HPV-positive Cervical Cancer Cells Research Articles

Abstract 4072: Molecular cytogenetic characterization of HPV types 16 and 18 cervical cancers: Acquired genomic instability by E6 and E7 oncoproteins

Abstract Human Papillomavirus (HPV) high-risk types 16 and 18 are directly associated with approximately 90% incidence of invasive cervical carcinoma. Epithelial cells infected with HPV become transformed and acquire genomic instability. As a result of this transformation, E6 and E7 oncogenes are perpetually expressed. E6 degrades the tumor suppressor p53; E7 inhibits tumor suppressor pRB, leading to disproportionate target cell growth and proliferation. While there have been studies exploring genomic instability induced by HPV oncoproteins E6/E7, the full scope of the genomic damage has not been clearly characterized. Three HPV positive cervical cancer cell lines, HeLa (HPV18), SiHa (HPV16) and CaSki (HPV16), were studied using Spectral Karyotyping (SKY) and Giemsa banding (G-banding). Our results using SKY and G-banding analysis showed HeLa cells exhibited frequent translocations on chromosomes 4 and 11, and deletions on chromosomes 11 and 20. The SiHa cells exhibited translocations primarily on chromosomes 9 and 20, with deletions on chromosome 10. CaSki revealed translocations on chromosomes 10 and 4, with deletions on chromosome 21. All three cell lines gained copies on chromosome 5. The aforementioned aneuploidy is demonstrative of the induced genomic instability acquired from HPV infection, which include tumor suppressor genes; p53 and pRB disruptions caused by E6 and E7 oncoproteins. HeLa cells showed extensive genomic instability on chromosome 11q22-23, where the ATM gene is located. Acquired genomic instability to the ATM chromosome region may further increase the rate of cervical cancer. Citation Format: Karen Tate, Lucy Tran, Melanie Baker, Kamilah Evans, Mechelle Rouse, Seyung Chung, Duy Nguyen, Juri Kim, Enijah Smith-Joe, Jay Vadgama, Mengtao Li, Yu-Ling Lin, Roland Patillo, Eva McGhee. Molecular cytogenetic characterization of HPV types 16 and 18 cervical cancers: Acquired genomic instability by E6 and E7 oncoproteins. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4072.

Proteomics-based identification of VDAC1 as a tumor promoter in cervical carcinoma.

We used oxidative isotope-coded affinity tags (OxICAT) to investigate the global redox status of proteins in human papillomavirus (HPV)-related cervical cancer cells, in order to identify a potential target for gene therapy. Voltage-dependent anion channel 1 (VDAC1) was found to be highly oxidized in HPV-positive cervical cancer cells. VDAC1 expression correlated significantly with the invasion of cervical cancer, the grade of cervical intraepithelial neoplasia (CIN) and the expression of HPV16 E7 in CIN. Knockdown of VDAC1 in cell lines increased the rate of apoptosis, while overexpression of the VDAC1 (respectively) partly reversed the effect. Thus, VDAC1 may promote the malignant progression of HPV-related disease, and treatments designed to suppress VDAC1 could prevent the progression of HPV-induced cervical disease.

Comprehensive analysis of lncRNAs microarray profile and mRNA-lncRNA co-expression in oncogenic HPV-positive cervical cancer cell lines.

Long non-coding RNAs are emerging to be novel regulators in gene expression. In current study, lncRNAs microarray and lncRNA-mRNA co-expression analysis were performed to explore the alternation and function of lncRNAs in cervical cancer cells. We identified that 4750 lncRNAs (15.52%) were differentially expressed in SiHa (HPV-16 positive) (2127 up-regulated and 2623 down-regulated) compared with C-33A (HPV negative), while 5026 lncRNAs (16.43%) were differentially expressed in HeLa (HPV-18 positive) (2218 up-regulated and 2808 down-regulated) respectively. There were 5008 mRNAs differentially expressed in SiHa and 4993 in HeLa, which were all cataloged by GO terms and KEGG pathway. With the help of mRNA-lncRNA co-expression network, we found that ENST00000503812 was significantly negative correlated with RAD51B and IL-28A expression in SiHa, while ENST00000420168, ENST00000564977 and TCONS_00010232 had significant correlation with FOXQ1 and CASP3 expression in HeLa. Up-regulation of ENST00000503812 may inhibit RAD51B and IL-28A expression and result in deficiency of DNA repair pathway and immune responses in HPV-16 positive cervical cancer cell. Up-regulation of ENST00000420168, ENST00000564977 and down-regulation of TCONS_00010232 might stimulate FOXQ1 expression and suppress CASP3 expression in HPV-18 positive cervical cancer cell, which lead to HPV-induced proliferation and deficiency in apoptosis. These results indicate that changes of lncRNAs and related mRNAs might impact on several cellular pathways and involve in HPV-induced proliferation, which enriches our understanding of lncRNAs and coding transcripts anticipated in HPV oncogenesis of cervical cancer.

