Vaccination of full-length HPV16 E6 or E7 protein inhibits the growth of HPV16 associated tumors

Cervical cancer is the second most common cancer in women worldwide. Human papillomavirus (HPV) is the primary etiologic agent of cervical cancer. Two HPV16 proteins, E6 and E7, are consistently expressed in tumor cells. Most therapeutic vaccines target one or both of these proteins. Taking the advantages of safety and no human leukocyte antigen restriction, protein vaccine has become the most popular form of HPV therapeutic vaccines. Here we demonstrate that immunization with full-length HPV16 E6 or E7 protein elicited specific immunological effect and inhibition of TC-1 cell growth using TC-1 mouse model. HPV16 E6 and E7 genes were cloned into pET-28a(+) and introduced into E. coli Rosetta. Expression of the genes was induced by IPTG. Proteins were purified by Ni-NTA agarose and they were detected by SDS-PAGE and Western blotting. C57BL/6 mice were vaccinated with 1.5 nmol HPV16 E6 or E7 protein. Then they were implanted with 1x10(5) TC-1 cells. No tumor was detected in any mouse vaccinated with E7 protein. Forty days later, the tumor-free mice and control mice were challenged with 2x10(5) TC-1 cells. All control mice developed tumors 6 days later, but E7 immunized mice were tumor free until 90 days. Tumor growth was slow in the E6 immunized mice, but 83% of the mice developed tumors and the survival percentage was not significantly different from the control. An adoptive immune model was used to demonstrate the therapeutic effect. Results showed that the development of TC-1 cells was obviously reduced by transfusion of T-cells but not serum from mice immunized with E7 protein. T-cells from E7 immunized mice also induced the lysis of TC-1 cells in the cytotoxic T lymphocyte assay. These findings show that immunization with HPV16 E6 or E7 protein was able to elicit specific protective immunity against TC-1 tumor growth.     

HPV16 E6-mediated stabilization of ErbB2 in neoplastic transformation of human cervical keratinocytes

Whether ErbB2 receptor tyrosine kinase contributes to cervical cancer is controversial. We have examined the effects of E6 and E7 genes of human papillomaviruses type 16 (HPV-16) on ErbB2 expression in primary human cervical keratinocytes (HCK) immortalized with hTERT (HCK1T). In E6-positive cells (HCK1T-E6 and HCK1T-E6E7), ErbB2 expression levels increased with the cell density. HCK1T-E6E7 showed impaired contact inhibition and anchorage-independent growth in soft agar which were abrogated with introduction of ErbB2-specific short hairpin RNA (shRNA) or an ErbB2 specific inhibitor AG825. Furthermore, increased ErbB2 expression was also observed in HPV16 positive cervical cancer cell lines and this was diminished by introduction of HPV16E6- or E6AP-shRNA. At post-confluence cell densities, ErbB2 protein was stabilized in the presence of E6 whereas increased ErbB2 expression was not obvious with E6 mutants incapable of degrading p53. Furthermore, introduction of p53-shRNA to HCK1T resulted in increased ErbB2 protein stability, indicating possible ErbB2 regulation through p53. Finally, we showed that tumor formation of ErbB2-shRNA introduced SiHa cells were almost abolished. Taken together, these data indicate an important role of ErbB2 regulation by HPV16 E6 in oncogenic transformation of human cervical keratinocytes.     

NF‐κ B‐dependent upregulation of ICAM‐1 by HPV16‐E6/E7 facilitates NK cell/target cell interaction

