GLUT1 Inhibitor Research Articles

Abstract 3594: GLUT1 as a metabolic target in glioblastoma therapy

Abstract Glioblastoma (GBM) is an aggressive diffuse glioma with poor prognosis due to lack of sound therapeutic options. As in many aggressive tumors, GBMs create a hypoxic microenvironment and switch to glycolytic metabolism from oxidative phosphorylation in what is known as the Warburg Effect. Here, we hypothesize that strategically inhibiting influx of glucose via targeting GLUT1 may reverse the Warburg Effect and selectively slow tumor progression. Nanostring data suggests an aberrant metabolic clincial profile in our cohort of GBM patients. Datamining studies and immunoblot analysis were conducted on these same human GBM specimens to demonstrate upregulated GLUT1. Mass spectrometric analysis was performed on GLUT1 precipitated from mesenchymal subtype patient-derived xenograft (PDX). Zonula Occludens adhesion studies were carried out to determine GLUT1 inhibition's effects on cell-cell adhesion structures. Finally, ATP, lactate release, and glucose uptake assays were performed on PDX and GLUT1-silenced PDX to demonstrate a normalized metabolic phenotype when GLUT1 is silenced. Our work suggests that GLUT1 overexpression plays a major role in the metabolic profile of mesenchymal subtype GBM, and that targeting its overexpression could prove effective in reversing the Warburg Effect in GBM cells and ultimately improving patient outcomes. Citation Format: Collin M. Labak, Maheedhara R. Guda, Neha Jain, Chase P. Smith, Charles P. Cain, Andrew J. Tsung, Kiran K. Velpula. GLUT1 as a metabolic target in glioblastoma therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3594.

Phloretin Suppresses Bone Morphogenetic Protein-2-Induced Osteoblastogenesis and Mineralization via Inhibition of Phosphatidylinositol 3-kinases/Akt Pathway.

Phloretin has pleiotropic effects, including glucose transporter (GLUT) inhibition. We previously showed that phloretin promoted adipogenesis of bone marrow stromal cell (BMSC) line ST2 independently of GLUT1 inhibition. This study investigated the effect of phloretin on osteoblastogenesis of ST2 cells and osteoblastic MC3T3-E1 cells. Treatment with 10 to 100 µM phloretin suppressed mineralization and expression of osteoblast differentiation markers, such as alkaline phosphatase (ALP), osteocalcin (OCN), type 1 collagen, runt-related transcription factor 2 (Runx2), and osterix (Osx), while increased adipogenic markers, peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), fatty acid-binding protein 4, and adiponectin. Phloretin also inhibited mineralization and decreased osteoblast differentiation markers of MC3T3-E1 cells. Phloretin suppressed phosphorylation of Akt in ST2 cells. In addition, treatment with a phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor, LY294002, suppressed the mineralization and the expression of osteoblast differentiation markers other than ALP. GLUT1 silencing by siRNA did not affect mineralization, although it decreased the expression of OCN and increased the expression of ALP, Runx2, and Osx. The effects of GLUT1 silencing on osteoblast differentiation markers and mineralization were inconsistent with those of phloretin. Taken together, these findings suggest that phloretin suppressed osteoblastogenesis of ST2 and MC3T3-E1 cells by inhibiting the PI3K/Akt pathway, suggesting that the effects of phloretin may not be associated with glucose uptake inhibition.

A chemical toolbox for the study of bromodomains and epigenetic signaling

Bromodomains (BRDs) are conserved protein interaction modules which recognize (read) acetyl-lysine modifications, however their role(s) in regulating cellular states and their potential as targets for the development of targeted treatment strategies is poorly understood. Here we present a set of 25 chemical probes, selective small molecule inhibitors, covering 29 human bromodomain targets. We comprehensively evaluate the selectivity of this probe-set using BROMOscan and demonstrate the utility of the set identifying roles of BRDs in cellular processes and potential translational applications. For instance, we discovered crosstalk between histone acetylation and the glycolytic pathway resulting in a vulnerability of breast cancer cell lines under conditions of glucose deprivation or GLUT1 inhibition to inhibition of BRPF2/3 BRDs. This chemical probe-set will serve as a resource for future applications in the discovery of new physiological roles of bromodomain proteins in normal and disease states, and as a toolset for bromodomain target validation.

Epigenetic loss of the endoplasmic reticulum-associated degradation inhibitor SVIP induces cancer cell metabolic reprogramming.

