A Novel Nuclear SRC and P300 Signaling Axis in Pancreatic Cancer

Abstract

Src is a non-receptor tyrosine kinase that has the distinction of being the first discovered oncogene. Its function at the cell membrane has been demonstrated in a wide array of cellular signaling pathways. Additionally, its role in promoting the oncogenic phenotype in a number of tumor types has been extensively researched, and its over activation is believed mainly to influence tumor migration and metastasis, as well as to a lesser extent survival and proliferation. Our lab has previously described a nuclear function of Src in which it associates with gene promoters and regulates their transcription. Indeed, nuclear localization of Src has long been observed, but its functional role has remained largely a mystery. In this thesis the role and significance of nuclear Src in pancreatic cancer was characterized. In doing so, a novel mechanism was delineated by which Src in the nucleus modulates gene expression. Src functional nuclear activity was demonstrated by multiple independent approaches. Src association with p300 was also identified by mass spectrometry and validated by inverse co-immunoprecipitation. Putative Src target genes in the Panc-1 cell line were then defined by ChIP-array including HMGA2 and SMYD3. Small molecule inhibitors of Src and p300 resulted in a decrease in the transcription and protein expression ofHMGA2 and SMYD3, indicating their regulation by Src and p300 enzymatic activity. Src and p300 inhibition both were found to regulate migration and invasion in Panc-1 cells, implicating their signaling in a major aspect of pancreatic cancer. Interestingly, these results were able to be replicated in a well-known series of mouse embryonic fibroblast (MEF) cell lines in which Src, and Src family kinase members Yes, and Fyn (SYF-/-) are knocked out or restored by over-expression of exogenous c-Src. The Src-dependent gene regulation and reliance on p300 was only observed in the Src-active (overexpressing) cell line. Together, these results indicate a novel signaling modality of Src in pancreatic cancer or in cells over-expressing Src that reveal an epigenetic regulatory function in coordination with p300.