Abstract
PurposeSpatiotemporal regulation of cell membrane dynamics is a major process that promotes cancer cell invasion by acting as a driving force for cell migration. Beta-Pix (βPix), a guanine nucleotide exchange factor for Rac1, has been reported to be involved in actin-mediated cellular processes, such as cell migration, by interacting with various proteins. As yet, however, the molecular mechanisms underlying βPix-mediated cancer cell invasion remain unclear.MethodsThe clinical significance of βPix was analyzed in patients with colorectal cancer (CRC) using public clinical databases. Pull-down and immunoprecipitation assays were employed to identify novel binding partners for βPix. Additionally, various cell biological assays including immunocytochemistry and time-lapse video microscopy were performed to assess the effects of βPix on CRC progression. A βPix-SH3 antibody delivery system was used to determine the effects of the βPix-Dyn2 complex in CRC cells.ResultsWe found that the Src homology 3 (SH3) domain of βPix interacts with the proline-rich domain of Dynamin 2 (Dyn2), a large GTPase. The βPix-Dyn2 interaction promoted lamellipodia formation, along with plasma membrane localization of membrane-type 1 matrix metalloproteinase (MT1-MMP). Furthermore, we found that Src kinase-mediated phosphorylation of the tyrosine residue at position 442 of βPix enhanced βPix-Dyn2 complex formation. Disruption of the βPix-Dyn2 complex by βPix-SH3 antibodies targeting intracellular βPix inhibited CRC cell invasion.ConclusionsOur data indicate that spatiotemporal regulation of the Src-βPix-Dyn2 axis is crucial for CRC cell invasion by promoting membrane dynamics and MT1-MMP recruitment into the leading edge. The development of inhibitors that disrupt the βPix-Dyn2 complex may be a useful therapeutic strategy for CRC.
Highlights
Colorectal cancer (CRC) is the third most common cancer (10.2 % of all cancer cases) and the second most common cause of cancer-related mortality (9.2 %) [1]
Analysis of βPix expression using 60 cell lines derived from different cancer tissues in the Gene Expression Omnibus (GEO) databases revealed that βPix expression was highest in cells derived from colorectal cancer (CRC) tissues (Supplementary Fig. S1d)
We found that the localization of lamellipodial membrane-type 1 matrix metalloproteinase (MT1-matrix metalloproteinases (MMPs)) was inhibited in Dynamin 2 (Dyn2)-silenced cells, and that overexpression of the Dyn2 proline-rich domain (PRD) mutant (R834A) or guanosine triphosphatases (GTPases)-deficient mutant (K44A), except for the wild-type, failed to restore the peripheral localization of MT1-MMP (Fig. 5d)
Summary
Colorectal cancer (CRC) is the third most common cancer (10.2 % of all cancer cases) and the second most common cause of cancer-related mortality (9.2 %) [1]. The high CRC-related mortality rate is closely related to the occurrence of metastasis, which is characterized by Membrane protrusions are spatiotemporally regulated by classical Rho family small guanosine triphosphatases (GTPases), including RhoA, Rac and Cdc, along with their downstream effectors [4,5,6]. The Dbl and DOCK families of proteins have been identified as specific types of GEFs. Among them, beta-Pix (βPix), which activates Rac and Cdc, is one of the most frequently investigated GEFs, in terms of cell spreading and migration [7,8,9]. Previous studies have revealed that βPix promotes collective migration of endoderm cells and neural tube formation by activating downstream signaling including Rac and p21-activated kinase 2a (Pak2a) during embryonic development [9,10,11]. ΒPix may exert certain intracellular functions related to its binding partner
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