Immunological ignorance of an E7-encoded cytolytic T-lymphocyte epitope in transgenic mice expressing the E7 and E6 oncogenes of human papillomavirus type 16.

Certain human papillomaviruses (HPV) have been implicated in the etiology of cervical malignancies, and the E7 and E6 gene products of HPV type 16 are frequently expressed in these lesions. However, cytolytic T-lymphocyte (CTL)-mediated responses to HPV are rarely detectable in patients with cervical cancer. To examine whether the T-cell response is deficient during the HPV-induced transformation, we produced lines of transgenic (Tg) mice that expressed the E6 and E7 oncogenes in keratinized epithelia. The mice developed severe hypertrophy of all keratinized epithelia, but no malignancies were observed. Although epithelial cells from Tg mice could present at least an E7-encoded CTL epitope (E7 49-57), CTLs from these mice were neither primed to nor made tolerant of this epitope. No quantitative or qualitative differences were seen in the CTL responses of the Tg mice compared to those of their littermates following immunization with the peptide E7 49-57. Immunization of Tg mice with the E7 49-57 peptide protected them against a subcutaneous challenge with tumor cells expressing a transfected E7 gene, yet the skin was unaffected, although the cultured skin epithelial cells from Tg mice expressed E7. Our results suggest that the Tg mice were immunologically ignorant of HPV oncoproteins with respect to a CTL response and that a similar type of ignorance may explain why HPV-associated cervical cancer cells can escape immunological destruction.     

E6 and E7 from beta HPV38 cooperate with ultraviolet light in the development of actinic keratosis-like lesions and squamous cell carcinoma in mice

Cutaneous beta human papillomavirus (HPV) types appear to be involved in the development of non-melanoma skin cancer (NMSC); however, it is not entirely clear whether they play a direct role. We have previously shown that E6 and E7 oncoproteins from the beta HPV type 38 display transforming activities in several experimental models. To evaluate the possible contribution of HPV38 in a proliferative tissue compartment during carcinogenesis, we generated a new transgenic mouse model (Tg) where HPV38 E6 and E7 are expressed in the undifferentiated basal layer of epithelia under the control of the Keratin 14 (K14) promoter. Viral oncogene expression led to increased cellular proliferation in the epidermis of the Tg animals in comparison to the wild-type littermates. Although no spontaneous formation of tumours was observed during the lifespan of the K14 HPV38 E6/E7-Tg mice, they were highly susceptible to 7,12-dimethylbenz(a)anthracene (DMBA)/12-0-tetradecanoylphorbol-13-acetate (TPA) two-stage chemical carcinogenesis. In addition, when animals were exposed to ultraviolet light (UV) irradiation, we observed that accumulation of p21(WAF1) and cell-cycle arrest were significantly alleviated in the skin of Tg mice as compared to wild-type controls. Most importantly, chronic UV irradiation of Tg mice induced the development of actinic keratosis-like lesions, which are considered in humans as precursors of squamous cell carcinomas (SCC), and subsequently of SCC in a significant proportion of the animals. In contrast, wild-type animals subjected to identical treatments did not develop any type of skin lesions. Thus, the oncoproteins E6 and E7 from beta HPV38 significantly contribute to SCC development in the skin rendering keratinocytes more susceptible to UV-induced carcinogenesis.     

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.     

Skin hyperproliferation and susceptibility to chemical carcinogenesis in transgenic mice expressing E6 and E7 of human papillomavirus type 38

The oncoproteins E6 and E7 of human papillomavirus type 38 (HPV38) display several transforming activities in vitro, including immortalization of primary human keratinocytes. To evaluate the oncogenic activities of the viral proteins in an in vivo model, we generated transgenic mice expressing HPV38 E6 and E7 under the control of the bovine homologue of the human keratin 10 (K10) promoter. Two distinct lines of HPV38 E6/E7-expressing transgenic mice that express the viral genes at different levels were obtained. In both lines, HPV38 E6 and E7 induced cellular proliferation, hyperplasia, and dysplasia in the epidermis. The rate of occurrence of these events was proportional to the levels of HPV38 E6 and E7 expression in the two transgenic lines. Exposure of the epidermis of nontransgenic mice to UV led to p21WAF1 accumulation and cell cycle arrest. In contrast, keratinocytes from transgenic mice continued to proliferate and were not positive for p21WAF1, indicating that cell cycle checkpoints are altered in keratinocytes expressing the viral genes. Although the HPV38 E6/E7-expressing transgenic mice did not develop spontaneous tumors during their life span, two-stage carcinogen treatment led to a high incidence of papillomas, keratoacanthomas, and squamous-cell carcinomas in HPV38 mice compared with nontransgenic animals. Together, these data show that HPV38 E6 and E7 display transforming properties in vivo, providing further support for the role of HPV38 in carcinogenesis.     

