Autophagy inhibition and antimalarials promote cell death in gastrointestinal stromal tumor (GIST)

Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 08/2010; 107(32):14333-8. DOI: 10.1073/pnas.1000248107
Source: PubMed


Although gastrointestinal stromal tumors (GISTs) harboring activating KIT or platelet-derived growth factor receptor A (PDGFRA) mutations respond to treatment with targeted KIT/PDGFRA inhibitors such as imatinib mesylate, these treatments are rarely curative. Most often, a sizeable tumor cell subpopulation survives and remains quiescent for years, eventually resulting in acquired resistance and treatment failure. Here, we report that imatinib induces autophagy as a survival pathway in quiescent GIST cells. Inhibiting autophagy, using RNAi-mediated silencing of autophagy regulators (ATGs) or antimalarial lysosomotrophic agents, promotes the death of GIST cells both in vitro and in vivo. Thus, combining imatinib with autophagy inhibition represents a potentially valuable strategy to promote GIST cytotoxicity and to diminish both cellular quiescence and acquired resistance in GIST patients.

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Available from: Maria Debiec-Rychter
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    • "However, when we focused on imatinib-induced cell cycle arrest and apoptosis, we observed that cotreatment of imatinib and lysosomotropic NH 4 Cl resulted in increased apoptosis. This phenomenon has been reported before in GIST (Gupta et al., 2010) and CML (Bellodi et al., 2009). The explanation may be that imatinib induces autophagy (Ertmer et al., 2007) as a survival mechanism. "
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    ABSTRACT: The intracellular uptake and retention (IUR) of imatinib is reported to be controlled by the influx transporter SLC22A1 (OCT1). We recently hypothesized that alternative uptake and/or retention mechanisms exist that determine intracellular imatinib levels. Here we systematically investigate the nature of these mechanisms. Imatinib uptake in cells was quantitatively determined by LC-MS-MS. Fluorescent microscopy was used to establish subcellular localization of imatinib. Immunoblotting, cell cycle analyses and apoptosis assays were done to evaluate functional consequences of imatinib sequestration. Uptake experiments revealed high intracellular imatinib concentrations in HEK293, the leukemic cell lines K562, SD-1, and a gastrointestinal stromal tumor cell line GIST-T1. We demonstrated that imatinib IUR is time, dose, temperature and energy dependent and provide evidence that SLC22A1 and other potential imatinib transporters do not substantially contribute to the IUR of imatinib. Prazosin, amantadine, NH4Cl and the V-ATPase inhibitor bafilomycin A1 significantly decreased the IUR of imatinib and likely interfere with lysosomal retention and accumulation of imatinib. Co-staining experiments with Lysotracker Red confirmed lysosomal sequestration of imatinib. Inhibition of the lysosomal sequestration had no effect on the inhibition of c-Kit signaling and imatinib mediated cell cycle arrest but significantly increased apoptosis in imatinib sensitive GIST-T1 cells. We conclude that intracellular imatinib levels are primarily determined by lysosomal sequestration and do not depend on SLC22A1 expression. The American Society for Pharmacology and Experimental Therapeutics.
    Full-text · Article · Jun 2015 · Molecular pharmacology
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    • "Cells with stable expression of GFP-mCherry-LC3 were grown overnight before treatment and fixed with 2% paraformaldehyde, washed several times with PBS, mounted using Vectashield, and analyzed using an HCX Plan Apo 63×/1.4N.A. oil immersion objective lens on a Leica TCS-SP2 confocal microscope (Leica Microsystems AG). LC3B puncta were quantified using the Red and Green Puncta Co-localization Macro with the Image J program, as described [29,30]. "
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    ABSTRACT: Macroautophagy is a catabolic process that can mediate cell death or survival. Apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment (TR) is known to induce autophagy. Here we investigated whether SQSTM1/p62 (p62) overexpression, as a marker of autophagic flux, was related to aggressiveness of human prostate cancer (PCa) and whether autophagy regulated the treatment response in sensitive but not resistant PCa cell lines. Immunostaining and immunoblotting analyses of the autophagic markers p62 [in PCa tissue microarrays (TMAs) and PCa cell lines] and LC3 (in PCa cell lines), transmission electron microscopy, and GFP-mCherry-LC3 were used to study autophagy induction and flux. The effect of autophagy inhibition using pharmacologic (3-methyladenine and chloroquine) and genetic [(short hairpin (sh)-mediated knock-down of ATG7 and LAMP2) and small interfering (si)RNA-mediated BECN1 