Induction of Senescence-like State and Suppression of Telomerase Activity through Inhibition of HPV E6/E7 Gene Expression in Cells Immortalized by HPV16 DNA

Hwang E.S.

Studies on the replicative senescence and premature senescence induced by various stresses in normal somatic cells have provided important clues on the role of telomere shortening and mechanisms involved in aging processes and carcinogenesis. Recent work revealed that cancer cells also are induced to undergo replicative senescence state via telomere shortening as well as to enter a senescence-like state by the activation of cell cycle inhibitory pathways. Although less relevant in terms of aging physiology, studies on these phenomena in cancer cells have yielded important information on telomerase regulation and the roles of tumor suppressors in senescence and immortalization, and are expected to generate valuable anti-cancer strategies. Several features of the phenotypes specific for the senescent and senescence-like states induced in cancer cells are discussed.

Hwang S.G., Lee D., Kim J., Seo T., Choe J.

Damm K.

Telomerase, the ribonucleoprotein enzyme maintaining the telomeres of eukaryotic chromosomes, is active in most human cancers and in germline cells but, with few exceptions, not in normal human somatic tissues. Telomere maintenance is essential to the replicative potential of malignant cells and the inhibition of telomerase can lead to telomere shortening and cessation of unrestrained proliferation. We describe novel chemical compounds which selectively inhibit telomerase in vitro and in vivo. Treatment of cancer cells with these inhibitors leads to progressive telomere shortening, with no acute cytotoxicity, but a proliferation arrest after a characteristic lag period with hallmarks of senescence, including morphological, mitotic and chromosomal aberrations and altered patterns of gene expression. Telomerase inhibition and telomere shortening also result in a marked reduction of the tumorigenic potential of drug-treated tumour cells in a mouse xenograft model. This model was also used to demonstrate in vivo efficacy with no adverse side effects and uncomplicated oral administration of the inhibitor. These findings indicate that potent and selective, non-nucleosidic telomerase inhibitors can be designed as novel cancer treatment modalities.

Zhou X.Z., Lu K.P.

Telomerase activity is critical for normal and transformed human cells to escape from crisis and is implicated in oncogenesis. Here we describe a novel Pin2/TRF1 binding protein, PinX1 that inhibits telomerase activity and affects tumorigenicity. PinX1 and its small TID domain bind the telomerase catalytic subunit hTERT and potently inhibit its activity. Overexpression of PinX1 or its TID domain inhibits telomerase activity, shortens telomeres, and induces crisis, whereas depletion of endogenous PinX1 increases telomerase activity and elongates telomeres. Depletion of PinX1 also increases tumorigenicity in nude mice, consistent with its chromosome localization at 8p23, a region with frequent loss of heterozygosity in a number of human cancers. Thus, PinX1 is a potent telomerase inhibitor and a putative tumor suppressor.

Gewin L., Galloway D.A.

ABSTRACT Human papillomavirus type 16 (HPV-16) E6 activates telomerase specifically in epithelial cells. The oncogene c- myc has also been shown to activate telomerase in several cell types. Here we show that while both HPV-16 E6 and c- myc require intact E boxes to transactivate the hTERT promoter, E6 does not induce hTERT transcription simply by inducing expression of c- myc . Moreover, hTERT transactivation by HPV-16 E6 correlates with its ability to bind the cellular E6-associated protein (E6AP), suggesting that E6 and E6AP may target a regulator of hTERT expression.

Oh S.T., Kyo S., Laimins L.A.

ABSTRACT High-risk human papillomaviruses (HPVs) immortalize keratinocytes by disrupting the retinoblastoma protein (Rb)/p16 pathway and activating telomerase. The E7 oncoprotein targets Rb, while the E6 oncoprotein induces telomerase activity in human keratinocytes. This study has examined the mechanism by which E6 activates telomerase. Expression of human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, was found to be increased in keratinocytes stably expressing HPV type 16 E6, suggesting that E6 acts to increase hTERT transcription. hTERT expression and telomerase activity were activated to significantly higher levels in cells expressing both E6 and E7 than in cells expressing E6 alone. This indicates that E7 may augment E6-mediated activation of hTERT transcription. In transient-transfection assays using hTERT reporters, the induction of hTERT expression by E6 was found to be mediated by a 258-bp fragment of the hTERT promoter, proximal to the ATG initiation codon. Previous studies have demonstrated that overexpression of Myc can activate hTERT expression, suggesting that Myc may be a mediator of E6-mediated hTERT induction. However, in cells stably expressing E6, no strict correlation between the level of Myc and the activation of hTERT was found. Consistent with this observation, mutation of the two Myc binding sites in the hTERT promoter only modestly reduced responsiveness to E6 in transient reporter assays. This indicates that activation of Myc-dependent transcription is not essential for E6-mediated upregulation of hTERT expression. The hTERT promoter also contains five GC-rich elements that can bind Sp1. Mutation of these sites within the 258-bp fragment partially reduced hTERT induction by E6. However, when mutations in the Sp1 sites were combined with the mutated Myc binding sites, all activation by E6 was lost. This indicates that it is the combinatorial binding of factors to Myc and Sp1 cis elements that is responsible for hTERT induction by E6.