NK cell recognition of tumor cells is mediated by a delicate balance of signals received by MHC class I‐binding inhibitory NK cell receptors and activating NK cell receptors, which mainly bind to virus‐, stress‐ or tumor‐induced ligands. In addition, adhesion molecules such as the intercellular adhesion molecule‐1 (ICAM‐1) and its receptors, the lymphocyte function‐associated antigen‐1 (LFA‐1) and Mac‐1, are crucial for immune synapse formation and NK cell‐mediated killing. In this study, we show that expression of the adhesion molecule ICAM‐1 was rapidly induced by E6 and ‐E7 oncoproteins of HPV16, ‐18, ‐5 and ‐8, but not of HPV38 and ‐6 in primary human keratinocytes after retroviral transduction. ICAM‐1 was upregulated in E6E7‐expressing keratinocytes both at mRNA and protein levels. The observed ICAM‐1 upregulation in HPV16‐E6E7‐expressing keratinocytes was partially dependent on activation of the NF‐κB pathway. Importantly, the upregulated ICAM‐1 expression in HPV16‐E6E7‐expressing keratinocytes led to enhanced conjugate formation with NK cells. We previously showed that HPV16‐positive cervical carcinomas frequently express low levels of inhibitory NK cell ligands and high levels of activating NK cell ligands. Moreover, levels of the adhesion molecule ICAM‐1 are enhanced by HPV16‐E6/E7. Therefore, strategies that aim at harnessing NK cells might be beneficial for the treatment of cervical carcinoma.     

Intratumor injection of small interfering RNA-targeting human papillomavirus 18 E6 and E7 successfully inhibits the growth of cervical cancer

Human papillomavirus (HPV) 18 is related not only to squamous cell carcinoma of the cervix, but also to adenocarcinoma and small cell carcinoma of the cervix, in which prognosis is known to be poor. Small interfering RNA (siRNA) that targets HPV18 E6 and E7 was tested in HPV18-positive cell lines to investigate its effect and investigate its mechanism of action. Nude mice were also tested in a combination of siRNA and atelocollagen to determine whether it might be useful as a new molecule-targeting therapy for cervical cancer. siRNAs targeting HPV18 E6 and E7 were transfected into cervical cancer cells in vitro and they were investigated for cell growth inhibition, expression of E6 and E7 mRNA, expression of retinoblastoma protein, and senescence-associated beta-galactosidase staining. Sequence-specific siRNA inhibited cell growth. Decreased expression of E6 and E7 mRNA followed with E7 protein was observed in the transfected cells, but the expression of retinoblastoma protein and the beta-galactosidase staining increased, suggesting cell growth inhibitory effect through senescence. Treatment of xenografts established from SKG-II cells with siRNA specific for E6 and E7 obviously suppressed tumor growth in vivo. These results indicate that atelocollagen-mediated delivery of siRNA HPV18 E6 and E7 can be used as a novel therapeutic approach for cervical cancer.     

宫颈癌细胞系中HPV16 E6、E7及E6/E7基因对STK31基因甲基化状态及表达的影响

背景与目的：丝氨酸/苏氨酸蛋白激酶31(serine/threonine kinases 31,STK31)基因在人类多种癌症中扮演重要角色,且STK31基因的表达受其启动子及第一外显子区甲基化状态的影响;病毒感染与肿瘤组织中某些抑癌基因启动子区高甲基化有关。本研究旨在探讨宫颈癌细胞系中HPV16 E6、E7及E6/E7癌基因对STK31基因甲基化状态及表达的影响,以及不同种类甲基转移酶(DNA methyltransferases,DNMTs)基因在STK31基因甲基化中的潜在作用。方法：构建外源性HPV16 E6、E7以及E6/E7基因共表达慢病毒,分别感染人乳头瘤病毒(human papillomavirus,HPV)阴性宫颈癌细胞系HT-3及C33A,获得稳定转染的细胞系;采用亚硫酸氢盐基因组测序法(bisulfite genomic sequencing,BGS)和甲基化特异性PCR (methylation-specific PCR,MSP)检测3种HPV阳性宫颈癌细胞系HeLa、SiHa和CasKi以及HPV阴性宫颈癌细胞系HT-3和C33A转染前后STK31基因的甲基化状态;RT-PCR及蛋白［质］印迹法(Western blot)检测上述宫颈癌细胞系中STK31基因的表达以及DNMT1、DNMT2、DNMT3a、DNMT3b和DNMT3L基因在HPV16转染前后宫颈癌细胞系及HPV阳性、HPV阴性宫颈癌组织中的表达情况。结果：外源性HPV16 E6、E7以及E6/E7基因可在HPV阴性宫颈癌细胞系中稳定表达。3种HPV阳性细胞系HeLa、SiHa和CasKi中STK31基因呈低甲基化状态,STK31 mRNA及蛋白质表达阳性;2种HPV阴性细胞系HT-3、C33A中STK31基因则表现为高甲基化状态,STK31 mRNA及蛋白质表达缺失;与未感染慢病毒HT-3和C33A细胞系比较,外源性HPV16 E7以及E6/E7表达的HT-3和C33A细胞系STK31基因甲基化程度降低,其mRNA及蛋白质重新表达。DNMT1、DNMT3a和DNMT3b基因在HT-3E6/E7和C33AE6/E7细胞系中mRNA水平分别高于HT-3空载细胞系和C33A空载细胞系,差异有统计学意义(P<0.001)。DNMT1、DNMT3a和DNMT3b基因的mRNA水平在HPV16阳性宫颈癌组织中的表达高于其在HPV阴性宫颈癌组织中的表达,差异有统计学意义(t=5.997,P<0.001;t=6.743,P<0.001;t=7.926,P<0.001)。DNMT2在HT-3E6/E7和C33AE6/E7细胞系中mRNA表达水平分别低于HT-3空载细胞系和C33A空载细胞系,差异有统计学意义(t=7.451,P<0.001;t=2.451,P<0.05);DNMT2基因转录水平在HPV16阳性宫颈癌组织中低于HPV阴性宫颈癌组织(t=9.134,P<0.001)。DNMT3LmRNA表达水平在宫颈癌细胞系转染前后及HPV阴阳性宫颈癌组织中的差异无统计学意义(P>0.05)。结论：HPV感染可导致STK31基因启动子及第1外显子区甲基化状态降低,低甲基化状态促进该基因表达。STK31基因的表达受其启动子及第1外显子区甲基化状态的调控。HPV16 E7、E6/E7基因可能通过影响DNMT2的表达参与调控癌基因STK31基因启动子及第1外显子区甲基化状态。     