CUL2 overexpression driven by CUL2/E2F1/miR-424 regulatory loop promotes HPV16 E7 induced cervical carcinogenesis.

It has been shown that HPV16 E7, but not other genotypes, can bind to scaffold protein CUL2 during inducing cervical carcinogenesis, but the expression level, associated regulating mechanism, and potential carcinogenicity of CUL2 itself is still unknown as yet. Here, we demonstrated that CUL2 was specifically overexpressed in HPV16 positive cervical cancer cells and tissues, and CUL2 expression was significantly increased along with the cervical lesion progression and positively correlated with HPV16 E7. CUL2 knockdown slowed the growth of xenograft tumors in mouse models. Importantly, CUL2 specifically bound to HPV16 E7, but not HPV18 E7. Moreover, CUL2 acted as a direct target of miR-424, and reversely suppressed miR-424; E2F transcription factor 1 (E2F1) suppressed miR-424 expression; CUL2 bound to E2F1 and promoted E2F1 expression. Our results indicate the existence of a regulatory loop among CUL2, E2F1, and miR-424 in HPV16 positive cervical cancer cells. Our results suggest that E7 recruited CUL2, driven by CUL2/E2F1/miR-424 regulatory loop, is overexpressed and accelerates HPV16-induced cervical carcinogenesis. Our findings may serve as one of the explanations for a clinical phenomenon that HPV16 possesses the strongest cervical carcinogenicity among high-risk HPV genotypes.

KLF13 regulates the differentiation-dependent human papillomavirus life cycle in keratinocytes through STAT5 and IL-8.

High-risk strains of human papillomavirus (HPV) are the causative agents of cervical and anogenital cancers and are associated with 5% of all human cancers. Although prophylactic vaccines targeting a subset of HPV types are available, they are ineffective in HPV-infected individuals. Elucidation of the mechanisms controlling HPV replication may allow development of novel anti-HPV therapeutics. Infectious HPV virions are produced during terminal differentiation of host cells. The process of viral maturation requires synergistic interactions between viral and cellular proteins that leads to amplification of the viral genome and expression of late viral genes. Here we show that the transcription factor Kruppel-like factor 13 (KLF13) has a critical role in the HPV life cycle. KLF13 is overexpressed in HPV-positive keratinocytes and cervical cancer cell lines. Expression of KLF13 in normal cervical epithelium is low but increases significantly in cervical intraepithelial neoplasia and invasive squamous cervical cancer. After HPV infection, the E7 protein suppresses ubiquitin ligase FBW7 expression leading to an increase in KLF13 expression. Reduction of KLF13 with short hairpin RNA in differentiating HPV-positive cells resulted in diminished levels of viral gene expression and genome amplification. Knockdown of KLF13 also reduced the level of the transcription factor signal transducer and activator of transcription 5, which led to the downregulation of the ataxia-telangiectasia mutated DNA damage pathway and the chemokine interleukin-8 (IL-8). In addition, neutralization of IL-8 diminished viral genome amplification in differentiating HPV-positive cells. Thus, KLF13 is critical for the activation of the HPV productive life cycle and is likely involved in initiation and progression of cervical cancer.

Cudrania tricuspidata Stem Extract Induces Apoptosis via the Extrinsic Pathway in SiHa Cervical Cancer Cells.