Steenbergen R.D., Kramer D., Meijer C.J., Walboomers J.M., Trott D.A., Cuthbert A.P., Newbold R.F., Overkamp W.J., Zdzienicka M.Z., Snijders P.J.

High-risk human papillomavirus (HPV) types play a major role in the development of cervical cancer in vivo and can induce immortalization of primary human keratinocytes in vitro. Activation of the telomere-lengthening enzyme telomerase constitutes a key event in both processes. Because losses of alleles from chromosome 6 and increased telomerase activity have been observed in high-grade premalignant cervical lesions, we analyzed whether human chromosome 6 harbors a putative telomerase repressor locus that may be involved in HPV-mediated immortalization.Microcell-mediated chromosome transfer was used to introduce chromosomes 6 and 11 to the in vitro generated HPV type 16 (HPV16)-immortalized keratinocyte cell line FK16A and to the in vivo derived HPV16-containing cervical cancer cell line SIHA: Hybrid clones were analyzed for growth characteristics, telomerase activity, human telomerase reverse transcriptase (hTERT) and HPV16 E6 expression, and telomere length. FK16A hybrid clones were also transduced with an hTERT-containing retrovirus to examine the effect of ectopic hTERT expression on growth. Statistical tests were two-sided.Introduction of human chromosome 6 but not of chromosome 11 to both cell lines yielded hybrid cells that demonstrated crisis-like features (i.e., enlarged and flattened morphology, vacuolation, and multinucleation) and underwent growth arrest after a marked lag period. In the chromosome 6 hybrid clones analyzed, telomerase activity and hTERT messenger RNA (mRNA) expression were statistically significantly reduced compared with those in the chromosome 11 hybrid clones (for telomerase activity, P =.004 for the FK16A hybrids and P =.039 for the SiHa hybrids; for hTERT mRNA expression, P =.003 for the FK16A hybrids). The observed growth arrest was associated with telomeric shortening. Ectopic expression of hTERT in FK16A cells could prevent the telomeric shortening-based growth arrest induced by chromosome 6.Chromosome 6 may harbor a repressor of hTERT transcription, the loss of which may be involved in HPV-mediated immortalization.

Veldman T., Horikawa I., Barrett J.C., Schlegel R.

ABSTRACT The E6 and E7 oncogenes of human papillomavirus type 16 (HPV-16) are sufficient for the immortalization of human genital keratinocytes in vitro. The products of these viral genes associate with p53 and pRb tumor suppressor proteins, respectively, and interfere with their normal growth-regulatory functions. The HPV-16 E6 protein has also been shown to increase the telomerase enzyme activity in primary epithelial cells by an unknown mechanism. We report here that a study using reverse transcription-PCR and RNase protection assays in transduced primary human foreskin keratinocytes (HFKs) shows that the E6 gene (but not the E7 gene) increases telomerase hTERT gene transcription coordinately with E6-induced telomerase activity. In these same cells, the E6 gene induces a 6.5-fold increase in the activity of a 1,165-bp 5′ promoter/regulatory region of the hTERT gene, and this induction is attributable to a minimal 251-bp sequence (−211 to +40). Furthermore, there is a 35-bp region (+5 to +40) within this minimal E6-responsive promoter that is responsible for 60% of E6 activity. Although the minimal hTERT promoter contains Myc-responsive E-box elements and recent studies have suggested a role for Myc protein in hTERT transcriptional control, we found no alterations in the abundance of either c-Myc or c-Mad in E6-transduced HFKs, suggesting that there are other or additional transcription factors critical for regulating hTERT expression.

Campisi J.

Can studying cultured cells inform us about the biology of aging? The idea that this may be was stimulated by the first formal description of replicative senescence. Replicative senescence limits the proliferation of normal human cells in culture, causing them to irreversibly arrest growth and adopt striking changes in cell function. We now know that telomere shortening, which occurs in most somatic cells as a consequence of DNA replication, drives replicative senescence in human cells. However, rodent cells also undergo replicative senescence, despite very long telomeres, and DNA damage, the action of certain oncogenes and changes in chromatin induce a phenotype similar to that of replicatively senescent cells. Thus, replicative senescence is an example of the more general process of cellular senescence, indicating that the telomere hypothesis of aging is a misnomer, Cellular senescence appears to be a response to potentially oncogenic insults, including oxidative stress. The growth arrest almost certainly suppresses tumorigenesis, at least in young organisms, whereas the functional changes may contribute to aging, although this has yet to be critically tested. Thus, cellular senescence may be an example of antagonistic pleiotropy. Cross-species comparisons suggest there is a relationship between the senescence of cells in culture and organismal life span, but the relationship is neither quantitative nor direct.

