Human papillomavirus 16E6 oncogene mutation in cervical cancer

Objective: Cervical cancer (CC) is the second most common type of cancer in women worldwide, after breast cancer. High-risk human papillomaviruses (HR-HPVs) are considered to be the major causes of cervical cancer. HPV16 is the most common type of HR-HPVs and HPV16 E6 gene is one of the major oncogenes. Specific mutations are considered as dangerous factors causing CC. This study was designed to find mutations of HPV16 E6 and the relationship between the mutations and the happening of CC.Methods: The tissue DNA was extracted from 15 biopsies of CC. Part of HPV16 E6 gene (nucleotide 201-523) was amplified by polymerase chain reaction (PCR) from the CC tissue DNA. The PCR fragments were sequenced and analyzed.Results: The result of PCR showed that the positive rate of HPV16 E6 was 93.33% (14/15). After sequencing and analyzing, in the 13 out of 14 PCR fragments, 4 maintained prototype (30.77%), 8 had a same 350G mutation (61.54%), and 1 had a 249G mutation (7.69%).Conclusion: This study suggest that there is a high infection rate of HPV in cervical cancer and most of the HPV16 E6 gene has mutations. Those mutations may have an association with the development of cervical cancer.     

Human papillomavirus oncoprotein E6 upregulates c-Met through p53 downregulation

PurposeHuman papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) carries a distinct clinical behaviour. c-Met oncogene is an important driver for tumour progression and its relationship with HPV in OPSCC was explored in the present study.Experimental designKnockdown of HPV oncogene E6 or p53 alone and in combination was performed to examine their effects on c-Met expression by Western blot and quantitative real-time polymerase chain reaction. The effects of c-Met inhibition on cell proliferation, migration, and colony formation were examined in HPV-positive head and neck squamous cell carcinoma (HNSCC) cells. Retrospectively collected OPSCC patient specimens (N = 78) were stained for c-Met by immunohistochemistry and the staining levels were correlated with HPV status and patient outcomes.ResultsE6 knockdown decreased c-Met protein and mRNA expression in HPV-positive HNSCC cells, which was partially abolished by the elimination of p53. Reducing c-Met decreased cell proliferation, migration, and colony formation in HPV-positive HNSCC cells. In OPSCC patient samples, high c-Met expression was associated with HPV-positive status (OR = 4.11, 95%CI: 1.16–14.55, P = 0.028) and tumour stage (OR = 0.27, 95%CI: 0.08–0.93, P = 0.039) by multivariable analysis. In T3/T4 stage patients, high c-Met expression was associated with HPV positivity and low p53 levels, supporting an axis of E6-p53-c-Met regulation. Furthermore, high c-Met expression was marginally associated with poor disease-free survival in HPV-positive patients.ConclusionsOur results suggest that c-Met may serve as a novel target for treating HPV-associated OPSCC. The data also demonstrate that HPV E6 upregulates c-Met expression partially through p53 downregulation.     

人乳头瘤病毒E6与宫颈癌

宫颈癌的病因与持续感染人乳头瘤病毒(human papillomavirus,HPV)相关.最近的研究表明,HPV E6在宫颈癌的发生过程中发挥重要作用.本文就HPV E6宫颈癌的发生机制及HPV E6蛋白的表达与宫颈癌的临床意义进行综述.     

Human papillomavirus type 16 E6/E7-specific cytotoxic T lymphocytes in women with cervical neoplasia

