Induction of senescence-like state and suppression of telomerase activity through inhibition of HPV E6/E7 gene expression in cells immortalized by HPV16 DNA

The E6 and E7 oncoproteins of human papillomavirus (HPV) play a major role in the development of cervical carcinoma. In this study, a recombinant adenovirus that expresses the bovine papillomavirus (BPV) E2, which has been shown to inhibit HPV early gene expression, was delivered to two HPV-immortalized cell lines as well as CaSki, a cervical carcinoma cell line. We tested whether the carcinoma and the immortal cells were equally affected by the expression of BPV E2. In all cell lines, BPV E2-mediated inhibition of HPV E6/E7 expression caused a dramatic suppression of cell growth, being preceded by the activation of the p53-Rb growth-inhibitory pathway, and a decrease in hTERT mRNA expression and telomerase activity. This suggests that the HPV E6 and E7 proteins are required not only for induction of the proliferative phenotype and telomerase activity, but also for their maintenance. In both the carcinoma and the immortal lines, the number of cells with enlarged cytoplasm and senescence-associated beta-galactosidase activity, which are markers for cellular senescence, was significantly increased. These results suggest that a senescence program exists in cells immortalized by HPV DNA as well as in cervical carcinoma cells.     

Stabilization of IGF2BP1 by USP10 promotes breast cancer metastasis via CPT1A in an m6A-dependent manner

Metastasis leads to the vast majority of breast cancer mortality. Increasing evidence has shown that N6-methyladenosine (m6A) modification and its associated regulators play a pivotal role in breast cancer metastasis. Here, we showed that overexpression of the m6A reader IGF2BP1 was clinically correlated with metastasis in breast cancer patients. Moreover, IGF2BP1 promoted distant metastasis in vitro and in vivo. Mechanistically, we first identified USP10 as the IGF2BP1 deubiquitinase. USP10 can bind to, deubiquitinate, and stabilize IGF2BP1, resulting in its higher expression level in breast cancer. Furthermore, by MeRIP-seq and experimental verification, we found that IGF2BP1 directly recognized and bound to the m6A sites on CPT1A mRNA and enhanced its stability, which ultimately mediated IGF2BP1-induced breast cancer metastasis. In clinical samples, USP10 levels correlated with IGF2BP1 and CPT1A levels, and breast cancer patients with high levels of USP10, IGF2BP1, and CPT1A had the worst outcome. Therefore, these findings suggest that the USP10/IGF2BP1/CPT1A axis facilitates breast cancer metastasis, and this axis may be a promising prognostic biomarker and therapeutic target for breast cancer.     

De-oncogenic HPV E6/E7 vaccine gets enhanced antigenicity and promotes tumoricidal synergy with cisplatin

In order to develop more effective therapeutic vaccines against cancers with high-risk human papillomavirus （HPV） infection, it is crucial to enhance the immunogenicity, eliminate the oncogenicity of oncoproteins, and take a combination of ET- and E6-containing vaccines. It has been shown recently that PE（AIII）-E7-KDEL3 （E7）, a fusion protein containing the HPVI6 oncoprotein E7 and the trans- location domain of Pseudomonas aeruginosa exotoxin A, is effective against TC-1 tumor cells inoculated in mice, there- fore, we engineered PE（AIII）-E6-CRL-KDEL3 （E6）, the deoncogenic versions of the E7 and E6 fusion proteins [i.e. PE（AIII）-E7（d）-KDEL3, E7（d）, and PE（AIII）-E6（d）-CRL- KDEL3, E6（d）] and tested the immunoefficacies of these fusion proteins as mono- and bivalent vaccines. Results indicated that the E7（d） get higher immunogenicity than its wild type and the E6 fusion proteins augmented the im- munogenicity and antitumor effects of their E7 counterparts. Furthermore, the bivalent vaccine system E7（d） plus E6（d）, in the presence of cisplatin, showed the best tumori- static and tumoricidal effects against established tumors in vivo. Therefore, it can be concluded that this novel therapeutic vaccine system, upon further optimization, may shed new light on clinical management of HPV-related carcinomas.     