Moon M.S., Lee C.J., Um S.J., Park J.S., Yang J.M., Hwang E.S.

E6 and E7 proteins of high-risk-type human papillomavirus are major etiological agents for cervical carcinomas and are continuously expressed in those cancer cells. They inhibit cell cycle control functions by inactivating p53 and Rb proteins and also immortalize cells through the induction of telomerase activity. Expression of E6 and E7 genes in HeLa, an HPV18-positive cell line, has been shown to be inhibited by the E2 protein of bovine papillomavirus (BPV1), and this resulted in the activation of the p53-mediated growth inhibitory pathway followed by an inhibition of cell proliferation. In this study, the effect of BPV1 E2-mediated inhibition of E6 and E7 expression was examined in HPV16-positive cervical carcinoma cell lines recently established from Korean patients.BPV1 E2 was expressed in the test cells through acute infection of an SV40-BPV1 recombinant virus. Its effect on cell proliferation was assessed through MTT and DNA synthesis assays, and the status of factors involved in cell cycle control was examined through Western blotting and reverse transcription-polymerase chain reaction.BPV1 E2 expression caused a significant decrease in E6/E7 transcription in all three cell lines. This was accompanied by an increase in the levels of p53 protein and activity and a decrease in the expression of Cdc25A, a Cdk2-activating phosphatase. Concomitantly, E2F1 activity and cellular DNA synthesis capacity were significantly reduced.These results indicate that inhibition of E6/E7 gene expression in the HPV16-positive cervical carcinoma cells induces suppression in cell proliferation by activating the growth inhibitory factors, p53 and Rb, and also by downregulating the cell cycle stimulatory factor, Cdc25A.

Goodwin E.C., DiMaio D.

Most cervical carcinomas express high-risk human papillomaviruses (HPVs) E6 and E7 proteins, which neutralize cellular tumor suppressor function. To determine the consequences of removing the E6 and E7 proteins from cervical cancer cells, we infected HeLa cells, a cervical carcinoma cell line that contains HPV18 DNA, with a recombinant virus that expresses the bovine papillomavirus E2 protein. Expression of the E2 protein resulted in rapid repression of HPV E6 and E7 expression, followed ≈12 h later by profound inhibition of cellular DNA synthesis. Shortly after E6/E7 repression, there was dramatic posttranscriptional induction of p53. Two p53-responsive genes, mdm2 and p21, were induced with slightly slower kinetics than p53 and appeared to be functional, as assessed by inhibition of cyclin-dependent kinase activity and p53 destabilization. There was also dramatic posttranscriptional induction of p105 Rb and p107 after E6/E7 repression, followed shortly thereafter by induction of p130. By 24 h after infection, only hypophosphorylated p105 Rb was detectable and transcription of several Rb/E2F-regulated genes was dramatically repressed. Constitutive expression of the HPV16 E6/E7 genes alleviated E2-induced growth inhibition and impaired activation of the Rb pathway and repression of E2F-responsive genes. This dynamic response strongly suggests that the p53 and Rb tumor suppressor pathways are intact in HeLa cells and that repression of HPV E6 and E7 mobilizes these pathways in an orderly fashion to deliver growth inhibitory signals to the cells. Strikingly, the major alterations in the cell cycle machinery underlying cervical carcinogenesis can be reversed by repression of the endogenous HPV oncogenes.

Wells S.I.

Xu D., Wang Q., Gruber A., Björkholm M., Chen Z., Zaid A., Selivanova G., Peterson C., Wiman K.G., Pisa P.

The p53 tumor suppressor protein inhibits the formation of tumors through induction of cell cycle arrest and/or apoptosis. In the present study we demonstrated that p53 is also a powerful inhibitor of human telomerase reverse transcriptase (hTERT), a key component for telomerase. Activation of either exogenous temperature-sensitive (ts) p53 in BL41 Burkitt lymphoma cells or endogenous wild type (wt) p53 at a physiological level in MCF-7 breast carcinoma cells triggered a rapid downregulation of hTERT mRNA expression, independently of the induction of the p53 target gene p21. Co-transfection of an hTERT promoter construct with wt p53 but not mutant p53 in HeLa cells inhibited the hTERT promoter activity. Furthermore, the activation of the hTERT promoter in Drosophila Schneider SL2 cells was completely dependent on the ectopic expression of Sp1 and was abrogated by wt p53. Finally, wt p53 inhibited Sp1 binding to the hTERT proximal promoter by forming a p53-Sp1 complex. Since activation of telomerase, widely observed in human tumor cell lines and primary tumors, is a critical step in tumorigenesis, wt p53-triggered inhibition of hTERT/telomerase expression may reflect yet another mechanism of p53-mediated tumor suppression. Our findings provide new insights into both the biological function of p53 and the regulation of hTERT/telomerase expression.