ErbB-2 Receptor Cooperates with E6/E7 Oncoproteins of HPV Type 16 in Breast Tumorigenesis

The ErbB-2 receptor is overexpressed in roughly 30% of human breast cancers. Moreover, approximately 50% of breast cancers are positive for high-risk human papillomaviruses (HPVs), specifically types 16 and 18. Recently, we reported that ErbB-2 cooperates with E6/E7 oncoproteins of HPV type 16 to induce neoplastic transformation of human normal oral epithelial cells. We also demonstrated that E6/E7 of HPV type 16 converts non-invasive breast cancer cells to an invasive form. In order to investigate the effect of ErbB-2/E6/E7 cooperation in breast carcinogenesis, we generated double transgenic mice carrying ErbB-2 and E6/E7 of HPV type 16 under mouse mammary tumor virus (MMTV) and human keratin 14 promoters, respectively. Within six months, these double transgenic mice developed large and extensive invasive breast cancer in comparison to ErbB-2 or E6/E7 singly transgenic mice. Histological analysis of ErbB-2/E6/E7 transgenic mice tumors showed the presence of invasive breast carcinomas. However, the breast tissues from ErbB-2 and E6/E7 transgenic mice showed only in-situ cancer and normal mammary phenotype, respectively. In parallel, we examined the cooperation effect of ErbB-2 and E6/E7 in the human breast cancer cell line, BT20; in comparison to ErbB-2 and E6/E7 alone as well as wild type cells, we found that ErbB 2/E6/E7 together stimulate colony formation and cell migration in the BT20 cell line. Furthermore, we found that β-catenin is constitutively phosphorylated by c-Src and consequently trans-located to the nucleus in ErbB-2/E6/E7-breast cancer cells. These findings provide evidence that the ErbB-2 receptor cooperates with high-risk HPVs in breast tumorigenesis via β-catenin activation.     

The E6 oncoprotein from HPV16 enhances the canonical Wnt/β-catenin pathway in skin epidermis in vivo

The contribution of the Wnt signaling pathway to human papilloma virus (HPV)-induced carcinogenesis is poorly understood. In high-grade dysplastic lesions that are caused by high-risk HPVs (HR-HPV), β-catenin is often located in the cell nucleus, which suggests that Wnt pathway may be involved in the development of HPV-related carcinomas. Most of the oncogenic potential of HR-HPVs resides on the PDZ-binding domain of E6 protein. We hypothesized that the PDZ-binding domain of the HPV16-E6 oncoprotein induces the nuclear accumulation of β-catenin due to its capacity to degrade PDZ-containing cellular targets. To test this hypothesis, we evaluated the staining pattern of β-catenin in the skin epidermis of transgenic mice expressing the full-length E6 oncoprotein (K14E6 mice) and measured LacZ gene expression in K14E6 mice that were crossed with a strain expressing LacZ that was knocked into the Axin2 locus (Axin2(+/LacZ) mice). Here, we show that the E6 oncoprotein enhances the nuclear accumulation of β-catenin, the accumulation of cellular β-catenin-responsive genes, and the expression of LacZ. None of these effects were observed when a truncated E6 oncoprotein that lacks the PDZ-binding domain was expressed alone (K14E6ΔPDZ mice) or in combination with Axin2(+/LacZ). Conversely, cotransfection with either E6 or E6ΔPDZ similarly enhanced canonical Wnt signaling in short-term in vitro assays that used a luciferase Wnt/β-catenin/TCF-dependent promoter. We propose that the activation of canonical Wnt signaling could be induced by the HPV16-E6 oncoprotein; however, the participation of the E6 PDZ-binding domain seems to be important in in vivo models only.     

Suppression in vivo of human papillomavirus type 18 E6-E7 gene expression in nontumorigenic HeLa X fibroblast hybrid cells.

The E6 and E7 genes of the cancer-associated human papillomavirus (HPV) types 16 (HPV16) and 18 (HPV18) can induce cell immortalization in vitro in normal human keratinocytes. This, however, is not associated with tumorigenicity in vivo. On the other hand, tumorigenicity of HPV18-positive HeLa cervical carcinoma cells can be suppressed by fusion of HeLa cells with normal human keratinocytes or fibroblasts. We have addressed the question of whether suppression of tumorigenicity in HeLa x fibroblast hybrid cells might be due to a reduced ability of these cells to express the HPV18 E6-E7 genes in vivo. Nontumorigenic hybrid cells and tumorigenic hybrid segregants were transplanted as organotypical cultures or injected subcutaneously into immunocompromised mice and were analyzed for HPV18 E6-E7 gene expression by RNA-RNA in situ hybridization. The tumorigenic hybrid cells showed a continuous and invasive growth that was associated with high levels of HPV18 E6-E7 mRNAs at all time points examined. In contrast, the nontumorigenic hybrid cells stopped cell proliferation approximately 3 days after transplantation. At this time they expressed the E6-E7 genes at low levels, whereas at day 2 high expression levels were observed. However, the mRNA levels of the cytoskeletal genes beta-actin and vimentin remained high for at least 14 days, demonstrating that inhibition of growth and of HPV18 E6-E7 gene expression was not due to cell death. These results suggest that growth inhibition of the nontumorigenic HeLa x fibroblast hybrid cells in vivo might be caused by suppression of HPV18 E6-E7 gene expression and are compatible with the idea of an intracellular surveillance mechanism for HPV gene expression existing in nontumorigenic cells.     

