Proteomic Signature of PDAC Cells with Mutant p53

The p53 tumor suppressor plays a crucial role in the prevention of oncogenic transformation through the removal or enduring growth arrest of potentially neoplastic cells. Upon cellular insults, activated p53 serves as a sequence-specific transcription factor to control the expression of specific genes, thereby stimulating various biological processes. In contrast to the tumor suppressive roles of wild-type p53, mutant p53 proteins have been shown to support cancer progression by enhancing the ability of cancer cells to invade and metastasize and to confer chemo-resistance. These remarks demonstrate that mutant p53 possesses gain-of-function (GOF) properties that promote oncogenesis. Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive disease with a five-year survival of less than 5%. The typical features of PDAC, as high genomic instability, chemo-resistance and invasiveness, constitute also the main peculiarities that cancer cells acquire when the tumor suppressor TP53 gene undergoes GOF mutations, which have been recognized in about 50–75% of PDAC patients. A better understanding of the functional communication between subpopulations of cells within a tumor is essential for the development of effective cancer treatment strategies. Tumor metastasis and invasion are strongly dependent on the composition of the extracellular medium, which is itself affected by the release of proteins by the tumor cells. Mutant p53 proteins are able to influence the secretion of enzymes involved in the modulation of extracellular matrix components, but also to alter the secretion of inflammatory cytokines and to increase the extracellular acidification and to regulate the crosstalk between cancer and stromal cells. The objective of this research was to recognize a specific signature of biomarkers secreted by PDAC cells carrying GOF mutant p53. To state the direct involvement of mutant p53, transfection of siRNAp53 to specifically knock-down (KD) the endogenous mutant p53 was performed in both wt and mutant p53 PDAC cell lines. To confirm the results the overexpression of mutant p53 was also performed. Using the shotgun proteomics approach and the sequential window acquisition of all theoretical fragment-ion spectra (SWATH) we were able to quantify hundreds of secreted proteins and to identify potential biomarkers of mutant p53. Furthermore the specific mRNA expression of selected biomarkers was investigated by qRT-PCR assay. We discovered a number of common proteins having opposite regulation after overexpression and knock-down of mutant p53. These proteins might constitute a sort of secreted signature driven by the hot-spot mutant p53 in PDAC and will be validated in serum samples of PDAC patients having WT or mutant TP53 gene. These data might also suggest the identification of targeted therapies specifically addressed to inhibit growth of PDACs carrying oncogenic mutant p53, which are strongly resistant to traditional chemotherapies.