The Protease Activated Receptor2 Promotes Rab5a Mediated Generation of Pro-metastatic Microvesicles

Kaushik Das,Ramesh Prasad,Sreetama Roy,Prosenjit Sen,Ashis Mukherjee

doi:10.1038/s41598-018-25725-w

Abstract

Metastasis, the hallmark of cancer propagation is attributed by the modification of phenotypic/functional behavior of cells to break attachment and migrate to distant body parts. Cancer cell-secreted microvesicles (MVs) contribute immensely in disease propagation. These nano-vesicles, generated from plasma membrane outward budding are taken up by nearby healthy cells thereby inducing phenotypic alterations in those recipient cells. Protease activated receptor 2 (PAR2), activated by trypsin, also contributes to cancer progression by increasing metastasis, angiogenesis etc. Here, we report that PAR2 activation promotes pro-metastatic MVs generation from human breast cancer cell line, MDA-MB-231. Rab5a, located at the plasma membrane plays vital roles in MVs biogenesis. We show that PAR2 stimulation promotes AKT phosphorylation which activates Rab5a by converting inactive Rab5a-GDP to active Rab5a-GTP. Active Rab5a polymerizes actin which critically regulates MVs shedding. Not only MVs generation, has this Rab5a activation also promoted cell migration and invasion. We reveal that Rab5a is over-expressed in human breast tumor specimen and contributes MVs generation in those patients. The involvement of p38 MAPK in MVs-induced cell metastasis has also been highlighted in the present study. Blockade of Rab5a activation can be a potential therapeutic approach to restrict MVs shedding and associated breast cancer metastasis.

Highlights

The contribution of protease activated receptor 2 (PAR2) in human breast cancer progression has been well established[21], its role in cancer propagation has not been focused yet
We implemented the same experiment in another human breast cancer cell line MCF-7 where we over-expressed all forms of Rab5a (WT, DN and CP) followed by the addition of trypsin and quantified MVs generation by MVs protein estimation by Bradford assay (Fig. 1J)
The severity of the disease results from the metastasis of tumor from initial tumor forming site to a secondary site which requires the detachment of cancer cells from its origin, degradation of extracellular matrix (ECM), migration through blood vessels and colonization at a different location

Summary

Introduction

The contribution of protease activated receptor 2 (PAR2) in human breast cancer progression has been well established[21], its role in cancer propagation has not been focused yet. The role of trypsin dependent PAR2 activation in pro-metastatic MVs generation from human breast cancer cells has not been studied its role in breast cancer progression is well-established[21]. AKT driven Rab5a activation is well reported[25], the role of Rab5a in the context of PAR2-mediated MVs generation has not been identified. The contributions of these PAR2-derived MVs in promoting breast cancer migration and invasion as well as its underlying mechanism have not been well-established. We have investigated the mechanism of MVs generation from PAR2-activated human breast cancer cells and the consequences of MVs shedding in the propagation of the disease

Methods

Results

Conclusion