Counteracting Autophagy Overcomes Resistance to Everolimus in Mantle Cell Lymphoma
ABSTRACT Mantle cell lymphoma (MCL) is an aggressive B-lymphoid neoplasm with poor response to conventional chemotherapy and short survival. The phosphatidylinositol 3-kinase/Akt/mTOR survival pathway is constitutively activated in MCL cells, thereby making the mTOR inhibition an attractive therapeutic strategy. The first clinical studies of everolimus (RAD001), an mTOR inhibitor, in relapsed MCL patients have reported a significant response. Our aim was to analyze the mechanism related to everolimus resistance/sensitivity in MCL cells.
Sensitivity to everolimus was analyzed in MCL cell lines and primary MCL cells. Everolimus mechanism of action was determined by flow cytometry and Western blot. Particularly, autophagy was studied by LC3BI/II expression, autophagolysosomes detection by flow cytometry and fluorescence microscopy, and siRNA-mediated gene silencing.
Everolimus exerted antitumoral effect on MCL cells while sparing normal cells. In MCL cell lines, this phenomenon was associated to G(1) cell-cycle arrest, dephosphorylation of the mTOR downstream targets, 4E-BP1 and S6RP, and rephosphorylation of Akt. A synergistic cytotoxic effect was observed between everolimus and an Akt inhibitor, which overcame the compensatory reactivation within the mTOR signaling pathway. Interestingly, MCL cells with low response to this combination showed high levels of autophagy. Accordingly, selective triple knockdown of the autophagy genes ATG7, ATG5 and ATG3, and pretreatment with the autophagy inhibitor hydroxychloroquine, efficiently overcame the resistance to Akt/mTOR inhibitors, leading to the activation of the mitochondrial apoptotic pathway.
These results suggest that autophagy induction protects MCL cells from Akt/mTOR targeting and counteracting autophagy may represent an attractive strategy for sensitizing MCL cells to everolimus-based therapy. Clin Cancer Res; 18(19); 5278-89. ©2012 AACR.
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ABSTRACT: Regulation of cellular death is a complex method to maintain cellular homeostasis by protecting against oncogenic development and by recycling damaged cellular debris. Dysregulation of autophagy cellular death is common among a wide range of cancers and presents challenges to current treatment options. This review will evaluate current methods to directly and indirectly modulate autophagy to prevent cancer and to overcome resistance to anticancer therapy. PubMed was searched for keywords: autophagy, hydroxychloroquine, chloroquine, and cell death for preclinical and clinical studies evaluating autophagy-modulating pathways and compounds. Clinicaltrials.gov was searched for keywords: autophagy, hydroxychloroquine, and chloroquine for clinical trials involving autophagy. The pathways of autophagy are highly prevalent in numerous cancers cell types including leukemia, renal cell cancer, non-small cell lung cancer, melanoma, and advanced solid tumor. Autophagy-inducing compounds represent various drug classes and include everolimus, bortezomib, vorinostat, and arsenic trioxide. The autophagy-inhibiting compounds include chloroquine, hydroxychloroquine, and bafilomycin. Clinicaltrials.gov search identified 32 currently ongoing clinical studies evaluating autophagy and included 14 and 3 studies involving hydroxychloroquine and chloroquine, respectively. These phase I and phase II studies, evaluating the therapeutic benefit of combining autophagy modulators with current anticancer treatments, demonstrate early evidence for application in resistant cancer therapy. Despite positive results, there remains a need to identify direct-acting autophagy inhibitors and for larger phase III trials to be conducted. The preclinical evidence for modulating autophagy describes a promising, novel mechanism for enhancing anticancer treatments and overcoming current challenges such as chemotherapy resistance.Cancer Chemotherapy and Pharmacology 11/2014; 75(3). DOI:10.1007/s00280-014-2637-z · 2.57 Impact Factor
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ABSTRACT: A new family of 4-aminopyrido[2,3-d]pyrimidines active against non-Hodgkin's lymphomas (NHLs) is described. Among these compounds, 19 inhibits the most upstream tyrosine kinases in the B cell receptor (BCR) signaling pathway which are involved in the mature B cell neoplasms. Thus, 19 showed antiproliferative activity at 24 h and 48 h against a panel of 20 NHLs cell lines with GI50 ranging from 1.3 to 6.9 μM at 24 h, and 1.4-7.2 μM at 48 h, being this effect related to a significant (20-90%) inhibition of the phosphorylation of the BCR-related kinases Btk, Syk, and Lyn. Most importantly, 19 was able to induce a 63% reduction in Rec-1 cell proliferation, which was significantly greater than the 31% and 3% blockade of proliferation observed after cell treatment with R406, a Syk inhibitor, and ibrutinib, a Btk inhibitor, respectively. The computational blind docking and ligand binding within the pockets of Btk, Syk and Lyn kinases showed that compound 19 presents the same kind of interactions of described cocrystallized inhibitors.European Journal of Medicinal Chemistry 09/2014; 86C:664-675. DOI:10.1016/j.ejmech.2014.09.018 · 3.43 Impact Factor
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ABSTRACT: Brucea javanica oil emulsion (BJOE), the petroleum ether extract of B. javanica emulsified by phospholipid, is widely used in China as an anticancer agent. The extracts from B. javanica induce cancer cell death by various mechanisms; however, it is not known whether these mechanisms involve autophagy, which is an important process in cancer development and treatment. Thus, the current study aimed to investigate whether BJOE modulates autophagy in HCT116 human colon cancer cells and whether modulation of autophagy is an anticancer mechanism of BJOE. Immunoblotting was employed to analyze the protein expression levels of microtubule-associated protein light-chain 3 (LC3), a specific protein marker of autophagy, in HCT116 cancer cells following exposure to BJOE. The apoptosis rate of the HCT116 cancer cells was detected by performing an Annexin V-fluorescein isothiocyanate/propidium iodide assay. According to the effect of BJOE administration on autophagy in the HCT116 cancer cells (induction or suppression), a functionally opposite agent (autophagy suppressor or inducer) was applied to counteract this effect, and the apoptosis rate of the cancer cells was detected again. The role of autophagy (pro-survival or pro-death) was demonstrated by comparing the rates of apoptotic cancer cells prior to and following the counteraction. The results revealed that BJOE suppressed the protein expression levels of LC3, including the LC3-I and LC3-II forms, and induced apoptosis in the HCT116 cancer cells with a high level of basal LC3. The apoptosis-inducing activity of BJOE was significantly attenuated when autophagy was induced by the administration of trehalose, an autophagy inducer. The data indicates that autophagy inhibition is involved in BJOE-induced cancer cell death, and that this inhibition may be a potential anticancer mechanism of BJOE.Oncology letters 03/2015; 9(3):1425-1431. DOI:10.3892/ol.2015.2875 · 0.99 Impact Factor