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: The phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway promotes melanoma tumor growth and survival while suppressing autophagy, a catabolic process through which cells collect and recycle cellular components to sustain energy homeostasis in starvation. Conversely, inhibitors of the PI3K/AKT/mTOR pathway, in particular the mTOR inhibitor temsirolimus (CCI-779), induce autophagy, which can promote tumor survival and thus, these agents potentially limit their own efficacy. We hypothesized that inhibition of autophagy in combination with mTOR inhibition would block this tumor survival mechanism and hence improve the cytotoxicity of mTOR inhibitors in melanoma. Here we found that melanoma cell lines of multiple genotypes exhibit high basal levels of autophagy. Knockdown of expression of the essential autophagy gene product ATG7 resulted in cell death, indicating that survival of melanoma cells is autophagy-dependent. We also found that the lysosomotropic agent and autophagy inhibitor hydroxychloroquine (HCQ) synergizes with CCI-779 and led to melanoma cell death via apoptosis. Combination treatment with CCI-779 and HCQ suppressed melanoma growth and induced cell death both in 3-dimensional (3D) spheroid cultures and in tumor xenografts. These data suggest that coordinate inhibition of the mTOR and autophagy pathways promotes apoptosis and could be a new therapeutic paradigm for the treatment of melanoma.PLoS ONE 01/2013; 8(1):e55096. DOI:10.1371/journal.pone.0055096 · 3.53 Impact Factor
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ABSTRACT: Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase and a key element in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. Moreover, it is a negative regulator of autophagy and acts as a central regulator in cell growth. For the treatment of cancer, mTOR is a novel and validated therapeutic target. Previous studies have shown that Akt is frequently activated in nasopharyngeal carcinoma (NPC) tissues; thus, the inhibition of mTOR may be a treatment strategy for this tumor type. To evaluate the effect of the mTOR inhibitor RAD001 on NPC cell lines, we performed 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assays, lactate dehydrogenase (LDH) assays, western blotting and flow cytometry to evaluate the mechanisms of cell death. The growth of both CNE-1 and HONE-1 cells was inhibited in a time- and dose-dependent manner. CNE-1 was more sensitive, with a 50% growth inhibition (GI50) of 30.0±1.0 µM compared to HONE-1, cells which had a GI50 of 56.9±13.1 µM. RAD001 induced apoptosis and autophagy in both cell lines. RAD001 induced a significant increase in growth inhibition in the two cell lines when used in combination with the autophagy inhibitor, 3-methyladenine; however, the percentages of apoptotic cells decreased when RAD001 was combined with the caspase inhibitor, z-VAD-fmk. In conclusion, the main mechanism of the mTOR inhibitor RAD001 in these two NPC cells was apoptotic, not autophagic cell death. The combination of RAD001 with autophagy inhibitors may be a useful therapeutic strategy for nasopharyngeal carcinoma.International Journal of Molecular Medicine 04/2013; 31(4):904-12. DOI:10.3892/ijmm.2013.1282 · 1.88 Impact Factor
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ABSTRACT: Most patients with mantle cell lymphoma (MCL) relapse within a few years of treatment. Conventional agents provide little benefit, thus identification of new therapies is critical to improve patient outcomes. Temsirolimus, an inhibitor of mammalian target of rapamycin, is an effective, well-tolerated option authorized in Europe for treatment of patients with relapsed/refractory MCL. Intravenous temsirolimus has been extensively studied in MCL and has consistently demonstrated single-agent antitumor activity. In the pivotal phase III trial, treatment with temsirolimus 175 mg weekly for 3 weeks followed by 75 mg weekly (175/75 mg) resulted in significant improvements in progression-free survival (P = .0009) and objective response rate (P = .002) vs. investigator's choice of therapy. Hematologic toxicities (thrombocytopenia, neutropenia) were the principal grade 3/4 adverse events associated with temsirolimus 175/75 mg. Other toxicities included increases in serum cholesterol and triglycerides, hyperglycemia, fatigue, and dyspnea. Overall, the safety profile of temsirolimus is acceptable in this setting, and most toxicities are manageable with dose modification or medical intervention. Clinical studies of temsirolimus in relapsed or refractory MCL patients aim to clarify the optimal treatment schedule and to assess rational combinations with other therapeutic agents, such as rituximab or chemotherapy. Practical considerations are discussed for the clinical use of temsirolimus in patients with MCL.Clinical lymphoma, myeloma & leukemia 06/2013; 13(4). DOI:10.1016/j.clml.2013.04.003 · 2.02 Impact Factor