The endoplasmic reticulum (ER) of cancer cells needs to adapt to the enhanced proteotoxic stress associated with the accumulation of unfolded, misfolded and transformation-associated proteins. One way by which tumors thrive in the context of ER stress is by promoting ER-Associated Degradation (ERAD), although the mechanisms are poorly understood. Here, we show that the Small p97/VCP Interacting Protein (SVIP), an endogenous inhibitor of ERAD, undergoes DNA hypermethylation-associated silencing in tumorigenesis to achieve this goal. SVIP exhibits tumor suppressor features and its recovery is associated with increased ER stress and growth inhibition. Proteomic and metabolomic analyses show that cancer cells with epigenetic loss of SVIP are depleted in mitochondrial enzymes and oxidative respiration activity. This phenotype is reverted upon SVIP restoration. The dependence of SVIP hypermethylated cancer cells on aerobic glycolysis and glucose was also associated with sensitivity to an inhibitor of the glucose transporter GLUT1. This could be relevant to the management of tumors carrying SVIP epigenetic loss, because these occur in high-risk patients who manifest poor clinical outcomes. Overall, our study provides insights into how epigenetics helps deal with ER stress and how SVIP epigenetic loss in cancer may be amenable to therapies that target glucose transporters.

The promise of GLUT1 inhibitors in preventing diabetes induced microvasculopathy

PurposeDiabetes induced microvasculopathy (DIM) is a leading cause of atherosclerosis, heart failure, stroke, blindness, and renal failure. Current therapeutic interventions still heavily rely on controlling systemic hyperglycemia. Yet, many diabetic patients develop microvasculopathies despite having good glycemic control. Endothelial activation is a common underlying causative factor; however, the molecular basis of such activation remains a big gap in our knowledge. Aberrant glucose transport and metabolism by endothelial cells are attracting great interest as disease‐related mechanisms with little attention in the context of DIM. In this study we aimed to evaluate the pre‐clinical efficacy of the glucose transporter (GLUT)‐1 inhibitor, which is a derivative of Fluoro‐catechol ester of 3‐Hydroxy‐benzoic acid (FCEHBA), to halt microvascular dysfunctions induced by diabetes.MethodsHuman retinal endothelial cells (HRECs) were used and treated with high glucose (HG, 30 mM) or osmotic control with or without low oxygen (1%O2) in presence or absence of FCEHBA (10 mM) or Vehicle (DMSO). Barrier function was assessed by Electric Cell‐substrate Impedance Sensing (ECIS) and immunofluorescence organization of Zonula Occluden‐1 (ZO‐1). The angiogenic potential of HRECs was evaluated by migration; sprouting and tube formation. Group differences were evaluated by ANOVA followed by Tukey Posthoc test.Results(1) Increased glucose influx in HRECs through the increased expression of GLUT1 and its translocation from the cytosol to the plasma membrane have been observed in response to simultaneous insults of diabetes and hypoxia. (2) The increase in glucose influx via recruitment of GLUT1 to the plasma membrane was associated with (a) an accelerated breakdown of HREC barrier integrity, indicated by the significant drop in transendothelial electric resistance (TER); (b) a marked disruption of ZO‐1, a key tight junction protein (TJP) regulating barrier integrity in HRECs; (c) a dramatic enhancement of HRECs migration; and (d) an induction of profound HREC neovascularization and tube formation. (3) Importantly, FCEHBA was able to remarkably restore the drop in barrier function induced by both diabetes and hypoxia and to reverse their disruptive effects on ZO‐1 integrity. (4) FCEHBA was also able to effectively mitigate angiogenic effects of both diabetes and hypoxia on HRECs, including increased cell migration and tube formation, without affecting cell viability.ConclusionTo our knowledge, this study is the first to report that FCEHBA protects against High Glucose/Hypoxia‐induced microvasculopathy. This finding may lead to a new therapeutic approach that could be clinically translated to not only patients with diabetic retinopathy but also those with other related microvascular conditions including atherosclerosis, stroke, and cardiovascular diseases.Support or Funding InformationAmerican Heart Association Grant 18CDA34080403 (ASI) and the National Eye Institute grant R01EY023315 (MA).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Apigenin Combined With Gefitinib Blocks Autophagy Flux and Induces Apoptotic Cell Death Through Inhibition of HIF-1α, c-Myc, p-EGFR, and Glucose Metabolism in EGFR L858R+T790M-Mutated H1975 Cells.

Cancer cells are characterized by abnormally increased glucose uptake and active bio-energy and biosynthesis to support the proliferation, metastasis, and drug resistant survival. We examined the therapeutic value of the combination of apigenin (a natural small-molecule inhibitor of Glut1 belonging to the flavonoid family) and gefitinib on epidermal growth factor receptor (EGFR)-resistant mutant non-small cell lung cancer, to notably damage glucose utilization and thus suppress cell growth and malignant behavior. Here, we demonstrate that apigenin combined with gefitinib inhibits multiple oncogenic drivers such as c-Myc, HIF-1α, and EGFR, reduces Gluts and MCT1 protein expression, and inactivates the 5′ adenosine monophosphate-activated protein kinase (AMPK) signaling, which regulates glucose uptake and maintains energy metabolism, leading to impaired energy utilization in EGFR L858R-T790M-mutated H1975 lung cancer cells. H1975 cells exhibit dysregulated metabolism and apoptotic cell death following treatment with apigenin + gefitinib. Therefore, the combined apigenin + gefitinib treatment presents an attractive strategy as alternative treatment for the acquired resistance to EGFR-TKIs in NSCLC.

