Abstract 3477: Tamoxifen leads to an increase in tumor cells with self-renewing capacity in a mouse model of estrogen dependent breast cancer
Authors: Raffo Diego , Sampayo Rocio , de Kier Joffé Elisa Bal , Simian Marina 
Published: August 2012

Abstract
About 75% of breast cancers are positive for estrogen receptor (ER). Tamoxifen (TAM) has been the main treatment for the last 30 years, however, 1/3 of patients that initially respond develop resistance. Recent discoveries have led to hypothesis that considers a direct involvement of stem cell populations (SCP) in cancer progression and resistance to therapy. The aim of this work was to analyze the impact of endocrine therapy on SCP in breast cancer. To do so we used the M05 mouse mammary tumor (Simian et al., BCRT, 2009), a syngeneic model of estrogen dependent, TAM sensitive breast cancer, together with the LM05-E cell line derived from it and MCF-7 cells. When treated with TAM for 30 days tumor growth was inhibited and immunofluorescence (IF) staining showed a decrease in ER+/E-cadherin+ tumor cells together with an increase in the stromal compartment and deposition of extracellular matrix (ECM) components, compared to vehicle treated controls. Tumors were digested until a single cell suspension was obtained. Mammosphere assays (MA) were carried out and quantified after 7 days in culture. A statistically significant increase in mammosphere formation was achieved in the cultures of tumors derived from TAM treated mice, compared to controls. Next we tested whether this was true for tumors derived from TAM treated and untreated animals that were subsequently passaged to untreated mice and allowed to regrow. MA revealed that this was the case. IF studies showed that those tumors that derived from the TAM treated mice contained higher levels of β1 and β6 integrins as well as increased phopho-ser118-ER. Next, to further establish a link between TAM and self-renewal capacity in vitro, we treated LM05-E and MCF-7 cells with 4-OH-TAM 10-6M or vehicle for 5 days and carried out MA. TAM led to an increase in mammosphere formation. However, when TAM was added directly to the MA no differences were detected between the treated and control groups suggesting that only those cells that resist the treatment are able to grow in suspension. Finally we had previously shown that fibronectin and laminin conferred TAM resistance through β1 and β6 integrins respectively. To test whether these factors lead to an increase in cells with self-renewing capacity we treated LM05-E and MCF-7 cells with these ECM components for 48 hours and then carried out MA and measured ALDH activity. Compared to the control group we found a decrease in the % of mammosphere forming cells as well as a decrease in ALDH activity in the treated groups. Our results show that TAM leads to an increase in cells with self-renewal capacity both in vitro and in vivo, and that microenvironmental factors seem to have the opposite effect. Understanding the effects of tamoxifen on the breast cancer SCP has important implications for the development of strategies that could lead to a decrease in recurrence in patients with ER+ tumors.
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 3477. doi:1538-7445.AM2012-3477