Severe abnormalities in the oral mucosa induced by suprabasal expression of epidermal keratin K10 in transgenic mice

Previous studies have demonstrated that keratin K10 plays an important role in mediating cell signaling processes, since the ectopic expression of this keratin induces cell cycle arrest in proliferating cells in vitro and in vivo. However, apart from its well known function of providing epithelial cells with resilience to mechanical trauma, little is known about its possible roles in nondividing cells. To investigate what these might be, transgenic mice were generated in which the expression of K10 was driven by bovine K6beta gene control elements (bK6(beta)hK10). The transgenic mice displayed severe abnormalities in the tongue and palate but not in other K6-expressing cells such as those of the esophagus, nails, and hair follicles. The lesions in the tongue and palate included the cytolysis of epithelial suprabasal cells associated with an acute inflammatory response and lymphocyte infiltration. The alterations in the oral mucosa caused the death of transgenic pups soon after birth, probably because suckling was impaired. These anomalies, together with others found in the teeth, are reminiscent of the lesions observed in some patients with pachyonychia congenita, an inherited epithelial fragility associated with mutations in keratins K6 and K16. Although no epithelial fragility was observed in the bK6(beta)hK10 oral epithelia of the experimental mice, necrotic processes were seen. Collectively, these data show that the carefully regulated tissue- and differentiation-specific patterns displayed by the keratin genes have dramatic consequences on the biological behavior of epithelial cells and that changes in the specific composition of the keratin intermediate filament cytoskeleton can affect their physiology, in particular those of the oral mucosa.     

Fusion of CTLA-4 with HPV16 E7 and E6 Enhanced the Potency of Therapeutic HPV DNA Vaccine

Preventive anti-HPV vaccines are effective against HPV infection but not against existing HPV-associated diseases, including cervical cancer and other malignant diseases. Therefore, the development of therapeutic vaccines is urgently needed. To improve anti-tumor effects of therapeutic vaccine, we fused cytotoxic T-lymphocyte antigen 4 (CTLA-4) with HPV16 E7 and E6 as a fusion therapeutic DNA vaccine (pCTLA4-E7E6). pCTLA4-E7E6 induced significantly higher anti-E7E6 specific antibodies and relatively stronger specific CTL responses than the nonfusion DNA vaccine pE7E6 in C57BL/6 mice bearing with TC-1 tumors. pCTLA4-E7E6 showed relatively stronger anti-tumor effects than pE7E6 in therapeutic immunization. These results suggest that fusing CTLA-4 with E7E6 is a useful strategy to develop therapeutic HPV DNA vaccines. In addition, fusing the C-terminal of E7 with the N-terminal of E6 impaired the functions of both E7 and E6.     

Beta-catenin accelerates human papilloma virus type-16 mediated cervical carcinogenesis in transgenic mice

Human papilloma virus (HPV) is the principal etiological agent of cervical cancer in women, and its DNA is present in virtually all of these tumors. However, exposure to the high-risk HPV types alone is insufficient for tumor development. Identifying specific collaborating factors that will lead to cervical cancer remains an unanswered question, especially because millions of women are exposed to HPV. Our earlier work using an in vitro model indicated that activation of the canonical Wnt pathway in HPV-positive epithelial cells was sufficient to induce anchorage independent growth. We therefore hypothesized that constitutive activation of this pathway might function as the "second hit." To address this possibility, we developed two double-transgenic (DT) mouse models, K14-E7/ΔN87βcat and K14-HPV16/ΔN87βcat that express either the proteins encoded by the E7 oncogene or the HPV16 early region along with constitutively active β-catenin, which was expressed by linking it to the keratin-14 (K14) promoter. We initiated tumor formation by treating all groups with estrogen for six months. Invasive cervical cancer was observed in 11% of the K14-ΔN87βcat mice, expressing activated β-catenin and in 50% of the animals expressing the HPV16 E7 oncogene. In double-transgenic mice, coexpression of β-catenin and HPV16 E7 induced invasive cervical cancer at about 7 months in 94% of the cases. We did not observe cervical cancer in any group unless the mice were treated with estrogen. In the second model, K14-HPV16 mice suffered cervical dysplasias, but this phenotype was not augmented in HPV16/ΔN87βcat mice. In summary, the phenotypes of the K14-E7/ΔN87βcat mice support the hypothesis that activation of the Wnt/β-catenin pathway in HPV-associated premalignant lesions plays a functional role in accelerating cervical carcinogenesis.     