Human keratin 14 driven HPV 16 E6/E7 transgenic mice exhibit hyperkeratinosis

Human papillomavirus type 16 (HPV16) has been known as a major causative factor for the development of uterine cervical carcinomas. To investigate the in vivo activity of HPV16 expressed in squamous epithelia, transgenic mice harboring HPV16 E6/E7 with human keratin 14 (hK14) promoter were generated. Grossly, hK14 driven HPV16 E6/E7 transgenic mice exhibited multiple phenotypes, including wrinkled skin that was apparent prior to the appearance of hair in neonates, thickened ears, and loss of hair in adults. Transgenic mice with phenotype exhibiting severe wrinkled skin and a lack of hair growth died at the age of 3-4 weeks. Histological analysis revealed that in transgenic mice survived beyond the initial 3-4 weeks, HPV16 E6/E7 causes epidermal hyperplasia in multiple transgenic lineages with high incidence of transgene penetration. This epithelial hyperplasia was characterized by an expansion of the proliferating compartment and keratinocytes, and was associated with hyperkeratosis. Such activities were significantly higher in the skin of transgenic mice than that of the normal mice. Thus, these transgenic mice appeared to be useful for the expression of HPV16 E6/E7 gene and subsequent analysis on hyperkeratosis.     

E5 and E6/E7 of high-risk HPVs cooperate to enhance cancer progression through EMT initiation

It is estimated that 10–20% of human carcinogenesis is linked to virus infection including papillomaviruses (HPVs). Moreover, since metastatic cancer disease is a major cause of morbidity and mortality in cancer patients, the role of onco-viruses in cancer progression to a metastatic form is of particular interest. Recent studies reported that E5 and E6/E7 onco-proteins of high-risk HPVs could enhance cancer progression via the initiation of the epithelial–mesenchymal transition (EMT) event. Herein, we discuss the association between E5 as well as E6/E7 of high-risk HPV and cancer progression.     

Up-regulation of MELK by E2F1 promotes the proliferation in cervical cancer cells

Cervical cancer is a common gynecologic cancer and a frequent cause of death. In this study, we investigated the role of MELK (maternal embryonic leucine zipper kinase) in cervical cancer. We found that HPV 18 E6/E7 promoted MELK expression by activating E2F1. MELK knockdown blocked cancer cells growth. Furthermore, we used MELK-8A to inhibit the kinase activity of MELK and caused the G2/M phase arrest of cancer cells. Under the treatment of inhibitors, Hela cells formed multipolar spindles and eventually underwent apoptosis. We also found that MELK is involved in protein translation and folding during cell division through the MELK interactome and the temporal proteomic analysis under inhibition with MELK-8A. Altogether, these results suggest that MELK may play a vital role in cancer cell proliferation and indicate a potential therapeutic target for cervical cancer.     

Increased IGF2BP3 expression promotes the aggressive phenotypes of colorectal cancer cells in vitro and vivo

Previous literatures reported insulin-like growth factor-2 messenger RNA-binding protein 3 (IGF2BP3) is a poor prognostic marker for colorectal cancer (CRC) patients. However, basic research on the effect and biological role of IGF2BP3 in CRC was still scare. Real-time quantitative polymerase chain reaction and western blot analysis were used to examine IGF2BP3 expression level in tumors and paired normal tissues from CRC patients. Tissue microarrays with 192 CRC patients were subjected to immunohistochemical staining to analyze the prognostic value of IGF2BP3. Proliferation assays, migration assays, and xenograft tumor formation in nude mice were performed to assess the biological role of IGF2BP3 in CRC cells. IGF2BP3 expression was significantly upregulated in tumor tissues compared with the matched normal tissues both in messenger RNA and protein level and was associated with worse prognosis. IGF2BP3 knockdown made cell cycle arrest to impair the proliferation ability of CRC cells and further inhibited the xenograft tumor growth in nude mice, also inhibited the migration ability of CRC cells via inducing epithelial–mesenchymal transition. Therefore, the research demonstrated that increased IGF2BP3 expression promoted the aggressive phenotypes of CRC cells. Targeted IGF2BP3 could be a novel and effective gene therapy for CRC patients to make a better prognosis.     

Suppression of HPV E6 and E7 expression by BAF53 depletion in cervical cancer cells

Deregulation of the expression of human papillomavirus (HPV) oncogenes E6 and E7 plays a pivotal role in cervical carcinogenesis because the E6 and E7 proteins neutralize p53 and Rb tumor suppressor pathways, respectively. In approximately 90% of all cervical carcinomas, HPVs are found to be integrated into the host genome. Following integration, the core-enhancer element and P105 promoter that control expression of E6 and E7 adopt a chromatin structure that is different from that of episomal HPV, and this has been proposed to contribute to activation of E6 and E7 expression. However, the molecular basis underlying this chromatin structural change remains unknown. Previously, BAF53 has been shown to be essential for the integrity of higher-order chromatin structure and interchromosomal interactions. Here, we examined whether BAF53 is required for activated expression of E6 and E7 genes. We found that BAF53 knockdown led to suppression of expression of E6 and E7 genes from HPV integrants in cervical carcinoma cell lines HeLa and SiHa. Conversely, expression of transiently transfected HPV18-LCR-Luciferase was not suppressed by BAF53 knockdown. The level of the active histone marks H3K9Ac and H4K12Ac on the P105 promoter of integrated HPV 18 was decreased in BAF53 knockdown cells. BAF53 knockdown restored the p53-dependent signaling pathway in HeLa and SiHa cells. These results suggest that activated expression of the E6 and E7 genes of integrated HPV is dependent on BAF53-dependent higher-order chromatin structure or nuclear motor activity.     

