Abstract P5-03-07: Targeting Hedgehog pathway to reverse chemoresistance in breast cancer stem cells
Authors: Mañu A , Fresquet V , Mena M , Sánchez S , Espinós J , Fernandez Hidalgo OA , Fernández-Zapico M , Knutson KL , Martínez-Climent JA , Santisteban M 
Published: August 2014

PURPOSE: Hedgehog (HH) pathway has been implicated in maintenace and survival of breast cancer and cancer stem cells, EMT regulation, breast carcinogenesis and resistance to chemoradiation in solid tumors. Dysregulation of this pathway occurs in more than 50% of breast cancers. We have already demonstrated that immune-induced epithelial to mesenchymal transition (EMT) leads to the generation of breast cancer stem cells (BCSCs) from a parental epithelial breast cancer cell line in an in vivo murine model (Cancer Research 2009). The aim of our study is to demonstrate that resistance in the BCSCs is linked to HH hyperactivation and that the selective blockade of this pathway could eradicate this subpopulation of BCSCs in preclinical models.

METHODS: One parental epithelial (E) and three mesenchymal BCSCs cell lines (M1, M2 and M3) were used for all the in vitro experiments. qRT-PCR was used to quantify the expression of the gene components of the pathway and gene targets. Measurement of Gli1, caspase 3 and Cyclin D1 protein expression was determined by Western blot. Cell viability assays were performed with Paclitaxel and the HH inhibitor GDC-0449. Apoptosis assays were performed when cell lines were treated with paclitaxel (10nM), GDC-0449 (25uM) or the combination of them. For the in vivo xenografted mouse model, 106 cells (E and M2) were inoculated sc respectively into the flank of 6–8 week-old syngeneic female FVB/N-TgN (MMTVneu) 202Mul/J mice. Treatment was started on daily GDC-0449 (ip), paclitaxel (ip on days 1, 4 and 8) or both drugs when tumors reached 0.5 cm2. Statistical analysis was performed using one-way ANOVA with the Bonferroni multiple comparison test.

RESULTS: Activity of the HH measured by the expression of Gli1 was increased in BCSC cell lines as compared to E cell line (p &lt; 0.001). This was antagonized as expected by treatment with GDC-0449. Reporter and expression assay showed that GDC-0449 decreases Gli1 transcriptional activity and levels of a HH downstream target gene, cyclin D1. In all BCSC lines (but not in the E cell line), a synergistic effect was showed with the combination treatment as compared to control (p ≤ 0.01) and to paclitaxel or GDC-0449 alone (p ≤ 0.05 respectively) as well as a highly increased apoptotic activity (p ≤ 0.001 with control and p ≤ 0.05 with monotherapy). Tumors derived from M2 injection regressed when the combination schedule was administered (p ≤ 0.05) at 2 weeks after the start of the treatment.

CONCLUSION: The inhibition of the HH pathway with GDC-0449 plus paclitaxel demonstrates a synergistic therapeutic effect in comparison to monotherapy, as shown by increased apoptotic activity, cell cycle arrest and significant reduction of tumor size in vivo. Together these results provide the rationale for future clinical trials including the blockade HH and standard chemotherapy in order to eradicate the whole population of tumor cells (also BCSCs) within the tumors and avoid disease relapse and metastasis in breast cancer patients.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-03-07.