Osteopontin Splice Variants In Pancreatic Cancer Cell Lines And Fine Needle Aspirates Of Pancreatic Tumors

Abstract

Introduction: Osteopontin (OPN) is a secreted phosphoprotein that confers on cancer cells a migratory phenotype and activates signaling pathways that induce cell survival, proliferation, invasion, and metastasis. High levels of tissue and serum OPN have been reported to correlate with poor survival in pancreatic ductal adenocarcinoma (PDA) patients. Three isoforms of OPN (OPNa, b and c) are known to exist as a result of alternative splicing of the OPN gene. In this study, we examined the expression of OPN isoforms in PDA cell lines and in fine needle aspirates (FNAs) from pancreatic tumors. We also evaluated their individual effect on PDA cell metastatic potential. Methods: FNAs from 24 patients were collected and analyzed for the expression of OPN isoforms by RT-PCR. Data were evaluated and correlated with different clinicopathological parameters. Basal levels of OPN isoforms in seven PDA cell lines were examined by RT-PCR. Transient transfection studies using cDNA plasmids specific to each isoform analyzed the functional impact of each isoform on PDA cell behavior using wound-induced migration, MTT, and transwell infiltration assays. The effect of knocking down OPN by small hairpin (sh) RNA on cell proliferation and migration potential was also evaluated. Results: FNAs were diagnosed as PDA (n= 21) or non-malignant (n=3). OPNa was highly expressed in 20 of 21 PDA FNAs (95%), compared to 1 of 3 (33%) in non-malignant FNA samples. High levels of OPNb were present in 14 of 21 (67%) of PDA samples, while OPNc was present in 5 of 21 (24%). No OPN b or c was detected in the non-malignant FNAs. In PDA cell lines, all three isoforms were present in BxPC-3, Hs766T, and PK9 cells, which express high levels of total OPN. OPNa and OPNb were found in Panc-1, HPAF-1 and AsPC-1 cells, which express moderate levels of total OPN, and only OPNa was found in MiaPaca cells, which express low levels of total OPN. Transwell infiltration and wound-induced migration assays revealed that OPNb induced MiaPaca cell migration, while OPNa and OPNc had no significant effects. By contrast, OPNc significantly (p<0.05) suppressed the migratory activity of AsPC-1 cells although no significant changes were induced by OPNa or b. OPNc overexpression also induced an early, but significant (P<0.05) reduction of cell proliferation compared to controls. Knocking down total OPN by shRNA resulted in significant (p<0.05) reduction in cell proliferation and migration. Conclusion: The findings of the present study show for the first time the consistent presence of OPNa in PDA FNAs and the consistent absence of OPNb and c in non-malignant FNAs, suggesting a discriminatory role for the individual isoforms. Our functional in vitro data demonstrate that OPN splice variants differentially modulate the migratory property of PDA cells and suggest that this may be one of the mechanisms underlying the pathologic heterogeneity of PDA progression.