The focus of this study is the anti-cancer effects of Cudrania tricuspidata stem (CTS) extract on cervical cancer cells. The effect of CTS on cell viability was investigated in HPV-positive cervical cancer cells and HaCaT human normal keratinocytes. CTS showed significant dose-dependent cytotoxic effects in cervical cancer cells. However, there was no cytotoxic effect of CTS on HaCaT keratinocytes at concentrations of 0.125–0.5 mg/mL. Based on this cytotoxic effect, we demonstrated that CTS induced apoptosis by down-regulating the E6 and E7 viral oncogenes. Apoptosis was detected by DAPI staining, annexin V-FITC/PI staining, cell cycle analysis, western blotting, RT-PCR, and JC-1 staining in SiHa cervical cancer cells. The mRNA expression levels of extrinsic pathway molecules such as Fas, death receptor 5 (DR5), and TNF-related apoptosis-inducing ligand (TRAIL) were increased by CTS. Furthermore, CTS treatment activated caspase-3/caspase-8 and cleavage of poly (ADP-ribose) polymerase (PARP). However, the mitochondrial membrane potential and expression levels of intrinsic pathway molecules such as Bcl-2, Bcl-xL, Bax, and cytochrome C were not modulated by CTS. Taken together, these results indicate that CTS induced apoptosis by activating the extrinsic pathway, but not the intrinsic pathway, in SiHa cervical cancer cells. These results suggest that CTS can be used as a modulating agent in cervical cancer.

Effect of nano-realgar on proliferation and apoptosis of human cervical carcinoma cells

To investigate the effect of nano-realgar on proliferation and apoptosis of cervical cancer cells. Different cervical cancer cell lines (Caski/HPV16+, adeno carcinoma; Hela/HPV18+, squmous carcinoma; C33A/HPV-, adeno carcinoma) were incubated with nano-realgar at different concentrations (5, 10, 20, 40 mg/L) for different times (24, 48, 72, 96 h). The morphology was observed under phase contrast microscope. The cell viability and apoptosis were examined by MTT and flow cytometry, respectively. The inhibitory effect of nano-realgar on the proliferation of cervical cancer cells was in a dose-dependent manner, with a range of inhibitory rate from 9.02% to 49.06%. Taking the group (20 mg/L) for an example, the inhibitory rates for Caski, Hela and C33A were 39.15%, 36.17% and 30.56%, respectively. The results of flow cytometry showed that the nano-realgar induced apoptosis in a concentration-dependent manner, with a range of apoptosis rate from 19.29% to 99.54%. Also taking the group (20 mg/L) for an example, the apoptosis rates for Caski, Hela and C33A were (60.43 ± 2.88)%, (41.95 ± 3.01)% and (43.49 ± 2.19)%, respectively. High concentration of nano-realgar (20 or 40 mg/L) could induce block of Hela and Caski at G2/M stage. Nano-realgar can inhibit the proliferation of different cervical carcinoma cell lines and can induce the cell apoptosis. The inhibitory effect on cell proliferation is strongest for Caski, followed by Hela and C33A. It can also induce G2/M stage block on HPV positive cervical cancer cells at high enough concentration.

E3 ligase EDD1/UBR5 is utilized by the HPV E6 oncogene to destabilize tumor suppressor TIP60.

Tat-interacting protein of 60 kDa (TIP60) is an essential lysine acetyltransferase implicated in transcription, DNA damage response and apoptosis. TIP60 protein expression is reduced in cancers. In cervical cancers, human papillomavirus (HPV) E6 oncogene targets cellular p53, Bak and some of the PDZ domain-containing proteins for proteasome-mediated degradation through E6AP ligase. Recently, E6 oncogene from high-risk and low-risk categories was also shown to target TIP60. However, the molecular mechanisms and whether destabilization of TIP60 contributes to HPV E6-mediated transformation remain unanswered. Our proteomic analyses revealed EDD1 (E3 identified by differential display), an E3 ligase generally overexpressed in cancers as a novel interacting partner of TIP60. By investigating protein turnover and ubiquitination assays, we show that EDD1 negatively regulates TIP60's stability through the proteasome pathway. Strikingly, HPV E6 uses this function of EDD1 to destabilize TIP60. Colony-formation assays and soft agar assays show that gain of function of TIP60 or depletion of EDD1 in HPV-positive cervical cancer cells significantly inhibits cell growth in vitro. This phenotype is strongly supported by the in-vivo studies where re-activation of TIP60 in cervical cancer cells dramatically reduces tumor formation. In summary, we have discovered a novel ligase through which E6 destabilizes TIP60. Currently, in the absence of an effective therapeutic vaccine for malignant cervical cancers, cervical cancer still remains to be a major disease burden. Hence, our studies implying a distinct tumor suppressor role for TIP60 in cervical cancers show that reactivation of TIP60 could be of therapeutic value.

The Curcumin Analogue 1,5-Bis(2-hydroxyphenyl)-1,4-pentadiene-3-one Induces Apoptosis and Downregulates E6 and E7 Oncogene Expression in HPV16 and HPV18-Infected Cervical Cancer Cells.

