Amphiregulin Interacts With Wnt Signaling To Regulate Cancer Cell Growth And Survival

Epidermal growth factor receptor (EGFR) initiates signaling via several ligands that include but are not limited to EGF, amphiregulin (Areg) and epiregulin. These ligands are believed to differentially affect various physiological and cellular processes such as cell survival, proliferation and differentiation. This study is designed to delineate how amphiregulin alters the biology of both breast and ovarian cancer cells that overexpress amphiregulin and activate EGFR in an autocrine manner. AREG knock down drastically reduces the rate of proliferation of both a breast cancer cell line, SUM149 and an ovarian cancer cell line, OV1 that overexpress amphiregulin. Stable, clonal populations of SUM149 cells with AREG knockdown grow poorly in serum free media, but can be rescued by addition of exogenous AREG or other EGFR ligands to the culture medium. Clones with the most effective AREG knock down are completely unable to survive or proliferate in serum free media. Our Micro array analyses show that inhibiting amphiregulin in SUM149 cells dysregulates the expression of several important genes, including up-regulating DKK1, an antagonist of Wnt signaling. DKK1 up regulation upon AREG knockdown was confirmed by ELISA, with one of the clones, C2 showing a 16 fold increase in DKK1 secretion following AREG knock-down. Flow cytometetric analysis of SUM-149 cells and AREG knock-down derivatives demonstrated that 48% of control cells expressing a non-silencing shRNA construct were CD44+/CD24- but only a small population (7%) expressed CD24 alone. AREG knockdown induced significant reduction in the fraction of CD44 positive cells, down to ∼4% while, simultaneously increasing the non-stem cell fraction, CD44-/CD24+ to 60% of the total population. Since CD44hi/CD24lo is a characteristic of undifferentiated cells that have the potential to initiate tumor formation, we are currently examining the in vivo growth potential of SUM-149 cells and AREG knock-down derivatives. To determine the tumorigenicity of SUM149 cells three different cell innocula of unsorted parental SUM149 cells and SUM149 cells expressing a non-silencing shRNA construct (SUM149-NS) were transplanted into the mammary fat pad of NOD-SCID mice. As expected, the tumorigenic activity of the cells is proportional to the cell titer. In the parental SUM149 cells, the injection of 5x10 5 cells resulted in measurable tumors beginning at day 28 while SUM149-NS displays tumors at day 24, while transplantation of 2.5x10 5 cells per fat pad yielded tumor formation in 31 and 28 days respectively. Experiments to measure the in vivo growth potential of AREG knock-down SUM-149 cells is in progress. Thus, amphiregulin has a unique biological function to enhance cell survival and differentiation in vitro and in vivo. Future studies will focus on the role of AREG mediated WNT signaling in the malignant potential of SUM-149 cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3248. doi:1538-7445.AM2012-3248