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Glutamate Secretion and Metabotropic Glutamate Receptor 1 Expression during Kaposi's Sarcoma-Associated Herpesvirus Infection Promotes Cell Proliferation

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      Kaposi's sarcoma associated herpesvirus (KSHV) is etiologically associated with endothelial Kaposi's sarcoma (KS) and B-cell proliferative primary effusion lymphoma (PEL), common malignancies seen in immunocompromised HIV-1 infected patients. The progression of these cancers occurs by the proliferation of cells latently infected with KSHV, which is highly dependent on autocrine and paracrine factors secreted from the infected cells. Glutamate and glutamate receptors have emerged as key regulators of intracellular signaling pathways and cell proliferation. However, whether they play any role in the pathological changes associated with virus induced oncogenesis is not known. Here, we report the first systematic study of the role of glutamate and its metabotropic glutamate receptor 1 (mGluR1) in KSHV infected cell proliferation. Our studies show increased glutamate secretion and glutaminase expression during de novo KSHV infection of endothelial cells as well as in KSHV latently infected endothelial and B-cells. Increased mGluR1 expression was detected in KSHV infected KS and PEL tissue sections. Increased c-Myc and glutaminase expression in the infected cells was mediated by KSHV latency associated nuclear antigen 1 (LANA-1). In addition, mGluR1 expression regulating host RE-1 silencing transcription factor/neuron restrictive silencer factor (REST/NRSF) was retained in the cytoplasm of infected cells. KSHV latent protein Kaposin A was also involved in the over expression of mGluR1 by interacting with REST in the cytoplasm of infected cells and by regulating the phosphorylation of REST and interaction with β-TRCP for ubiquitination. Colocalization of Kaposin A with REST was also observed in KS and PEL tissue samples. KSHV infected cell proliferation was significantly inhibited by glutamate release inhibitor and mGluR1 antagonists. These studies demonstrated that elevated glutamate secretion and mGluR1 expression play a role in KSHV induced cell proliferation and suggest that targeting glutamate and mGluR1 is an attractive therapeutic strategy to effectively control the KSHV associated malignancies.

      Author Summary

      Kaposi's sarcoma associated herpesvirus (KSHV), prevalent in immunosuppressed HIV infected individuals and transplant recipients, is etiologically associated with cancers such as endothelial Kaposi's sarcoma (KS) and B-cell primary effusion lymphoma (PEL). Both KS and PEL develop from the unlimited proliferation of KSHV infected cells. Increased secretion of various host cytokines and growth factors, and the activation of their corresponding receptors, are shown to be contributing to the proliferation of KSHV latently infected cells. Glutamate, a neurotransmitter, is also involved in several cellular events including cell proliferation. In the present study, we report that KSHV-infected latent cells induce the secretion of glutamate and activation of metabotropic glutamate receptor 1 (mGluR1), and KSHV latency associated LANA-1 and Kaposin A proteins are involved in glutaminase and mGluR1 expression. Our functional analysis showed that elevated secretion of glutamate and mGluR1 activation is linked to increased proliferation of KSHV infected cells and glutamate release inhibitor and glutamate receptor antagonists blocked the proliferation of KSHV infected cells. These studies show that proliferation of cancer cells latently infected with KSHV in part depends upon glutamate and glutamate receptor and therefore could potentially be used as therapeutic targets for the control and elimination of KSHV associated cancers.

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      Most cited references 71

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      Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma.

      Representational difference analysis was used to isolate unique sequences present in more than 90 percent of Kaposi's sarcoma (KS) tissues obtained from patients with acquired immunodeficiency syndrome (AIDS). These sequences were not present in tissue DNA from non-AIDS patients, but were present in 15 percent of non-KS tissue DNA samples from AIDS patients. The sequences are homologous to, but distinct from, capsid and tegument protein genes of the Gammaherpesvirinae, herpesvirus saimiri and Epstein-Barr virus. These KS-associated herpesvirus-like (KSHV) sequences appear to define a new human herpesvirus.
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        Glutamate uptake.

