Abstract 267: The effect of amphiregulin knockdown on the expression of transformed phenotypes in triple-negative breast cancer

Abstract

Abstract Amphiregulin (AREG) and epidermal growth factor receptor (EGFR) co-expression leads to aggressive growth phenotypes observed in some triple negative breast tumors. In SUM-149 cells, a triple negative breast cancer cell line with an AREG/EGFR autocrine loop, we have shown that these cells are more invasive and motile than MCF10A cells grown with EGF and that altered signaling occurs when AREG is the stimulating ligand. To provide additional insight into the role of AREG in the SUM-149 cells, we have successfully knocked down AREG expression in the SUM-149 cells. Q-RT-PCR analysis showed an approximately 40-fold knock-down of AREG mRNA following stable transduction of the most effective shRNA vector. In these knock-down cells, secreted AREG was undetectable by ELISA, and cellular AREG was undetectable by western blot. Knock-down of AREG in SUM-149 cells resulted in a dramatic decrease in the growth rate of these cells, resulting in a doubling time of about half that of the parental SUM149 cell line. Soft agar assays comparing the anchorage-independent growth potential of SUM-149 cells with AREG knock-down SUM-149 cells demonstrated an approximately 50% decrease in soft agar colony forming ability. To examine the migration and invasion capacity of the AREG knock-down cells relative to parental cells, Matrigel invasion assays were carried out and showed that knocking down AREG resulted in a 40-fold decrease in the cells ability to invade the matrix. Our lab has previously shown that in SUM-149 cells, NF-κB DNA binding activity is initiated as a result of AREG activation of EGFR. NF-κB activity in the AREG knock-down cells was altered suggesting that AREG affects cellular signaling by altering transcription factors. Addressing the continued proliferation of the AREG knock-down cells, we examined the requirement for EGFR activation for growth of these cells. Phosphotyrosine western blot analysis demonstrated detectable EGFR activation in the AREG knock-down cells. Growth assays where these cells were treated with an EGFR tyrosine kinase inhibitor or with a neutralizing EGFR antibody demonstrated the dependency of EGFR for growth of these cells. Q-RT-PCR analysis for expression of all seven EGFR family ligands suggested that epigen or EGF may be factors contributing to the continued growth of these cells, in the absence of AREG. In summary, knock-down of AREG expression in SUM-149 cells has a dramatic affect on the cells ability to proliferate, invade, and form colonies in soft agar, which links this growth factor with the aggressive properties associated with the SUM-149 cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 267.