Tumorigenicity of the E6 and E6-E7 gene constructions derived from human papillomavirus type 33.

Human papillomavirus type 33 (HPV33) belongs to the group of HPV types frequently found in severe cervical dysplasias and carcinomas. By analogy with HPV types 16 and 18 selectively expressing E6 and E7 genes in malignant tissues, we studied the HPV33 E6 and E7 open reading frames in various configurations with upstream promoter and noncoding region (NCR) known to contain a particular 78-bp tandem repeat. HPV DNA fragments were cloned into expression vectors between the SV40 or the mouse metallothionein I promoter and the neo gene, transfected into NIH3T3 cells, selected by neo resistance, and inoculated into nude mice. In these bioassays, a weak transforming activity was detected for E6 open reading frame, and could be significantly enhanced either by the NCR or the E7 open reading frame. No tumorigenicity could be detected for E7 alone or in configuration with the upstream NCR. Further analyses of tumor cells showed that HPV33-derived genes were not sufficient to induce an anchorage-independent phenotype and, interstingly, there was no requirement for virus-transfected tumor cells to retain HPV sequences during tumor progression. We concluded that the transformation function of HPV33 resides in E6 gene as assayed by tumorigenicity. An enhancer of the E6 promoter is located in the NCR. On the other hand, in the absence of the NCR, E6 tumorigenicity may be augmented by the E7.     

Cloning and characterization of human papillomavirus type 52 from cervical carcinoma in Indonesia.

Human papillomavirus (HPV) type 52 from a cervical carcinoma in Indonesia was molecularly cloned and characterized. By hybridization with cervical carcinoma DNAs from Indonesian patients, HPV 52 was detected in 3 of 52 cases (6%), whereas HPV 16 and 18 were detected in 8 and 7 cases, respectively (15% and 13%). Sequence analysis revealed that the E6-E7 ORFs contained several DNA binding motifs (Cys-X-X-Cys) like previously sequenced HPVs. The E6 ORF also contained splice donor and acceptor signals, which may allow the expression of E6* protein. The E7 ORF encoded an amino acid sequence that is conserved in some DNA tumor viruses and is involved in binding to Rb protein and in cellular transformation. Transfection of a subgenomic fragment of HPV 52 under the control of a heterologous promoter showed that the E7 ORF alone induced anchorage-independent growth of established rodent cells and immortalized primary rat embryo fibroblasts (REF), and that in cooperation with activated ras, it induced malignant transformation of REF. The E6 ORF also induced, less efficiently, anchorage-independent growth. These results strongly suggest that HPV 52, like HPV 16 and 18, has oncogenic potential.     