knock-down] approaches on TR-induced cell death was assessed by clonogenic survival, sub-G1 DNA content, and annexinV/PI staining by flow cytometry. Caspase-8 activation was determined by immunoblotting. We found that increased cytoplasmic expression of p62 was associated with high-grade PCa, indicating that autophagy signaling might be important for survival in high-grade tumors. TR-resistant cells exhibited high autophagic flux, with more efficient clearance of p62-aggregates in four TR-resistant PCa cell lines: C4-2, LNCaP, DU145, and CWRv22.1. In contrast, autophagic flux was low in TR-sensitive PC3 cells, leading to accumulation of p62-aggregates. Pharmacologic (chloroquine or 3-methyladenine) and genetic (shATG7 or shLAMP2) inhibition of autophagy led to cell death in TR-resistant C4-2 cells. shATG7-expressing PC3 cells, were less sensitive to TR-induced cell death whereas shLAMP2-expressing were as sensitive as shControl-expressing PC3 cells. Inhibition of autophagic flux using chloroquine prevented clearance of p62 aggregates, leading to caspase-8 activation and cell death in C4-2 cells. In PC3 cells, inhibition of autophagy induction prevented p62 accumulation and hence caspase-8 activation. We show that the p62 overexpression correlates with advanced stage human PCa. Pharmacologic and genetic inhibition of autophagy in PCa cell lines indicate that autophagic flux can determine the cellular response to TR by regulating caspase-8 activation. Thus, combining various autophagic inhibitors may have a differential impact on TR-induced cell death.
    Full-text · Article · Mar 2014 · Molecular Cancer
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    • "We postulated that the metabolic stress caused by PFKFB3 inhibition might activate autophagy as a survival pathway, which in turn might confer resistance to 3PO. Chloroquine (CQ), an anti-malarial agent that has been used in humans since the 1940’s, has been shown to inhibit autophagy and potentiate cancer cell death and is now being added to a number of other drugs as a part of several human cancer clinical trials [21-26]. We hypothesized that the combination of the PFKFB3 inhibitor 3PO with the autophagy inhibitor CQ might lead to a significant improvement in the anti-cancer effects of 3PO in vitro and that this combination might also increase efficacy of 3PO as an anti-tumor agent in vivo. "
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    ABSTRACT: Unlike glycolytic enzymes that directly catabolize glucose to pyruvate, the family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFBs) control the conversion of fructose-6-phosphate to and from fructose-2,6-bisphosphate, a key regulator of the glycolytic enzyme phosphofructokinase-1 (PFK-1). One family member, PFKFB3, has been shown to be highly expressed and activated in human cancer cells, and derivatives of a PFKFB3 inhibitor, (3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), are currently being developed in clinical trials. However, the effectiveness of drugs such as 3PO that target energetic pathways is limited by survival pathways that can be activated by reduced ATP and nutrient uptake. One such pathway is the process of cellular self-catabolism termed autophagy. We hypothesized that the functional glucose starvation induced by inhibition of PFKFB3 in tumor cells would induce autophagy as a pro-survival mechanism and that inhibitors of autophagy could increase the anti-tumor effects of PFKFB3 inhibitors. We found that selective inhibition of PFKFB3 with either siRNA transfection or 3PO in HCT-116 colon adenocarcinoma cells caused a marked decrease in glucose uptake simultaneously with an increase in autophagy based on LC3-II and p62 protein expression, acridine orange fluorescence of acidic vacuoles and electron microscopic detection of autophagosomes. The induction of autophagy caused by PFKFB3 inhibition required an increase in reactive oxygen species since N-acetyl-cysteine blocked both the conversion of LC3-I to LC3-II and the increase in acridine orange fluorescence in acidic vesicles after exposure of HCT-116 cells to 3PO. We speculated that the induction of autophagy might protect cells from the pro-apoptotic effects of 3PO and found that agents that disrupt autophagy, including chloroquine, increased 3PO-induced apoptosis as measured by double staining with Annexin V and propidium iodide in both HCT-116 cells and Lewis lung carcinoma (LLC) cells. Chloroquine also increased the anti-growth effect of 3PO against LLCs in vivo and resulted in an increase in apoptotic cells within the tumors. We conclude that PFKFB3 inhibitors suppress glucose uptake, which in turn causes an increase in autophagy. The addition of selective inhibitors of autophagy to 3PO and its more potent derivatives may prove useful as rational combinations for the treatment of cancer.
    Full-text · Article · Jan 2014
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