The Biguanides Metformin and Buformin in Combination with 2-Deoxy-glucose or WZB-117 Inhibit the Viability of Highly Resistant Human Lung Cancer Cells.

The biguanides metformin (MET) and to a lesser extent buformin (BUF) have recently been shown to exert anticancer effects. In particular, MET targets cancer stem cells (CSCs) in a variety of cancer types but these compounds have not been extensively tested for combination therapy. In this study, we investigated in vitro the anticancer activity of MET and BUF alone or in combination with 2-deoxy-D-glucose (2-DG) and WZB-117 (WZB), which are a glycolysis and a GLUT-1 inhibitor, respectively, in H460 human lung cancer cells growing under three different culture conditions with varying degrees of stemness: (1) routine culture conditions (RCCs), (2) floating lung tumorspheres (LTSs) that are enriched for stem-like cancer cells, and (3) adherent cells under prolonged periods (8-12 days) of serum starvation (PPSS). These cells are highly resistant to conventional anticancer drugs such as paclitaxel, hydroxyurea, and colchicine and display an increased level of stemness markers. As single agents, MET, BUF, 2-DG, and WZB-117 potently inhibited the viability of cells growing under RCCs. Both MET and BUF showed a strong synergistic effect when used in combination with 2-DG. A weak potentiation was observed when used with WZB-117. Under RCCs, H460 cells were more sensitive to MET and BUF and WZB-117 compared to nontumorigenic Beas-2B cells. While LTSs were less sensitive to each single drug, both MET and BUF in combination with 2-DG showed a strong synergistic effect and reduced cell viability to similar levels compared to the parental H460 cells. Adherent cells growing under PPSS were also less sensitive to each single drug, and MET and BUF showed a strong synergistic effect on cell viability in combination with 2-DG. Overall, our data demonstrates that the combination of BGs with either 2-DG or WZB-117 has “broad-spectrum” anticancer activities targeting cells growing under a variety of cell culture conditions with varying degrees of stemness. These properties may be useful to overcome the chemoresistance due to intratumoral heterogeneity found in lung cancer.

Involvement of GLUT1-mediated glucose transport and metabolism in gefitinib resistance of non-small-cell lung cancer cells.

Use of epidermal growth factor receptor (EGFR) inhibitors represented by gefitinib and erlotinib has become the standard of treatment for non-small-cell lung cancers (NSCLCs) with activating EGFR mutations. However, the majority of NSCLCs, which overexpress EGFR without such mutations, are resistant to EGFR inhibitors, and the mechanism(s) behind such primary resistance of NSCLCs without activating EGFR mutations to EGFR inhibitors still remains poorly understood. Here in this study, we show that glucose metabolism mediated by GLUT1, a facilitative glucose transporter, is involved in gefitinib resistance of NSCLC cells. We found that GLUT1 expression and glucose uptake were increased in resistant NSCLC cells after gefitinib treatment and that genetic as well as pharmacological inhibition of GLUT1 sensitized not only NSCLC cells with primary resistance but also those with acquired resistance to gefitinib. In vivo, the combination of systemic gefitinib and a GLUT1 inhibitor, both of which failed to inhibit tumor growth when administered alone, significantly inhibited the growth of xenograft tumors formed by the implantation of NSCLC cells with wild-type EGFR (wt-EGFR). Since our data indicated that GLUT1 was similarly involved in erlotinib resistance, our findings suggest that the activity of GLUT1-mediated glucose metabolism could be a critical determinant for the sensitivity of NSCLC cells to EGFR inhibitors and that concurrent GLUT1 inhibition may therefore be a mechanism-based approach to treating NSCLCs resistant to EGFR inhibitors, including those with wt-EGFR.

Abstract 4922: The combination of MK-2206 and WZB117 exerts a synergistic cytotoxic effect against breast cancer cells

