P229

Introduction: Mesothelin (MSLN), a cell-surface glycoprotein, is found to be overexpressed in many pancreatic cancer cell lines and pancreatic cancer clinical tissue specimens. MSLN has been suggested to play a role in tumor progress but the mechanism of this enhanced tumor progression caused by overexpression of MSLN is unknown. Here, we examined the effect of MSLN on pancreatic cancer cell proliferation, cell cycle progression and signal transduction pathway involvement in three pancreatic cancer cell lines. Methods: Stable overexpression of MSLN in the cell lines MIA PaCa-2 (MIA-MSLN) and Panc-1 (Panc-MSLN) and stable siRNA MSLN silencing in the cell line BxPC-3 (BxPC-siMSLN) was confirmed at mRNA level by real-time RT-PCR and at protein level by western blot. Cell viability assay (MTT), thymidine incorporation and cell cycle analysis were used to assess the cell growth parameters. Activation of transcription factor STAT3 was examined by western blot. Results: MSLN overexpression led to significantly increased cell proliferation (p<0.001) in both MIA-MSLN and Panc-MSLN as detected by MTT and thymidine incorporation assays. After 6 days of proliferation, the cell numbers of MIA PaCa-2 increased 23-fold whereas MIA-MSLN increased by 53 folds. Parental BxPC3, normally expressing high MSLN proliferated 28-fold whereas BxPC-siMSLN cell line proliferated to only 14 fold. After initial G0 synchronization by serum starvation and subsequent release by medium containing 2% serum for 4 h, there is about 50% of MIA-MSLN cells enter the S phase whereas only 14% of MIA PaCa-2 cells enter the S phase. Similar cell cycle analysis trend was seen in the Panc-MSLN cells. In contrast, while there were about 43% of BxPC-3 cells entering the S phase after initial serum starvation and release by 2% serum-medium for 24 h, only 21% of BxPC-siMSLN entered the S phase. To further examine which signaling pathway maybe responsible for the MSLN induced cell proliferation, we found that signal transducer and activator of transcription protein 3 (STAT3) was constitutively activated in MIA-MSLN cell line but not in the parental MIA PaCa-2 cell line. Exposing the MIA-MSLN cells to a JAK-specific inhibitor, tyrphostin AG490, markedly inhibited STAT3 activation in MIA-MSLN within 12 h of treatment followed by a marked decrease in proliferation of MIA-MSLN cells (p<0.001) but not the parental cell MIA PaCa-2. Conclusions: Overexpression of MSLN in pancreatic cancer cells leads to increased cellular proliferation by rapid DNA synthesis and faster progression through cell cycles. This effect is mediated by the constitutive activation of the transcription factor STAT3 in these cells. Our results provide evidence of MSLN contribution to tumorigenesis and aberrant STAT3 activation in pancreatic cancer cells.