New highly potent and specific E6 and E7 siRNAs for treatment of HPV16 positive cervical cancer

Persistent infection by high-risk types of human papillomaviruses (HPV) is a necessary cause of cervical cancer, with HPV16 the most prevalent, accounting for more than 50% of reported cases. The virus encodes the E6 and E7 oncoproteins, whose expression is essential for maintenance of the malignant phenotype. To select efficacious siRNAs applicable to RNAi therapy for patients with HPV16+ cervical cancer, E6 and E7 siRNAs were designed using siDirect computer software, after which 10 compatible with all HPV16 variants were selected, and then extensively examined for RNAi activity and specificity using HPV16+ and HPV16-cells. Three siRNAs with the highest RNAi activities toward E6 and E7 expression, as well as specific and potent growth suppression of HPV16+ cancer cells as low as 1 nM were chosen. Growth suppression was accompanied by accumulation of p53 and p21(WAF1/CIP1), as well as morphological and cytochemical changes characteristic of cellular senescence. Antitumor activity of one of the selected siRNAs was confirmed by retarded tumor growth of HPV16+ cells in NOD/SCID mice when locally injected in a complex with atelocollagen. Our results demonstrate that these E6 and E7 siRNAs are promising therapeutic agents for treatment of virus-related cancer.     

Induction of apoptosis in human keratinocytes containing mutated p53 alleles and its inhibition by both the E6 and E7 oncoproteins

Induction of apoptosis is a function of external stimuli and cellular gene expression. Many cells respond to DNA damage by the induction of apoptosis, which depends on a functional p53 protein and is signaled by elevation of p53 levels. We have investigated the response of immortalized human keratinocytes (HaCaT) bearing mutated alleles of p53 to genotoxic stress and the effect of human papillomavirus (HPV) 16 E6 and E7 on this response. UVC irradiation triggered HaCaT's cell death with several characteristics of apoptosis, including DNA laddering, chromatin condensation and fragmentation, and the appearance of cells with a low content of DNA (categorized as sub-G₁ by cell sorter analysis). This response was accompanied by accumulation of cells in S phase of the cell cycle. HaCaT cells infected with retroviruses carrying HPV16 E6 or E7 showed a significant reduction in their apoptotic response, which was not observed in cells infected with the LXSN vector DNA-carrying virus. Reduced apoptosis in HaCaT cells expressing E6 or E7 also was observed after treatment with the alkylating agent mitomycin C. Western blot analysis of p53 and p21/WAF-1/CIP-1, a downstream effector of p53, did not reveal any changes in the levels of these proteins after UVC irradiation in either HaCaT cells or HaCaT cells expressing HPV16 E6 or E7. Int. J. Cancer 75:96–104, 1998.© 1998 Wiley-Liss, Inc.     

Immortalization without neoplastic transformation of human mesenchymal stem cells by transduction with HPV16 E6/E7 genes

hMSCs derived from bone marrow are useful as a species-specific cell culture system for studying cell lineage differentiation and tissue remodeling. However, hMSCs usually have a short in vitro life span due to replicative senescence. We therefore used a high dose of retroviral vector LXSN-16E6E7 to transduce hMSCs of an aging donor and obtained an actively proliferating cell line, designated KP-hMSCs, which expressed HPV16 E6/E7 mRNA. Whereas parental hMSCs ceased to grow after 30 PDs, KP-hMSCs could be propagated beyond 100 PDs. With culture procedures to avoid selection pressure and crowded cell growth, KP-hMSCs showed no signs of neoplastic transformation as examined by soft-agar anchorage-independent growth and NOD-SCID mouse tumorigenicity assays. KP-hMSCs gave similar cytofluorimetric profiles of 31 CD markers to those of the parental primary hMSCs, except with some morphologic changes and expansion of an originally very minor CD34(dim)CD38(+)CD50+ cell population. Upon exposure to specific stimulating conditions in vitro, KP-hMSCs could respond and differentiate along the mesenchymal (bone, fat and cartilage) and nonmesenchymal (neuron) cell lineages. Our results indicated that hMSCs could be immortalized by transduction with HPV16 E6/E7, maintained without neoplastic transformation by careful culture procedures and thus useful for stem cell research and clinical application.     