Abstract Breast cancer is the most commonly diagnosed cancer and the second leading cause of cancer death in women. Hormone receptor-positive breast cancer is usually subjected to hormone therapy, while triple-negative breast cancer is more formidable and poses a therapeutic challenge. The PI3K/Akt/mTOR signaling pathway plays a crucial role in cancer cell growth, survival, and metabolism. It has been demonstrated that MK-2206, a potent allosteric Akt inhibitor, is cytotoxic against many cancer cell lines and is currently in clinical development. Cancer cells consume more glucose and rely on glycolysis for energy production even in the presence of abundant oxygen, a phenomenon called the Warburg effect. Therefore, glucose transporters responsible for delivering glucose into cells have become targets for the development of anticancer drugs. In search for anticancer agents whose effect could be enhanced by a GLUT1 inhibitor WZB117, we found that MK-2206, when combined with WZB117, showed a synergistic effect on growth inhibition of breast cancer cells, including ER(+) MCF-7 cells and triple-negative MDA-MB-231 cells. The combination index values at 50% growth inhibition were 0.45 and 0.21, respectively. Western blot analysis of proteins involved in the Akt/mTOR pathway revealed that Akt phosphorylation was markedly suppressed in both cell lines by MK-2206 and WZB117 treatment. Phosphorylation of mTOR and its downstream effectors p70S6K and 4E-BP-1 was also dramatically downregulated in MCF-7 but the effect was not as significant in MDA-MB-231 cells, suggesting that other mechanisms might also contribute to cytotoxicity induced by MK-2206 and WZB117. Further investigation revealed that the combination of MK-2206 and WZB117 induced γ-H2AX, a DNA damage marker, in both cell lines. Results from the comet assay confirmed that MK-2206 and WZB117 induced DNA damage. Protein levels of Rad51 and Ku80, participating in homologous recombination and non-homologous end joining repair respectively, were also decreased in MCF-7 cells, suggesting that MK-2206 and WZB117 may not only cause DNA damage, but also impair DNA repair. Furthermore, MK-2206 or WZB117 alone significantly induced ROS, and the combination of both further increased the ROS level within a short time period. In conclusion, this new finding indicates that MK-2206 and WZB117 may exert a synergistic cytotoxic effect in both ER(+) MCF-7 and triple-negative MDA-MB-231 breast cancer cells via ROS induction, which may in turn cause DNA damage. The combination may also compromise DNA damage repair, and ultimately lead to cell death. This finding may have clinical implications. Citation Format: Yu-Liang Li, Hao-Cheng Weng, Lih-Ching Hsu. The combination of MK-2206 and WZB117 exerts a synergistic cytotoxic effect against breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4922.

Abstract 2799: New-generation glucose transporter inhibitors targeting non-small cell lung cancer and triple-negative breast cancer

Abstract Cancer is a top killer second only to cardiovascular diseases. Cellular metabolic reprogramming and altered energetics are key features of cancer. Opportunistic uptake of extracellular nutrients has recently been named as an emerging hallmark of cancer metabolism. Glucose is a key nutrient taken in by cancer cells to meet their high energy and biosynthesis needs. Drastic upregulation of glucose uptake and glycolytic activity, a phenomenon called the Warburg effect, is prevalent in most cancers, including non-small cell lung cancer (NSCLC) and triple-negative breast cancer (TNBC). Cancer cells overexpress specific glucose transporters (GLUTs), particularly GLUT1, on their membranes to transport glucose via facilitated diffusion. Considering the addiction of cancer cells for glucose and their sensitivity and vulnerability to changes in glucose supply, GLUTs, especially GLUT1, have been recognized as an attractive anticancer target. We previously reported the identification of first-generation lead GLUT1 inhibitor WZB117. WZB117 treatment of human NSCLC A549 cells resulted in inhibition of glucose uptake, glycolysis and induction of apoptosis and necrosis (1). In addition, WZB-117 reduced tumor growth by more than 70% in an A549 tumor mouse model (1). Through a structure activity relationship (SAR) study of WZB117, a second-generation lead, DRB18, has been identified. DRB-18 is much more stable and 5 to 10 times more potent than WZB117 in inhibiting growth and proliferation in more than 90% of the 60 different cancer cell lines in 9 major cancer types as revealed by NCI-60 screenings. The compound shows particularly higher potency against cell lines with more metastatic potential such as HOP92 (NSCLC) and MDA-MB-231 (TNBC) with IC50 values in high nM range. DRB-18 also demonstrates a favorable liver microsomal metabolic profile and a maximal tolerance dose (MTD). All these indicate that DRB-18 is a potential therapeutic candidate for treating NSCLC and TNBC. Reference: 1. Liu Y, Cao Y, Zhang W, et al. A small-molecule inhibitor of glucose transporter 1 downregulates glycolysis, induces cell-cycle arrest, and inhibits cancer cell growth in vitro and in vivo. Mol Cancer Ther 2012;11(8):1672-82. Citation Format: Pratik Shriwas, Yanrong Qian, Xuan Wang, Dennis Roberts, Stephen Bergmeier, Xiaozhuo Chen. New-generation glucose transporter inhibitors targeting non-small cell lung cancer and triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2799.

