A humanized mouse model of HPV-associated pathology driven by E7 expression.

Human papillomavirus (HPV) is the causative agent of human cervical cancer and has been associated with oropharyngeal squamous cell carcinoma development. Although prophylactic vaccines have been developed, there is a need to develop new targeted therapies for individuals affected with malignant infected lesions in these locations, which must be tested in appropriate models. Cutaneous beta HPV types appear to be involved in skin carcinogenesis. Virus oncogenicity is partly achieved by inactivation of retinoblastoma protein family members by the viral E7 gene. Here we show that the E7 protein of cutaneous beta HPV5 binds pRb and promotes its degradation. In addition, we described an in vivo model of HPV-associated disease in which artificial human skin prepared using primary keratinocytes engineered to express the E7 protein is engrafted onto nude mice. Expression of E7 in the transplants was stably maintained for up to 6 months, inducing the appearance of lesions that, in the case of HPV16 E7, histologically resembled human anogenital lesions caused by oncogenic HPVs. Moreover, it was confirmed through biomarker expression analysis via immunodetection and/or quantitative PCR from mRNA and miRNA that the 16E7-modified engrafted skin shares molecular features with human HPV-associated pretumoral and tumoral lesions. Finally, our findings indicate a decrease of the in vitro capacity of HPV5 E7 to reduce pRb levels in vivo, possibly explaining the phenotypical differences when compared with 16E7-grafts. Our model seems to be a valuable platform for basic research into HPV oncogenesis and preclinical testing of HPV-associated antitumor therapies.

Gene expression profiling of mouse p53-deficient epidermal carcinoma defines molecular determinants of human cancer malignancy.

BACKGROUND: The epidermal specific ablation of Trp53 gene leads to the spontaneous development of aggressive tumors in mice through a process that is accelerated by the simultaneous ablation of Rb gene. Since alterations of p53-dependent pathway are common hallmarks of aggressive, poor prognostic human cancers, these mouse models can recapitulate the molecular features of some of these human malignancies. RESULTS: To evaluate this possibility, gene expression microarray analysis was performed in mouse samples. The mouse tumors display increased expression of cell cycle and chromosomal instability associated genes. Remarkably, they are also enriched in human embryonic stem cell gene signatures, a characteristic feature of human aggressive tumors. Using cross-species comparison and meta-analytical approaches, we also observed that spontaneous mouse tumors display robust similarities with gene expression profiles of human tumors bearing mutated TP53, or displaying poor prognostic outcome, from multiple body tissues. We have obtained a 20-gene signature whose genes are overexpressed in mouse tumors and can identify human tumors with poor outcome from breast cancer, astrocytoma and multiple myeloma. This signature was consistently overexpressed in additional mouse tumors using microarray analysis. Two of the genes of this signature, AURKA and UBE2C, were validated in human breast and cervical cancer as potential biomarkers of malignancy. CONCLUSIONS: Our analyses demonstrate that these mouse models are promising preclinical tools aimed to search for malignancy biomarkers and to test targeted therapies of prospective use in human aggressive tumors and/or with p53 mutation or inactivation.

A functional role of RB-dependent pathway in the control of quiescence in adult epidermal stem cells revealed by genomic profiling.

Continuous cell renewal in mouse epidermis is at the expense of a pool of pluripotent cells that lie in a well defined niche in the hair follicle known as the bulge. To identify mechanisms controlling hair follicle stem cell homeostasis, we developed a strategy to isolate adult bulge stem cells in mice and to define their transcriptional profile. We observed that a large number of transcripts are underexpressed in hair follicle stem cells when compared to non-stem cells. Importantly, the majority of these downregulated genes are involved in cell cycle. Using bioinformatics tools, we identified the E2F transcription factor family as a potential element involved in the regulation of these transcripts. To determine their functional role, we used engineered mice lacking Rb gene in epidermis, which showed increased expression of most E2F family members and increased E2F transcriptional activity. Experiments designed to analyze epidermal stem cell functionality (i.e.: hair regrowth and wound healing) imply a role of the Rb-E2F axis in the control of stem cell quiescence in epidermis.

Molecular Signature of HPV-Induced Carcinogenesis: pRb, p53 and Gene Expression Profiling.

The infection by mucosal human papillomavirus (HPV) is causally associated with tumor development in cervix and oropharynx. The mechanisms responsible for this oncogenic potential are mainly due to the product activities of two early viral oncogenes: E6 and E7. Although a large number of cellular targets have been described for both oncoproteins, the interaction with tumor suppressors p53 and retinoblastoma protein (pRb) emerged as the key functional activities. E6 degrades tumor suppressor p53, thus inhibiting p53-dependent functions, whereas E7 binds and degrades pRb, allowing the transcription of E2F-dependent genes. Since these two tumor suppressors exert their actions through transcriptional modulation, functional genomics has provided a large body of data that reflects the altered gene expression of HPVinfected cells or tissues. Here we will review the similarities and differences of these findings, and we also compare them with those obtained with transgenic mouse models bearing the deletion of some of the viral oncogene targets. The comparative analysis supports molecular evidences about the role of oncogenes E6 and E7 in the interference with the mentioned cellular functions, and also suggests that the mentioned transgenic mice can be used as models for HPV-associated diseases such as human cervical, oropharynx, and skin carcinomas.

Spontaneous tumor formation in Trp53-deficient epidermis mediated by chromosomal instability and inflammation.

BACKGROUND: The specific ablation of Trp53 gene in mouse epidermis leads to the spontaneous development of aggressive squamous cell carcinoma, a process that is accelerated by the subsequent loss of Rb gene. MATERIALS AND METHODS: The possible mechanisms leading to spontaneous tumor formation in epidermis in the absence of Trp53 were studied focusing on hair cycle defects, inflammation and possible chromosomal instability (CIN). RESULTS: Loss of p53 induces tumorigenesis primarily by mediating early CIN and, to a minor extent, nuclear factor kappaB activation. Notably, CIN occurs not only in p53-deficient skin, but also in epidermis lacking both Rb and Tp53 tumor suppressors, indicating a predominant role of this process in spontaneous tumorigenesis. CONCLUSION: These data identify CIN as a major mechanism in tumorigenesis originated by Trp53 loss in stratified epithelia and imply that therapies aimed to counterbalance CIN might be of relevance for the treatment of human cancer bearing impaired p53 functions.