In an effort to study curcumin analogues as an alternative to improve the therapeutic efficacy of curcumin, we screened the cytotoxic potential of four diarylpentanoids using the HeLa and CaSki cervical cancer cell lines. Determination of their EC50 values indicated relatively higher potency of 1,5-bis(2-hydroxyphenyl)-1,4-pentadiene-3-one (MS17, 1.03 ± 0.5 μM; 2.6 ± 0.9 μM) and 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadiene-3-one (MS13, 2.8 ± 0.4; 6.7 ± 2.4 μM) in CaSki and HeLa, respectively, with significantly greater growth inhibition at 48 and 72 h of treatment compared to the other analogues or curcumin. Based on cytotoxic and anti-proliferative activity, MS17 was selected for comprehensive apoptotic studies. At 24 h of treatment, fluorescence microscopy detected that MS17-exposed cells exhibited significant morphological changes consistent with apoptosis, corroborated by an increase in nucleosomal enrichment due to DNA fragmentation in HeLa and CaSki cells and activation of caspase-3 activity in CaSki cells. Quantitative real-time PCR also detected significant down-regulation of HPV18- and HPV16-associated E6 and E7 oncogene expression following treatment. The overall data suggests that MS17 treatment has cytotoxic, anti-proliferative and apoptosis-inducing potential in HPV-positive cervical cancer cells. Furthermore, its role in down-regulation of HPV-associated oncogenes responsible for cancer progression merits further investigation into its chemotherapeutic role for cervical cancer.

Human telomerase reverse transcriptase regulates vascular endothelial growth factor expression via human papillomavirus oncogene E7 in HPV-18-positive cervical cancer cells.

Human papillomavirus (HPV) infection induces chronic and precancerous lesions and results in invasive cervical cancer. Human telomerase as well as inflammatory and angiogenic factors such as telomerase reverse transcriptase (hTERT) or vascular endothelial growth factor (VEGF) could play a role in regulating HPV-induced cervical cancer. This study investigated underlying molecular events in HPV-induced HPV-positive cervical cancer through hTERT and VEGF in vitro. Expressions of hTERT, a rate-limiting subunit of telomerase, and VEGF mRNA and proteins were, respectively, assessed by qRT-PCR, ELISA, and TRAP-ELISA in HPV-positive tissue samples and cervical cancer cell lines. To assess hTERT and VEGF secretion, hTERT overexpression and knockdown were conducted in HPV-18-positive Hela cells by hTERT cDNA and shRNA transfection, respectively. Then, the effect of HPV E6 and E7 on VEGF expressions was assessed in HPV-negative cervical cancer cells. Data have shown that VEGF expression levels are associated with hTERT expressions and telomerase activity in HPV-positive cervical cancer tissues and cells. Knockdown of hTERT expression down-regulated VEGF expressions, whereas overexpression of hTERT up-regulated VEGF expressions in HPV-18-positive Hela cells. Furthermore, HPV E7 oncoprotein was necessary for hTERT to up-regulate VEGF expressions in HPV-negative cervical cancer cells. Data from this current study indicate that HPV oncoproteins up-regulated hTERT and telomerase activity and in turn promoted VEGF expressions, which could be a key mechanism for HPV-induced cervical cancer development and progression.

Human Papillomavirus E6/E7-Specific siRNA Potentiates the Effect of Radiotherapy for Cervical Cancer in Vitro and in Vivo.

The functional inactivation of TP53 and Rb tumor suppressor proteins by the HPV-derived E6 and E7 oncoproteins is likely an important step in cervical carcinogenesis. We have previously shown siRNA technology to selectively silence both E6/E7 oncogenes and demonstrated that the synthetic siRNAs could specifically block its expression in HPV-positive cervical cancer cells. Herein, we investigated the potentiality of E6/E7 siRNA candidates as radiosensitizers of radiotherapy for the human cervical carcinomas. HeLa and SiHa cells were transfected with HPV E6/E7 siRNA; the combined cytotoxic effect of E6/E7 siRNA and radiation was assessed by using the cell viability assay, flow cytometric analysis and the senescence-associated β-galactosidase (SA-β-Gal) assay. In addition, we also investigated the effect of combined therapy with irradiation and E6/E7 siRNA intravenous injection in an in vivo xenograft model. Combination therapy with siRNA and irradiation efficiently retarded tumor growth in established tumors of human cervical cancer cell xenografted mice. In addition, the chemically-modified HPV16 and 18 E6/E7 pooled siRNA in combination with irradiation strongly inhibited the growth of cervical cancer cells. Our results indicated that simultaneous inhibition of HPV E6/E7 oncogene expression with radiotherapy can promote potent antitumor activity and radiosensitizing activity in human cervical carcinomas.

