Multiple myeloma is one of numerous malignancies characterized by increased glucose consumption, a phenomenon with significant prognostic implications in this disease. Few studies have focused on elucidating the molecular underpinnings of glucose transporter (GLUT) activation in cancer, knowledge that could facilitate identification of promising therapeutic targets. To address this issue, we performed gene expression profiling studies involving myeloma cell lines and primary cells as well as normal lymphocytes to uncover deregulated GLUT family members in myeloma. Our data demonstrate that myeloma cells exhibit reliance on constitutively cell surface-localized GLUT4 for basal glucose consumption, maintenance of Mcl-1 expression, growth, and survival. We also establish that the activities of the enigmatic transporters GLUT8 and GLUT11 are required for proliferation and viability in myeloma, albeit because of functionalities probably distinct from whole-cell glucose supply. As proof of principle regarding the therapeutic potential of GLUT-targeted compounds, we include evidence of the antimyeloma effects elicited against both cell lines and primary cells by the FDAapproved HIV protease inhibitor ritonavir, which exerts a selective off-target inhibitory effect on GLUT4. Our work reveals critical roles for novel GLUT family members and highlights a therapeutic strategy entailing selective GLUT inhibition to specifically target aberrant glucose metabolism in cancer.
BackgroundMultiple myeloma (MM) is a fatal plasma cell malignancy exhibiting enhanced glucose consumption associated with an aerobic glycolytic phenotype (i.e., the Warburg effect). We have previously demonstrated that myeloma cells exhibit constitutive plasma membrane (PM) localization of GLUT4, consistent with the dependence of MM cells on this transporter for maintenance of glucose consumption rates, proliferative capacity, and viability. The purpose of this study was to investigate the molecular basis of constitutive GLUT4 plasma membrane localization in MM cells.FindingsWe have elucidated a novel mechanism through which myeloma cells achieve constitutive GLUT4 activation involving elevated expression of the Rab-GTPase activating protein AS160_v2 splice variant to promote the Warburg effect. AS160_v2-positive MM cell lines display constitutive Thr642 phosphorylation, known to be required for inactivation of AS160 Rab-GAP activity. Importantly, we show that enforced expression of AS160_v2 is required for GLUT4 PM translocation and activation in these select MM lines. Furthermore, we demonstrate that ectopic expression of a full-length, phospho-deficient AS160 mutant is sufficient to impair constitutive GLUT4 cell surface residence, which is characteristic of MM cells.ConclusionsThis is the first study to tie AS160 de-regulation to increased glucose consumption rates and the Warburg effect in cancer. Future studies investigating connections between the insulin/IGF-1/AS160_v2/GLUT4 axis and FDG-PET positivity in myeloma patients are warranted and could provide rationale for therapeutically targeting this pathway in MM patients with advanced disease.
The incurable plasma cell malignancy multiple myeloma (MM) is characterized by the progressive development of chemoresistance, thus warranting the design of new therapeutic strategies tailored to unique molecular mechanisms driving tumor propagation. One aspect of myeloma pathogenesis which has not been exploited therapeutically is an abnormal avidity for glucose. Inhibition of glucose metabolism has broad applicability in cancer and has demonstrated in vitro potency; unfortunately, previous attempts focusing on hexokinase inhibition have been largely unsuccessful. Targeting the upstream process of glucose transport has not been a major area of investigation due to the common association between GLUT1 and cancer and the importance of GLUT1 in many normal tissues. However, the specific glucose transporters responsible for maintaining the glycolytic phenotype in myeloma have not been identified, leaving open the possibility that family members other than GLUT1 play important roles in this cellular context. Therefore, we undertook an unbiased real time PCR-based screen of GLUT gene expression profiles in myeloma cell lines and control B lymphocytes. GLUTs 4, 8, and 11 exhibit widespread overexpression in MM cells with no significant difference noted in GLUT1 levels. Examination of microarray studies of MM patient samples and western blot analyses of MM cell lines and normal B cells confirm the upregulation of GLUT8 and GLUT11 in clinical specimens and at the protein level. However, we did not find further evidence for GLUT4 overexpression. After evaluating the subcellular localization of GLUT4 through microscopy, we determined that a substantial fraction of