Goodwin E.C., Yang E., Lee C., Lee H., DiMaio D., Hwang E.

Expression of the bovine papillomavirus E2 regulatory protein in human cervical carcinoma cell lines repressed expression of the resident human papillomavirus E6 and E7 oncogenes and within a few days caused essentially all of the cells to synchronously display numerous phenotypic markers characteristic of cells undergoing replicative senescence. This process was accompanied by marked but in some cases transient alterations in the expression of cell cycle regulatory proteins and by decreased telomerase activity. We propose that the human papillomavirus E6 and E7 proteins actively prevent senescence from occurring in cervical carcinoma cells, and that once viral oncogene expression is extinguished, the senescence program is rapidly executed. Activation of endogenous senescence pathways in cancer cells may represent an alternative approach to treat human cancers.

Duensing S., Lee L.Y., Duensing A., Basile J., Piboonniyom S., Gonzalez S., Crum C.P., Münger K.

Loss of genomic integrity is a defining feature of many human malignancies, including human papillomavirus (HPV)-associated preinvasive and invasive genital squamous lesions. Here we show that aberrant mitotic spindle pole formation caused by abnormal centrosome numbers represents an important mechanism in accounting for numeric chromosomal alterations in HPV-associated carcinogenesis. Similar to what we found in histopathological specimens, HPV-16 E6 and E7 oncoproteins cooperate to induce abnormal centrosome numbers, aberrant mitotic spindle pole formation, and genomic instability. The low-risk HPV-6 E6 and E7 proteins did not induce such abnormalities. Whereas the HPV-16 E6 oncoprotein has no immediate effects on centrosome numbers, HPV-16 E7 rapidly induces abnormal centrosome duplication. Thus our results suggest a model whereby HPV-16 E7 induces centrosome-related mitotic disturbances that are potentiated by HPV-16 E6.

Khasawneh A.I., Al Shboul S., Himsawi N., Al Rousan A., Shahin N.A., El-Sadoni M., Alhesa A., Abu Ghalioun A., Khawaldeh S., Shawish B., Mahfouz S.A., Al-Shayeb M., Dawoud S.A., Tlilan R., Nuseir M., et. al.

Oncogene-Induced Senescence (OIS) is a form of senescence that occurs as a consequence of oncogenic overstimulation and possibly infection by oncogenic viruses. Whether senescence plays a role in the pathogenesis of cervical cancer (CC) is not well understood. Moreover, whether cervical epithelial cells that are part of the premalignant cervical intraepithelial neoplasia (CIN), exhibit markers of OIS in Human Papillomavirus (HPV)-infected tissue, has not been investigated. We utilized a set of patient-derived premalignant and malignant tissue samples to investigate the protein (Ki67 and Lamin B1) and gene (TP53, IL1A, CCL2, and MMP9) expression of several OIS-associated biomarkers using immunohistochemistry (IHC) and qRT-PCR, respectively. Furthermore, we characterized the HPV status of all tissue samples. Most of the CC samples (34/37) were positive for HPV, mainly HPV-16 which was observed in 62.2% of the CC samples. Among CINs, HPV infection was found in 60.2% of the 32 samples with HPV-16 as the dominant genotype in 58.5% of the CINs. IHC analysis revealed a significant increase in the expression levels of both Ki67 and Lamin B1 proteins in CC tissue compared to CIN. On average, 93% of tumor cells were positive for Ki67 in comparison to only 25% of premalignant cells in CIN samples. Similarly, Lamin B1 expression was observed in 89% of tumor cells in malignant tissue on average, compared to 60% in CIN samples. Importantly, Lamin B1 expression was elevated in nonmalignant cervical tissue suggesting that its downregulation is more predominant in the premalignant state. Furthermore, RT-PCR revealed a significant decrease in the expression of TP53, IL1a, CCL2, and MMP9 markers in CC samples compared to CINs. Specifically, 84% of CC samples showed reduced TP53 expression, 90% showed reduced IL1a expression, 74% showed reduced CCL2 expression, and 76% showed reduced MMP9 expression when compared with their premalignant baseline. Infection of HPV was confirmed in 61% of the tumor tissues while only 25% of the CINs were positive for HPV. This work shall provide an opportunity to further examine the role of OIS in the process of HPV-driven CC development.

Luna A.J., Young J.M., Sterk R.T., Bondu V., Schultz F.A., Kusewitt D.F., Kang H., Ozbun M.A.

