Intrinsic Chemoresistance To Gemecitabine Is Associated With Constitutive And LN-induced Phosphorylation Of FAK In Pancreatic Cancer Cell Lines

Treatment of pancreatic cancer remains to be an unsolved problem. One of the major reasons for poor prognosis of pancreatic cancer is its highly resistance to currently available agents including gemcitabine which is considered as the most effective first-line drug for the treatment of advanced pancreatic cancer. Gemcitabine alone and gemcitabine-based combination chemotherapy are likely to be accompanied by much higher toxicity and not likely to achieve great success according to clinical trials.Target therapy is emerging as a new era for tumor therapy. As for pancreatic cancer, a combination of conventional chemotherapies with target therapy targeted at the molecular changes in pancreatic cancer seems to be the most promising way to go. Links between tyrosine kinases and pancreatic cancer chemoresistance have attracted more and more attention in recent years, and target therapy agaist tyrosine kinases increases the effectiveness of radiation and cytotoxic drugs in the treatment of pancreatic cancer.In recent years, non-receptor tyrosine kinase focal adhesion kinase (FAK), a central molecule in extracellular matrix (ECM)/integrin-mediated signaling, has been accepted as a determinant of chemoresistance and ECM proteins including laminin (LN) have also been thought to be associated with the intrinsic chemoresistance to different drugs in a viariety of cancers, which is named as cell adhesion-mediated drug resistance (CAM-DR). Besides the regulation of FAK expression, another well-understood mode of FAK regulation in cancer cells is phosphorylation.To determine the roles of FAK and LN in the intrinsic chemoresistance of pancreatic cancer and whether these effects are mediated by constitutive and induced FAK phosphorylation and subsequent downstream signal pathway, the following aspects were studied:1. The relationship between constitutive FAK expression or phosphorylation level and the intrinsic resistance to gemcitabine (Gem) or 5-fluorouracil (5-FU) in four pancreatic cancer cell lines(BxPC-3, AsPC-1, MiaPaCa-2 and Panc-1).2. Construction of recombinant plasmids, including FAK RNAi plasmid (FAK RNAi1 and FAK RNAi2), and FRNK expression plasmid (pcDNA3.1-FRNK) Selection of stable clones.3. Effect of FAK RNAi and FRNK overexpression on the expression and phosphorylation of FAK and its downstream signaling kinases (Akt and ERK1/2) in Panc-1 cells.4. Effects of FAK RNAi and FRNK overexpression on Gem-induced cytotoxicity and apoptosis and apoptosis associated proteins in Panc-1 cells5. Effect of LN on FAK phosphorylation and its downstream signaling kinases (Akt and ERK1/2) activity in AsPC-1 cells6. Effect of FAK RNAi and FRNK overexpression on the expression and phosphorylation of FAK and its downstream signaling kinase Akt in AsPC-1 cells.7. Effect of LN on Gem-induced cytotoxicity and apoptosis and apoptosis associated proteins expression in AsPC-1 cells.8. Effects of FAK RNAi and FRNK overexpression on LN mediated apoptosis resistance to gemcitabine in AsPC-1 cells.The results showed that:1. In pancreatic cancer cell lines (BxPC-3, AsPC-1, MiaPaCa-2 and Panc-1), the level of constitutive phosphorylation of FAK at Tyr-397 is correlated with the extent of intrinsic resistance to Gem.2. In Panc-1 cells, FAK RNAi inhibits FAK both at mRNA and protein levels, and suppresses constitutive FAK and Akt phosphorylation, while FRNK overexpression suppresses constitutive FAK and Akt phosphorylation without total FAK expression alteration. 3. In Panc-1 cells, specific inhibition of constitutive FAK phosphorylation by either FAK RNAi or FRNK overexpression renders Panc-1 cells more sensitive to Gem-induced cytotoxicity and apoptosis and causes a decrease in the expression of survivin and the phosphorylation of Bad at serl36.4. In AsPC-1 cells. LN reduces Gem-induced cytotoxicity and apoptosis, and increases the expression of survivin and the phosphorylation of Bad at serl36.5. In AsPC-1 cells. LN induces FAK and Akt activation in a time-dependent manner.6. In AsPC-1 cells, specific inhibition of constitutive FAK phosphorylation by either FAK RNAi or FRNK overexpression leads to the inhibition of FAK and Akt activation stimulated by LN.7. In AsPC-1 cells, when plated on plastic, FAK RNAi or FRNK overexpression has few effects on Gem-induced cytotoxicity and apoptosis; However, FAK RNAi or FRNK overexpression enhances Gem-induced cytotoxicity and apoptosis in AsPC-1 cells plated on LN.Conclusion:In conclusion, our research demonstrates that both constitutive and LN-induced phosphorylation of FAK contributes to increased intrinsic chemoresistance to Gem in pancreatic cancer cell lines. FAK RNAi and FRNK overexpression both can render pancreatic cancer cells more sensitive to Gem-induced cytotoxicity and apoptosis. These effects are partly due to the regulation of Akt and Bad phosphorylation and survivm expression.