Abstract
Background: Pancreatic ductal adenocarcinoma (PDAC) is the most lethal solid malignancy, mainly because of its metastatic spread and multifactorial resistance to chemotherapy. c-Met is a tyrosine kinase receptor, which is overexpressed in PDAC and pancreatic-CSCs. Aberrations in c-Met signaling pathway have been shown to be associated with poor prognosis, invasive behavior and intrinsic resistance of PDAC to chemotherapy. Aim: To evaluate the therapeutic potential of crizotinib, a novel c-Met/ALK inhibitor, to suppress critical signaling pathways in order to overcome PDAC chemoresistance. Methods: To achieve this goal, the expression of CSC markers (CD24, CD44, CD133, and CD326/ESA), gemcitabine determinants (hENT1, hCNT1, dCK, CDA, RRM1, RRM2) and invasiveness markers/EMT (E-cadherin and vimentin) were evaluated by quantitative-RT-PCR in PANC-1, PP109 (primary cell culture), Capan-1 and Capan-1-gemcitabineresistant cells (Capan-1-R). The Capan-1-R were established after continuous exposure to gemcitabine and maintained with gemcitabine 10 μM. In Capan-1-R and Capan-1 cells we also evaluated total cytosolic adenosine and phosphorylated deoxynucleosides, using Liquid chromatography-mass spectrometry (LC-MS/MS). The expression of c-Met and phospho-c-Met was investigated at protein level using both Western blotting (WB) and immunocytochemistry (ICC). Cell growth inhibitory effects of the crizotinib and gemcitabine were determined by sulforhodamine B (SRB) assay. Perturbation of cell cycle and cell death was studied before/after treatment with drugs using flow cytometry, while cellular migration was evaluated by wound-healing assay. Results: c-Met protein expression was detected by WB and ICC in all PDAC cells, and was significantly increased in Capan-1-R cells with respect to Capan-1 (approximately 2-fold). In addition, the mRNA expression of the gemcitabine catabolism enzyme CDA and vimentin were increased in Capan-1-R, compared to Capan-1, whereas gemcitabine nucleotides were significantly reduced in Capan-1-R compared to Capan-1. The expression of CSC markers was detectable by FACS analysis, and CD24+, CD44+, CD133+, and CD326+ cells were significantly reduced after treatment with crizotinib at 50% growth inhibitory concentration (IC50), as well as after its combination with gemcitabine. Crizotinib inhibited cell growth within the micromolar range (2.5-7.3 μM), and synergistically enhanced the antiproliferative activity of gemcitabine, with combination index values of 0.43 (Capan-1), 0.65 (PANC-1) and 0.8 (PP109). Crizotinib induced cell cycle arrest in the G1-S boundary (e.g., in Capan-1-R from 43 to 20%, P Moreover, crizotinib reduced cell migration, which was additionally reduced by crizotinib/ gemcitabine combination (e.g., >50% reduction of cell migration in Capan-1-R after 4 hours of exposure compared to controls). This reduced migration was associated with increased E-cadherin mRNA expression. Conclusion: Taken together, all this data showed the activity of crizotinib against PDAC cells, unraveling its ability to specifically target CSC-like subpopulations, interfere with cell proliferation, induce apoptosis, reduce migration and interact positively with gemcitabine. These results provide evidence that c-Met is a viable target in pancreatic cancer cells, and several molecular mechanisms underline the activity of crizotinib against PDAC cells, supporting further studies on this novel therapeutic approach for pancreatic ductal adenocarcinoma. Note: This abstract was not presented at the conference. Citation Format: Amir Avan, Karl Quint, Francesco Nicolini, Mina Maftouh, Niccola Funel, Godefridus J. Peters, Elisa Giovannetti. c-MET as a potential therapeutic target in pancreatic cancer: Implications in cancer-stem-like cell (CSC) population and gemcitabine resistance in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A46.
Published Version
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