Human papillomaviruses (HPVs) are a significant public health concern due to their widespread transmission, morbidity, and oncogenic potential. Despite efficacious vaccines, millions of unvaccinated individuals and those with existing infections will develop HPV-related diseases for the next two decades and beyond. The continuing burden of HPV-related diseases is exacerbated by the lack of effective therapies or cures for infections, highlighting the need to identify and develop antivirals. The experimental murine papillomavirus type 1 (MmuPV1) model provides opportunities to study papillomavirus pathogenesis in cutaneous epithelium, the oral cavity, and the anogenital tract. However, to date the MmuPV1 infection model has not been used to demonstrate the effectiveness of potential antivirals. We previously reported that inhibitors of cellular MEK/ERK signaling suppress oncogenic HPV early gene expression in three-dimensional tissue cultures. Herein, we adapted the MmuPV1 infection model to determine whether MEK inhibitors have anti-papillomavirus properties in vivo. We demonstrate that oral delivery of a MEK1/2 inhibitor promotes papilloma regression in immunodeficient mice that otherwise would have developed persistent infections. Quantitative histological analyses reveal that inhibition of MEK/ERK signaling reduces E6/E7 mRNA, MmuPV1 DNA, and L1 protein expression within MmuPV1-induced lesions. These data suggest that MEK1/2 signaling is essential for both early and late MmuPV1 replication events supporting our previous findings with oncogenic HPVs. We also provide evidence that MEK inhibitors protect mice from developing secondary tumors. Thus, our data suggest that MEK inhibitors have potent antiviral and anti-tumor properties in a preclinical mouse model and merit further investigation as papillomavirus antiviral therapies.

Luna A.J., Young J.M., Sterk R.T., Bondu V., Schultz F.A., Kusewitt D.F., Kang H., Ozbun M.A.

AbstractHuman papillomaviruses (HPVs) are a significant public health concern due to their widespread transmission, morbidity, and oncogenic potential. Despite efficacious vaccines, millions of unvaccinated individuals and those with existing infections will develop HPV-related diseases for the next two decades. The continuing burden of HPV-related diseases is exacerbated by the lack of effective therapies or cures for most infections, highlighting the need to identify and develop antivirals. The experimental murine papillomavirus type 1 (MmuPV1) model provides opportunities to study papillomavirus pathogenesis in cutaneous epithelium, the oral cavity, and the anogenital tract. However, to date the MmuPV1 infection model has not been used to demonstrate the effectiveness of potential antivirals. We previously reported that inhibitors of cellular MEK/ERK signaling suppress oncogenic HPV early gene expressionin vitro. Herein, we adapted the MmuPV1 infection model to determine whether MEK inhibitors have anti-papillomavirus propertiesin vivo. We demonstrate that oral delivery of a MEK1/2 inhibitor promotes papilloma regression in immunodeficient mice that otherwise would have developed persistent infections. Quantitative histological analyses revealed that inhibition of MEK/ERK signaling reduces E6/E7 mRNAs, MmuPV1 DNA, and L1 protein expression within MmuPV1-induced lesions. These data suggest that MEK1/2 signaling is essential for both early and late MmuPV1 replication events supporting our previous findings with oncogenic HPVs. We also provide evidence that MEK inhibitors protect mice from developing secondary tumors. Thus, our data suggest that MEK inhibitors have potent anti-viral and anti-tumor properties in a preclinical mouse model and merit further investigation as papillomavirus antiviral therapies.Significance StatementPersistent human papillomavirus (HPV) infections cause significant morbidity and oncogenic HPV infections can progress to anogenital and oropharyngeal cancers. Despite the availability of effective prophylactic HPV vaccines, millions of unvaccinated individuals, and those currently infected will develop HPV-related diseases over the next two decades and beyond. Thus, it remains critical to identify effective antivirals against papillomaviruses. Using a mouse papillomavirus model of HPV infection, this study reveals that cellular MEK1/2 signaling supports viral tumorigenesis. The MEK1/2 inhibitor, trametinib, demonstrates potent antiviral activities and promotes tumor regression. This work provides insight into the conserved regulation of papillomavirus gene expression by MEK1/2 signaling and reveals this cellular pathway as a promising therapeutic target for the treatment of papillomavirus diseases.

Han Y., Micklem G., Kim S.Y.

