4). similar to the treatment of recombinant human AREG. Small interference RNA targeting AREG inhibited ExeR proliferation, confirming that AREG is truly functioning as a growth factor of ExeR cells. The specific inhibitors to ER (ICI 182,780), EGF receptor (EGFR; AG1478), and mitogen-activated protein kinase (MAPK; U0126) all showed dose-dependent suppression of the proliferation of ExeR cells, indicating the involvement of the ER, EGFR, and MAPK pathways. Based on these findings, we propose a possible mechanism that underlies exemestane resistance: exemestane induces AREG in an ER-dependent manner. AREG then activates the EGFR pathway and prospects to the activation of the MAPK pathway that drives cell proliferation. Introduction Exemestane, letrozole, and Vadadustat anastrozole are Food and Drug Administrationapproved aromatase inhibitors. Aromatase inhibitors are proved to be very effective in treating hormone-dependent breast malignancy in postmenopausal women (1). However, for prolonged treatment, resistance becomes a major concern. To study the mechanisms of aromatase inhibitor resistance, our lab has developed several aromatase inhibitorresistant cell lines that are derived Vadadustat from MCF-7 cells that overexpress aromatase (MCF-7aro). These resistant cell lines were selected under the following conditions: testosterone plus letrozole (T + LetR), testosterone plus anastrozole (T + AnaR), anastrozole only (AnaR), testosterone plus exemestane (T + ExeR), exemestane only (ExeR), or Vadadustat long-term estrogen deprivation (LTEDaro). MCF-7aro cells cultured in testosterone (in which testosterone was converted to 17-estradiol) were used as positive controls (2, 3). Although they all target aromatase specifically and potently, it is known that different aromatase inhibitors behave differently based on their structures. Rabbit Polyclonal to RBM34 Letrozole and anastrozole are nonsteroidal inhibitors and interact with the heme group. Exemestane is a steroidal inhibitor and is also known as a mechanism-based aromatase inactivator. Exemestane binds to aromatase irreversibly and causes time-dependent aromatase inactivation (4). A recent study from our lab showed that exemestane could destabilize aromatase protein, in addition to inhibiting its activity (5). As an unbiased and systemic approach that could give important information about the resistance mechanisms of different aromatase inhibitors, our laboratory has generated three to six replicates of resistant cell lines to each aromatase inhibitor and examined the gene expression profiles using cDNA microarray analysis. From our analysis, we have noticed high levels of amphiregulin (AREG) expression in ExeR cells. Because the expression of AREG is known to be up-regulated by estrogen (6, 7), we were not surprised to find that the expression of AREG was high in MCF-7aro cells treated with testosterone and was detected in all testosterone + aromatase inhibitortreated cells (T + LetR, T + AnaR, and T + ExeR) but not in AnaR and LTEDaro cells. Whereas the microarray analysis will be discussed in detail in a separate article, this present study will be focused on AREG expression in ExeR cells. AREG was originally isolated from conditioned medium of phorbol 12-myristate 13-acetate (PMA)stimulated MCF-7 cells (8). AREG caught our attention for the following reasons (test (Microsoft Excel). Results AREG is highly expressed in ExeR cells Microassay analysis has shown high levels of AREG expression in ExeR cells. In Fig. 1to 0.01, compared with LTEDaro. Exemestane induces AREG expression in an ER-dependent manner Parental MCF-7aro cells were used to determine if high levels of AREG in ExeR cells were due to the presence of exemestane. ICI is known as a pure antiestrogen that degrades ER (15). Here we first checked the effect of ICI on ER protein. There are two types of ER: ER and ER. Our microarray analysis revealed that there is.