Isoform-Selective Targeting of Facilitative Glucose Transporters with Small Molecule Inhibitors

There are currently 14 known mammalian facilitative glucose transporters (GLUTs) that serve a variety of critical roles in maintaining glucose homeostasis. The ability to potently target glucose transport in an isoform-selective manner is of paramount importance to investigate the normal function of these transporter isoforms and their individual contributions to disease pathogenesis. A number of GLUT isoform-selective small molecule inhibitors have been identified. To investigate the structural basis of GLUT inhibition and facilitate further refinement of the efficacy and selectivity of these antagonists, we performed kinetic analysis of the effects of WBZ-117, C-20, BAY-876 and several HIV protease inhibitors, on 3H-2-deoxyglucose uptake into cell lines selectively over-expressing GLUT1, GLUT4 or a T30I H160N GLUT1 mutant transporter. Several important findings were made: 1) based on Ki(app) measurements, indinavir, lopinavir, WZB-117 and C-20 selectively inhibit GLUT4 13-55 fold vs. GLUT1. Although structurally divergent, the potency of these 4 compounds against GLUT1 can be markedly enhanced to levels approximating those for GLUT4 by substituting just two GLUT1 amino acids located in the glucose permeation pathway to those found in GLUT4 (T30I and H160N); 2) nelfinavir is a weak GLUT4 inhibitor but acts as a GLUT1 activator, increasing transport activity 1.4-fold; 3) saquinavir selectively inhibits GLUT4 and the T30I H160N GLUT1 mutant, and exhibits complex kinetic patterns in the mutant cell line consistent with a two site model for partial inhibition; 4) kinetic analysis revealed that selective GLUT1 inhibitor, BAY-876, is a noncompetitive inhibitor of this transporter. Taken together, these data provide key structural insights into the ability of small molecules to alter GLUT function that will aid in the development of novel isoform-selective drugs for glucose-dependent diseases. Disclosure M. Heitmeier: None. R.C. Hresko: None. M. Shanmugam: None. P.W. Hruz: None.

Expression of glycolytic enzymes in ovarian cancers and evaluation of the glycolytic pathway as a strategy for ovarian cancer treatment

BackgroundNovel therapeutic approaches are required to treat ovarian cancer and dependency on glycolysis may provide new targets for treatment. This study sought to investigate the variation of expression of molecular components (GLUT1, HKII, PKM2, LDHA) of the glycolytic pathway in ovarian cancers and the effectiveness of targeting this pathway in ovarian cancer cell lines with inhibitors.MethodsExpression of GLUT1, HKII, PKM2, LDHA were analysed by quantitative immunofluorescence in a tissue microarray (TMA) analysis of 380 ovarian cancers and associations with clinicopathological features were sought. The effect of glycolysis pathway inhibitors on the growth of a panel of ovarian cancer cell lines was assessed by use of the SRB proliferation assay. Combination studies were undertaken combining these inhibitors with cytotoxic agents.ResultsMean expression levels of GLUT1 and HKII were higher in high grade serous ovarian cancer (HGSOC), the most frequently occurring subtype, than in non-HGSOC. GLUT1 expression was also significantly higher in advanced stage (III/IV) ovarian cancer than early stage (I/II) disease. Growth dependency of ovarian cancer cells on glucose was demonstrated in a panel of ovarian cancer cell lines. Inhibitors of the glycolytic pathway (STF31, IOM-1190, 3PO and oxamic acid) attenuated cell proliferation in platinum-sensitive and platinum-resistant HGSOC cell line models in a concentration dependent manner. In combination with either cisplatin or paclitaxel, 3PO (a novel PFKFB3 inhibitor) enhanced the cytotoxic effect in both platinum sensitive and platinum resistant ovarian cancer cells. Furthermore, synergy was identified between STF31 (a novel GLUT1 inhibitor) or oxamic acid (an LDH inhibitor) when combined with metformin, an inhibitor of oxidative phosphorylation, resulting in marked inhibition of ovarian cancer cell growth.ConclusionsThe findings of this study provide further support for targeting the glycolytic pathway in ovarian cancer and several useful combinations were identified.

Quercetin inhibits glucose transport by binding to an exofacial site on GLUT1

Quercetin, a common dietary flavone, is a competitive inhibitor of glucose uptake and is also thought to be transported into cells by GLUT1. In this study, we confirm that quercetin is a competitive inhibitor of GLUT1 and also demonstrate that newly synthesized compounds, WZB-117 and BAY-876 are robust inhibitors of GLUT1 in L929 cells. To measure quercetin interaction with L929 cells, we develop a new fluorescent assay using flow cytometry. The binding of quercetin and its inhibitory effects on 2-deoxyglucose (2DG) uptake showed nearly identical dose dependent effects, with both having maximum effects between 50 and 100 μM and similar half maximum effects at 8.9 and 8.5 μM respectively. The interaction of quercetin was rapid with t1/2 of 54 s and the onset and loss of its inhibitory effects on 2DG uptake were equally fast. This suggests that either quercetin is simply binding to surface GLUT1 or its transport in and out of the cell reaches equilibrium very quickly. If quercetin is transported, the co-incubation of quercetin with other glucose inhibitors should block quercetin uptake. However, we observed that WZB-117, an exofacial binding inhibitor of GLUT1 reduced quercetin interaction, while cytochalasin B, an endofacial binding inhibitor, enhanced quercetin interaction, and BAY-876 had no effect on quercetin interaction. Taken together, these data are more consistent with quercetin simply binding to GLUT1, but not actually being transported into L929 cells via the glucose channel in GLUT1.

