An investigation into the role of Rab-Coupling Protein and EphA2 during cancer cell migration

[摘要] The gene encoding Rab-Coupling Protein (RCP), a Rab11 GTPase effector, is found on a chromosomal locus that is frequently amplified in cancer. We have previously shown that RCP drives alpha5beta1-integrin and EGFR recycling to the plasma membrane, thus contributing to the invasive migration of tumour cells. Using MALDI-TOF mass spectrometry, I have identified EphA2 and two Rab GTPases, Rab6 and Rab14, as novel RCP-associated proteins. Immunoprecipitation-based studies confirm these associations using several different cancer cell lines. EphA2 is a receptor tyrosine kinase (RTK) that is required for contact inhibition of locomotion (CIL), and this process is thought to contribute to cancer cell invasion. To determine whether the EphA2-RCP association has functional significance, I tested whether RCP, Rab GTPases and other Rab11 effector proteins are required for CIL. siRNA knockdown of either RCP or Rab14 prevented efficient CIL, but depletion of Rab11 or Fip2 expression was ineffective in this regard. Likewise, HGF-induced scattering of cell colonies was opposed by suppression of EphA2, RCP or Rab14 expression. Since RCP and Rab14 are involved in RTK trafficking, EphA2 internalisation kinetics were investigated. HGF increased EphA2 trafficking in an RCP- and Rab14-dependent fashion. Live cell imaging demonstrated that EphA2 is delivered to an RCP and Rab14 positive compartment upon HGF treatment. Furthermore, HGF drove phosphorylation of RCP on Serine435, which enhanced the association between EphA2, RCP and Rab14. Indeed, mutating the phosphor-acceptor site on RCP (RCPS435A) reduced its association with EphA2 and Rab14, and blocked HGF-driven cell scattering.EphA2 is frequently overexpressed in human pancreatic cancer and this is associated with poor patient prognosis. The role of EphA2 and RCP in metastasis was investigated in an autochthonous model of pancreatic ductal adenocarcinoma (PDAC) by crossing EphA2-/- and RCPfl/fl mice into the KPC (Pdx1-Cre, KrasG12D/+, p53R172H/+) PDAC model. Indeed, ablation of EphA2 or RCP expression in the mouse pancreas reduced the formation of liver metastases. Furthermore, PDAC lines from KPC EphA2-/- and KPC RCPfl/fl mice had a less scattered phenotype and were less invasive in vitro thus corroborating the observations indicating that EphA2 and RCP have an important role in cell-cell repulsion.

[发布机构] University:University of Glasgow;Department:Institute of Cancer Sciences

[关键词] EphA2, RCP, Rab14, contact inhibition of locomotion, cell-cell repulsion