Abstract
BackgroundResistance to anoikis, apoptosis triggered by a loss of cellular adhesion to the underlying extracellular matrix, is a hallmark of metastatic cancer. Previously we have shown re-establishment of CXCL12 expression in colorectal carcinoma cells inhibits metastasis by enhancing anoikis sensitivity. The objective of these studies was to define the signaling mechanisms regulating CXCL12-mediated anoikis.Methodology/Principal FindingsAdhesion, examined by crystal violet staining, immunofluorescence microscopy, and immunoblot analysis indicated decreased focal adhesion signaling corresponding with loss of adhesion in cells constitutively simulated by CXCL12. Loss of adhesion was inhibited by pertussis toxin treatment, indicating CXCL12 regulating anoikis through Gαi-protein coupled receptors. Non-adherent HCT116 and HT29 colorectal carcinoma cells expressing CXCL12 exhibited enhanced anoikis sensitivity by propidium iodide staining, caspase activity assays, and immunoblot compared to GFP control cells. CXCL12 producing carcinomas cultured on poly-HEMA displayed heightened Bim and loss of Mcl-1 and Bcl-2 preceding cytochrome c release, and caspase-9 activation. RNAi knockdown of Bim reversed anoikis sensitivity of CXCL12-expressing cells and fostered increased soft-agar foci formation and hepatic tumors in an orthotopic mouse model of metastasis.Conclusions/SignificanceThese data indicate CXCL12 provides a barrier to metastasis by increasing anoikis via activation of a Bim-mediated intrinsic apoptotic pathway. These results underscore the importance of retaining CXCL12 expression to sensitize colorectal carcinomas to anoikis and minimize tumor progression.
Highlights
Intestinal epithelial cells migrate along the crypt-villus axis where their survival is dependent on integrin binding to the underlying extracellular matrix (ECM)
Our previous studies have shown that autocrine CXCL12 reduces cellular colorectal carcinoma cell adherence compared to wildtype colonic carcinoma cells
Reflecting our previous studies in breast cancer carcinomas, CXCR4 surface expression was unaltered between control and CXCL12-expressing cells (Supplemental Data Figure S1) [23]
Summary
Intestinal epithelial cells migrate along the crypt-villus axis where their survival is dependent on integrin binding to the underlying extracellular matrix (ECM). Anoikis has primarily been described as an intrinsic apoptotic pathway with cell fate being dictated through mitochondrial outer membrane permeabilization by Bcl-2 family member proteins [7]. Multi-domain pro-apoptotic proteins, Bak and Bax, contain transmembrane domains that permeabilize the mitochondrial outer membrane to release cytochrome c resulting in activation of caspase-9-dependent apoptosis. Resistance to anoikis, apoptosis triggered by a loss of cellular adhesion to the underlying extracellular matrix, is a hallmark of metastatic cancer. We have shown re-establishment of CXCL12 expression in colorectal carcinoma cells inhibits metastasis by enhancing anoikis sensitivity. The objective of these studies was to define the signaling mechanisms regulating CXCL12-mediated anoikis
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