PD08-04: Inhibition of the TGFb/TGFbR2 Pathway Prevents Enrichment of Drug-Resistant Breast Cancer Stem Cells by Anti-Cancer Chemotherapy.

Abstract

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 a good 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) β and TGFβ type II receptor (TGFβR2) mRNA expression compared to the non-CSC population. Furthermore, breast CSCs exhibit characteristics of an epithelial to mesenchymal transition (EMT), a process driven by the TGFβ signaling and associated with metastatic progression. Therefore, we hypothesize that inhibiting the TGFβ/TGFβR2 pathway can abrogate the breast CSC population and sensitize TNBC to chemotherapy, thus reducing metastatic recurrences and tumor progression. To address this hypothesis, we used the TNBC cell lines SUM159 and BT549. We examined the effect of TGFβ1, the TGFβ receptor I/II kinase inhibitor LY2157299, the TGFbRII receptor and TGFb1 neutralizing antibodies TR1, and LY2424087 and paclitaxel on the CSC population of these cell lines by 1) flow cytometric analysis of stem cell markers (ALDH, CD44, and PROCR); and 2) mammosphere formation assays. In both cell lines, LY2157299, LY2424087, and TR1 abrogated TGFβ-mediated CSC enrichment and mammosphere formation (p≤0.01). Treatment with paclitaxel resulted in an enrichment of CSCs as measured by FACS and mammosphere formation (p≤0.005) both in vitro and in SUM159 xenografts established in athymic mice. Further, treatment with paclitaxel upregulated TGFβR2 and TGFβ1 mRNAs and phosphorylated SMAD2 levels in CSCs sorted from post-therapy SUM159 xenografts. RNAi-mediated knockdown of TGFβR2 in BT549 cells decreased mammosphere formation but did not affect the proportion of CSCs analyzed by FACS (p≤0.005 and p=0.03, respectively). TGFβR2 and SMAD4 siRNA decreased the CD44hi/CD24lo population in both cell lines. SMAD4 siRNA also decreased CSCs, mammosphere formation, and viability in SUM159, BT549, and MDA-231 basal-like and HCC1954 (HER2+) cells. SMAD4 knockdown resulted in a significant decrease in the CSC-associated genes interleukin-8 (IL-8) and Nanog. Finally, a combination of either LY2157299 or SMAD4 siRNA with paclitaxel decreased both stem cell marker expression, IL-8 and mammosphere formation compared to each treatment alone (p≤0.006). These findings suggest that autocrine TGFb signaling plays a maintenance role in breast CSCs viability. Second, blockade of the TGFb/TGFbR2 pathway with genetic or pharmacological inhibitors can ameliorate or prevent the enrichment of drug-resistant CSCs by anti-cancer chemotherapy. These studies provide a rationale for studies of chemotherapy ± TGFb inhibitors in patients with TNBC using stem cell markers as surrogates of clinical response. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD08-04.