Tumorigenicity by human papillomavirus type 16 E6 and E7 in transgenic mice correlates with alterations in epithelial cell growth and differentiation.

The human papillomavirus type 16 (HPV-16) E6 and E7 oncogenes are thought to play a role in the development of most human cervical cancers. These E6 and E7 oncoproteins affect cell growth control at least in part through their association with and inactivation of the cellular tumor suppressor gene products, p53 and Rb. To study the biological activities of the HPV-16 E6 and E7 genes in epithelial cells in vivo, transgenic mice were generated in which expression of E6 and E7 was targeted to the ocular lens. Expression of the transgenes correlated with bilateral microphthalmia and cataracts (100% penetrance) resulting from an efficient impairment of lens fiber cell differentiation and coincident induction of cell proliferation. Lens tumors formed in 40% of adult mice from the mouse lineage with the highest level of E6 and E7 expression. Additionally, when lens cells from neonatal transgenic animals were placed in tissue culture, immortalized cell populations grew out and acquired a tumorigenic phenotype with continuous passage. These observations indicate that genetic changes in addition to the transgenes are likely necessary for tumor formation. These transgenic mice and cell lines provide the basis for further studies into the mechanism of action of E6 and E7 in eliciting the observed pathology and into the genetic alterations required for HPV-16-associated tumor progression.     

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.     

The Human Papillomavirus Type 16 E6 Gene Alone Is Sufficient To Induce Carcinomas in Transgenic Animals

High-risk human papillomaviruses (HPVs) are the causative agents of certain human cancers. HPV type 16 (HPV16) is the papillomavirus most frequently associated with cervical cancer in women. The E6 and E7 genes of HPV are expressed in cells derived from these cancers and can transform cells in tissue culture. Animal experiments have demonstrated that E6 and E7 together cause tumors. We showed previously that E6 and E7 together or E7 alone could induce skin tumors in mice when these genes were expressed in the basal epithelia of the skin. In this study, we investigated the role that the E6 gene plays in carcinogenesis. We generated K14E6 transgenic mice, in which the HPV16 E6 gene was directed in its expression by the human keratin 14 promoter (hK14) to the basal layer of the epidermis. We found that E6 induced cellular hyperproliferation and epidermal hyperplasia and caused skin tumors in adult mice. Interestingly, the tumors derived from E6 were mostly malignant, as opposed to the tumors from E7 mice, which were mostly benign. This result leads us to hypothesize that E6 may contribute differently than E7 to HPV-associated carcinogenesis; whereas E7 primarily contributes to the early stages of carcinogenesis that lead to the formation of benign tumors, E6 primarily contributes to the late stages of carcinogenesis that lead to malignancy.     

E6/E7 expression of human papillomavirus type 20 (HPV-20) and HPV-27 influences proliferation and differentiation of the skin in UV-irradiated SKH-hr1 transgenic mice

The functional role of UV irradiation, in combination with the E6 and E7 proteins of the cutaneous human papillomavirus (HPV) types in the malignant conversion of benign papillomatous lesions, has not been elucidated. Transgenic SKH-hr1 hairless mice expressing HPV-20 and HPV-27 E6 and E7 proteins in the suprabasal compartment were generated and exposed to chronic UV irradiation. Histological and immunohistochemical examination of skin samples revealed enhanced proliferation of the epidermal layers and papilloma formation in both transgenic strains in comparison to what was observed with nontransgenic mice. Squamous cell carcinoma developed in the HPV-20 E6/E7 transgenic line as well as in the HPV-27 E6/E7 transgenic line. Several weeks after cessation of UV-B exposure, enhanced proliferation, as measured by BrdU incorporation, was maintained only in HPV-20 transgenic skin. Keratin 6 expression was increased in the transgenic mice throughout all cell layers. Expression of the differentiation markers involucrin and loricrin was reduced and disturbed. p63alpha expression was differentially regulated with high levels of cytoplasmic expression in clusters of cells in the granular layer of the skin in the transgenic lines 20 weeks after cessation of UV-B exposure, in contrast to uninterrupted staining in the nontransgenic lines. p53 was expressed in clusters of cells in nontransgenic and HPV-27 transgenic mice, in contrast to an even distribution in a higher number of cells in HPV-20 transgenic animals.