PI3K/AKT/mTOR and Sonic hedgehog pathways cooperate together to inhibit human pancreatic cancer stem cell characteristics and tumor growth

Cancer stem cells (CSCs) play major roles in cancer initiation, progression, and metastasis. It is evident from growing reports that PI3K/Akt/mTOR and Sonic Hedgehog (Shh) signaling pathway are aberrantly reactivated in pancreatic CSCs. Here, we examined the efficacy of combining NVP-LDE-225 (PI3K/mTOR inhibitor) and NVP-BEZ-235 (Smoothened inhibitor) on pancreatic CSCs characteristics, microRNA regulatory network, and tumor growth. NVP-LDE-225 cooperated with NVP-BEZ-235 in inhibiting pancreatic CSC’s characteristics and tumor growth in NOD/SCID IL2R null mice by acting at the level of Gli. Combination of NVP-LDE-225 and NVP-BEZ-235 inhibited self-renewal capacity of CSCs by suppressing the expression of pluripotency maintaining factors Nanog, Oct-4, Sox-2 and cMyc. NVP-LDE-225 cooperated with NVP-BEZ-235 to inhibit Lin28/Let7a/Kras axis in pancreatic CSCs. Furthermore, synergistic interaction of these drugs was observed on spheroid formation by pancreatic CSCs isolated from Pankras/p53 mice. The combination of these drugs also showed synergistic effects on the expression of proteins involved in cell survival and apoptosis. In addition, synergistic effects of these drugs were observed on inhibition of epithelial-to-mesenchymal transition (EMT) through modulation of cadherin, vimentin and transcription factors Snail, Slug and Zeb1. In conclusion, these data suggest that the combined inhibition of PI3K/Akt/mTOR and Shh pathways may be an effective strategy for the treatment of pancreatic cancer. Pancreatic CSCs were treated with NVP-BEZ-235 (1 µM) and NVP-LDE-225 (5 µM) in duplicate for 36 h. RNAs were isolated using the trizol reagent (invitrogen, CA, USA). The expression levels of expressed miRNAs were measured using Affymetrix GeneChip miRNA 3.0 arrays. Only probesets of human small-RNAs in the array were used for the analysis.