Oncogene-induced senescence (OIS) is highly heterogeneous, varying by oncogenic signals and cellular context. While its dual role, in the initial inhibition potentially later leading to promotion of tumors through the senescence-associated secretory phenotype, is still a matter of debate, it is undeniable that OIS is critical to understanding tumorigenesis. A major obstacle to OIS research is the absence of a universally accepted marker. Here, we present a robust OIS-specific transcriptomic secretory phenotype, termed oncogene-induced senescence secretory phenotype (OIS-SP), which can identify OIS across multiple biological contexts from in vitro datasets to in vivo human samples. We apply a meta-analytic machine learning pipeline to harmonize a deliberately varied selection of Ras-Raf-MEK-induced senescence datasets of differing origins, oncogenic signals and cell types. Finally we make use of bypass data to identify key genes and eliminate genes associated with quiescence, so identifying 40 OIS-SP genes. Within this set, we determined a robust core of five OIS-SP genes (FBLN1, CXCL12, EREG, CST1 and MMP10). Importantly, these 5 OIS-SP genes showed clear, consistent regulation patterns across various human Ras-Raf-MEK-mutated tumor tissues, which suggests that OIS-SP may be a novel cancer driver phenotype with an unexpectedly critical role in tumorigenesis.

Saleh T., Khasawneh A.I., Himsawi N., Abu-Raideh J., Ejeilat V., Elshazly A.M., Gewirtz D.A.

Senescence represents a unique cellular stress response characterized by a stable growth arrest, macromolecular alterations, and wide spectrum changes in gene expression. Classically, senescence is the end-product of progressive telomeric attrition resulting from the repetitive division of somatic cells. In addition, senescent cells accumulate in premalignant lesions, in part, as a product of oncogene hyperactivation, reflecting one element of the tumor suppressive function of senescence. Oncogenic processes that induce senescence include overexpression/hyperactivation of H-Ras, B-Raf, and cyclin E as well as inactivation of PTEN. Oncogenic viruses, such as Human Papilloma Virus (HPV), have also been shown to induce senescence. High-risk strains of HPV drive the immortalization, and hence transformation, of cervical epithelial cells via several mechanisms, but primarily via deregulation of the cell cycle, and possibly, by facilitating escape from senescence. Despite the wide and successful utilization of HPV vaccines in reducing the incidence of cervical cancer, this measure is not effective in preventing cancer development in individuals already positive for HPV. Accordingly, in this commentary, we focus on the potential contribution of oncogene and HPV-induced senescence (OIS) in cervical cancer. We further consider the potential utility of senolytic agents for the elimination of HPV-harboring senescent cells as a strategy for reducing HPV-driven transformation and the risk of cervical cancer development.

Luna A.J., Sterk R.T., Griego-Fisher A.M., Chung J., Berggren K.L., Bondu V., Barraza-Flores P., Cowan A.T., Gan G.N., Yilmaz E., Cho H., Kim J., Hewitt S.M., Bauman J.E., Ozbun M.A.

Intracellular pathogens have evolved to utilize normal cellular processes to complete their replicative cycles. Pathogens that interface with proliferative cell signaling pathways risk infections that can lead to cancers, but the factors that influence malignant outcomes are incompletely understood. Human papillomaviruses (HPVs) predominantly cause benign hyperplasia in stratifying epithelial tissues. However, a subset of carcinogenic or “high-risk” HPV (hr-HPV) genotypes are etiologically linked to nearly 5% of all human cancers. Progression of hr-HPV-induced lesions to malignancies is characterized by increased expression of the E6 and E7 oncogenes and the oncogenic functions of these viral proteins have been widely studied. Yet, the mechanisms that regulate hr-HPV oncogene transcription and suppress their expression in benign lesions remain poorly understood. Here, we demonstrate that EGFR/MEK/ERK signaling, influenced by epithelial contact inhibition and tissue differentiation cues, regulates hr-HPV oncogene expression. Using monolayer cells, epithelial organotypic tissue models, and neoplastic tissue biopsy materials, we show that cell-extrinsic activation of ERK overrides cellular control to promote HPV oncogene expression and the neoplastic phenotype. Our data suggest that HPVs are adapted to use the EGFR/MEK/ERK signaling pathway to regulate their productive replicative cycles. Mechanistic studies show that EGFR/MEK/ERK signaling influences AP-1 transcription factor activity and AP-1 factor knockdown reduces oncogene transcription. Furthermore, pharmacological inhibitors of EGFR, MEK, and ERK signaling quash HPV oncogene expression and the neoplastic phenotype, revealing a potential clinical strategy to suppress uncontrolled cell proliferation, reduce oncogene expression and treat HPV neoplasia.

Bourguignon L.Y., Earle C., Shiina M.

