Cervical cancer (CC) is a leading cause of mortality in females, especially in developing countries. The two viral oncoproteins E6 and E7 mediate the oncogenic activities of high-risk human papillomavirus (hrHPV), and hrHPV, especially HPV16 or/and HPV18 (HPV16/18) play critical roles in CC through different pathways. STK31 gene of which the expression has been proven to be regulated by the methylation status of its promoter, is one of the novel cancer/testis (CT) genes and plays important roles in human cancers. Reasearches have indicated that viral infection is correlated to the methylation statuses of some genes. Herein, we detected methylation status of the STK31 gene in cervical tumors and explored its interaction with HPV16 or/and HPV18 (HPV16/18) infection.
Bisulfite genomic sequencing PCR (BGS) combined with TA clone, methylation-specific PCR (MSP) were used to analyze methylation statuses of the STK31 gene promoter/exon 1 region in HPV16/18-positive, HPV-negative CC cell lines; ectopically expressed HPV16 E6, -E7, and -E6/E7 CC cells; normal cervical tissues and cervical tumor tissues of different stages. The mRNA and protein expressions of STK31 were detected by RT-PCR and western blotting.
The STK31 gene promoter/exon 1 was hypomethylated in the HPV16/18-positive cell lines HeLa, SiHa and CaSki, and the mRNA and protein expression were detected. In contrast, the STK31 gene exhibited hypermethylation and silenced expression in the HPV-negative CC cells C33A and HT-3. Compared with the primary HPV-negative CC cell lines, the STK31 methylation was downregulated, and STK31 expression was induced in the HPV16E7/E67 transfected cells. The methylation statuses and expressions of STK31 were verified in the cervical tumor samples at different stages. Additionally, chemotherapy treatment may influence STK31 expression by regulating its methylation status.
STK31 may be a novel cellular target gene for the HPV16 oncogeneE7. The HPV16 oncogene E7 may affect STK31 expression through a methylation-mediated mechanism. The aberrant methylation of the STK31 promoter/exon 1 region may be a precursor of human cervical carcinogenesis and a potential DNA aberrant methylation biomarker of conditions ranging from precancerous disease to invasive cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/s12985-016-0515-5) contains supplementary material, which is available to authorized users.
Cervical cancer; STK31; Human papillomavirus; DNA methylation; 5-aza-2′deoxycytidine; Biomarker
Worldwide, cervical cancer is the second most common causes of cancer in women and represents an important mortality rate. Cisplatin (CIS) is a very important antitumoral agent and can lead tumor cells toward two important cellular states: apoptosis and senescence. In some types of cancers pentoxifylline (PTX) sensitizes these cells to the toxic action of chemotherapeutics drugs such as adriamycin, inducing apoptosis. In the present work, we studied in vitro whether PTX alone or in combination with CIS induces apoptosis and/or senescence in cervix cancer HeLa and SiHa cell lines infected with HPV types 16 and 18, respectively, as well as in immortalized keratinocytyes HaCaT cells.
HeLa (HPV 18+), SiHa (HPV 16+) cervix cancer cells and non-tumorigenic immortalized HaCaT cells (control) were treated with PTX, CIS or both. The cellular toxicity and survival fraction of PTX and CIS were determinate by WST-1 and clonogenic assays respectively. Apoptosis, caspase activation and phosphorylation of ERK1/2, p38, p65 (NF-κB), Bcl-2 and Bcl-XL anti-apoptotic proteins were determinated by flow cytometry. Senescence by microscopy. Phosphorylation of IκBα and IκB total were measured by ELISA. Pro-apoptotic, anti-apoptotic and senescence genes, as well as HPV-E6/7 mRNA expression, were detected by RT-PCR.
Our results show that after 24 hours of incubation PTX per se is toxic for cancer cells affecting cell viability and inducing apoptosis. The toxicity in HaCaT cells was minimal. CIS induces apoptosis in HeLa and SiHa cells and its effect was significantly increases when the cells were treated with PTX + CIS. In all studies there was a direct correlation with levels of caspases (-3, -6, -7, -9 and -8) activity and apoptosis. CIS induces important levels of senescence and phosphorylation of ERK1/2, p38, p65/RELA, and IκBα, and decreased the expression of anti-apoptotic protein Bcl-XL. Surprisingly these levels were significantly reduced by PTX in tumor cells, and at the same time, increases the expression of pro-apoptotic genes.
PTX sensitizes cervical cancer cells to CIS-induced apoptosis and decreases the CIS-induced senescence in these cells via inhibition of NF-κB signaling pathway; diminishes expression of antiapoptotic proteins and the activation of caspases.
Cervical cancer is the third most commonly diagnosed cancer worldwide and the fourth leading cause of cancer-related mortality in females worldwide, accounting for 10–15% of cancer-related mortalities. Cytological screening and DNA testing for high-risk human papillomavirus (HPV) types have markedly decreased the rates of cervical cancer in developed countries, however, for vulnerable populations without access to health care, cervical cancer remains a considerable problem. Chemotherapeutic agents such as cisplatin (DDP) are considered as first-line treatment for cervical carcinoma. Although initially patients often exhibit high responsiveness, the majority eventually develop DDP resistance. However, the mechanisms underlying this process remain unclear. Furthermore, patients with metastatic cancer and those exhibiting persistent or recurrent disease after platinum-based chemoradiotherapy have limited options and thus, non-platinum combination chemotherapy has been proposed as a strategy to circumvent platinum resistance, however, novel therapeutic strategies are required. In the present study, P16 expression was analyzed by quantitative-polymerase chain reaction and western blot analysis in SiHa and SiHa-DDP cells and the interaction between P16 and CDK4 was detected via co-immunoprecipitation. In addition, the proliferation and apoptosis rates of P16 knockdown SiHa-DDP cells were measured by MTT assay and Annexin V flow cytometry and the subsequent changes in cyclin D1 and pRb expression were analyzed by western blot analysis. In this study, a high level of P16INK4A expression and its enhanced interaction with cyclin-dependent kinase-4 in cervical carcinoma DDP-resistance cells (SiHa-DDP) was identified, which was associated with the inactivation of phosphorylated retinoblastoma protein (pRb). Knockdown of P16INK4A significantly induced cellular growth, when compared with the control cells, via the upregulation of pRb, and also promoted apoptosis following treatment with DDP. The results of this study indicated, for the first time, that P16INK4A is required for DDP resistance in cervical carcinoma SiHa cells and, thus, these results may lead to the development of novel strategies for the treatment of chemoresistant cervical carcinoma.