Differential glucose requirement in skin homeostasis and injury identifies a therapeutic target for psoriasis.

Proliferating cells depend on glucose uptake more than quiescent cells for their growth. While glucose transport in keratinocytes is mediated largely by the Glut1 facilitative transporter, the keratinocyte-specific ablation of Glut1 did not compromise mouse skin development and homeostasis. Ex vivo metabolic profiling revealed altered sphingolipid, hexose, amino acid, and nucleotide metabolism in Glut1-deficient keratinocytes, suggesting metabolic adaptation. On the other hand, Glut1-deficient keratinocytes in culture displayed metabolic and oxidative stress and impaired proliferation. Similarly, Glut1 deficiency impaired in vivo keratinocyte proliferation and migration within wounded or UV-damaged mouse skin. Notably, both genetic and pharmacological Glut1 inactivation reduced hyperplasia in mouse models of psoriasis-like disease. Topical application of a Glut1 inhibitor also reduced inflammation in these models. Glut1 inhibition decreased expression of pathology-associated genes in human psoriatic skin organoids. Thus, Glut1 is selectively required for injury- and inflammation-associated keratinocyte proliferation, and its inhibition offers a novel treatment strategy for psoriasis.

Abstract B56: Potential biomarkers for personalized oncology radiation in uterine cervical cancer

Abstract Uterine cervical cancer (UCC) is one of the most prevalent malignant neoplasms in the world. UCC develops beyond the stage in situ and is frequently treated by a combination of intracavitary radiation therapy and external beam radiation therapy; 30-40% of patients with similar prognosis factors do not respond equally to a comparable standard treatment. Therefore, the study and identification of prognostic biomarkers, which indicate the probable course of the disease in an untreated individual, and predictive biomarkers, which allow identification of subpopulations of patients most likely to respond to a given therapy, would be extremely useful in the selection of patients for the development of innovative and effective therapies for locally advanced, metastatic, and refractory uterine cervical cancer. Within work that we have been developing, we reported that gene expression of IGF1R is a strong predictive marker for lack of response to radiotherapy (p=0.018, 95% CI (1.7-41.2)), and patients HPV16 (+) have 28.6 times higher risk of failure treatment; consequently, with the present study, we proposed to determine whether expression of IGF-IR, GAPDH, HIF-1 alpha, Survivin, GLUT1, CAIX, HKII, and clinic-pathologic parameters can be used as prognostic and predictive biomarkers and as possible molecular targets for individualized treatment. Patients and Methods: This prospective cohort study included 149 patients with squamous cell carcinomas of the uterine cervix in FIGO stages IIB (n= 53) and IIIB (n=96). Of the 149 patients, 61 were treated with radiotherapy and 88 with concurrent radio-chemotherapy. Expression of the proteins CAIX, GLUT-1, HIF1α, HKII, IGF-IRα, IGF-IRβ, and Survivin was determined by immunohistochemistry in biopsies taken before treatment. Results: An overexpression was found for GAPDH (100%), followed by Survivin (87%), IGF-IRα (76.5%), IGF-IRβ (74.5%), HIF1α (74.1%), and IGF-IRα and IGF-IRβ (73%); strong expression was observed with low frequency for GLUT-1 (31.1%), CAIX (16.2%), and HKII (10.6%). Hgb level was significantly correlated with treatment response (p=0.01). With a median follow-up of 2.1 years, OS was decreased for patients overexpressing IGF-1R β (p=0.04). The OS of the subgroup of patients with anemia (Hgb < 11 g/dL) and concomitantly overexpressing IGF-1R and GLUT-1 was significantly decreased compared to the opposite control group (p=0.015). Conclusions: The expression of GLUT-1, IGF-1R, and Hgb (≤ 11 g/dl) is associated with poor prognosis, and thus appear to be interesting biomarkers of radiation resistance. In order to contribute to appropriate therapeutic management as a personalized neoadjuvant treatment, a prospective phase II study should be designed in the near future to evaluate the effect of turmeric (curcumin) as an inhibitor of IGF-1Rβ and GLUT1. Note: This abstract was not presented at the conference. Citation Format: Pablo Moreno-Acosta, Schyrly Carrillo, Oscar Gamboa, Alfredo Romero-Rojas, Jinneth Acosta, Diana Mayorga, Mónica Molano, Martha Cotes, Nicolas Magne. Potential biomarkers for personalized oncology radiation in uterine cervical cancer [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B56.