MiR-34a Inhibits Viability and Invasion of Human Papillomavirus-Positive Cervical Cancer Cells by Targeting E2F3 and Regulating Survivin.

Previous studies confirmed that high-risk human papillomavirus (HR-HPV) infection is a risk factor of cervical cancer, and the infection was associated with significantly reduced miR-34a expression during carcinogenesis. However, the downstream targets of miR-34a and their roles are still not well understood. This study explored the regulative role of miR-34a on E2F3 and survivin expression and the viability and invasion of HPV-positive cervical cancer cells. MiR-34a and survivin expression in 56 cases of HR-HPV-positive patients, 28 cases of HR-HPV-negative patients, and 28 normal cases without HR-HPV infections were measured. Human papillomavirus-18-positive HeLa cervical cancer cells and HPV-16-positive SiHa cells were used to explore the effect of miR-34a on cell viability and invasion. The molecular target of miR-34a was also explored in cervical cancer cells. The results showed that miR-34a overexpression could inhibit HPV-positive cancer cell viability, whereas its downregulation promoted cell viability. E2F3 is a direct target of miR-34a in HPV-positive cervical cancer cells. By targeting E2F3, miR-34a could regulate the expression of survivin. Thus, through regulating E2F3 and survivin, miR-34a could reduce the viability and invasion of HPV-positive cervical cancer cells. This study confirmed a novel miR-34a-E2F3-survivin axis in the tumor suppressor role of miR-34a in cervical cancer.

Two less common human microRNAs miR-875 and miR-3144 target a conserved site of E6 oncogene in most high-risk human papillomavirus subtypes.

Human papillomaviruses (HPVs) including high-risk (HR) and low-risk (LR) subtypes have distinguishable variation on both genotypes and phenotypes. The co-infection of multiple HR-HPVs, headed by HPV16, is common in cervical cancer in female. Recently accumulating reports have focused on the interaction between virus and host, particularly the role of human microRNAs (miRNAs) in anti-viral defense by targeting viral genome. Here, we found a well-conserved target site of miRNAs in the genomes of most HR-HPVs, not LR-HPVs, by scanning all potential target sites of human miRNAs on 24 HPVs of unambiguous subtypes of risk. The site is targeted by two less common human miRNAs, miR-875 and miR-3144, and is located in E6 oncogene open reading frame (ORF) and overlap with the first alternative splice exon of viral early transcripts. In validation tests, miR-875 and miR-3144 were identified to suppress the target reporter activity markedly and inhibit the expression of both synthetically exogenous E6 and endogenous E6 oncogene. High level of two miRNAs can inhibit cell growth and promote apoptosis in HPV16-positive cervical cancer cells. This study provides a promising common target of miRNAs for most HR-HPVs and highlights the effects of two low expressed human miRNAs on tumour suppression.

HPV-16 impairs the subcellular distribution and levels of expression of protein phosphatase 1γ in cervical malignancy.

BackgroundThe high risk Human Papillomavirus (HPV) E6 oncoproteins play an essential role in the development of cervical malignancy. Important cellular targets of E6 include p53 and the PDZ domain containing substrates such as hScrib and Dlg. We recently showed that hScrib activity was mediated in part through recruitment of protein phosphatase 1γ (PP1γ).MethodsExpression patterns of hScrib and PP1γ were assessed by immunohistochemistry of HPV-16 positive cervical intraepithelial neoplasm (CIN), classified as CIN1 (n = 4), CIN2 (n = 8), CIN3 (n = 8), cervical carcinoma tissues (n = 11), and HPV-negative cervical tissues (n = 8), as well as by subfractionation assay of the HPV-16 positive cervical cancer cell lines, CaSki and SiHa. To explore the effects of the HPV-16 oncoproteins, we have performed siRNA knockdown of E6/E7 expression, and monitored the effects on the expression patterns of hScrib and PP1γ.ResultsWe show that PP1γ levels in HPV-16 positive tumour cells are reduced in an E6/E7 dependent manner. Residual PP1γ in these cells is found mostly in the cytoplasm as opposed to the nucleus where it is predominantly found in normal cells. We have found a striking concordance with redistribution in the pattern of expression (9/11; 81.8%) and loss of PP1γ expression in HPV-16 positive cervical tumours (2/11; 18.2%). Furthermore, this loss of PP1γ expression and redistribution in the pattern of expression occurs progressively as the lesions develop (8/8; 100%).ConclusionTogether, these results suggest that PP1γ may be a novel target of the HPV-16 oncoproteins and indicate that it might be a potential novel biomarker for HPV-16 induced malignancy.