RNA interference against HPV16 E7 oncogene leads to viral E6 and E7 suppression in cervical cancer cells and apoptosis via upregulation of Rb and p53

The simultaneous expression of human papillomavirus type 16 (HPV16) E6 and E7 oncogenes is pivotal for malignant transformation and maintenance of malignant phenotypes. Silencing these oncogenes is considered to be applicable in molecular therapies of human cervical cancer. However, it remains to be determined whether HPV16 E6 and E7 could be both silenced to obtain most efficient antitumor activity by using RNA interference (RNAi) technology. Herein, we designed a small interfering RNA (siRNA) targeting HPV16-E7 region to degrade either E6, or truncated E6 (E6*) and E7 mRNAs and to simultaneously knockdown both E6 and E7 expression. Firstly, the sequence targeting HPV16-E7 region was inserted into the shRNA packing vector pSIREN-DNR, yielding pSIREN-16E7 to stably express corresponding shRNA. HPV16-transformed SiHa and CaSki cells were used as a model system; RT-PCR, Western Blotting, MTT assay, TUNEL staining, Annexin V apoptosis assay and flow cytometry were applied to examine the effects of pSIREN-16E7. Our results indicated that HPV16-E7 specific shRNA (16E7-shRNA) induced selective degradation of E6 and E7 mRNAs and proteins. E6 silencing induced accumulation of cellular p53 and p21. In contrast, E7 silencing induced hypophosphorylation of retinoblastoma (Rb) protein. The loss of E6 and E7 reduced cell growth and ultimately resulted in massive apoptotic cell death selectively in HPV-positive cancer cells, compared with the HPV-negative ones. We demonstrated that 16E7-shRNA can induce simultaneous E6 and E7 suppression and lead to striking apoptosis in HPV16-related cancer cells by activating cellular p53, p21 and Rb. Therefore, RNAi using E7 shRNA may have the gene-specific therapy potential for HPV16-related cancers.     

Differential m6A RNA landscapes across hematopoiesis reveal a role for IGF2BP2 in preserving hematopoietic stem cell function

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EphA2 promotes tumorigenicity of cervical cancer by up-regulating CDK6

Erythropoietin-producing hepatocellular receptor A2 (EphA2) receptor tyrosine kinase plays an important role in tissue organization and homeostasis in normal organs. EphA2 is overexpressed in a variety of types of solid tumours with oncogenic functions. However, the role of EphA2 in cervical cancer (CC) is still needed to be further explored. Here, we examined the role of EphA2 by establishing a stable EphA2 knock-down CC cell lines or a stable EphA2-overexpressed CC cells lines. Overexpression of EphA2 increased cell proliferation and migration of CC while EphA2 knock-down decreased the CC tumorigenicity. In addition, EphA2 knock-down suppressed CC tumour development in the xenograft mouse model. Inhibition of EphA2 by AWL-II-41-27, EphA2-specific tyrosine kinase inhibitor, or knock-down of EphA2 decreased mRNA and protein expression of cyclin-dependent kinase (CDK) 6 in CC cells, which increased cellular susceptibility to epirubicin (EPI), an anti-cancer chemotherapy drug. A clinicopathological study of EphA2 was conducted on a cohort of 158 human CC patients. EphA2 protein expression was positively correlated with CDK6 protein expression, invasion depth, lymph node metastasis and clinicopathological stage (P < .05). This study demonstrates the oncogenic activity of EphA2 in vitro and in vivo, which provides insights into the relevant mechanisms that might lead to novel treatments for CC.     

The HPV16 E6/E7 oncogene sensitizes human ovarian surface epithelial cells to low-dose but not high-dose 5-FU and 5-FUdR

To evaluate the effect of HPV16 E6/E7 on drug sensitivity, primary human OSE cells were infected with HPV16 E6/E7 expressing retrovirus and then exposed to chemotherapeutic agents. Apoptosis induced by mitomycin C was dose-dependent in both primary OSE and E6E7/OSE cells. E6E7/OSE cells were more sensitive to mitomycin C than parental OSE cells. HPV16 E6/E7 also sensitized OSE cells to 5-FU and its derivative 5-FUdR, but only at low doses. This phenomenon was also observed in cervical cancer cells and was independent of thymidylate synthase, a target of thymine and thymidine analogues. We conclude that HPV16 E6/E7 specifically modulates the activity of 5-FU and 5-FUdR, and confers OSE cells hypersensitivity to low-dose but not high-dose 5-FU and 5-FUdR. Molecular analysis indicates that induction of p53 and p21, and suppression of pRB are associated with apoptosis induced by 5-FUdR and may partly explain the hypersensitivity of E6E7/OSE cells to low-dose 5-FUdR.     

