TGF-β1 enhances pancreatic cancer invasiveness by regulating CXCR4 expression through SMAD signal transduction

Abstract

Introduction: Chemokine receptor CXCR4 appears to have a critical role in cancer metastasis. We hypothesize that TGF-β1, a ubiquitous cytokine, directs the invasive properties of pancreatic cancer through regulatory control of CXCR4 expression. Methods: Established pancreatic cancer cell lines (SU.86.86, BxPC-3, PANC-1, CFPAC-1, COLO357, and MIAPaCa-2) were treated with TGF-β1. Changes in CXCR4 gene expression were measured by a quantitative real-time RT-PCR (qRT) assay. CXCR4 protein was verified by flow cytometry. The role of established mediators of TGF-β1 signal transduction--Smad proteins and p38/mitogen-activated protein kinase (MAPK)--was investigated by transfecting pancreatic cancer cells with Smad4 and Smad7 short-interfering RNA (siRNA) or treating pancreatic cancer cells with SB203580 (p38/MAPK inhibitor), respectively, to determine the specific contributions of each to TGF-β1 regulation of CXCR4. Demethylation of CXCR4 gene promoter region by TGF-β1 was assessed. The functional consequences of altered CXCR4 expression were measured by an in vitro invasion assay. Results: qRT demonstrated 1- to 8-fold upregulation of CXCR4 gene expression in SU.86.86, PANC-1, COLO357, and MIA PaCa-2 after treatment with TGF-β1 (all p<0.05). Flow cytometry corroborated an increase in CXCR4 protein expression. Interestingly, TGF-β1 did not alter CXCR4 gene expression in cell lines with absent Smad4 expression (BxPC3 and CFPAC-1). Transfection of pancreatic cancer cells (SU.86.86, PANC-1, COLO357, and MIA PaCa-2) with Smad4 siRNA significantly enhanced TGF-β1-mediated CXCR4 expression by 1- to 3-fold (all p<0.05), demonstrating the inhibitory function of Smad4 for CXCR4 expression. Treatment with Smad7 siRNA did not significantly change CXCR4 expression. Inhibition of p38/MAPK by SB203580 reduced TGF-β1 upregulation of CXCR4. TGF-β1-treated pancreatic cancer cells were evaluated by methylation-specific PCR, which demonstrated no change in DNA methylation. A modified Boyden chamber assay exhibited a measurable increase in pancreas cell invasion in TGF-β1-treated cells that were stimulated with CXCL12, the specific ligand for CXCR4. Conclusions: This report demonstrates that TGF-β1 is a critical component of the pancreatic cancer microenvironment by regulating CXCR4 expression and directing the invasive properties of pancreatic cancer. The mechanism of this regulation appears to occur through displacement of Smad4, an inhibitor of CXCR4 gene expression. There also appears to be an important cross-talk between Smad4 and p38/MAPK.