Human Papillomavirus 16 Oncoprotein Expression Is Controlled by the Cellular Splicing Factor SRSF2 (SC35).

High-risk human papillomaviruses (HR-HPV) cause anogenital cancers, including cervical cancer, and head and neck cancers. Human papillomavirus 16 (HPV16) is the most prevalent HR-HPV. HPV oncogenesis is driven by two viral oncoproteins, E6 and E7, which are expressed through alternative splicing of a polycistronic RNA to yield four major splice isoforms (E6 full length, E6*I, E6*II, E6*X). The production of multiple mRNA isoforms from a single gene is controlled by serine/arginine-rich splicing factors (SRSFs), and HPV16 infection induces overexpression of a subset of these, SRSFs 1, 2, and 3. In this study, we examined whether these proteins could control HPV16 oncoprotein expression. Small interfering RNA (siRNA) depletion experiments revealed that SRSF1 did not affect oncoprotein RNA levels. While SRSF3 knockdown caused some reduction in E6E7 expression, depletion of SRSF2 resulted in a significant loss of E6E7 RNAs, resulting in reduced levels of the E6-regulated p53 proteins and E7 oncoprotein itself. SRSF2 contributed to the tumor phenotype of HPV16-positive cervical cancer cells, as its depletion resulted in decreased cell proliferation, reduced colony formation, and increased apoptosis. SRSF2 did not affect transcription from the P97 promoter that controls viral oncoprotein expression. Rather, RNA decay experiments showed that SRSF2 is required to maintain stability of E6E7 mRNAs. These data show that SRSF2 is a key regulator of HPV16 oncoprotein expression and cervical tumor maintenance. Expression of the HPV16 oncoproteins E7 and E6 drives HPV-associated tumor formation. Although increased transcription may yield increased levels of E6E7 mRNAs, it is known that the RNAs can have increased stability upon integration into the host genome. SR splicing factors (SRSFs) control splicing but can also control other events in the RNA life cycle, including RNA stability. Previously, we demonstrated increased levels of SRSFs 1, 2, and 3 during cervical tumor progression. Now we show that SRSF2 is required for expression of E6E7 mRNAs in cervical tumor but not nontumor cells and may act by inhibiting their decay. SRSF2 depletion in W12 tumor cells resulted in increased apoptosis, decreased proliferation, and decreased colony formation, suggesting that SRSF2 has oncogenic functions in cervical tumor progression. SRSF function can be targeted by known drugs that inhibit SRSF phosphorylation, suggesting a possible new avenue in abrogating HPV oncoprotein activity.

HPV16 early gene E5 specifically reduces miRNA-196a in cervical cancer cells.

High-risk human papillomavirus (HPV) type 16, which is responsible for greater than 50% of cervical cancer cases, is the most prevalent and lethal HPV type. However, the molecular mechanisms of cervical carcinogenesis remain elusive, particularly the early steps of HPV infection that may transform normal cervical epithelium into a pre-neoplastic state. Here, we report that a group of microRNAs (microRNAs) were aberrantly decreased in HPV16-positive normal cervical tissues, and these groups of microRNAs are further reduced in cervical carcinoma. Among these miRNAs, miR196a expression is the most reduced in HPV16-infected tissues. Interestingly, miR196a expression is low in HPV16-positive cervical cancer cell lines but high in HPV16-negative cervical cancer cell lines. Furthermore, we found that only HPV16 early gene E5 specifically down-regulated miRNA196a in the cervical cancer cell lines. In addition, HoxB8, a known miR196a target gene, is up-regulated in the HPV16 cervical carcinoma cell line but not in HPV18 cervical cancer cell lines. Various doses of miR196a affected cervical cancer cell proliferation and apoptosis. Altogether, these results suggested that HPV16 E5 specifically down-regulates miR196a upon infection of the human cervix and initiates the transformation of normal cervix cells to cervical carcinoma.

Deregulation of microRNAs Let-7a and miR-21 mediate aberrant STAT3 signaling during human papillomavirus-induced cervical carcinogenesis: role of E6 oncoprotein.

