Abstract 3475: Inhibition of the TGFβ pathway inhibits chemotherapy-induced enrichment of breast cancer stem cells in triple-negative breast cancer
Authors: Bhola Neil E. , Balko Justin , Arteaga Carlos 
Published: August 2012

Abstract
Triple-negative breast cancer (TNBC) is the most virulent form of breast cancer and is associated with a worse prognosis compared to hormone receptor- and HER2-positive tumors. The standard treatment of TNBC is cytotoxic chemotherapy. TNBC patients tend to display an initial response to chemotherapy; however, they exhibit higher recurrence rates and overall poor long term survival. Reasons for this high metastatic recurrence rate and mortality are believed to be the existence of a chemo-resistant tumor-initiating population called cancer stem cells (CSCs). CSCs are defined as cells with the ability to self-renew, differentiate into non-tumorigenic cells, and initiate tumors in vivo. CSCs isolated from breast cancer tissue display increased transforming growth factor (TGF) ≤ and TGFβ type 2 receptor (TGFβR2) mRNA expression compared to the non-CSC population. Furthermore, breast CSCs exhibit characteristics of the TGFβ-driven epithelial to mesenchymal transition (EMT). Therefore, we hypothesize that inhibiting the TGFβ pathway can abrogate the breast CSC population and sensitize TNBC to chemotherapy, thus reducing metastatic recurrences and tumor progression. From a cohort of pre and post-chemotherapy treated TNBC patient samples we observed increased TGFβ signaling receptors and ligands, CD44 and ALDH1 levels in post-chemotherapy treated patients. We examined the effect of TGFβ1, the TGFβ receptor 1/2 kinase inhibitor LY2157299, the TGFβR2 receptor neutralizing antibody TR1, and paclitaxel on the CSC population in TNBC cell lines by 1) FACS analysis of CSC markers (ALDH, CD44, and PROCR); and 2) mammosphere formation assays. Paclitaxel treatment resulted in an enrichment of CSCs as measured by FACS and mammosphere formation (p≤0.005) both in vitro and in SUM159 xenografts. Further, treatment with paclitaxel upregulated TGFβR2 and TGFβ1 mRNAs and phosphorylated SMAD2 levels from post-therapy SUM159 xenografts. LY2157299 and TR1 abrogated TGFβ-mediated CSC enrichment and mammosphere formation (p≤0.01). Specific targeting of TGFβR2 and SMAD4 by RNAi decreased the CD44hi/CD24lo population in both cell lines. SMAD4 downmodulation also decreased mammosphere formation and interleukin-8 (IL-8) expression in three TNBC cell lines. Finally, a combination of either LY2157299 or SMAD4 siRNA with paclitaxel decreased CSC marker expression, IL-8, and mammosphere formation compared to each treatment alone (p≤0.006) from bulk and sorted CSC populations. These findings suggest that TGFβ signaling plays a maintenance role in breast CSCs viability. Second, blockade of the TGFβ pathway with genetic or pharmacological inhibitors can ameliorate or prevent the enrichment of drug-resistant CSCs by chemotherapy. These studies provide a rationale for studies of chemotherapy ± TGFβ inhibitors in patients with TNBC using CSC markers as surrogates of clinical response.
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 3475. doi:1538-7445.AM2012-3475