Head and neck squamous cell carcinoma (HNSCC) is a malignancy that often involves the oral cavity, pharynx, larynx, or paranasal sinuses. There is a compelling evidence of the human papilloma virus including HPV16 E6 oncogene drives cell transformation and oncogenic processes of HPV positive (HVP+) HNSCC [in particular, Oropharyngeal Squamous Cell Carcinoma (OPSCC)]. In this study, we determined that human OPSCC-derived, HPV16 E6+ cells (UMSCC-104 and UMSCC-47 cell lines) express CD44 and a regulatory transcription factor, c-Jun. Importantly, interaction between matrix hyaluronan (HA) and CD44 (an HA receptor) promotes c-Jun phosphorylation followed by phospho-c-Jun nuclear translocation and co-localization with HPV16 E6 in the nucleus of both UMSCC-104 and UMSCC-47 cells. Further analyses revealed that HPV16 E6 expression is regulated by an upstream promoter containing AP1/c-Jun binding site(s), and chromatin immunoprecipitation (ChIP) assays demonstrated that stimulation of HPV16 E6 expression by HA-CD44 interaction is phospho-c-Jun dependent in these HPV16+ UMSCC-104 and UMSCC-47 cells. This process results in an upregulation of survival proteins, inhibitors of the apoptosis family of proteins (IAPs) and chemoresistance in these HPV16+ cells. Treatment of UMSCC-104 or UMSCC-47 cells with c-Jun-specific or HPV16 E6-specific small interfering RNAs effectively blocks HA/CD44-mediated c-Jun signaling and abrogates HPV16 E6 expression as well as causes downregulation of survival proteins (cIAP-1 and cIAP-2) expression and enhancement of chemosensitivity. Together, these findings suggest that the HA/CD44-induced c-Jun signaling plays a pivotal role in HPV16 E6 upregulation leading to survival protein (cIAP-1/cIAP-2) production and chemoresistance in HPV16+ UMSCC-104 and UMSCC-47 cells. Most importantly, using a mouse xenograft model, we have observed that Cisplatin chemotherapy combined with the suppression of CD44, c-Jun and HPV16 E6 (by treating both UMSCC-104 cells and UMSCC-47 cells with CD44shRNA or c-Jun shRNA or HPV16 E6 shRNA) appears to be more effective in tumor size reduction than chemotherapy alone. Thus, these newly-discovered HA/CD44-c-Jun/HPV16E6 signaling pathways may provide new drug targets for overcoming cisplatin chemoresistance in HPV16E6-positive OPSCC cells.

Stone S.C., Rossetti R.A., Alvarez K.L., Carvalho J.P., Margarido P.F., Baracat E.C., Tacla M., Boccardo E., Yokochi K., Lorenzi N.P., Lepique A.P.

Cervical cancer continues to be a public health problem in developing countries. Previous studies have shown that cervical cancer cells display markers of aerobic glycolysis, indicating that these tumors are likely to secrete lactate. Mostly, lactate is recognized as a molecule capable of suppressing immune responses, through inhibition of T cells, Mϕs, and dendritic cells. We and others have previously shown that Mϕs are frequent cells infiltrating cervical cancers with the ability to inhibit antitumor immune responses and promote tumor growth through angiogenesis. Here, we have tested the hypothesis that lactate, secreted by cervical cancer cells, can modulate Mϕ phenotype. First, we showed higher lactate plasma concentrations in patients with increasing cervical lesion grades, with maximum concentration in the plasma of cancer patients, which supported our hypothesis. We then inhibited lactate production in tumor cell spheroids established from cervical cancer derived cell lines, using the lactate dehydrogenase inhibitor, oxamate, prior to co-culture with monocytes. Lactate mediated part of the crosstalk between tumor cells and Mϕs, promoting secretion of IL-1β, IL-10, IL-6, and up-regulation of hypoxia induced factor-1α expression, and down-regulation of p65-NFκB phosphorylation in Mϕs. We also showed that Mϕs from co-cultures treated with oxamate were better inducers of T cell activation. Of note, experiments performed with inhibition of the monocarboxylate transporters rendered similar results. Our data confirms the hypothesis that lactate, secreted by cervical tumor cells, influences the phenotype of tumor Mϕs, promoting a suppressive phenotype.

Kim Y.H., Park T.J.

Lin M., Xue X., Liang S., Li Y., Lv Y., He L., Xu K., Zhang L., Chen J., Niu L.

Aberrantly expressed microRNAs contribute to the initiation and progression of human cancer. MiRNA-187 has been reported in nasopharyngeal, renal, pancreatic, prostate, and esophageal cancer, and acts as a tumor suppressor or oncogene. However, the underlying function of miRNA-187 in cervical cancer remains largely unexplored. In the present study, we demonstrated significantly miRNA-187 down-regulation in cervical cancer tissues and cell lines compared to their normal counterparts. Kaplan-Meier analysis revealed that decreased miRNA-187 was closely associated with shorter overall survival and relapse-free survival. Gain- and loss-of-function studies showed that miRNA-187 suppressed cervical cancer cell proliferation, migration, and invasion, and promoted cervical cancer cell apoptosis. Furthermore, luciferase reporter assay determined that human papillomavirus 16 E6 was a direct functional target of miRNA-187. Taken together, our findings indicate the essential role of miRNA-187 in suppressing cervical cancer progression and indicate a novel link between miRNA-187 and human papillomavirus 16 E6 in cervical cancer.