P16INK4A; cervical carcinoma; cisplatin resistance; SiHa-cisplatin; cell cycle
A new therapeutic approach of looking at the expression of glucose-regulated protein (GRP) 58 as an indication of cisplatin sensitivity may eradicate fruitless treatment and side effects in patients with cervical cancer. Thymoquinone, the bioactive compound in Nigella sativa, has been reported to have an antiproliferative effect on cervical cancer cells. This study compared the cytotoxic effects of cisplatin, a drug commonly used in the treatment of cervical cancer, and thymoquinone in cervical cancer (HeLa and SiHa) cell lines by 3-(4,5-Dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and measured GRP58 expression in the cells by quantitative real-time polymerase chain reaction and Western blotting. Cisplatin had higher antiproliferative activity towards the cervical cancer cell lines than thymoquinone in a dose-dependent and time-dependent manner. However, cisplatin was more toxic to normal 3T3 and Vero cell lines than thymoquinone. The half maximal inhibitory concentration (IC50) of cisplatin in HeLa and SiHa cells at 72 hours was 13.3±2.52 μM and 19.5±2.12 μM, respectively. Meanwhile, the IC50 of thymoquinone in HeLa and SiHa cells was 29.57±5.81 μM and 23.41±1.51 μM, respectively (P<0.05). A significant correlation was found between the cytotoxicity of cisplatin and expression of GRP58, but this relationship was not significant for thymoquinone. Therefore, the response of cervical cancer cells to cisplatin can be predicted on the basis of GRP58 expression.
glucose-regulated protein 58; cervical cancer; cisplatin; thymoquinone
Cisplatin is the main chemotherapeutic drug for the treatment of cervical cancers, however resistance to cisplatin is increasingly common and therefore has limited the efficacy and use of this drug in the clinic. Dose-dependent toxicity poses an additional challenge since patients suffer long-term and often permanent side-effects after treatment. Bcl-2 up-regulation has been implicated in the resistance to cisplatin in a variety of cancer cell lines, however its role in cervical cancer is confounding.
A low, non-cytotoxic concentration of cisplatin was used in the treatment of HeLa and CaSki cells. Bcl-2 expression was determined through Western blotting and immunocytochemistry before and after treatment with cisplatin. To assess the reliance of the cervical cancer cells on Bcl-2 in the presence of cisplatin, Bcl-2 knock-down was achieved through RNA interference, where after apoptosis was assessed through PARP cleavage (Western blotting), Caspase activity (Caspase-Glo©) and PI inclusion analysis (Flow cytometry). Finally, pre-malignant and malignant cervical tissue was analysed for the presence of Bcl-2 through Western blotting and immunofluorescence.
Cervical cancer cells upregulate Bcl-2 when treated with a non-cytotoxic concentration of cisplatin, which when silenced, effectively enhanced cisplatin sensitivity, and therefore significantly induced apoptosis. Analysis of the expression profile of Bcl-2 in cervical tissue revealed its up-regulation in cervical carcinoma, which agrees with results obtained from the in vitro data.
Our data strongly suggest that utilising a lower dose of cisplatin is feasible when combined with Bcl-2 silencing as an adjuvant treatment, thereby improving both the dose-dependent toxicity, as well as cervical cancer resistance.
Bcl-2; Beclin-1; Cisplatin; Cervical cancer; Apoptosis
A substantial number of microRNAs (miRNAs) is subject to epigenetic silencing in cancer. Although epigenetic silencing of tumour suppressor genes is an important feature of cervical cancer, little is known about epigenetic silencing of miRNAs. Since DNA methylation-based silencing of hsa-miR-124 occurs in various human cancers, we studied the frequency and functional effects of hsa-miR-124 methylation in cervical carcinogenesis.
Quantitative MSP analysis of all 3 loci encoding the mature hsa-miR-124 (hsa-miR-124-1/-2/-3) showed methylation in cervical cancer cell lines SiHa, CaSki and HeLa as well as in late passages of human papillomavirus (HPV) type 16 or 18 immortalised keratinocytes. Treatment of SiHa cells with a demethylating agent reduced hsa-miR-124 methylation levels and induced hsa-miR-124 expression. In HPV-immortalised keratinocytes increased methylation levels were related to reduced hsa-miR-124 expression and higher mRNA expression of IGFBP7, a potential hsa-miR-124 target gene. Ectopic hsa-miR-124 expression in SiHa and CaSki cells decreased proliferation rates and migratory capacity. Combined hsa-miR-124-1 and/or hsa-miR-124-2 methylation analysis of 139 cervical tissue specimens showed an increasing methylation frequency from 0% in normal tissues up to 93% in cervical carcinomas. Increased methylation levels of hsa-miR-124-1 and hsa-miR-124-2 were significantly correlated with reduced hsa-miR-124 expression in cervical tissue specimens. Combined hsa-miR-124-1 and/or hsa-miR-124-2 methylation analysis of 43 cervical scrapes of high-risk HPV positive women was predictive of underlying high-grade lesions.
DNA methylation-based silencing of hsa-miR-124 is functionally involved in cervical carcinogenesis and may provide a valuable marker for improved detection of cervical cancer and its high-grade precursor lesions.
