Functional study of popx2 phosphatase in breast cancer metastasis

• Main Author: Zhang, Songjing
• Other Authors: Koh Cheng Gee
• Format: Theses and Dissertations
• Language: English
• Published: 2014
• Subjects: DRNTU::Science::Biological sciences::Molecular biology
• Online Access: https://hdl.handle.net/10356/58892
• Institution: Nanyang Technological University

POPX2, a serine/threonine phosphatase belonging to the PP2C family, is involved in the regulation of actin cytoskeleton, mDia-SRF (serum response factor) mediated transcription and breast cancer cell motility and invasiveness. In this project, we propose to use three different approaches to explore the biological functions of POPX2 in breast cancer. Firstly, we used an integrative transcriptome and proteome approach to decipher the roles of POPX2 in cancer motility and related signaling. Our study suggests that POPX2 is involved in cancer cell migration through modulating MAPK1/3-stathmin-mediated microtubule dynamics, in collaboration with other possible signaling molecules to modulate actin cytoskeleton and focal adhesions. POPX2 is also implicated in transcriptome regulation possibly through modulating MAPK1/3 and GSK3β as well as the downstream transcription factors. To further explore the role of POPX2 in angiogenesis, we also initiated a combined phosphoproteome and secretome study. Our phosphoproteome data implicate the participation of POPX2 in CDK1-mediated cell cycle progression. Secretome analysis combined with extensive biological validation verified that there is exosome enrichment and accelerated EMT progression in response to POPX2 silencing, which could further contribute to angiogenesis. Apart from the –omics approaches to understand the signaling network regulated by POPX2, we have also taken a candidate approach to specifically study candidate proteins which might be regulated by POPX2. RhoGDIs, the candidate proteins related to POPX2-regulated signaling, are also examined as we have earlier found that the levels of RhoGDI are affected by POPX2 knockdown1. The effects of RhoGDIs on actin cytoskeleton are studied. Overexpression of RhoGDI1 and RhoGDI2 result in cell rounding with concomitant loss of stress fiber and focal adhesions to different extent. RhoGDI1 appears to exert a more prominent effect compared to that of RhoGDI2. This might be due to their differential binding affinities to RhoGTPases, one of the major regulators of the actin cytoskeleton. Further characterization of RhoGDIs’ interaction with the different RhoGTPases reveals two conserved leucine residues, which are critical to RhoGTPases binding. We also verify other mutants, which affect RhoGDI binding affinity to various RhoGTPases. These mutants help to elucidate that RhoGDIs affect the actin cytoskeleton via RhoGTPases.