Sun L., Xu S., Liang L., Zhao L., Zhang L.

Cervical carcinoma is a multifactorial malignant tumor and diagnosis is therefore crucial. The aim of the present study was to examine the value of E6 oncoprotein, in human papillomavirus type 16 (HPV16), in the diagnosis of early stage cervical carcinoma and precancerous lesions. Receiver operating characteristic curve was used to analyze accuracy of diagnosis. A total of 124 patients infected with HPV16 were included in the study. The patients had an average age of 46.7±6.9 years and duration of disease of 10.5±3.4 months. To determine the expression level of HPV16 E6 the immunohistochemical Elivision method was performed. Proportion/horizon positive cells were used to count the cells, and pathologic diagnosis was employed for analysis of the results. The average follow-up time was 2.6±0.7 years. Sensitivity and specificity of diagnosing HPV16 E16 at 1 and 2 years, respectively, were calculated. The diagnostic rate of cervical carcinoma increased with time, and the positive expression of HPV16 E6 was also increased with the development of the disease. Differences among groups were statistically significant (P

Wei L., Griego A.M., Chu M., Ozbun M.A.

High-risk human papillomavirus (HR-HPV) infections are necessary but insufficient agents of cervical and other epithelial cancers. Epidemiological studies support a causal, but ill-defined, relationship between tobacco smoking and cervical malignancies. In this study, we used mainstream tobacco smoke condensate (MSTS-C) treatments of cervical cell lines that maintain either episomal or integrated HPV16 or HPV31 genomes to model tobacco smoke exposure to the cervical epithelium of the smoker. MSTS-C exposure caused a dose-dependent increase in viral genome replication and correspondingly higher early gene transcription in cells with episomal HPV genomes. However, MSTS-C exposure in cells with integrated HR-HPV genomes had no effect on genome copy number or early gene transcription. In cells with episomal HPV genomes, the MSTS-C-induced increases in E6 oncogene transcription led to decreased p53 protein levels and activity. As expected from loss of p53 activity in tobacco-exposed cells, DNA strand breaks were significantly higher but apoptosis was minimal compared with cells containing integrated viral genomes. Furthermore, DNA mutation frequencies were higher in surviving cells with HPV episomes. These findings provide increased understanding of tobacco smoke exposure risk in HPV infection and indicate tobacco smoking acts more directly to alter HR-HPV oncogene expression in cells that maintain episomal viral genomes. This suggests a more prominent role for tobacco smoke in earlier stages of HPV-related cancer progression.

Hu Z., Yu L., Zhu D., Ding W., Wang X., Zhang C., Wang L., Jiang X., Shen H., He D., Li K., Xi L., Ma D., Wang H.

High-risk human papillomavirus (HR-HPV) has been recognized as a major causative agent for cervical cancer. Upon HPV infection, early genes E6 and E7 play important roles in maintaining malignant phenotype of cervical cancer cells. By using clustered regularly interspaced short palindromic repeats- (CRISPR-) associated protein system (CRISPR/Cas system), a widely used genome editing tool in many organisms, to target HPV16-E7 DNA in HPV positive cell lines, we showed for the first time that the HPV16-E7 single-guide RNA (sgRNA) guided CRISPR/Cas system could disrupt HPV16-E7 DNA at specific sites, inducing apoptosis and growth inhibition in HPV positive SiHa and Caski cells, but not in HPV negative C33A and HEK293 cells. Moreover, disruption of E7 DNA directly leads to downregulation of E7 protein and upregulation of tumor suppressor protein pRb. Therefore, our results suggest that HPV16-E7 gRNA guided CRISPR/Cas system might be used as a therapeutic strategy for the treatment of cervical cancer.

Magaldi T.G., Almstead L.L., Bellone S., Prevatt E.G., Santin A.D., DiMaio D.

Repression of human papillomavirus (HPV) E6 and E7 oncogenes in established cervical carcinoma cell lines causes senescence due to reactivation of cellular tumor suppressor pathways. Here, we determined whether ongoing expression of HPV16 or HPV18 oncogenes is required for the proliferation of primary human cervical carcinoma cells in serum-free conditions at low passage number after isolation from patients. We used an SV40 viral vector expressing the bovine papillomavirus E2 protein to repress E6 and E7 in these cells. To enable efficient SV40 infection and E2 gene delivery, we first incubated the primary cervical cancer cells with the ganglioside GM1, a cell-surface receptor for SV40 that is limiting in these cells. Repression of HPV in primary cervical carcinoma cells caused them to undergo senescence, but the E2 protein had little effect on HPV-negative primary cells. These data suggest that E6 and E7 dependence is an inherent property of human cervical cancer cells.

Nguyen M., Blaho J.