Lactate dehydrogenase A promotes the invasion and proliferation of pituitary adenoma

Lactate dehydrogenase A (LDHA) has been reported to be involved in the initiation and progression of tumors. However, the potential role of LDHA in pituitary adenoma (PA) remains unknown. In this study, we showed that the expression levels of LDHA mRNA and protein were significantly elevated in invasive PA samples, and positively correlated with higher Ki-67 index. Overexpression of LDHA in a PA cell line (GH3) promoted glucose uptake through the upregulation of glucose transporter-1 (Glut1), lactate secretion and induced cellular invasion by upregulation of matrix metalloproteinase2 (MMP2). LDHA also promoted GH3 cell proliferation through induction of cell cycle progression via activation of the Akt-GSK-3β-cyclinD1 pathway. Accordingly, oxamate-induced inhibition of LDHA suppressed glucose uptake, lactate secretion, invasion and proliferation in GH3 cells via down regulation of Glut1 and MMP2 expression and inhibition of the Akt-GSK-3β-cyclinD1 pathway. Moreover, oxamate induced GH3 cell apoptosis by increasing mitochondrial reactive oxygen species (ROS) generation. In vivo, LDHA overexpression promoted tumor growth, and oxamate delayed tumor growth. In primary PA cell cultures, oxamate also effectively suppressed invasion and proliferation. Our data indicate that LDHA is involved in promoting the progression of PA, and oxamate might be a promising therapeutic agent for the treatment of PA.

Abstract 5433: β-Tubulin inhibitors reduce GLUT1 membrane trafficking to attenuate tumorigenesis in glioblastoma subtypes

Abstract Glioblastoma is an aggressive, high-grade tumor with poor prognosis due to lack of sound therapeutic options. Mesenchymal subtype GBMs are particularly difficult to treat because they tend to proliferate in the sub ventricular zone, an area that breeds stem-like neural cells and proves nearly impossible to access for surgical resection. They also contribute to creating a hypoxic microenvironment and switch to glycolytic metabolism in what is known as the Warburg Effect. Here, we hypothesize that strategically inhibiting the glucose transporter, GLUT1, through cytoskeletal components that traffic it to the cell membrane may reverse the Warburg Effect, attenuating further tumorigenesis and decreasing the stemness of such cells. Datamining studies and immunoblot analysis conducted on human glioblastoma patient specimens (hGBM) demonstrated that GLUT1 is highly upregulated. Limiting dilution assay conducted using GLUT1 inhibitors- fasentin and 2-Deoxy-D-glucose reduced the proliferation in mesenchymal cancer stem cell (CSC) subtype in in vitro culture. Simultaneously, mass spectrometric analysis revealed significant association between GLUT1 and β-tubulin 4 (TUBB4) in mesenchymal subtyped cells. This association was further confirmed by large-sample datamining and in immunoprecipitation studies from hGBM specimens, suggesting that TUBB4 may be a viable target in deterring the trafficking of upregulated GLUT1 to the membrane. Collectively, these studies confirm that GLUT1 is associated with TUBB4, and that targeting TUBB4 via siRNA or colchicine derivatives could prove effective in reversing the Warburg Effect in GBM cells and ultimately improve patient outcomes. Citation Format: Collin M. Labak, Maheedhara R. Guda, Swapna Asuthkar, Neha Jain, Yining Lu, Ian Purvis, Jack Tuszynski, Ichiro Nakano, Andrew J. Tsung, Kiran K. Velpula. β-Tubulin inhibitors reduce GLUT1 membrane trafficking to attenuate tumorigenesis in glioblastoma subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5433. doi:10.1158/1538-7445.AM2017-5433

Abstract 1157: Screening and identification of new generation glucose transporter inhibitors as anticancer therapeutics

Abstract Cancer is a top killer second only to cardiovascular diseases. Cellular metabolic reprogramming and altered energetics are key features of cancer. Opportunistic uptake of extracellular nutrients has recently been named as an emerging hallmark of cancer metabolism. Glucose is a key nutrient taken in by cancer cells for satisfying their high energy and biosynthesis needs. Drastic upregulation of glucose uptake and glycolytic activity, a phenomenon called the Warburg effect, is prevalent in cancers. Cancer cells overexpress specific glucose transporters (Gluts), particularly Glut1, on cell membranes for taking in glucose via facilitated diffusion. Considering the addiction of cancer cells for glucose and their sensitivity and vulnerability to changes in glucose supply, Gluts, especially Glut1, have been recognized as an attractive anticancer target. We previously reported the identification of first generation lead Glut1 inhibitor WZB117 (1). WZB117 treatment of human nonsmall cell lung cancer (NSCLC) A549 cells resulted in inhibition of glucose uptake, glycolysis and induction of apoptosis and necrosis. In addition, WZB-117 reduced tumor growth by ~70% in an A549 tumor mouse model. WZB117 inhibit cell growth of 7 cancer cell lines in 4 cancer types with IC50 values lower than 10 μM. Through a structure activity relationship (SAR) study of WZB117, a second generation lead, DRB18, has been identified. DRB-18 is much more stable and 5 to 10 times more potent than WZB117 in inhibiting growth and proliferation in about 90% of the 60 different cancer cell lines in 9 major cancer types as revealed in NCI-60 screenings. And its IC50s in many cancer cell lines are in the high nM range. In this study, we also report new compounds screening results for the objective of identifying novel compounds with higher glucose transport-inhibitory and cancer cell growth-inhibitory activities using nonsmall cell lung cancer cell lines as models.

