Abstract 1945: TORC inhibitors increase the cancer stem cell (CSC) population and Notch signaling in triple negative breast cancer

Tumor-initiating cells (TICs) or cancer stem cells (CSCs) are resistant to chemotherapy and have been associated with metastatic recurrences and poor patient outcome particularly among patients with triple negative breast cancer (TNBC). Genomic and proteomic data have indicated more than 30% of TNBC patients have PI3K/mTOR pathway lesions making this pathway a promising therapeutic target. Recent publications have demonstrated mechanisms of resistance (JAK2/STAT5 and MYC amplification) to PI3K pathway inhibitors. We hypothesized that resistance to TORC inhibition is due to the survival of a CSC population and that targeting pathways that sustain these cells can provide a significant therapeutic benefit. Treatment of TNBC cell lines with the PI3K/mTOR inhibitor NVP-BEZ235 or the TORC1/2 inhibitor MLN128 resulted in a significant reduction of proliferation in vitro. However, we observed that both BEZ235 and MLN128 enriched for a CSC population as assessed by FACS analysis of cancer stem-like markers and mammosphere formation. This observation was specific to TNBC cell lines since BEZ235 and MLN128 significantly abrogated the CSC population in ER+ (MCF7) and HER2+ (HCC1954) breast cancer cell lines. To determine the mechanisms involved in this CSC enrichment we used a Stem Cell specific PCR Array. We observed an increase in Notch1, FGF1 and ABCG2 mRNA levels in TNBC cells treated with BEZ235 and MLN128. Treatment with these inhibitors also increased the expression of the active Notch intracellular domain, the Notch ligand Jagged1, and the Notch1 target genes Hes1 and Hey1 by qRT-PCR and transcriptional reporter activity. In addition to Hes1 and Hey1, c-myc, another Notch target gene, expression was augmented in 2 of the 3 TNBC cell lines tested. Treament with the γ-secretase inhibitor, DAPT and transfection with Notch1 siRNA oligonucleotides abrogated BEZ235 and INK128-mediated enrichment of CSC populations as measured by FACS analysis and mammosphere formation assays. To determine whether inhibition of either TORC1 or TORC2 enriched for the CSC population, we used RNAi against Rictor (TORC2), Raptor (TORC2) or both. We observed that only the combined knockdown of Rictor and raptor increased the CSC population in TNBC cell lines. These observations suggest that treatment of TNBC harboring PI3K pathway aberrations with TORC1/2 inhibitors results in an initial reduction of tumor burden but do not eradicate the drug-resistant, slow cycling CSC population driven by Notch signaling. Thus, combination of a Notch inhibitor with TORC1/2 inhibitors and chemotherapy may be an effective therapeutic strategy to decrease primary tumor growth and prevent recurrences in patients with TNBC. Citation Format: Neil E. Bhola, Valerie Jansen, Carlos Arteaga. TORC inhibitors increase the cancer stem cell (CSC) population and Notch signaling in triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1945. doi:10.1158/1538-7445.AM2014-1945