GLUT4 protein is constitutively mislocalized to the plasma membrane in myeloma cells, an event normally regulated by insulin in non-malignant tissues. Indeed, cell fractionation studies demonstrate a dramatic increase in plasma membrane GLUT4 content in MM cell lines relative to PBMC. Lentiviral transduction of GLUT4-targeted shRNA verifies the functional relevance of GLUT4 activity, as MM cells with reduced GLUT4 expression exhibit dramatic deficiencies in glucose transport and lactate production rates, which leads to growth inhibition and significant cell death. Similar studies with a GLUT1-targeted shRNA reveal comparable trends but less potent effects. Next, we investigated a class of HIV protease inhibitors which elicit off-target inhibitory effects on GLUT4 in vivo. Treatment with these compounds recapitulates the effects of GLUT4 knockdown, thus supporting the relevance of GLUT4 in myeloma. These studies represent the first time a prominent, functional role for GLUT4 has been demonstrated in any cancer and highlight a therapeutic strategy involving the repositioning of an FDA-approved class of drugs to target myeloma cell metabolism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1268. doi:10.1158/1538-7445.AM2011-1268
1850 Multiple Myeloma (MM) is an incurable heterogeneous B cell malignancy characterized by hyperdiploidy and/or aberrant chromosomal translocations warranting strategies that can target diverse molecular subtypes. One such unifying feature in MM entails an increased avidity for glucose that forms the basis for clinical imaging of myeloma by 18fluoro-deoxyglucose positron emission tomography. We are however not yet able to target abnormal glucose utilization for therapy. Given that glucose entry is a key rate-limiting step in glycolysis we performed an unbiased multi-cell line gene-expression profiling study combined with functional knock-down experiments to determine critical glucose transporters facilitating glucose entry in myeloma. These studies revealed a critical dependence of myeloma on the insulin-responsive glucose transporter GLUT4, and GLUTs 8 and 11. Our data demonstrate that myeloma cells exhibit a significant reliance on constitutively cell surface-localized GLUT4 for basal glucose consumption and viability. The functional significance of these transporters was further bolstered by detection of similar patterns of expression in myeloma patient samples. We also tested the therapeutic utility of targeting plasma-membrane localized GLUT4 in MM cell lines and patient samples with the FDA approved HIV protease inhibitor ritonavir that has an off-target inhibitory effect on GLUT4, providing proof of principle that GLUTs can be targeted for therapeutic benefit. In this study we have investigated the mechanism by which GLUT4 suppression elicits cell death in myeloma. GLUT4 contributes to a significant fraction of glucose entry in myeloma while GLUTs 8 and 11 appear to have a significant impact on myeloma cell viability relating to alternative enigmatic subcellular functions not reliant on glucose entry per se. To further elucidate the mechanisms associated with cell death upon glucose deprivation in response to GLUT4 suppression, we examined expression levels of key apoptotic effectors in three myeloma cell lines expressing GLUT 1, 4, 8 or 11 shRNAs. Suppression of GLUT4 appears to selectively engage an apoptotic cascade involving suppression of MCL-1 and BCL-xL in addition to an increase in pro-apoptotic BAX expression. We do not detect up-regulation of PUMA or NOXA that have previously been demonstrated to play a role in glucose-deprivation induced cell death in other cell types. The suppression of MCL-1 is associated with an upstream activation of GSK-3. Expression of an MCL-1 ubiquitination-resistant mutant prevents reduction of MCL-1 protein upon GLUT4 knockdown, and reverses the toxicity elicited by GLUT4 suppression. In addition we do not detect the appearance of the cleaved pro-apoptotic fragment of MCL-1. These observations suggest glucose-deprivation induced cell death in response to GLUT4 reduction is mediated by ubiquitination and proteasomal degradation of MCL-1. In sum we have discovered a novel myeloma specific method to target glucose entry and elicit apoptosis via suppression of MCL-1 expression. MCL-1 is a key effector of chemo-resistance in MM with levels of expression correlating to disease severity. Therefore strategies that can target MCL-1 will have significant utility in treatment of this fatal malignancy. These observations provide further rationale for the development of GLUT4-specific biologics to target aberrant glucose metabolism in myeloma and potentially other glucose-driven cancers. Disclosures: No relevant conflicts of interest to declare.
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