Mechanistic Investigation On Rab34 Regulating Migration And Invasion Of Breast Cancer Cells

Rab proteins are important regulators for mediating every aspect of vesicle trafficking through combining with GTP or GDP to carry out various physiological functions.The dysfunction of Rab proteins is related to many diseases since the tight association between Rab proteins and vesicle trafficking which has important role in normal physiological activities in cells.Previously,our lab found that Rab34 has high expression level in tumor samples of distal metastasis breast cancer based on immunohistochemistry experiments.Furthermore,screening the expression profiles of 58 Rab proteins in different breast cancer cell lines showed that Rab34 was high expressed in high invasive breast cancer cell lines specifically.Therefore,we further studied the effect of Rab34 on cell migration/invasion of breast cancer cells and related mechanisms by means of cell biology so as to explore the possibility of Rab34 as drug target or diagnosis marker for breast cancer.The experimental results show that knocking down Rab34 expression by shRNA would inhibit high invasive breast cancer cells migration and invasion significantly.Moreover,knocked down Rab34 expression in MDA-MB-231 could change cell morphology(shorter,rounded and degenerated lamellipodia)which is unfavorable for cell migration.Paradoxically,over-expressing Rab34-WT in low invasive breast cancer cells MCF7 could not enhance cell migration.The results above suggest that Rab34 may mediate the process of cell migration/invasion upon Rab34 is modified or interaction with other specific effector in high invasive breast cancer cells.Cell migration relys on cell pseudopodia which offers force and direction together with focal adhesion which interacts with microfilaments to drive the whole cell body forward.Cancer cell moving is faster because they have more obvious pseudopodia and stronger adhesion ability to adhere at extracellular matrix than normal cells.We focus on studying the underlying mechanisms involved in Rab34 regulating cell migration through mediating pseudopodia activity and focal adhesion formation.Mechanistic investigation shows that Rab34 is phosphorylated by Src at 247 Tyrosine upon EGF stimulation or the process of cell adhesion.On the one hand,phosphorylated Rab34 enhances the activity of membrane ruffling to promote lamellipodia extending.On the other hand,phosphorylated Rab34 regulates internalization of integrin β3 through macropinocytosis which contributes to Rab4-dependent recycling back to leading edge for forming new adhesions.We found that activity of Src is higher in high invasive breast cancer cells than in low invasive breast cancer cells.The result explains why transfection of Rab34-WT in MCF7 has no effect on cell migration while over-expressing Rab34-Y247D mimicking phosphorylated status could enhance MCF7 cell migration.So Rab34 has positive effects on cell migration through promoting lamellipodia protrusion and adhesion formation mentioned above.Our work demonstrates that the potential relationship between Rab34 and breast cancer metastasis,which lays a good foundation for studying breast cancer further and provides a theroretical basis for the follow-up study of Rab34 as biomarker or a new interventional target to therapy and diagonose breast cancer.