MicroRNA-184 suppresses cell growth and survival via targeting c-Myc and Bcl- 2. We recently reported that miR-184 promotes tumor progression in non-small cell lung cancer via targeting CDC25A and c-Myc. We here hypothesized that miR-184 could be down-regulated by E6 oncoprotein to confer cisplatin resistance in NSCLC. Human papillomavirus (HPV) 16-positive lung cancer TL-1 and cervical cancer SiHa cells compared with HPV16-negative TL-10 and C33A cells were enrolled for E6 manipulation. MiR-184 expression levels were increased by E6-knockdown in TL-1 and SiHa cells, but decreased by E6-overexpression in TL-10 and C33A cells. The MTT assay showed that the inhibition concentration of cisplatin yielding for 50% cell viability was dependent on miR-184 levels. Bcl-2 de-targeted by E6-mediated miR- 184 reduction was responsible for cisplatin resistance. Luciferase reporter assay and real- time PCR analysis indicated that the miR-184 promoter activity and its expression were modulated by E6 and/or p53 manipulation. Chromatin immunoprecipitation (ChIP) assay confirmed that p53 was bound onto the miR-184 promoter and its binding activity was modulated by E6 and/or p53 manipulation. Among patients, high miR184 and high Bcl-2 mRNA expression was more commonly occurred in E6- positive tumors than in E6-negative tumors. Fifty-nine out of 136 patients receiving cisplatin-based chemotherapy were available for the retrospective study. Patients with low-mR-184, E6-positive, high-Bcl-2 tumors, and both combinations were more prevalently occurred unfavorable response to cisplatin-based chemotherapy than their counterparts. In conclusion, a decrease in miR-184 level by E6 oncoprotein may predict unfavorable response to cisplatin-based chemotherapy in HPV-infected NSCLC patients via increasing Bcl-2 expression.
miR-184; HPV; cisplatin resistance
Viral oncoproteins E6/E7 play key oncogenic role in human papillomavirus (HPV)-mediated cervical carcinogenesis in conjunction with aberrant activation of cellular signaling events. GLI-signaling has been implicated in metastasis and tumor recurrence of cervical cancer. However, the interaction of GLI-signaling with HPV oncogenes is unknown. We examined this relationship in established HPV-positive and HPV-negative cervical cancer cell lines using specific GLI inhibitor, cyclopamine and HPVE6/E7 siRNAs. Cervical cancer cell lines showed variable expression of GLI-signaling components. HPV16-positive SiHa cells, overexpressed GLI1, Smo and Patch. Inhibition by cyclopamine resulted in dose-dependent reduction of Smo and GLI1 and loss of cell viability with a higher magnitude in HPV-positive cells. Cyclopamine selectively downregulated HPVE6 expression and resulted in p53 accumulation, whereas HPVE7 and pRb level remained unaffected. siRNA-mediated silencing of HPV16E6 demonstrated reduced GLI1 transcripts in SiHa cells. Cervical cancer stem-like cells isolated by side population analysis, displayed retention of E6 and GLI1 expression. Fraction of SP cells was reduced in cyclopamine-treated cultures. When combined with E6-silencing cyclopamine resulted in loss of SP cell’s sphere-forming ability. Co-inhibition of GLI1 and E6 in cervical cancer cells showed additive anti-cancer effects. Overall, our data show existence of a cooperative interaction between GLI signaling and HPVE6.
Many advanced human tumors, including hepatocellular carcinomas (HCC) are auxotrophic for arginine due to down-regulation of argininosuccinate synthetase (ASS1), the rate-limiting enzyme in arginine synthesis. The arginine-lowering agent PEGylated arginine deiminase (ADI-PEG 20) has shown efficacy as a monotherapy in clinical trials for treating arginine-auxotrophic tumors and is currently being evaluated in combination with cisplatin in other cancer types. Epigenetic silencing via methylation of the ASS1 promoter has been previously demonstrated in other cancer types, and a reciprocal relationship between ASS1 expression and cisplatin resistance has also been observed in ovarian cancer. However, the mechanism of ASS1 down-regulation, as well as the correlation with cisplatin resistance has not been explored in HCC. The present study investigates ADI-PEG 20 and cisplatin sensitivities in relation to ASS1 expression in HCC. In addition, we show how this biomarker is regulated by cisplatin alone and in combination with ADI-PEG 20.
ASS1 protein expression in both untreated and drug treated human HCC cell lines was assessed by western blot. The correlation between ASS1 protein levels, ADI-PEG 20 sensitivity and cisplatin resistance in these cell lines was established using a luminescence-based cell viability assay. Epigenetic regulation of ASS1 was analyzed by bisulfite conversion and methylation-specific PCR.
A good correlation between absence of ASS1 protein expression, ASS1 promoter methylation, sensitivity to ADI-PEG 20 and resistance to cisplatin in HCC cell lines was observed. In addition, cisplatin treatment down-regulated ASS1 protein expression in select HCC cell lines. While, at clinically relevant concentrations, the combination of ADI-PEG 20 and cisplatin restored ASS1 protein levels in most of the cell lines studied.
ASS1 silencing in HCC cell lines is associated with simultaneous cisplatin resistance and ADI-PEG 20 sensitivity which suggests a promising combination therapeutic strategy for the management of HCC.
Arginine; Argininosuccinate synthetase; Arginine deiminase; Cisplatin; Hepatocellular carcinoma; Combination therapy
Placental like alkaline phosphate (PLAP), an oncofetal antigen, is highly expressed in germ cell, cervical, ovarian and several other tumour types but minimally in normal tissues. The expression of a PLAP promoter based transcriptional unit following antigen mediated cell specific delivery is a possible approach for tumour targeting.
PLAP promoter alone or in combination with NFκB DNA response elements was used for expressing shRNA targeting the long control region (LCR) of human papillomavirus (HPV)-16 oncogenes E6 and E7 via transcriptional gene silencing in PLAP expressing cervical cancer cell lines, SiHa and CaSki. This was packaged in a Sendai virus envelope incorporating a single chain variable fragment antibody (scFv) for antibody mediated targeting. Specificity and efficacy of the shRNA was assessed by studying the heterochromatization, down regulation of the HPV-16 E6/E7 genes and subsequent effects on their targets and cell growth properties.
Reduction of HPV-16 E6 and E7 expression by TGS led to the activation of the previously suppressed target genes of p53 (PUMA and NOXA) and Rb (cyclins A2 and E). Cell death was seen only in PLAP expressing HPV-16 infected SiHa and CaSki cells but not in the HPV-18 integrated HeLa and non-PLAP CHO cells. There was reduction in the enhancer associated transcripts of the long control region (LCR) of HPV-16 E6/E7 genes. Also, an increase in the enrichment of dimethylated histone three lysine nine (H3K9Me2) and trimethylated histone three lysine twenty-seven (H3K27Me3) was observed by ChIP assay, which decreased upon trichostatin A treatment, indicating a possible mechanism for the heterochromatization of the target LCR region.
