Abstract 4240: TORC inhibition enriches for a cancer stem cell-like population with FGFR-dependent Notch1 activation
Authors: Bhola Neil E. , Jansen Valerie M. , Arteaga Carlos L. 
Published: August 2015

Abstract
Background. Cancer stem cells (CSCs) are associated with metastatic recurrences and poor prognosis in triple negative breast cancer (TNBC) patients. Genomic and proteomic data suggest that &gt;30% of TNBC harbor PI3K/mTOR pathway aberrations. Furthermore, PI3K/mTOR inhibitors have shown antitumor activity in preclinical TNBC models. However, resistance to PI3K/mTOR inhibitors in breast and ovarian cancer has been recently shown. We hypothesized that resistance to TORC inhibition in TNBC is driven by a CSC-like population and targeting this population can improve antitumor action of TORC inhibitors.
Methods and Results. Treatment of TNBC cells, SUM159 (PIK3CA-H1047L), BT549 (PTEN-del), and MDA468 (PTEN-del), with the PI3K/mTOR inhibitor BEZ235 or the TORC1/2 inhibitor MLN128 reduced growth. However, cells that survived BEZ235 and MLN128 treatment displayed CSC properties as assessed by FACS analysis for CSC markers and mammosphere formation. Using a Stem Cell-specific PCR Array and qRT-PCR, Notch1, Jagged1 and FGF1 mRNA levels were elevated in BEZ235 and MLN128-treated TNBC cells. Transient and long-term treatment with BEZ235 and MLN128 also increased expression of the active Notch intracellular domain (NICD), Notch target genes Hes1, Hey1 and c-Myc, and NICD-luciferase reporter activity. Nanostring analysis of post-chemotherapy primary breast cancers displayed increased expression of CSC markers (ALDH1A1 and CD44), Notch1, and Jagged1. Cells surviving the combination of the chemotherapeutic paclitaxel with BEZ235 or MLN128 displayed increased NICD and CSC-like properties. Similar changes were observed in OVCAR8 ovarian cancer cells treated with MLN128 ± cisplatin. Notch1 siRNA and the γ-secretase inhibitor (GSI) DAPT abrogated BEZ235- and MLN128-mediated enrichment of CSC markers and mammosphere formation. Treatment of SUM159 and MDA468 xenografts established in nude mice with MLN128 and DAPT did not significantly decrease tumor growth compared to MLN128 alone. However, MLN128 and DAPT-treated tumors displayed decreased tumorigenicity compared to MLN128-treated tumors as assessed by an in vivo limiting dilution assay. Furthermore, TNBC cells stably expressing mTOR shRNA exhibited increased NICD levels, CSC markers and tumorigenicity in vivo which was reduced with DAPT. With the increased FGF1 levels observed with BEZ235 and MLN128 treatment, we examined the effect of FRS2 siRNA and the FGFR1 kinase inhibitor lucitanib. Both approaches resulted in a reduction of the CSCs and NICD expression in MLN128-treated cells.
Conclusions. These data suggest that treatment of TNBC harboring PI3K pathway aberrations with TORC1/2 inhibitors results in decreased tumor growth but may spare a drug-resistant CSC population with FGFR-Notch signaling. Thus, combination of Notch and TORC1/2 inhibitors in addition to chemotherapy may effectively decrease primary tumor growth and prevent recurrences in TNBC patients.
Citation Format: Neil E. Bhola, Valerie M. Jansen, Carlos L. Arteaga. TORC inhibition enriches for a cancer stem cell-like population with FGFR-dependent Notch1 activation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4240. doi:10.1158/1538-7445.AM2015-4240