solute carrier family 25 member 19 (Slc25a19)] involved in pyruvate oxidation and reported to be individually mutated in human metabolic syndromes. E4F1 dysfunction results in 80% decrease of PDH activity and alterations of pyruvate metabolism. Genetic inactivation of murine E4f1 in striated muscles results in viable animals that show low muscle PDH activity, severe endurance defects, and chronic lactic acidemia, recapitulating some clinical symptoms described in PDC-deficient patients. These phenotypes were attenuated by pharmacological stimulation of PDH or by a ketogenic diet, two treatments used for PDH deficiencies. Taken together, these data identify E4F1 as a master regulator of the PDC.

E4F1-mediated control of pyruvate dehydrogenase activity is essential for skin homeostasis

The multifunctional protein E4 transcription factor 1 (E4F1) is an essential regulator of epidermal stem cell (ESC) maintenance. Here, we found that E4F1 transcriptionally regulates a metabolic program involved in pyruvate metabolism that is required to maintain skin homeostasis. E4F1 deficiency in basal keratinocytes resulted in deregulated expression of dihydrolipoamide acetyltransferase (Dlat), a gene encoding the E2 subunit of the mitochondrial pyruvate dehydrogenase (PDH) complex. Accordingly, E4f1 knock-out (KO) keratinocytes exhibited impaired PDH activity and a redirection of the glycolytic flux toward lactate production. The metabolic reprogramming of E4f1 KO keratinocytes associated with remodeling of their microenvironment and alterations of the basement membrane, led to ESC mislocalization and exhaustion of the ESC pool. ShRNA-mediated depletion of Dlat in primary keratinocytes recapitulated defects observed upon E4f1 inactivation, including increased lactate secretion, enhanced activity of extracellular matrix remodeling enzymes, and impaired clonogenic potential. Altogether, our data reveal a central role for Dlat in the metabolic program regulated by E4F1 in basal keratinocytes and illustrate the importance of PDH activity in skin homeostasis.

Cervical Cancer Stem Cells Selectively Overexpress HPV Oncoprotein E6 that Controls Stemness and Self-Renewal through Upregulation of HES1.

Perturbation of keratinocyte differentiation by E6/E7 oncoproteins of high-risk human papillomaviruses that drive oncogenic transformation of cells in squamocolumnar junction of the uterine cervix may confer "stem-cell like" characteristics. However, the crosstalk between E6/E7 and stem cell signaling during cervical carcinogenesis is not well understood. We therefore examined the role of viral oncoproteins in stem cell signaling and maintenance of stemness in cervical cancer. Isolation and enrichment of cervical cancer stem-like cells (CaCxSLCs) was done from cervical primary tumors and cancer cell lines by novel sequential gating using a set of functional and phenotypic markers (ABCG2, CD49f, CD71, CD133) in defined conditioned media for assessing sphere formation and expression of self-renewal and stemness markers by FACS, confocal microscopy, and qRT-PCR. Differential expression level and DNA-binding activity of Notch1 and its downstream targets in CaCxSLCs as well as silencing of HPVE6/Hes1 by siRNA was evaluated by gel retardation assay, FACS, immunoblotting, and qRT-PCR followed by in silico and in vivo xenograft analysis. CaCxSLCs showed spheroid-forming ability, expressed self-renewal and stemness markers Oct4, Sox2, Nanog, Lrig1, and CD133, and selectively overexpressed E6 and HES1 transcripts in both cervical primary tumors and cancer cell lines. The enriched CaCxSLCs were highly tumorigenic and did recapitulate primary tumor histology in nude mice. siRNA silencing of HPVE6 or Hes1 abolished sphere formation, downregulated AP-1-STAT3 signaling, and induced redifferentiation. Our findings suggest the possible mechanism by which HPVE6 potentially regulate and maintain stem-like cancer cells through Hes1. Clin Cancer Res; 22(16); 4170-84. ©2016 AACR.

Human papillomavirus oncoproteins differentially modulate epithelial-mesenchymal transition in 5-FU-resistant cervical cancer cells.

Etiological role of viral proteins E6 and E7 of high-risk HPV in cervical carcinogenesis is well established. However, their contribution in chemoresistance and epithelial-mesenchymal transition (EMT) that leads to advanced metastatic lesions and chemoresistance is poorly defined. In the present study, contribution of viral oncoproteins in acquisition of EMT character during onset of chemoresistance was assessed. A chemoresistant cell line (SiHaCR) was developed from an established HPV16-positive cervical cancer cell line, SiHa, by escalating selection pressure of 5-fluorouracil (5-FU). Expression of Survivin, ABCG2, Snail, Slug, Twist, and Vimentin was examined in SiHa and SiHaCR cells by reverse transcriptase-PCR (RT-PCR) and immunoblotting assays. Mesenchymal phenotype in SiHaCR cells was confirmed by assessment of migration and invasion potentials. SiHaCR cells displayed elevated level of functional and molecular markers associated with chemoresistance (Survivin, ABCG2) and EMT (Snail, Slug, Twist, Vimentin) and reduced E-cadherin. SiHaCR also showed increased levels of HPV16 E6 and E7 transcripts. Specific silencing of HPV16 E6, but not E7 using corresponding siRNA, demonstrated a differential involvement of HPV oncogenes in manifestation of EMT. HPV16 E6 silencing resulted in reduction of Slug and Twist expression. However, the expression of Snail and Vimentin was only marginally affected. In contrast, there was an increase in the expression of E-cadherin. A reduced migration and invasion capabilities were observed only in E6-silenced SiHaCR cells, which further confirmed functional contribution of HPV16 E6 in manifestation of EMT. Taken together, our study demonstrated an active involvement of HPV16 E6 in regulation of EMT, which promotes chemoresistance in cervical cancer.

5-aza-2'-deoxycytidine (DAC) treatment downregulates the HPV E6 and E7 oncogene expression and blocks neoplastic growth of HPV-associated cancer cells.

High-risk human papillomaviruses (hr HPVs) may cause various human cancers and associated premalignant lesions. Transformation of the host cells is triggered by overexpression of the viral oncogenes E6 and E7 that deregulate the cell cycle and induce chromosomal instability. This process is accompanied by hypermethylation of distinct CpG sites resulting in silencing of tumor suppressor genes, inhibition of the viral E2 mediated control of E6 and E7 transcription as well as deregulated expression of host cell microRNAs. Therefore, we hypothesized that treatment with demethylating agents might restore those regulatory mechanisms. Here we show that treatment with 5-aza-2′-deoxycytidine (DAC) strongly decreases the expression of E6 and E7 in a panel of HPV-transformed cervical cancer and head and neck squamous cell carcinoma cell lines. Reduction of E6 and E7 further resulted in increased target protein levels including p53 and p21 reducing the proliferation rates and colony formation abilities of the treated cell lines. Moreover, DAC treatment led to enhanced expression of tumor the suppressive miRNA-375 that targets and degrades E6 and E7 transcripts. Therefore, we suggest that DAC treatment of HPV-associated cancers and respective precursor lesions may constitute a targeted approach to subvert HPV oncogene functions that deserves testing in clinical trials.