A combination of novel PLAP promoter and antibody based specificities has the potential for being developed as a possible therapeutic strategy for PLAP positive neoplasia.
Electronic supplementary material
The online version of this article (doi:10.1186/s12967-015-0602-1) contains supplementary material, which is available to authorized users.
Placental like alkaline phosphate; Promoter; HPV-16; Transcriptional gene silencing; Recombinant antibody; scFv; Sendai virosome; Gene therapy; Germ cell alkaline phosphate (GCAP)
Specific types of human papillomaviruses (HPVs) cause cervical cancer. Cervical cancers exhibit aberrant cellular microRNA (miRNA) expression patterns. By genome-wide analyses, we investigate whether the intracellular and exosomal miRNA compositions of HPV-positive cancer cells are dependent on endogenous E6/E7 oncogene expression. Deep sequencing studies combined with qRT-PCR analyses show that E6/E7 silencing significantly affects ten of the 52 most abundant intracellular miRNAs in HPV18-positive HeLa cells, downregulating miR-17-5p, miR-186-5p, miR-378a-3p, miR-378f, miR-629-5p and miR-7-5p, and upregulating miR-143-3p, miR-23a-3p, miR-23b-3p and miR-27b-3p. The effects of E6/E7 silencing on miRNA levels are mainly not dependent on p53 and similarly observed in HPV16-positive SiHa cells. The E6/E7-regulated miRNAs are enriched for species involved in the control of cell proliferation, senescence and apoptosis, suggesting that they contribute to the growth of HPV-positive cancer cells. Consistently, we show that sustained E6/E7 expression is required to maintain the intracellular levels of members of the miR-17~92 cluster, which reduce expression of the anti-proliferative p21 gene in HPV-positive cancer cells. In exosomes secreted by HeLa cells, a distinct seven-miRNA-signature was identified among the most abundant miRNAs, with significant downregulation of let-7d-5p, miR-20a-5p, miR-378a-3p, miR-423-3p, miR-7-5p, miR-92a-3p and upregulation of miR-21-5p, upon E6/E7 silencing. Several of the E6/E7-dependent exosomal miRNAs have also been linked to the control of cell proliferation and apoptosis. This study represents the first global analysis of intracellular and exosomal miRNAs and shows that viral oncogene expression affects the abundance of multiple miRNAs likely contributing to the E6/E7-dependent growth of HPV-positive cancer cells.
Oncogenic human papillomaviruses (HPVs) are major human carcinogens of broad biomedical importance. The growth of HPV-positive cervical cancer cells is critically dependent on sustained E6/E7 oncogene expression from endogenous viral DNA sequences. We here addressed the question of whether this process is linked to specific, E6/E7-dependent alterations of the cellular micro(mi)RNA network. By comprehensive deep sequencing analyses we show that endogenous E6/E7 expression significantly affects the concentrations of abundant intracellular miRNAs in HPV-positive cervical cancer cells, which are linked to the control of cell proliferation, senescence and apoptosis. These include members of the miR-17~92 cluster, which are expressed at increased levels by sustained E6/E7 expression and repress the anti-proliferative p21 gene in HPV-positive cancer cells. Moreover, we identified an E6/E7-dependent seven-miRNA-signature in exosomes secreted from HPV-positive cancer cells. These small vesicles are involved in intercellular communication and may serve as novel diagnostic markers. Taken together, our results show that continuous E6/E7 expression in HPV-positive cancer cells is linked to significant alterations in the amounts of intracellular and exosomal miRNAs with growth-promoting, anti-senescent and anti-apoptotic potential.
This study aimed to investigate the methylation status of the promoter region of spalt-like transcription factor 3 (SALL3) and the expression of SALL3 in cervical cancer to explore the function of this gene in cervical cancer carcinogenesis.
The methylation status of SALL3 was detected by methylation-specific PCR, and SALL3 gene expression was assessed by real-time quantitative PCR in the cervical cancer cell lines, SiHa, HeLa and C33A, as well as in cervical cancer tissue samples (n = 23), matched pericarcinomatous tissue samples (n = 23) and normal cervix tissue samples (n = 17). MTT was used to measure the cell viability and proliferation capacity of SiHa and HeLa cells.
The SALL3 promoter was completely methylated in SiHa cells, unmethylated in C33A cells and partially methylated in HeLa cells. After treatment of SiHa and HeLa cells with 5 μM and 10 μM of 5-Azacytidine (5-Aza), respectively, the methylation level of the SALL3 promoter decreased and observed increase in the degree of unmethylation in a dose-dependent manner. Moreover, the relative expression of SALL3 mRNA increased as the concentration of 5-Aza increased in SiHa (p<0.05) and HeLa (p<0.05) cells. This above-mentioned increase in SALL3 mRNA in SiHa cells was more remarkable than that observed in HeLa cells. Cell proliferation capacity also decreased after administration of 5-Aza to SiHa and HeLa cells (p<0.05). Methylation of the SALL3 promoter was observed in 15 of 23 (65.21%) cervical cancer tissue samples, 15 of 23 (65.21%) matched pericarcinomatous tissue samples and 5 of 17 (29.41%) normal cervical tissue samples (p<0.05). SALL3 mRNA expression was significantly lower in cervical cancer and pericarcinomatous tissues compared with normal cervical tissues (p<0.05). In all cervix tissue samples, HPV infection was positively associated with hypermethylation of the promoter region of SALL3 (p<0.05, r = 0.408), and the expression of SALL3 mRNA in HPV-positive tissues was lower than that in HPV-negative tissues (p<0.05).
The aberrant hypermethylation of SALL3 together with HPV involvement inactivated its function as a tumor suppressor and contributed to carcinogenesis in cervical cancer.