Infection with oncogenic human papillomavirus (HPV) types is associated with the development of cervical neoplasia (CIN). The E6 and E7 oncoproteins are constitutively expressed in these lesions and are therefore putative targets for the immune response against HPV. The relation between HPV 16-specific memory cytotoxic T-cell precursor (mCTLp) activity to both oncoproteins and the natural course of cervical dysplasia was analyzed in 38 patients participating in a nonintervention cohort study of women with CIN and 11 HPV 16-positive cervical carcinoma patients. In a cross-sectional study at the end of follow-up prior to biopsy, 8 of 20 patients with a persistent HPV 16 infection had specific mCTLp against at least one of the two oncoproteins. By contrast, no specific mCTLp activity was detected in 11 HPV-negative patients or in 7 patients who had cleared an HPV 16 infection at the end of follow-up. However, 5 of 11 cervical carcinoma patients showed mCTLp activity against the E7 protein only. This study demonstrates that HPV 16 oncogene-specific mCTLp are present in women with HPV 16-positive CIN prior to any intervention. Since HPV-specific mCTLp were detected predominantly in women with high-grade lesions or invasive cervical carcinoma and not in women who cleared the virus, the role of naturally occurring mCTLp in the protection against HPV-associated cervical neoplasia remains to be established.     

Human papillomavirus type 16 reduces the expression of microRNA-218 in cervical carcinoma cells

Human papillomaviruses (HPVs) are involved in the pathogenesis of cancer of the cervix (CaCx). MicroRNA (miRNA) expression analysis using Ambion (Austin, TX, USA) arrays showed that three miRNAs were overexpressed and 24 underexpressed in cervical cell lines containing integrated HPV-16 DNA compared to the normal cervix. Furthermore, nine miRNAs were overexpressed and one underexpressed in integrated HPV-16 cell lines compared to the HPV-negative CaCx cell line C-33A. Based on microarray and/or quantitative real-time PCR and northern blot analyses, microRNA-218 (miR-218) was specifically underexpressed in HPV-positive cell lines, cervical lesions and cancer tissues containing HPV-16 DNA compared to both C-33A and the normal cervix. Expression of the E6 oncogene of high-risk HPV-16, but not that of low-risk HPV-6, reduced miR-218 expression, and conversely, RNA interference of E6/E7 oncogenes in an HPV-16-positive cell line increased miR-218 expression. We also demonstrate that the epithelial cell-specific marker LAMB3 is a target of miR-218. We also show that LAMB3 expression is increased in the presence of the HPV-16 E6 oncogene and this effect is mediated through miR-218. These findings may contribute to a better understanding of the molecular mechanisms involved in cervical carcinogenesis.     

Human papillomavirus type 16 E6 variants and HLA class II alleles among Japanese women with cervical cancer

The enhanced oncogenicity of particular human papillomavirus type 16 (HPV16) E6 variants is population-dependent, implying the involvement of additional genetic cofactors. This study was designed to investigate the association between E6 variants and human leukocyte antigen (HLA) polymorphism within a Japanese population. Fifty-seven women with HPV16-positive cervical cancer were analyzed for E6 sequence variation and its relationship to HLA class II alleles. Compared with local controls (n = 138) and published controls (n = 916), DRB1*1501 and DQB1*0602 frequencies were significantly increased among patients with HPV16 E6 prototype (n = 11). Additionally, DRB1*1502 was positively associated with a particular E6 variant designated D25E (n = 25), although we could not find a significant association between HLA class II alleles and L83V variants (n = 16). Our observations suggest that a specific match between E6 variant proteins and HLA types may contribute to HPV16-related cervical carcinogenesis.     

p53 codon 72 polymorphism and various human papillomavirus 16 E6 genotypes are risk factors for cervical cancer development

Risk factors other than human papillomavirus (HPV) infection per se for cervical cancer development have been investigated recently. It was suggested that HPV 16 E6 variants and the p53 codon 72 arginine polymorphism could be progression markers. Indeed, it has been demonstrated that specific E6 variants and p53 arginine were both enriched in cancer. However, especially with regard to the latter, divergent results have been reported. Our aim was thus to investigate whether p53 arginine is important for cervical carcinogenesis by scaling up samples of the two European cohorts, the initial results of which were reported previously. In addition, we have assessed the occurrence of p53 codon 72 arginine, in combination with specific HPV 16 E6 genotypes. We found p53 arginine to be increased in cancer of both cohorts, consistent with our previous concept. Although specific E6 genotypes increased gradually with the severity of the lesion, p53 arginine was enriched in cancer only. Moreover, the frequency of the arginine allele was similar in groups with different E6 genotypes. It is concluded that p53 arginine is a risk factor for cervical cancer but probably acts independently of E6 variants.     

