Étude de Necdin par un modèle de carcinogénèse lié à l’antigène grand T du Virus du polyome
Viruses have been used extensively in cancer research and have contributed greatly to the advancement of knowledge as well as to the establishment of important concepts still valid in the field today.  Currently, one of the challenges in cancer research is to better define the individual steps that contribute to the transition of a normal cell to a transformed cell.  To address this important issue, we characterized a model system based on the expression of polyomavirus large-T antigen (PyLT), derived from a virus capable of inducing tumors in rodents.  Importantly, PyLT is able to induce tumors in transgenic mice, although only after a latent period, suggesting that additional transforming events are necessary.  Hence, the PyLT viral oncogene possesses several interesting properties, which suggest that, beside its role in immortalization, PyLT can contribute to the early events of carcinogenesis.  Here, we used a cell culture model to dissect the early changes associated with the presence of PyLT.   The establishment of a gene expression profile associated with PyLT expression in a mouse cell line model allowed us to select a number of genes whose levels were modulated by the presence of this viral oncogene.  Among candidate genes, we chose to further study Necdin in more details because even if only a limited number of reports existed for this protein, there was evidence suggesting that Necdin displays either tumor suppressor or oncogenic functions within different contexts.  We demonstrated that, despite the proposed growth suppressor function of Necdin, its expression was not incompatible with the proliferation in the mouse NIH 3T3 cell line and in human IMR90 primary cells.  Nonetheless, the inhibition of Necdin expression by shRNA confered a proliferative advantage.  We confirmed that Necdin was a p53 target gene inducible by different genotoxic stresses, although its expression was also regulated in a p53-independent manner.  Moreover, Necdin acted negatively on cell cycle arrest in response to p53 activation.  These results suggest that Necdin is involved in a negative feedback loop of the p53 pathway and that abnormal elevation of Necdin expression could contribute to the disruption of the p53 tumor suppressor pathway. p53 activation allows transitory cell cycle arrest under stress conditions and it is also involved in the establishment of a permanent growth arrest called senescence.  Senescence represents a protective mechanism preventing the accumulation of mutations that could contribute to cancer initiation.  As our research supports a role for Necdin in the regulation of p53 activity, we transposed the knowledge acquired from our mouse model to a human model more suitable to study senescence.  The characterization of Necdin expression in human primary fibroblasts at different passages revealed that Necdin was expressed in actively proliferating young cells and its expression decreased gradually during the establishment of replicative senescence.  The same phenomenon was observed during premature senescence induced by both oncogene expression and ionizing radiation exposure.  Moreover, in normal growth conditions, Necdin modulation by gain- and loss-of-function assays did not affect the life span of primary cells.  However, in a genotoxic stress conditions caused by irradiation, Necdin overexpressing cells presented an increase radioresistance comparable to when p53 was directly inactivated.  These results in human cells supports the effect observed in mouse cells relative to the impact of Necdin levels on a p53 response under stress conditions.  We initiated preliminary experiments to address whether our results in cell culture could be translated to human cancer models.  We characterized Necdin expression in two types of human cancers.  First, in ovarian cancer, we observed elevated levels of Necdin expression in low malignant potential serous ovarian cancers (LMP) when compared to aggressive serous ovarian cancers.  Our results suggest that Necdin expression was limited to LMPs, which usually present a wild type p53 gene.  Necdin expression was also found in two ovarian cancer cell lines, which were both non-tumorigenic in a mouse xenograft assay, and interestingly one of the cell line had a functional p53.  Moreover, the characterization of Necdin expression in four prostate cancer cell lines also suggested a relationship between its expression and the presence of functional p53.  In prostate cancer, as in ovarian cancer, Necdin expression seems to be detected in cell lines representing less aggressive forms of the disease.   The use of the PyLT viral oncoprotein allowed us to reveal interesting properties for Necdin.  We propose that, in some contexts, the constitutive expression of Necdin could contribute to cancer promotion by delaying appropriate p53 responses and possibly promoting genomic instability.  The potential oncogenic function of Necdin, and its relationship with p53 as revealed by the research described in this dissertation, requires more investigation.  Preliminary results suggest that human ovarian and prostate cancers could be good models to address the role of Necdin in carcinogenesis.
Necdin p53 Polyomavirus murin Large T antigen Murin polyomavirus Carcinogenesis Senescence Ovarian cancer Prostate cancer