The Transcription Factor Iroquois Homeobox 2 (Irx2) Is A Candidate Metastasis Suppressor That Acts As Regulator Of Cellular Motility And Chemokine Expression In Breast Cancer Cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The onset of the metastatic cascade -the main cause of cancer related death- is the dissemination of single tumor cells into distant organs. The presence of disseminated tumor cells is an independent prognostic factor for the risk of recurrence and overall survival of cancer patients in different cancer entities. We found that low expression of the transcription factor iroquois homeobox 2 (IRX2) in primary breast and lung tumors is associated with the presence of disseminated tumor cells in the bone marrow. Accordingly, IRX2 represents a candidate metastasis suppressor gene. The purpose of this study is to gain insights into the significance of IRX2 expression in the progression of breast cancer. To assess its physiological relevance in breast cancer, we evaluated IRX2 expression in large publically available breast cancer cohorts. Additionally, we established two breast cancer cell lines with ectopic IRX2 protein expression and subjected those cell lines to a variety of functional assays. IRX2 mRNA expression was found to correlate with tumor grading and estrogen receptor status in primary breast tumors. Western blot analysis of IRX2 protein expression in breast cancer cells lines revealed that luminal and HER2 positive cell lines expressed IRX2, whereas cell lines belonging to the basal molecular subtype did not express the IRX2 transcription factor. SAM-analysis in combination with gene ontology analysis revealed that 6 out of the 40 genes, whose expression is most strongly correlated with IRX2 expression belong to the chemokine signaling pathway. Coincidently, in BT-549 and Hs578T with ectopic IRX2 expression we found markedly reduced levels of CCL5 and CXCL10 mRNA expression. A gene reporter assay also revealed a decreased trans-activation of the CCL5 proximal promoter in BT-549 cells in the presence of ectopic IRX2 expression. Furthermore, we could detect significantly lower CCL5 protein levels in cell culture supernatants obtained from this cell line. When used as chemoattractant, conditioned cell culture medium from this cell line also diminished migration of parental MDA-MD-231 and BT-549 breast cancer cells. BT-549 cells with IRX2 over expressing also showed clearly reduced motility in both wound healing as well as Boyden chamber assays. Our results imply that the IRX2 transcription factor might represent a novel metastasis suppressor protein that also acts a repressor of CCL5 gene transcription. Loss of IRX2 expression could contribute in particular to early hematogenous dissemination of breast and lung cancer cells by sustaining CCL5 secretion and enabling autocrine stimulated cell motility. Citation Format: Stefan Werner, Hauke Stamm, Mutiha Pandjaitan, Dirk Kemming, Klaus Pantel, Harriet Wikman. The transcription factor iroquois homeobox 2 (IRX2) is a candidate metastasis suppressor that acts as regulator of cellular motility and chemokine expression in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 117. doi:10.1158/1538-7445.AM2014-117