Maspin retards cell detachment via a novel interaction with the urokinase-type plasminogen activator/urokinase-type plasminogen activator receptor system

It is well documented that tumor suppressive maspin inhibits tumor cell invasion and extracellular matrix remodeling. Maspin is a cytosolic, cell surface-associated, and secreted protein in the serine protease inhibitor superfamily. Although several molecules have been identified as candidate intracellular maspin targets, the extracellular maspin target(s) remains elusive. Although maspin does not directly inhibit urokinase-type plasminogen activator (uPA) activity, we have shown evidence that maspin may block the pericellular proteolysis mediated by cell surface-associated uPA. In the current study, maspin significantly inhibited the Ca2+ reduction-induced detachment of DU145 cells. This maspin effect was associated with increased and sustained levels of mature focal adhesion contacts (FAC). We noted that maspin (a) colocalized with uPA and uPA receptor (uPAR), (b) enhanced the interaction between uPAR and low-density lipoprotein receptor related protein, and (c) induced rapid internalization of uPA and uPAR. The maspin effects on surface-associated uPA and uPAR required the interaction between uPA and uPAR. Further biochemical and biophysical analyses revealed that maspin specifically bound to pro-uPA with a deduced K(d) of 270 nmol/L and inhibited the plasmin-mediated pro-uPA cleavage. Interestingly, substitution of maspin p1' site Arg340 in the reactive site loop (RSL) with alanine not only abolished the binding to pro-uPA but also diminished the maspin effects on pro-uPA cleavage and cell detachment. These data show an important role of maspin RSL in regulating the uPA/uPAR-dependent cell detachment. Together, our data led to a new hypothesis that maspin may stabilize mature FACs by quenching localized uPA/uPAR complex before uPA activation.     

宫颈癌组织中人乳头瘤病毒16E6基因突变分析

背景与目的：高危型人乳头瘤病毒（humanpa pillomavirus,HPV）以16型最为常见,HPV16E6是宫颈癌主要的致癌基因之一,特定的E6突变是宫颈癌发生的主要因素之一。本研究主要观察兰州地区宫颈癌组织中是否存在E6突变,并探讨了突变与宫颈癌发生的关系。方法：以23例宫颈癌手术切除标本及5例正常宫颈组织的DNA作为模板,PCR扩增HPV-16E6基因201-523位,PCR产物直接测序．分析其HPV16E6基因的突变规律。结果：PCR扩增结果表明5例正常宫颈组织中HPV16E6阳性率为0％（0／5）,宫颈癌组织中HPV16E6阳性率为82．61％（19／23）,对18例样本PCR产物的测序和序列分析结果表明,6例（33．33％）E6基因与原型相同,12例（66．67％）E6基因发生突变,其中11例（61．11％）发生了350G突变。同时,在1例样本（5．56％）发现了249G突变。结论：兰州地区宫颈癌组织中存在着非常高的HPV感染率,且多数HP116E6发生了突变。     

宫颈癌组织中HPV16E6序列多态性及同源性分析

目的：探讨广东地区宫颈癌组织中HPV16肿瘤相关性抗原E6基因序列的多态性及同源性。方法：采用通用引物PCR直接测序法对宫颈癌标本中的HPV分型，从舍有HPV16型的标本中采用自行设计的多重引物通过巢式PCR扩增出HPV16E6，经DNA序列测定法检测其基因变异，进而分析其同源性。结果：50例宫颈癌组织HPV-DNA的检出率为78％．其中HPV16和HPV18型混合感染18例，单纯HPV16型感染15例。含有HPV16型的标本34例中扩增出HPV16E 625例。其中178位核苷酸变异较大，变异率为72％，其相应氨基酸均由天冬氨酸变为谷氨酸。结论：HPV16E6 DNA序列发生碱基替换的区域主要在氨基端94～241位，羧基端相对保守，未见变异。广东地区宫颈癌组织中HPV16E6的热点突变为Nt178。     

