Tyrosine Kinase Fyn Modulates Bcl-X Alternative Splicing In Pancreatic Cancer

Background:Tumor metastasis is a major cause of mortality in patients with pancreatic cancer. It is now clear that deregulation of cell proliferation and anti-apoptosis can lead to tumor initiation, progression or metastasis. Compelling evidences indicate that the deregulation of several signal molecules is associated with progression of pancreatic cancer. In these signaling molecules, the non-receptor-tyrosine kinase c-Src is over expressed in 70% of human pancreatic cancers and its activity is an important property of tumor progression.Src family kinase (SFK) transmits signals between cell surface proteins and other intracellular proteins. Recent studies suggest that Src activity may also modulate cell apoptosis and influence tumor progression. Fyn, a critical member of SFKs, mediates a variety of cellular processes, including T-cell receptor signaling, regulation of brain function and adhesion-mediated signaling, but little has been reported regarding its role in apoptosis regulation.Alternative splicing has been shown to play a role in apoptosis. A clear example is the Bcl-X transcript, which has two alternatively spliced products including the antiapoptotic Bcl-X(L) and the proapoptotic Bcl-X(s). Cancer cell often changes Bcl-X splicing to escape from apoptosis, resulting in tumor formation, metastasis and poor prognosis. Recent reports have shown that the RNA-binding protein Sam68 binds to Bcl-X Mrna and affects its alternative splicing. The roles of Sam68 in Bcl-X splicing are regulated by phosphorylation. In normal cells, the unphosphorylated Sam68 is predominantly localized in nuclear, participating in Bcl-X pre-mRNA processing. However, in cancer cells, highly phosphorylated Sam68 by SFK distributed in the cytoplasm and led to decrease its role in regulating Bcl-X splicing. Therefore, it is still unknown whether other RNA-binding proteins participate in the regulation of alternative splicing of Bcl-X and resulting in cancer metastasis and poor prognosis. As a group of RNA-binding proteins, heterogeneous nuclear ribonucleoproteins (HnRNPs) regulate splicing and transporting of messenger RNA (mRNA) and participate in growth regulation and carcinogenesis. It has been shown that an increased expression of HnRNP A2/B1 is detected in lung cancer and involved in cancer progression. However, whether hnRNP A2B1 participates in pancreatic cancer metastasis and its related mechanism is unclear yet.Methods:Immunohistochemistry was performed in a total of 28 pancreatic cancer tissues from our institute. The tumor specimens included 11 metastatic pancreatic cancer (liver metastasis) specimens and 17 non-metastatic pancreatic cancers as well as pancreatic tissues adjacent to cancer were obtained from surgery. We examined the expression of Fyn and correlate the result with pathological and clinical parameters.By using MTT, TUNEL, Transwell invasive activity assay and nude mice xenograft model, we examined the effect of Fyn kinase in pancreatic cancer in vitro and in vivo. RT-PCR was used to investigate the role of Fyn kinase activity in apoptosis related gene Bcl-X alternative splicing. 2-D gel electrophoresis was performed to search for the possible downstream target protein which is regulated by Fyn kinase activity. RNA-immunoprecipitation was used to explore the direct connection of Fyn signaling pathway downstream protein and Bcl-X pre-mRNA. Full length of hnRNP A2/B1 and its RNAi plasmids were subcloned into pAdTrack-CMV vector. The resultant plasmid was linearized and subsequently cotransformed into E.coli BJ5183 containing adenovirus backbone plasmid pAdEasy-1. The adenovirus vector was then transferred into 293N cells by liposome to get recombinant adenovirus particles. Finally, the recombined adenovirus were used to infect BxPC3 cell line. Then the alternative splicing of Bcl-X in BxPC3 cells was evaluated by RT-PCR assay.Results:We found that the up-regulation of Fyn expression and Fyn activity were associated with the metastasis of pancreatic cancer. In BxPC3 pancreatic cancer cells, inhibition of Fyn activity by kinase dead Fyn(KD-Fyn) not only decreased the cell proliferation and liver metastasis, but also down-regulated HnRNP A2/B1 expression. Further analysis showed that HnRNP A2/B1 expression was associated with pancreatic cancer progression. In BxPc3 cell, the HnRNP A2/B1 and Sam68 were bound with Bcl-X mRNA and affected its splicing. Suppressed expression of HnRNP A2/B1 by RNAi increased the formation of proapoptotic Bcl-X(s) and promoted apoptosis of BxPc3 cell. In addition, in BxPc3 cell, inhibition of Fyn activity decreased the phosphorylation of Sam68 and decreased its binding to Bcl-X mRNA, thereby increased antiapoptotic Bcl-XL formation. Finally, overexpression of HnRNP A2/B1 could rescue pancreatic cancer cell from apoptosis induced by KD-Fyn.Conclusions:In conclusion, our results provide a possible explanation of Fyn-A2B1/Sam68 signaling pathway in regulating apoptosis of pancreatic cancer cells. In pancreatic cancer, activation of Fyn is via two pathway to regulate apopotosis, one is via upregulation hnRNP A2/B1 expression to decrease formation of proapoptotic Bcl-X(s) isoforms. In another aspect, activation of Fyn also results in tyrosine phosphorylation of Sam68, promotes formation of the antiapoptotic Bcl-X(L) mRNA. Thus, by changing Bcl-X splicing, Fyn-A2B1/Sam68 signaling pathway can modulate survival of pancreatic cancer cells to influence the proliferation and metastasis of pancreatic cancer.