Abstract 4731: Phospho-akt: a potential resistance marker to chemotherapy and a therapy-target to restore sensitivity in pancreatic cancer

Abstract Oncogenic KRAS signaling is the main driving force behind pancreatic ductal adenocarcinoma (PDAC); however, targeting this pathway has proven to be difficult. Conversely, the PI3K/Akt pathway represents an exciting new target, because it has been associated with poor prognosis and chemoresistance, and several inhibitors are under development. In particular, perifosine prevents Akt translocation to the cell membrane, while MK-2206 is an Akt allosteric inhibitor and BEZ-235 is a dual PI3K/mTOR inhibitor. Therefore, we investigated the prognostic role of phospho (p)-Akt in PDAC tissues, as well as the molecular mechanisms underlying the interaction of Akt inhibitors with gemcitabine, using PDAC cells, primary cultures and spheroids. Immunohistochemistry of tissue microarrays with specimens from radically-resected patients (n=100) revealed a correlation between high p-Akt1 expression and worse outcome. Patients with low p-Akt expression (as detected by digital scoring) had a median overall survival (OS) of 16.2 months (95% CI, 14.8-20.1), while patients with high expression had a median OS of 12.0 months (95% CI, 9.0-14.9, P=0.03). Parallel immunocytochemistry studies revealed high expression levels in LPC028 primary cells, while LPC006 were characterized by low p-Akt1. Akt inhibitors reduced cancer cell growth in monolayers and spheroids, and synergistically enhanced the antiproliferative activity of gemcitabine in LPC028 (e.g., combination index CI of 0.2, in the gemcitabine-perifosine combination for 72h, at fixed IC50 ratio), while this combination was antagonistic in LPC006 cells. The synergistic effect was paralleled by a 5-fold reduction in the expression of the main gemcitabine target ribonucleotide reductase. Inhibition of Akt decreased cell migration and invasion, which was additionally reduced by the combination with gemcitabine. However, the combination of Akt inhibitors with gemcitabine 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. The Akt signaling is involved in the expression/localization of the key glucose transporter Glut1, and increased glucose metabolism was associated to resistance to axitinib (Hudson et al, Cell Death Dis 2014). Remarkably, the resistant LPC006 cells were characterized by overexpression of Glut1, which was not reduced after exposure to Akt inhibitors. However, the novel Glut1 inhibitor PGL13 enhanced perifosine and perifosine/gemcitabine-induced cell death. In conclusion, our findings support the analysis of phospho-Akt1 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. Citation Format: Daniela Massihnia, Amir Avan, Niccola Funel, Carlotta Granchi, Filippo Minutolo, Leticia Leon, Godefridus Peters, Elisa Giovannetti. Phospho-akt: a potential resistance marker to chemotherapy and a therapy-target to restore sensitivity in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4731. doi:10.1158/1538-7445.AM2017-4731

Overcoming the Heat Endurance of Tumor Cells by Interfering with the Anaerobic Glycolysis Metabolism for Improved Photothermal Therapy.

In this study, we developed a general method to decorate plasmonic gold nanorods (GNRs) with a CD44-targeting functional polymer, containing a hyaluronic acid (HA)-targeting moiety and a small molecule Glut1 inhibitor of diclofenac (DC), to obtain GNR/HA-DC. This nanosystem exhibited the superiority of selectively sensitizing tumor cells for photothermal therapy (PTT) by inhibiting anaerobic glycolysis. Upon specifically targeting CD44, sequentially time-dependent DC release could be achieved by the trigger of hyaluronidase (HAase), which abundantly existed in tumor tissues. The released DC depleted the Glut1 level in tumor cells and induced a cascade effect on cellular metabolism by inhibiting glucose uptake, blocking glycolysis, decreasing ATP levels, hampering heat shock protein (HSP) expression, and ultimately leaving malignant cells out from the protection of HSPs to stress (e.g., heat), and then tumor cells were more easy to kill. Owing to the sensitization effect of GNR/HA-DC, CD44 overexpressed tumor cells could be significantly damaged by PTT with an enhanced therapeutic efficiency in vitro and in vivo.