BackgroundAberrantly expressed and constitutively active STAT3 signaling plays a pivotal role in initiation and progression of human papillomavirus-induced cervical carcinogenesis. However, the underlying mechanism(s) responsible for pleiotropic effects of STAT3 signaling is poorly understood. In view of emerging regulatory role of microRNAs, Let-7a and miR-21 that may interact with STAT3 signaling and/or its downstream effectors, present study was designed in HPV16-positive cervical cancer cells to assess the functional contribution of these miRs in STAT3 signaling in cervical cancer.MethodsFunctional silencing of STAT3 signaling and HPV16 oncoprotein expression in SiHa cells was done by STAT3-specific and 16 E6 siRNAs. Pharmacological intervention of STAT3 was done using specific inhibitors like curcumin and stattic. Loss-of-function study of miR-21 using miR-21 inhibitor and gain-of-function study of let-7a was done using let-7a mimic in SiHa cells.ResultsFunctional silencing of STAT3 signaling in SiHa cells by STAT3-specific siRNA resulted in a dose-dependent decrease in cellular miR-21 level. Pharmacological intervention of STAT3 using specific inhibitors like curcumin and Stattic that abrogated STAT3 activation resulted in loss of cellular miR-21 pool. Contrary to this, specific targeting of miR-21 using miR-21 inhibitor resulted in an increased level of PTEN, a negative regulator of STAT3, and reduced active pSTAT3 level. Besides miR-21, restoration of cellular Let-7a using chemically synthesized Let-7a mimic reduced overall STAT3 level. Abrogation of HPV oncoprotein E6 by specific siRNA resulted in increased Let-7a but loss of miR-21 and a correspondingly reduced pSTAT3/STAT3 and elevated the level of cellular PTEN.ConclusionsOur results demonstrate existence of a functional loop involving Let-7a, STAT3 and miR-21 which were found potentially regulated by viral oncoprotein E6. Implications: miR-21 and Let-7a along with STAT3 may prove useful targets for pharmacological intervention for management of cervical cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-996) contains supplementary material, which is available to authorized users.

Up-Regulation of FOXM1 by E6 Oncoprotein through the MZF1/NKX2-1 Axis Is Required for Human Papillomavirus–Associated Tumorigenesis

PURPOSE: Foxhead box M1 (FOXM1) expression has been shown to be linked with human papillomavirus (HPV) 16/18–infected cervical cancer. However, the mechanism underlying the induction of FOXM1 in HPV 16/18–infected cancers remains elusive. EXPERIMENTAL DESIGN: The mechanistic actions of FOXM1 induced by the E6/NKX2-1 axis in tumor aggressiveness were elucidated in cellular and animal models. The prognostic value of FOXM1 for overall survival (OS) and relapse-free survival (RFS) in HPV-positive oral and lung cancers was assessed using Kaplan-Meier and Cox regression models. RESULTS: Herein, FOXM1 expression is upregulated by E6-mediated NKX2-1 in HPV-positive cervical, oral, and lung cancer cells. Induction of FOXM1 by E6 through the MZF1/NKX2-1 axis is responsible for HPV-mediated soft agar growth, invasiveness, and stemness through activating Wnt/β-catenin signaling pathway. In a nude mice model, metastatic lung tumor nodules in HPV 18 E6-positive GNM or HPV 16 E6-positive TL-1–injected nude mice were markedly decreased in both cell types with E6 knockdown, FOXM1 knockdown, or treatment with FOXM1 inhibitor (thiostrepton). Among the four subgroup patients, the worst FOXM1 prognostic value for OS and RFS was observed in HPV 16/18–positive patients with tumors with high-expressing FOXM1. CONCLUSIONS: Induction of FOXM1 by E6 oncoprotein through the MZF1/NKX2-1 axis may be responsible for HPV 16/18–mediated tumor progression and poor outcomes in HPV-positive patients.

