T. Gan1, P. D. Stevens1, Y. Wen1, X. Xing1, S. Golshani1, B. M. Evers1, T. Gao1 1University Of Kentucky,Markey Cancer Center,Lexington, KY, USA
Introduction: Wnt signaling is a highly regulated and evolutionarily conserved pathway that plays an important role in normal intestinal stem cell renewal and differentiation. Hyperactivation of Wnt signaling is associated with 90% of colorectal cancers (CRCs). Many signaling molecules in the Wnt pathway are known to be regulated by protein kinase-mediated phosphorylation; however, it remains unclear whether the Wnt pathway in CRCs is regulated by protein phosphatases. In this study, we investigated the role of a transmembrane receptor tyrosine phosphatase, PTPRF, in promoting the activation of Wnt signaling in CRC.
Methods: The expression of endogenous PTPRF was silenced using lentivirus-mediated RNAi in CRC cell lines: SW480, HCT116, and Caco2. Stable PTPRF knockdown and control cell lines were generated by puromycin selection. Two different shRNA targeting sequences for PTPRF were used to control for specificity. The knockdown efficiency of each shRNA targeting sequence was confirmed using western blot and real-time PCR (RT-PCR) analyses. The expression of Wnt target genes was determined using Quantitative RT-PCR analysis. Growth assays over 72 hours was performed assessing cell proliferation in control and PTPRF knockdown lines. Furthermore, we developed an orthotopic murine model of CRC by endoscopic colonic submucosal injection of 1 x 105 live human cancer cells. Endoscopic evaluation of tumor size was performed weekly.
Results: Knockdown of PTPRF in CRC cells significantly decreased Wnt target gene expression (eg, LGR5, Axin2, TCF7, Cyclin D1 and CD44) as noted by RT-PCR, suggesting a positive role of PTPRF in regulating the canonical Wnt pathway. Moreover, growth assays consistently demonstrated a marked reduction in cell proliferation in PTPRF knockdown cells. The levels of phosphorylated LRP6, a co-receptor for Wnt, and activated β-catenin proteins were downregulated in PTPRF knockdown cells as noted by western blot, demonstrating a potential interaction of PTPRF with LRP6. Since hyperactivation of Wnt signaling is associated with cancer stem cells, studies are underway to evaluate if PTPRF inhibition reduces the “stemness” of CRC cells. In addition, control and PTPRF knockdown CRC cells were endoscopically injected into NSG mice, this novel model allowed us to evaluate the effect of silencing PTPRF on tumor growth in vivo.
Conclusion: Our studies are the first to demonstrate that PTPRF functions as an oncogenic factor in CRC by promoting the activation of Wnt signaling. Additional studies on the role of PTPRF in cancer stem cell regulation will provide novel insights into the regulation of Wnt signaling in CRC. Since Wnt signaling is a major driver of CRC development and progression, inhibition of PTPRF may represent an exciting new therapeutic option for future clinical applications.