A protocol for cervical cancer screening among sexually active women 25 to 65 years of age was introduced in 2006 in Catalonia, Spain to increase coverage and to recommend a 3-year-interval between screening cytology. In addition, Human Papillomavirus (HPV) was offered as a triage test for women with a diagnosis of atypical squamous cells of undetermined significance (ASC-US). HPV testing was recommended within 3 months of ASC-US diagnosis. According to protocol, HPV negative women were referred to regular screening including a cytological exam every 3 years while HPV positive women were referred to colposcopy and closer follow-up. We evaluated the implementation of the protocol and the prediction of HPV testing as a triage tool for cervical intraepithelial lesions grade two or worse (CIN2+) in women with a cytological diagnosis of ASC-US.
During 2007-08 a total of 611 women from five reference laboratories in Catalonia with a novel diagnosis of ASC-US were referred for high risk HPV (hrHPV) triage using high risk Hybrid Capture version 2. Using routine record linkage data, women were followed for 3 years to evaluate hrHPV testing efficacy for predicting CIN2+ cases. Logistic regression analysis was used to estimate the odds ratio for CIN2 +.
Among the 611 women diagnosed with ASC-US, 493 (80.7%) had at least one follow-up visit during the study period. hrHPV was detected in 48.3% of the women at study entry (mean age 35.2 years). hrHPV positivity decreased with increasing age from 72.6% among women younger than 25 years to 31.6% in women older than 54 years (p < 0.01).
At the end of the 3 years follow-up period, 37 women with a diagnosis of CIN2+ (18 CIN2, 16 CIN3, 2 cancers, and 1 with high squamous intraepithelial lesions -HSIL) were identified and all but one had a hrHPV positive test at study entry. Sensitivity to detect CIN2+ of hrHPV was 97.2% (95%confidence interval (CI) = 85.5-99.9) and specificity was 68.3% (95%CI = 63.1-73.2). The odds ratio for CIN2+ was 45.3 (95% CI: 6.2-333.0), when among ASC-US hrHPV positive women were compared to ASC-US hrHPV negative women.
Triage of ASC-US with hrHPV testing showed a high sensitivity for the detection of CIN2+ and a high negative predictive value after 3 years of follow-up. The results of this study are in line with the current guidelines for triage of women with ASC-US in the target age range of 25-65. Non adherence to guidelines will lead to unnecessary medical interventions. Further investigation is needed to improve specificity of ASC-US triage.
Human papillomavirus (HPV); Diagnosis of atypical squamous cell of undetermined significance (ASC-US); Triage; cervical cancer screening; hrHC2 testing
The infection with high-risk human papillomavirus is linked to cervical cancer, nevertheless, the role of miRNAs regulated by HPV oncogenes in cancer progression remain largely unknown. Here, we knocked down endogenous E6/E7 in HPV16-positive CaSki cell lines, screened differences in miRNA expression profile with control using miRNA array. 38 miRNAs were down-regulated and 6 miRNAs were up-regulated in the E6/E7 silenced CaSki cells (>2-fold changes with P <0.05). The levels of miR-27b, miR-20a, miR-24, miR-93, and miR-106b were verified by qPCR in E6/E7 silenced CaSki and SiHa cells. MiR-27b, up-regulated by E7, promoted CaSki and SiHa cell proliferation and invasion, inhibit paclitaxel-induced apoptosis. Dual-luciferase experiment confirmed miR-27b down-regulated its target gene PLK2 through the “seed regions”. The tumor suppressor PLK2 inhibited SiHa cell proliferation, reduced cell viability, and promoted paclitaxel/cisplatin -induced apoptosis. Furthermore, DGCR8 was found to mediate the up-regulation of miR-27b by HPV16 E7. Our study demonstrated that HPV16 E7 could increase DGCR8 to promote the generation of miR-27b, which accelerated cell proliferation and inhibited paclitaxel-induced cell apoptosis through down-regulating PLK2. These findings provide an insight into the interaction network of viral oncogene, miR-27b and PLK2, and support the potential strategies using antisense nucleic acid of miR-27b for therapy of cervical cancer in the future.
microRNA-27b; HPV16 E7; cervical cancer; polo-like kinase2; DGCR8
Saikosaponin-a and -d, two naturally occurring compounds derived from Bupleurum radix, have been shown to exert anti-cancer activity in several cancer cell lines. However, the effect of combination of saikosaponins with chemotherapeutic drugs has never been addressed. Thus, we investigated whether these two saikosaponins have chemosensitization effect on cisplatin-induced cancer cell cytotoxicity.
Two cervical cancer cell lines, HeLa and Siha, an ovarian cancer cell line, SKOV3, and a non-small cell lung cancer cell line, A549, were treated with saikosaponins or cisplatin individually or in combination. Cell death was quantitatively detected by the release of lactate dehydrogenase (LDH) using a cytotoxicity detection kit. Cellular ROS was analyzed by flow cytometry. Apoptosis was evaluated by AO/EB staining, flow cytometry after Anexin V and PI staining, and Western blot for caspase activation. ROS scavengers and caspase inhibitor were used to determine the roles of ROS and apoptosis in the effects of saikosaponins on cisplatin-induced cell death.
Both saikosaponin-a and -d sensitized cancer cells to cisplatin-induced cell death in a dose-dependent manner, which was accompanied with induction of reactive oxygen species (ROS) accumulation. The dead cells showed typical apoptotic morphologies. Both early apoptotic and late apoptotic cells detected by flow cytometry were increased in saikosaponins and cisplatin cotreated cells, accompanied by activation of the caspase pathway. The pan-caspase inhibitor z-VAD and ROS scanvengers butylated hydroxyanisole (BHA) and N-acetyl-L-cysteine (NAC) dramatically suppressed the potentiated cytotoxicity achieved by combination of saikosaponin-a or -d and cisplatin.
These results suggest that saikosaponins sensitize cancer cells to cisplatin through ROS-mediated apoptosis, and the combination of saikosaponins with cisplatin could be an effective therapeutic strategy.