HPV16E6 siRNA联合5-Aza-CdR对宫颈癌SiHa细胞中E-钙黏蛋白表达和基因甲基化的影响

目的:探讨HPV16E6 siRNA联合5-Aza-CdR(5-aza-2'-deoxycytidine,5-氮-2'-脱氧胞苷,又称地西他滨)对宫颈癌SiHa细胞中E-钙黏蛋白表达和基因甲基化的影响。方法:采用SiHa细胞构建的HPV16E6 沉默细胞株及5-Aza-CdR细胞株,检测细胞中E-cadherin mRNA和蛋白表达,以及E-cadherin甲基化状态。采用细胞黏附、Transwell体外侵袭、迁移实验检测5-Aza-CdR和siRNA E6对SiHa细胞黏附、侵袭迁移的影响。结果:5-Aza-CdR+siRNA E6组较5-Aza-CdR组、siRNA E6组中E-cadherin mRNA 及蛋白表达均上升,细胞的黏附率、侵袭抑制率和迁移抑制率均升高;5-Aza-CdR+siRNA E6组较5-Aza-CdR组、siRNA E6组中E-cadherin基因启动子区甲基化指数明显下降。结论:HPV16E6 siRNA联合5-Aza-CdR可显著引起宫颈癌细胞中E-cadherin 基因低甲基化,并可导致E-cadherin mRNA 及蛋白的表达水平明显上调,肿瘤细胞黏附能力升高,侵袭迁移能力下降,两者在一定程度起到协同作用。     

The state of immortalized, transforming and suppressor genes in rat-cells, transfected by polyomavirus large-T and hpv18 e6+e7 genes

Several novel cell clones (A1-A6) derived from the rat embryo fibroblasts immortalized by the polyoma virus T-antigen (LT) gene and transformed by HPV18 E6 and E7 genes were explored. Using E6 or E7 peptide antisera we detected E6 and E7 proteins with approximate molecular masses of 16 kDa and 20 kDa, respectively. Monoclonal antibody to p53 PAb421 but not PAb240 precipitated different but appreciable amounts of p53 protein in all cell clones, indicating that wild-type p53 is expressed in these cells. So expression of HPV18 E6 protein in cells does not always lead to a complete reduction in p53 levels. The quantity of p53 protein in cell clones did not correlate with the level of polyoma virus large T-antigen expression. Variations in levels of p53 protein in clones did not influence on such cell biological properties as anchorage-independence and tumorigenicity for nude mice which were similar in all cell clones.     

Selective targeting of HPV-16 E6/E7 in cervical cancer cells with a potent oncolytic adenovirus and its enhanced effect with radiotherapy in vitro and vivo

Recent studies have shown that oncolytic adenovirus specifically targeted tumor cells while sparing normal cells. Here, we report a novel E1A-mutant adenovirus (M6) with antisense HPV16 E6 E7 DNA inserted into the deleted 6.7 K/gp19 K region of E3. The target effects of M6 on HPV16-positive cervical cancer cells were evaluated in vivo and in vitro. By using cytopathic effect (CPE) and viral replication assays, we verified M6 was competent to selectively replicate in cervical cancer cells in vitro. Moreover, we found infection of M6 was able to inhibit the expression of HPV16 E6 and E7 oncogenes and induce apoptosis of HPV16-positive cervical cancer cells. Further analysis in vitro revealed that the invasive ability of SiHa cells was significantly inhibited by M6. To determine if M6 synergized with radiotherapy-induced anti-tumor activity against HPV16-related cancer cells, we transfected SiHa cells with M6 followed by a single exposure to radiation. A significantly suppression of cell growth and induced apoptosis was observed in SiHa cells received M6 transfection combined with radiotherapy. Animal experiments showed that M6 transfection notably improved the survival of tumor-bearing mice in combination with radiotherapy, much superior to that of those treated by Adv5/dE1A plus radiation or M6 alone. These findings indicated the anti-tumoral efficacy of M6 on HPV16-positive cervical cancer cells and its synergic therapeutic application in radiation for cervical cancer.