Wild-type p53 reactivation by small-molecule Minnelide™ in human papillomavirus (HPV)-positive head and neck squamous cell carcinoma

The incidence of high-risk human papillomavirus (HR-HPV) head and neck squamous cell carcinoma (HNSCC) continues to increase, particularly oropharyngeal squamous cell carcinoma (OPSCC) cases. The inactivation of the p53 tumor suppressor gene promotes a chain of molecular events, including cell cycle progression and apoptosis resistance. Reactivation of wild-type p53 function is an intriguing therapeutic strategy. The aim of this study was to investigate whether a novel compound derived from diterpene triepoxide (Minnelide™) can reactivate wild-type p53 function in HPV-positive HNSCC. For all of our in vitro experiments, we used 2 HPV-positive HNSCC cell lines, University of Michigan squamous cell carcinoma (UM-SCC) 47 and 93-VU-147, and 2 HPV-positive human cervical cancer cell lines, SiHa and CaSki. Cells were treated with different concentrations of triptolide and analyzed for p53 activation. Mice bearing UM-SCC 47 subcutaneous xenografts and HPV-positive patient-derived tumor xenografts were treated with Minnelide and evaluated for tumor growth and p53 activation. In HPV-positive HNSCC, Minnelide reactivated p53 by suppressing E6 oncoprotein. Activation of apoptosis followed, both in vitro and in vivo. In 2 preclinical HNSCC animal models (a subcutaneous xenograft model and a patient-derived tumor xenograft model), Minnelide reactivated p53 function and significantly decreased tumor progression and tumor volume. Triptolide and Minnelide caused cell death in vitro and in vivo in HPV-positive HNSCC by reactivating wild-type p53 and thus inducing apoptosis. In addition, in 2 HPV-positive HNSCC animal models, Minnelide decreased tumor progression and induced apoptosis.

Abstract 2753: Ormeloxifene inhibits cervical cancer cell growth through intrinsic apoptotic pathway

Abstract Background: Cervical cancer (CxCa) remains the fourth leading cause of cancer related death among women worldwide. Cervical cancer is mainly (∼ 99.7%) derived from high risk Human papillomavirus (HR HPV). HPV E6/E7 oncoproteins interfere with p53 and pRb (retinoblastoma) cell cycle regulatory proteins and hinder their efficacy of controlling cell growth. Advanced stage cervical cancer is difficult to treat and patients diagnosed with metastatic disease have a poor survival rate of only 16%. Therefore, there is an urgent need to develop newer treatment modalities. Ormeloxifene is a non-hormonal, anti-estrogen, oral contraceptive for human use that effectively suppresses the proliferation of decidual cells in the endometrium to inhibit egg implantation. Growing evidences also suggest that ormeloxifene has anti-cancerous properties in breast, head and neck, and chronic myeloid leukemia cancers. Hence, considering its strong anti-proliferative/cancerous effect, we hypothesized that ormeloxifene may also inhibit the growth of cervical cancer cells and may suppress the expression of E6 and E7 HPV oncoproteins. Methods: We performed in vitro anti-cancer studies with ormeloxifene (dose range: 5 µM to 25 µM) in the HPV positive cervical cancer cell lines (Caski and SiHa) by utilizing MTS and colony formation assays. To further determine the effects of ormeloxifene on the cellular and molecular level, immunoblotting, flow cytometry and real time PCR analyses were performed using the Caski cell line model. Results: Ormeloxifene effectively inhibited cervical cancer cell growth as determined by proliferation and colony formation assays in a dose dependent manner. Ormeloxifene decreased mitochondrial membrane potential and induced apoptosis as shown by flow cytometry analysis of cells stained with TMRE (tetramethylrhodamine, ethyl ester) and 7AAD/Annexin V, respectively. Apoptosis was also confirmed by immunoblotting for cleaved PARP (Poly ADP ribose polymerase), Caspase 3 and Caspase 9. Additionally, ormeloxifene treatment arrested cell cycle in G1-S transition and decreased the expression of p-Akt while total Akt remained the same. Specifically relevant to cervical cancer, ormeloxifene decreased the expression of HPV E6/E7 at mRNA and protein levels as determined by quantitative PCR analysis and immunoblotting, respectively. Interestingly, ormeloxifene restored the levels of p53, Rb and PTPN 13 (Protein tyrosine Phosphatase non receptor type 13) tumor suppressor proteins as shown by immunoblotting. Conclusion: Our findings suggest that ormeloxifene inhibits cervical cancer cell growth and decreases mitochondrial membrane potential which clearly demonstrates that apoptosis is induced via intrinsic pathway. Importantly, ormeloxifene downregulates the expression of HPV E6/E7 oncoproteins. Thus, ormeloxifene appears to be a novel treatment modality for cervical cancer. Citation Format: Neeraj Chauhan, Mohd S. Zaman, Murali M. Yallapu, Diane M. Maher, Mara C. Ebeling, Subhash C. Chauhan, Meena Jaggi. Ormeloxifene inhibits cervical cancer cell growth through intrinsic apoptotic pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2753. doi:10.1158/1538-7445.AM2014-2753