There is increasing evidence of a constitutive activation of Akt in pancreatic ductal adenocarcinoma (PDAC), associated with poor prognosis and chemoresistance. Therefore, we evaluated the expression of phospho-Akt in PDAC tissues and cells, and investigated molecular mechanisms influencing the therapeutic potential of Akt inhibition in combination with gemcitabine. Phospho-Akt expression was evaluated by immunohistochemistry in tissue microarrays (TMAs) with specimens tissue from radically-resected patients (n = 100). Data were analyzed by Fisher and log-rank test. In vitro studies were performed in 14 PDAC cells, including seven primary cultures, characterized for their Akt1 mRNA and phospho-Akt/Akt levels by quantitative-RT-PCR and immunocytochemistry. Growth inhibitory effects of Akt inhibitors and gemcitabine were evaluated by SRB assay, whereas modulation of Akt and phospho-Akt was investigated by Western blotting and ELISA. Cell cycle perturbation, apoptosis-induction, and anti-migratory behaviors were studied by flow cytometry, AnnexinV, membrane potential, and migration assay, while pharmacological interaction with gemcitabine was determined with combination index (CI) method. Immunohistochemistry of TMAs revealed a correlation between phospho-Akt expression and worse outcome, particularly in patients with the highest phospho-Akt levels, who had significantly shorter overall and progression-free-survival. Similar expression levels were detected in LPC028 primary cells, while LPC006 were characterized by low phospho-Akt. Remarkably, Akt inhibitors reduced cancer cell growth in monolayers and spheroids and synergistically enhanced the antiproliferative activity of gemcitabine in LPC028, while this combination was antagonistic in LPC006 cells. The synergistic effect was paralleled by a reduced expression of ribonucleotide reductase, potentially facilitating gemcitabine cytotoxicity. Inhibition of Akt decreased cell migration and invasion, which was additionally reduced by the combination with gemcitabine. This combination significantly increased apoptosis, associated with induction of caspase-3/6/8/9, PARP and BAD, and inhibition of Bcl-2 and NF-kB in LPC028, but not in LPC006 cells. However, targeting the key glucose transporter Glut1 resulted in similar apoptosis induction in LPC006 cells. These data support the analysis of phospho-Akt expression as both a prognostic and a predictive biomarker, for the rational development of new combination therapies targeting the Akt pathway in PDAC. Finally, inhibition of Glut1 might overcome resistance to these therapies and warrants further studies.
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.
The V‐Ki‐ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) is frequently dysregulated in colorectal cancer (CRC). It is involved in the modulation of several downstream effectors, that include: Raf/Mek/Erk, PI3K/Akt, RalGDS/p38MAPK, and Rac/Rho, and thereby influences tumorigenesis, the invasive behaviors of tumor cell, and resistance to therapy. There is growing evidence exploring the use of drugs that target these pathways in the treatment of CRC. Cetuximab has been approved for CRC patients without a KRAS mutation, or for EGFR‐expressing metastatic CRC, although some of the patients have a mutation of KRAS and NRAS. This review summarizes the recent knowledge about the therapeutic potential of targeting RAS with particular emphasis on recent preclinical and clinical studies in treatment of CRC.
Lung cancer is among the leading causes of cancer-related-death. Non-small cell lung cancer (NSCLC) is the most common form of lung cancer. More than 70% of NSCLC patients have locally advanced or metastatic disease in diagnosis stage, which are then being treated with platinum-based chemotherapy or epidermal-growthfactor- receptor (EGFR) inhibitors. Several molecules which target multiple ErbB receptors and EGFR have been developed, including gefitinib and erlotinib. Identification of novel agents with less toxicity is warranted. Several interesting data have been reported about the antitumor activity of curcumin in several tumors, including lung, breast and colorectal cancers. In particular, a recent phase I trial evaluated the activity of curcumin in combination with FOLFOX chemotherapy in patients with inoperable colorectal cancer. They showed that curcumin added benefit in subsets of patients when administered with FOLFOX and was well-tolerated chemotherapy adjunct. Another ongoing trial is now investigating the beneficial effects of curcumin plus gefitinib or erlotinib for EGFRmutant NSCLC. Improved understanding of molecular mechanisms behind resistance to EGFR tyrosine kinase inhibitors suggests the importance of a genotype-guided approach to therapy and inhibition of parallel and downstream pathways, using agents which target heat-shock-protein-90, poly (ADP-ribose) polymerase and PI3K/mTOR pathway. The aim of the current review is to give an overview of the possible molecular mechanisms of curcumin in the preclinical and clinical investigations in solid tumors, with particular emphasis on its combination with other chemotherapeutic agents in lung cancers.
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