Cervical cancer is one of the most common gynecological cancers worldwide. Aberrant expression of E3 ubiquitin ligase isolated by differential display (EDD) has been detected in various types of tumor and has been demonstrated to have an important role in carcinogenesis, tumor growth and drug resistance. However, the role of EDD in cervical cancer and its underlying molecular mechanisms remains unknown. The present study aimed to investigate the role of EDD in the tumorigenicity of cervical cancer. EDD expression levels were measured using reverse transcription-quantitative polymerase chain reaction and western blotting in SiHa, HeLa, CaSki, c-41 and c-33A cervical cancer cell lines and cervical cancer tissue specimens. A functional study was performed using cell proliferation, colony formation, cell apoptosis assays in vitro and tumor growth assays in vivo with EDD either overexpressed or silenced. In the present study, EDD expression levels were significantly upregulated in cervical cancer cell lines and tissue samples. EDD knockdown significantly inhibited colony formation, cell proliferation and tumor growth and accelerated cell apoptosis in the cervical cancer cell lines and tissue samples. Furthermore, microRNA (miR)-143 expression levels were low in cervical cancer tissue samples and were negatively correlated with EDD expression. miR-143 silencing eliminated the effect of EDD on cell proliferation, colony formation and cell apoptosis in the cervical cancer cells, which suggested that miR-143 is critical for EDD-mediated regulation of cervical cancer cell growth. The results of the present study indicated that EDD may promote cervical cancer growth in vivo and in vitro by targeting miR-143. In conclusion, EDD may have an oncogenic role in cervical cancer and may serve as a potential therapeutic target for the treatment of patients with cervical cancer.
apoptosis; cervical cancer; E3 ubiquitin ligase isolated by differential display; microRNA-143; tumor growth
Cervical cancer is a common and devastating female cancer worldwide. The etiology of cervical cancer has been largely attributed to human papillomavirus (HPV) infection and activation of the P13K/AKT/mTOR (mammalian target of rapamycin) pathway. However, the limited HPV-directed therapy, as well as therapeutic approach targeting P13K/AKT/mTOR pathway, has not yet been established or effective. A deeper understanding of cervical carcinogenesis and finding of novel candidate molecules for cervical cancer therapeutics is largely warranted. The unconventional prefoldin RPB5 interactor (URI or URI1), a known transcription factor involving the TOR signaling pathway, has recently been implicated a role in multiple tumorigenesis. We recently reported significant upregulation of URI in precancerous cervical intra-epithelial neoplasia (CIN) and invasive cervical cancer, suggesting its role in cervical carcinogenesis. However, the effect and underlying mechanism of URI in cervical cancer development have never been elucidated. Here, we aimed to investigate the in vitro effect of URI on cervical cancer using two cervical cancer cell lines CaSki and C33A, which are HPV-positive and HPV-negative respectively. We have shown that forced over-expression of URI in C33A and CaSki cells markedly promoted cell growth, while down-regulation of URI mediated by siRNA inhibited cell proliferation. We have found that URI over-expression enhanced resistance of cervical cancer cells to cisplatin. In contrast, knockdown of URI promoted apoptosis by influencing cell response to cisplatin, supporting URI as an oncogenic protein for cervical cancer cells. We have also shown that URI promoted the migration and invasive capacity of cervical cancer cells by up-regulation of Vimentin, a mesenchymal cell migration marker relating to the epithelial-mesenchymal transition (EMT) program. Our data support an important function of URI in the biological behavior of cervical cancer cells and provide novel mechanistic insights into the role of URI in cervical cancer progression and possibly, metastasis.
URI; C33A and CaSki cell lines; cisplatin; cervical cancer invasive capacity; vimentin; epithelial-mesenchymal transition
Cervical carcinomas result from cellular transformation by the human papillomavirus (HPV) E6 and E7 oncogenes which are constitutively expressed in cancer cells. The E6 oncogene degrades p53 thereby modulating a large set of p53 target genes as shown previously in the cervical carcinoma cell line HeLa. Here we show that the TAp63β isoform of the p63 transcription factor is also a target of E6. The p63 gene plays an essential role in skin homeostasis and is expressed as at least six isoforms. One of these isoforms, ΔNp63α, has been found overexpressed in squamous cell carcinomas and is shown here to be constitutively expressed in Caski cells associated with HPV16. We therefore explored the role of p63 in these cells by performing microarray analyses after repression of endogenous E6/E7 expression. Upon repression of the oncogenes, a large set of p53 target genes was found activated together with many p63 target genes related to cell adhesion. However, through siRNA silencing and ectopic expression of various p63 isoforms we demonstrated that TAp63β is involved in activation of this cell adhesion pathway instead of the constitutively expressed ΔNp63α and β. Furthermore, we showed in cotransfection experiments, combined with E6AP siRNA silencing, that E6 induces an accelerated degradation of TAp63β although not through the E6AP ubiquitin ligase used for degradation of p53. Repression of E6 transcription also induces stabilization of endogenous TAp63β in cervical carcinoma cells that lead to an increased concentration of focal adhesions at the cell surface. Consequently, TAp63β is the only p63 isoform suppressed by E6 in cervical carcinoma as demonstrated previously for p53. Down-modulation of focal adhesions through disruption of TAp63β therefore appears as a novel E6-dependent pathway in transformation. These findings identify a major physiological role for TAp63β in anchorage independent growth that might represent a new critical pathway in human carcinogenesis.
High-risk human papillomavirus infection can cause cancer of the uterine cervix. The viral proteins leading to transformation of the infected keratinocytes are the E6 and E7 oncogenes which interact with and induce degradation of the cell cycle regulators p53 and pRB. In cervical carcinoma cells, repression of E6/E7 stabilizes the p53 transcription factor leading to activation of a large group of cellular p53 target genes. Here we show that repression of E6/E7 also induces transcriptional activation of an additional large set of genes involved in cell adhesion including previously described p63 target genes. Indeed, we further demonstrated that these p63 target genes are activated by TAp63β and not by p53 or by the ΔNp63α or β isoforms, even though these transcription factors are also expressed in these cells. In cervical carcinoma cells, E6 expression therefore leads to TAp63β degradation thereby allowing anchorage independent growth. Our work describes a new E6-dependent transformation pathway in HPV-associated carcinogenesis. TAp63β inhibition may also represent a common pathway to activate anchorage independent growth in cancers.