北京地区宫颈癌HPV16上游调控序列、E6、E7癌基因序列初步分析

为检测HPV16上游调控序列(Upstream regulatory region, URR)、E6、E7癌基因变异在北京地区宫颈癌患者癌组织中的分布特征, 探讨该地区宫颈癌发生同HPV16变异株间的相关性, 文章以提取的31例HPV16检测阳性宫颈癌组织DNA为模板, 设计针对性引物扩增URR、E6、E7 3个目的片段, PCR产物直接测序并通过GenBank对比分析变异和分支鉴定情况。在所分析的宫颈癌组织中, URR是突变频率最高的片段, 其次为E7, 最保守的序列为E6。共发现热突变位点8个, 分别为URR序列上G7521A(100%)、C7435G(96.77%)、C24T(45.16%)、A7729C(45.16%)、G7839A(45.16%); E6序列上T178G(41.94%); E7序列上A647G(45.16%)、T846C(45.16%)。HPV16分支分布频率最广的是As型(54.84%), 其次为E型(45.16%)。研究结果提示, HPV16URR序列上G7521A、A7729C、G7839A, E6序列上T178G、T350G, E7序列上A647G、G658A等位点的变异可能与病毒致癌潜能及宫颈癌的发生相关。北京地区宫颈癌患者中As和E型可能是两种最主要的HPV16分支, 这有可能会为HPV疫苗的研制和感染治疗提供有价值的信息。As型和E型病毒在不同年龄组和不同肿瘤分期组的患者中分布频率有差异, 这可能会为揭示宫颈癌年轻化趋势提供新的线索。     

IGF2BP3 facilitates cell proliferation and tumorigenesis via modulation of JAK/STAT signalling pathway in human bladder cancer

Insulin‐like growth factor‐2 messenger RNA‐binding protein 3 (IGF2BP3) has been reported to contribute to tumorigenesis in several human cancers. However, the biological functions of IGF2BP3 in bladder cancer are poorly understood. We investigated the relation between IGF2BP3 expression and prognosis of bladder cancer patients. Cell proliferation, cell cycle and cell apoptosis assays were performed to assess IGF2BP3 functions. The results showed that IGF2BP3 was overexpressed in bladder cancer tissues compared with that in normal bladder tissues, and its higher expression was closely correlated with poor prognosis in bladder cancer patients. Overexpression of IGF2BP3 markedly promoted cell proliferation and cell cycle progression and inhibited cell apoptosis, while knockdown of IGF2BP3 notably suppressed the proliferation, promoted cell apoptosis and induced cell cycle arrest at the G0/G1 phase. Mechanistically, we revealed that IGF2BP3 promotes the activation of the JAK/STAT pathway in bladder cancer cells. Moreover, the JAK/STAT inhibitor dramatically blocked the tumour‐promoting activity of IGF2BP3. Tumour growth in vivo was also suppressed by knocking down of IGF2BP3. Hence, IGF2BP3 facilitated bladder cancer cell proliferation by activating the JAK/STAT signalling pathway. These findings suggest that IGF2BP3 exhibits an oncogenic effect in human bladder cancer progression.     

Proteomics strategies to analyze HPV-transformed cells: relevance to cervical cancer

Cervical cancer is the second most common malignant tumor among women worldwide. The initiating event of cervical cancer is the infection with certain types of human papillomavirus (HPV). Interestingly, viral oncogene expression is necessary but not per se sufficient to promote cervical cancer and other factors are involved in neoplastic progression. Thus, major research efforts should be focused to identify novel co-carcinogenic factors and to understand the mechanisms played into tumor development. To reach this goal, proteomics strategies are powerful tools and a number of studies performed by following this approach have contributed to unravel the interplay between viral infection and protein dysfunction that ultimately results in cancer. The present review summarizes the most relevant findings obtained by applying proteomics technologies to both cell culture models and human tissue specimens. The results suggest that viral oncogenes selectively interact with a subset of intracellular proteins mainly involved in apoptosis resistance, cell growth and differentiation and cell transformation.     

R-2-hydroxyglutarate attenuates aerobic glycolysis in leukemia by targeting the FTO/m6A/PFKP/LDHB axis

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