CXCR4 alters ID family and ITF2B/E2-2/TCF-4 dynamics to confer invasive phenotype to breast cancer cells.

Abstract

Abstract Abstract #2045 Introduction: Breast cancer is characterized by a distinct metastatic pattern involving lymph nodes, bone marrow, lung, liver and brain. The chemokine CXCL12 and its receptor CXCR4 are suggested to play a role in invasion and organ-specific metastasis. Signaling pathways downstream of CXCL12:CXCR4 involved in invasion and/or metastasis are not known.
 Methods: MDA-MB-231 breast cancer cells were sorted for CXCR4-positive and CXCR4-negative subpopulation by FACS and subjected to microarray gene expression analysis. Differentially expressed genes were further verified by RT-PCR or qRT-PCR. siRNA or shRNA expression studies were used to evaluate the role of genes overexpressed in CXCR4-positive cells in invasion through Matrigel. Orthotopic xenograft model is being used to evaluate primary tumor growth and metastasis pattern of CXCR4-positive and CXCR4-negative subpopulation with various manipulations.
 Results: ITF2B, a class I bHLH transcription factor, is expressed at ∼4-fold higher level in CXCR4-positive cells. ITF2B mRNA level was lower in CXCR4 siRNA treated or CXCR4shRNA expressing CXCR4-positive cells suggesting that ITF2B is the direct target of CXCL12:CXCR4. ITF2B siRNA reduced invasion of CXCR4-positive cells whereas ITF2B overexpression increased invasive capacity of CXCR4-negative cells. ITF2B is a Wnt β-catenin regulated gene with transforming ability and controls the function of Inhibitor of DNA binding family of transcription factors (ID1 to ID4). ID1 is essential for lung metastasis whereas ID2 induces differentiation of breast cancer cells and higher levels of ID2 correlates with favorable prognosis in breast cancer. ID2 expression was higher in CXCR4-negative cells and its overexpression in CXCR4-positive cells reduced invasion. Both CXCR4 and ITF2B are involved in reducing ID2 expression as siRNA against both of these genes increased ID2 mRNA in CXCR4-positive cells. Interestingly, CXCR4-negative cells appear to have adapted to alternative mode of invasion/metastasis as these cells express higher levels of metastasis-associated genes such as TWIST and MT1-MMP. Xenograft studies with cells overexpressing ITF2B and ID2 are currently underway.
 Conclusions: CXCL12:CXCR4 activated signaling pathways target ID family/bHLH transcription factor network to promote invasion of breast cancer cells. Inducers of ID2 may help to overcome CXCR4- mediated invasion and/or metastasis of cancer cells. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2045.