宫颈癌组织中HPV16E6序列多态性及同源性分析

目的探讨广东地区宫颈癌组织中HPV16肿瘤相关性抗原E6基因序列的多态性及同源性。方法采用通用引物PCR直接测序法对宫颈癌标本中的HPV分型,从含有HPV16型的标本中采用自行设计的多重引物通过巢式PCR扩增出HPV16E6,经DNA序列测定法检测其基因变异,进而分析其同源性。结果50例宫颈癌组织HPV-DNA的检出率为78%,其中HPV16和HPV18型混合感染18例,单纯HPV16型感染15例。含有HPV16型的标本34例中扩增出HPV16E625例。其中178位核苷酸变异较大,变异率为72%,其相应氨基酸均由天冬氨酸变为谷氨酸。结论HPV16E6DNA序列发生碱基替换的区域主要在氨基端94~241位,羧基端相对保守,未见变异。广东地区宫颈癌组织中HPV16E6的热点突变为Nt178。     

Human papillomavirus type 16 E6 and E7 genotypes in head-and-neck carcinomas

Human papillomavirus type 16 (HPV16) is associated with squamous cell carcinomas of the head and neck (HNSCC) particularly from the Waldeyer's tonsillar ring. A causal role of HPV16 in carcinogenesis is linked to the activity of the viral oncoproteins E6 and E7 which inactivate the cellular tumor suppressors p53 and pRB, respectively. Lack of E6 expression in HPV16-positive HNSCC has been reported, in some cases caused by disruption of the E6 gene. We have examined the status of the HPV16 E6-E7 gene region in tumor and metastasis samples of 24 HNSCC patients employing genomic PCR. No cases with a disrupted E6-E7 region could be identified. Sequence analysis of the E6-E7 segments revealed three different HPV16 E6-E7 genotypes: the HPV16 prototype (6 of 21 cases), the E6 variant T350G (8 of 21 cases), and the E6-E7 variant A131G/C712A (7 of 21 cases). The E6 variants T350G and A131G have been associated with increased oncogenic potential in cervical cancer patients depending on host genetic factors. Their high prevalence in the HNSCC samples studied indicates that they may be important also in HNSCC development.     

Rapamycin induces p53-independent apoptosis through the mitochondrial pathway in non-small cell lung cancer cells

The mammalian target of rapamycin (mTOR) is a key kinase acting downstream of growth factor receptor PI3K and AKT signaling, leading to processes resulting in increased cell size and proliferation through translation control. Rapamycin, a specific inhibitor of mTOR, results predominately in G1 cell cycle arrest through translation control and occasionally, cell type-dependent apoptosis by an unknown mechanism. In this study, we investigated the effect and mechanism of action of rapamycin on non-small cell lung cancer (NSCLC) cell lines with p53 mutations. Cell proliferation was evaluated by modified MTT assay. The apoptotic effect of rapamycin was measured by caspase-3 activation and flow cytometric analysis of Annexin V binding. The expression of Bcl-2 and the release of cytochrome?c from mitochondria were evaluated by western blotting. We found that rapamycin induced apoptosis in NSCLC cell lines with p53 mutations. Western blot analysis demonstrated that rapamycin downregulates the expression levels of Bcl-2, which leads to increased cytochrome c release from mitochondria and subsequent activation of caspase cascades. These findings suggest that rapamycin induces p53-independent apoptosis through downregulation of Bcl-2 and the mitochondrial pathway in NSCLC cell lines as a novel antitumor mechanism.     