Specific types of high risk Human papillomaviruses (HR-HPVs) particularly, HPV types 16 and 18 cause cervical cancer and while the two recently developed vaccines against these HPV types are prophylactic in nature, therapeutic options for treatment and management of already existing HPV infection are not available as yet. Because transcription factor, Activator Protein-1 (AP-1) plays a central role in HPV-mediated cervical carcinogenesis, we explored the possibility of its therapeutic targeting by berberine, a natural alkaloid derived from a medicinal plant species, Berberis which has been shown to possess anti-inflammatory and anti-cancer properties with no known toxicity; however, the effect of berberine against HPV has not been elucidated.
We studied the effect of berberine on HPV16-positive cervical cancer cell line, SiHa and HPV18-positive cervical cancer cell line, HeLa using electrophoretic mobility gel shift assays, western and northern blotting which showed that berberine could selectively inhibit constitutively activated AP-1 in a dose- and time-dependent manner and downregulates HPV oncogenes expression. Inhibition of AP-1 was also accompanied by changes in the composition of their DNA-binding complex. Berberine specifically downregulated expression of oncogenic c-Fos which was also absent in the AP-1 binding complex. Treatment with berberine resulted in repression of E6 and E7 levels and concomitant increase in p53 and Rb expression in both cell types. Berberine also suppressed expression of telomerase protein, hTERT, which translated into growth inhibition of cervical cancer cells. Interestingly, a higher concentration of berberine was found to reduce the cell viability through mitochondria-mediated pathway and induce apoptosis by activating caspase-3.
These results indicate that berberine can effectively target both the host and viral factors responsible for development of cervical cancer through inhibition of AP-1 and blocking viral oncoproteins E6 and E7 expression. Inhibition of AP-1 activity by berberine may be one of the mechanisms responsible for the anti-HPV effect of berberine. We propose that berberine is a potentially promising compound for the treatment of cervical cancer infected with HPV.
The expression of the human papillomavirus (HPV) E6/E7 oncogenes is crucial for HPV-induced malignant cell transformation. The identification of cellular targets attacked by the HPV oncogenes is critical for our understanding of the molecular mechanisms of HPV-associated carcinogenesis and may open novel therapeutic opportunities. Here, we identify the Lens Epithelial-Derived Growth Factor (LEDGF) gene as a novel cellular target gene for the HPV oncogenes. Elevated LEDGF expression has been recently linked to human carcinogenesis and can protect tumor cells towards different forms of cellular stress. We show that intracellular LEDGF mRNA and protein levels in HPV-positive cancer cells are critically dependent on the maintenance of viral oncogene expression. Ectopic E6/E7 expression stimulates LEDGF transcription in primary keratinocytes, at least in part via activation of the LEDGF promoter. Repression of endogenous LEDGF expression by RNA interference results in an increased sensitivity of HPV-positive cancer cells towards genotoxic agents. Immunohistochemical analyses of cervical tissue specimens reveal a highly significant increase of LEDGF protein levels in HPV-positive lesions compared to histologically normal cervical epithelium. Taken together, these results indicate that the E6/E7-dependent maintenance of intracellular LEDGF expression is critical for protecting HPV-positive cancer cells against various forms of cellular stress, including DNA damage. This could support tumor cell survival and contribute to the therapeutic resistance of cervical cancers towards genotoxic treatment strategies in the clinic.
Specific types of human papillomaviruses (HPVs) are closely linked to the development of malignant tumors, such as cervical cancer. Virtually all cervical cancers contain HPV DNA and the tumorigenic growth behavior of cervical cancer cells is dependent on the activity of two viral oncogenes, called E6 and E7. It is important to study the activities by which the HPV oncogenes can support the growth of tumor cells. This should allow new insights into the molecular mechanisms of virus-induced carcinogenesis and could also be useful for developing novel approaches for cancer therapy. We here show that the HPV oncogenes stimulate and maintain expression of the cellular LEDGF gene in HPV-positive cancer cells. Consistently, pre-malignant and malignant lesions of the cervix exhibit significantly increased LEDGF protein levels. LEDGF is crucial for the protection of tumor cells against various forms of cellular stress, including DNA damage. LEDGF stimulation by the viral oncogenes could be a critical survival mechanism by which HPVs support the growth of cervical cancer cells and provide resistance towards chemo- and radiotherapy in the clinic.
Cervical cancer and a subset of anogenital and head-and-neck carcinomas are caused by high-risk types of the human papillomavirus (hrHPV). During hrHPV-induced malignant transformation keratinocytes become able to grow anchorage independently, a tumorigenic trait at least partly associated with inactivation of tumor suppressor genes. We used hrHPV-containing keratinocytes to investigate the role of DNA methylation-mediated silencing of microRNAs (miRNAs) in the acquisition of anchorage independence.
Anchorage dependent (n=11) and independent passages (n=19) of 4 hrHPV-immortalized keratinocyte cell lines were treated with 2′-deoxy-5-azacytidine (DAC). Genome-wide miRNA expression profiles before and after treatment were compared to identify miRNAs silenced by methylation. Bisulfite sequencing and methylation-specific PCR showed increased methylation of hsa-mir-129-2/-137/-935/-3663/-3665 and -4281 in anchorage independent HPV-transformed keratinocytes and cervical cancer cell lines. Mature miRNAs derived from hsa-mir-129-2/-137/-3663 and -3665 showed functional relevance as they decreased anchorage independence in cervical cancer cell lines. Cervical (pre)cancerous lesions demonstrated increased methylation of hsa-mir-129-2/-935/-3663/-3665 and -4281, underlining the clinical relevance of our findings.
