Abstract
In vitro, cellular immortalization and transformation define a model for multistep carcinogenesis and current ongoing challenges include the identification of specific molecular events associated with steps along this oncogenic pathway. Here, using NIH3T3 cells, we identified transcriptionally related events associated with the expression of Polyomavirus Large-T antigen (PyLT), a potent viral oncogene. We propose that a subset of these alterations in gene expression may be related to the early events that contribute to carcinogenesis. The proposed tumor suppressor Necdin, known to be regulated by p53, was within a group of genes that was consistently upregulated in the presence of PyLT. While Necdin is induced following p53 activation with different genotoxic stresses, Necdin induction by PyLT did not involve p53 activation or the Rb-binding site of PyLT. Necdin depletion by shRNA conferred a proliferative advantage to NIH3T3 and PyLT-expressing NIH3T3 (NIHLT) cells. In contrast, our results demonstrate that although overexpression of Necdin induced a growth arrest in NIH3T3 and NIHLT cells, a growing population rapidly emerged from these arrested cells. This population no longer showed significant proliferation defects despite high Necdin expression. Moreover, we established that Necdin is a negative regulator of p53-mediated growth arrest induced by nutlin-3, suggesting that Necdin upregulation could contribute to the bypass of a p53-response in p53 wild type tumors. To support this, we characterized Necdin expression in low malignant potential ovarian cancer (LMP) where p53 mutations rarely occur. Elevated levels of Necdin expression were observed in LMP when compared to aggressive serous ovarian cancers. We propose that in some contexts, the constitutive expression of Necdin could contribute to cancer promotion by delaying appropriate p53 responses and potentially promote genomic instability.
Highlights
Carcinogenesis is a multistep process defined by uncontrolled cell growth and neoplastic progression leading to invasive tumors and metastasis
Selected clones were used for microarray analysis comparing Polyomavirus Large-T antigen (PyLT)-expressing clones to a second group composed of parental NIH3T3 cells as well as clones that did not express a detectable amount of PyLT
It should be noted that the mere overexpression of Necdin did not confer to NIH cells the equivalent response to nutlin-3 seen in the NIH3T3 cells expressing PyLT (NIHLT) cells (Figure 5C and 5D). These results suggest that the acquired resistance to growth arrest in PyLT-expressing NIH3T3 cells was in part mediated by Necdin expression and that other factors were presumably involved
Summary
Carcinogenesis is a multistep process defined by uncontrolled cell growth and neoplastic progression leading to invasive tumors and metastasis. Cancer progression models dictate that normal cells undergo a variety of genetic/epigenetic alterations which can be summarized in vitro by two major phenotypic changes: immortalization and transformation. Normal cells need to overcome cell cycle checkpoints and their limited division potential to achieve immortalization. Interlaced with this process, additional events contribute to cellular transformation and move cells toward the complete neoplastic phenotype [1]. Human lung and colon cancers, genetically altered mice, mouse and human cell culture models, have all been extensively used to support the multistep progression model [2,3,4]. Despite the importance of these domains, the characterization of other viral oncogenic domains involved in transformation remains incomplete and additional activities could contribute to the carcinogenesis process
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