Human papillomavirus type 16 E7 up-regulates AKT activity through the retinoblastoma protein

Human papillomaviruses (HPV) are small DNA tumor viruses causally associated with cervical cancer. The early gene product E7 from high-risk HPV is considered the major transforming protein expressed by the virus. Although many functions have been described for E7 in disrupting normal cellular processes, we describe in this study a new cellular target in primary human foreskin keratinocytes (HFK), the serine/threonine kinase AKT. Expression of HPV type 16 E7 in HFK caused inhibition of differentiation, hyperproliferation, and up-regulation of AKT activity in organotypic raft cultures. The ability of E7 to up-regulate AKT activity is dependent on its ability to bind to and inactivate the retinoblastoma (Rb) gene product family of proteins. Furthermore, we show that knocking down Rb alone, with short hairpin RNAs, was sufficient to up-regulate AKT activity in differentiated keratinocytes. Up-regulation of AKT activity and loss of Rb was also observed in HPV-positive cervical high-grade squamous intraepithelial lesions when compared with normal cervical tissue. Together, these data provide evidence linking inactivation of Rb by E7 in the up-regulation of AKT activity during cervical cancer progression.     

The human papillomavirus type 16 E6 oncogene induces premature mitotic chromosome segregation

Expression of the human papillomavirus type 16 E6 and E7 oncogenes initiates and maintains abnormal cell replication, by interacting with the p53 and retinoblastoma (Rb) gene products. Subsequent changes in host cell gene expression, as a consequence of genetic instability, can result in progression to invasive carcinoma. In addition to previously described effects of these viral oncogenes on centrosome synthesis, primarily associated with the expression of E7, the results described herein demonstrate that the E6 oncogene can induce premature chromosome segregation in human cells.     

Dispensability of p53 degradation for tumorigenicity and decreased serum requirement of human papillomavirus type 16 E6

Certain types of human papillomavirus (HPV), such as types 16 and 18, are etiological agents for carcinogenesis of the uterine cervix. These HPVs have two oncogenes, E6 and E7, that have transforming activities in established murine cells. Tumorigenicity and decreased serum requirement for cell growth are conferred by the E6 gene, whereas anchorage-independent growth is mainly governed by the E7 gene. To understand the mechanism of cellular transformation by the HPV16 E6 gene, we examined three mutant E6 proteins defective for p53 binding, p53 degradation, or transactivation of the adenovirus E2 promoter for the ability to induce tumorigenicity and decreased serum requirement. The results showed that tumorigenicity and decreased serum requirement were associated with the ability of E6 to bind to p53, although the subsequent degradation of p53 was not required for these functions. Mol. Carcinog. 21:215–222, 1998. © 1998 Wiley-Liss, Inc.     

特异性siRNA抑制宫颈癌细胞CaSKi中HPV16 E6基因的表达

目的：研究RNAi技术抑制宫颈癌细胞CaSKi中HPV16 E6癌基因的表达以及对细胞生物学特性的影响。方法：构建真核细胞表达特异性shRNA的pGENESIL-1/E6（PS6）重组质粒,脂质体转染细胞后用半定量RT-PCR技术检测CaSKi细胞中E6mRNA水平的变化,然后用western blotting检测RNA干扰前后p53和p21蛋白的表达来验证RNAi效应。用透射电子显微镜观察CaSKi/PS6细胞形态变化;应用流式细胞仪检测细胞的凋亡和细胞周期的变化,最后利用细胞计数和裸鼠成瘤实验测定CaSKi/PS6细胞增殖能力。结果：成功构建了真核细胞表达特异性siRNA的PS6重组质粒;在RNA干扰24、48和72小时后CaSKi/PS6细胞中E6mRNA分别下降了21.50%、52.60%和65.60%;CaSKi/PS6细胞出现凋亡的特征性改变;RNA干扰24h、48h和72h后CaSKi/PS6细胞的凋亡率分别为27.94%、31.95%和56.63%,细胞生长周期停滞于S期。结论：应用RNA干扰技术能有效地、特异地抑制宫颈癌细胞中HPV16 E6基因的表达,为RNA干扰应用于研究宫颈癌发病分子机制、临床治疗和干预性预防提供了新的方法。