In conclusion, methylation-mediated silencing of tumor suppressive miRNAs contributes to acquisition of an anchorage independent phenotype. This study further substantiates the importance of miRNAs during early stages of carcinogenesis and underlines their potential as both disease markers and therapeutic targets.
anoikis; cervical cancer; DNA methylation; CIN lesion
PURPOSE: Human papillomavirus (HPV) type 16 is one of the major etiologic factors of cervical cancer. Our study aims to investigate the potentiality of the antiviral clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated Cas9 system (CRISPR/Cas9) targeting the E6 and E7 oncogenes of HPV16 as a potential chemosensitizer of cisplatin (cis-diaminedichloroplatinum II; CDDP) for cervical cancer. METHODS: Specifically, the therapeutic efficacy of combination of CDDP and HPV16 E6 + E7-CRISPR/Cas9 was assessed in cervical cancer cells and cervical cancer xenograft models. RESULTS: In vitro experiments showed that long-term exposure of SiHa cells to the HPV16 E6 + E7-CRISPR/Cas9 induced apoptosis, and its pro-apoptosis effect became more obvious when combined with CDDP. In vivo study found the efficacy of the combination of HPV16 E6 + E7-CRISPR/Cas9 and CDDP were superior to either of the treatments in term of apoptosis induction and metastasis inhibition. CONCLUSION: Collectively, our results suggested that HPV16 E6 + E7-CRISPR/Cas9 could be an effective sensitizer of CDDP chemotherapy in cervical cancer.
High-risk human papillomavirus (hrHPV) infections are causally related to cervical cancer development. The additional (epi)genetic alterations driving malignant transformation of hrHPV-infected cells however, are not yet fully elucidated. In this study we experimentally assessed the role of the PI3-kinase pathway and its regulator PIK3CA, which is frequently altered in cervical cancer, in HPV-induced transformation.
Cervical carcinomas and ectocervical controls were assessed for PIK3CA mRNA and protein expression by quantitative RT-PCR and immunohistochemical staining, respectively. A longitudinal in vitro model system of hrHPV-transfected keratinocytes, representing the immortal and anchorage independent phenotype, was assayed for PI3-kinase activation and function using chemical pathway inhibition i.e. LY294002 treatment, and PIK3CA RNA interference. Phenotypes examined included cellular viability, migration, anchorage independent growth and differentiation. mRNA expression of hTERT and HPV16 E6E7 were studied using quantitative RT-PCR and Northern blotting.
Cervical carcinomas showed significant overexpression of PIK3CA compared to controls. During HPV-induced transformation in vitro, expression of the catalytic subunit PIK3CA as well as activation of downstream effector PKB/AKT progressively increased in parallel. Inhibition of PI3-kinase signalling in HPV16-transfected keratinocytes by chemical interference or siRNA-mediated silencing of PIK3CA resulted in a decreased phosphorylation of PKB/AKT. Moreover, blockage of PI3-kinase resulted in reduced cellular viability, migration, and anchorage independent growth. These properties were accompanied with a downregulation of HPV16E7 and hTERT mRNA expression. In organotypic raft cultures of HPV16- and HPV18-immortalized cells, phosphorylated PKB/AKT was primarily seen in differentiated cells staining positive for cytokeratin 10 (CK10). Upon PI3-kinase signalling inhibition, there was a severe impairment in epithelial tissue development as well as a dramatic reduction in p-PKB/AKT and CK10.
The present data indicate that activation of the PI3-kinase/PKB/AKT pathway through PIK3CA regulates various transformed phenotypes as well as growth and differentiation of HPV-immortalized cells and may therefore play a pivotal role in HPV-induced carcinogenesis.
A user-friendly self-sampling method for collecting representative cervical cell material would lower the threshold for women to respond to the invitation for cervical screening. In the present article, we introduce such a device; we have evaluated its sensitivity and specificity to detect high-grade cervical intraepithelial neoplasia (CIN), via high-risk human papillomavirus (hrHPV) detection and liquid-based cytology (LBC), compared to endocervical brush samples obtained by gynecologists. Women who had a cervical smear reading of moderate dyskaryosis or worse or a repeat equivocal Pap smear result in the cervical screening program (n = 64) and healthy volunteers (n = 32) took a self-obtained sample at home prior to their visit to the gynecological outpatient department. At the outpatient department, an endocervical brush smear was taken, followed by colposcopy and biopsy whenever applicable. Both self-obtained samples and endocervical brush samples were immediately collected in Surepath preservation solution and used for LBC and hrHPV testing (by general primer-mediated GP5+/6+ PCR). hrHPV test results showed a good concordance between the two sample types (87%; κ = 0.71), with sensitivities for prevalent high-grade CIN that did not differ significantly (92% and 95%; P = 1.0). The hrHPV test on self-obtained samples proved to be at least as sensitive for high-grade CIN as cytology on endocervical brush samples (34/37 versus 31/37; P = 0.5). LBC showed a poor concordance between self-obtained and endocervical brush samples (60%; κ = 0.27). In conclusion, self-obtained samples taken by this novel device are highly representative of the hrHPV status of the cervix. In combination with hrHPV testing, the use of this device may have implications for increasing the attendance rate for cervical screening programs.
Cisplatin is one of the effective chemotherapeutics in the treatment of several types of cancers. However, in addition to the efforts against to its toxicity, the amelioration of cisplatin sensitivity is an important point in treatment of cervical cancer. To do so, additional substances such as epigallocatechin gallate (EGCG), a polyphenol in green tea, have been used in combination with chemotherapeutics. We aimed to investigate the possible molecular pathways to potentiate cervical cancer cell (HeLa) growth inhibition by combination therapy of cisplatin and EGCG. HeLa cells were treated with EGCG (25 μM), cisplatin (250 nM), and their combination for 24 h. Cell viability was determined by MTS Assay. We analyzed the expressions of NF-κB p65, COX-2, Nrf2, HO-1, p-mTOR, p-p70S6K1, p-4E-BP1, and p-Akt by Western blot analysis. Herein, we have demonstrated that EGCG works synergistic with cisplatin in inhibiting growth of cervical cancer cells. EGCG improved efficacy of cisplatin treatment in HeLa cells by regulating NFκB p65, COX-2, p-Akt, and p-mTOR pathways, whereas it increased the expression levels of Nrf2/HO-1 in combined therapy. Our observations revealed that EGCG increases the sensitization of cisplatin to cervical cancer cells by inhibiting cell survival and inducing apoptosis.
cisplatin; epigallocatechin gallate; HeLa cells; human cervical cancer; sensitization