         Niels Danbolt (2001)
        Brain tissue has a remarkable ability to accumulate glutamate. This ability is due to glutamate transporter proteins present in the plasma membranes of both glial cells and neurons. The transporter proteins represent the only (significant) mechanism for removal of glutamate from the extracellular fluid and their importance for the long-term maintenance of low and non-toxic concentrations of glutamate is now well documented. In addition to this simple, but essential glutamate removal role, the glutamate transporters appear to have more sophisticated functions in the modulation of neurotransmission. They may modify the time course of synaptic events, the extent and pattern of activation and desensitization of receptors outside the synaptic cleft and at neighboring synapses (intersynaptic cross-talk). Further, the glutamate transporters provide glutamate for synthesis of e.g. GABA, glutathione and protein, and for energy production. They also play roles in peripheral organs and tissues (e.g. bone, heart, intestine, kidneys, pancreas and placenta). Glutamate uptake appears to be modulated on virtually all possible levels, i.e. DNA transcription, mRNA splicing and degradation, protein synthesis and targeting, and actual amino acid transport activity and associated ion channel activities. A variety of soluble compounds (e.g. glutamate, cytokines and growth factors) influence glutamate transporter expression and activities. Neither the normal functioning of glutamatergic synapses nor the pathogenesis of major neurological diseases (e.g. cerebral ischemia, hypoglycemia, amyotrophic lateral sclerosis, Alzheimer's disease, traumatic brain injury, epilepsy and schizophrenia) as well as non-neurological diseases (e.g. osteoporosis) can be properly understood unless more is learned about these transporter proteins. Like glutamate itself, glutamate transporters are somehow involved in almost all aspects of normal and abnormal brain activity.
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          c-Myc suppression of miR-23 enhances mitochondrial glutaminase and glutamine metabolism

          Altered glucose metabolism in cancer cells is termed the Warburg effect, which describes the propensity for most cancer cells to take up glucose avidly and convert it primarily to lactate, despite available oxygen 1,2. Notwithstanding the renewed interest in the Warburg effect, cancer cells also depend on continued mitochondrial function for metabolism, specifically glutaminolysis that catabolizes glutamine to generate ATP and lactate3. Glutamine, which is highly transported into proliferating cells4,5, is a major source for energy and nitrogen for biosynthesis, and a carbon substrate for anabolic processes in cancer cells, but the regulation of glutamine metabolism is not well understood1,6. Here, we report that the c-Myc (or Myc) oncogenic transcription factor, which is known to regulate miRNAs7,8 and stimulate cell proliferation9, transcriptionally represses miR-23a and miR-23b, resulting in greater expression of their target protein, mitochondrial glutaminase (GLS). This leads to up-regulation of glutamine catabolism10. GLS converts glutamine to glutamate that is further catabolized through the TCA cycle for the production of ATP or serves as substrate for glutathione synthesis11. The unique means by which Myc regulates GLS reported herein uncovers a previously unsuspected link between Myc regulation of miRNAs, glutamine metabolism, and energy and reactive oxygen species (ROS) homeostasis. Oncogenes and tumor suppressors have now been linked to the regulation of glucose metabolism, thereby connecting genetic alterations in cancers to their glucose metabolic phenotype1,2. In particular, the MYC oncogene produces Myc protein that directly regulates glucose metabolic enzymes as well as genes involved in mitochondrial biogenesis9,12. In this regard, we sought to determine the role of the MYC in altering the mitochondrial proteome to further understand regulation of tumor metabolism. We studied the human P-493 B cells that bear a tetracycline-repressible MYC construct, such that tetracycline withdrawal results in rapid induction of Myc and mitochondrial biogenesis, followed by cell proliferation12,13. By comparing the mitochondrial proteome from tetracycline-treated and untreated cells with high Myc expression, we found 8 mitochondrial proteins that are distinctly differentially expressed in response to Myc (Figures 1a, 1b and Tables S1). We note that mitochondrial glutaminase or GLS (MW∼58kDa) was increased ∼10-fold in response to Myc. As such, we determined the response of glutaminase to Myc induction in a time-course study using anti-GLS antibody10 (Figure 1c) and found that GLS levels diminish with decreased Myc expression and recover upon Myc re-induction. However, the level of the mitochondrial protein TFAM remained virtually unaltered. GLS levels also correlate with Myc levels in another human B cell line (CB33) and one (CB33-Myc) with constitutive Myc expression14. Since human prostate cancer is linked to Myc expression15, we sought to determine whether reduction of Myc expression by siRNA in the human PC3 prostate cancer cell line was also associated with reduction of GLS expression (Figure 1d). Similar to the human lymphoid cells, the PC3 cells also displayed a correlation between Myc and GLS levels. We then sought to determine whether the dramatic alteration of GLS levels in response to Myc is functionally linked to Myc-induced cell proliferation. While there are two major known tissue-specific GLS isoforms, GLS1 and GLS216,17, our data showed that only GLS1 is predominantly expressed in P493-6 or PC3 cells (Figure S1). We first determined whether gain of GLS1 function through overexpression in PC3 cells would rescue the diminished growth rate associated with siRNA-mediated reduction of Myc (Figure S2) and found that ectopic GLS1 expression alone is insufficient to stimulate growth. In light of the observation that no single gene could substitute for Myc 18,19 and that Myc is a pleiotropic transcription factor9, this outcome was not particularly surprising. As such, we reduced the expression of GLS1 (herein referred to as GLS) by RNA interference (siGLS) and found that P-493-6 cell proliferation is markedly attenuated by siGLS but not by control siRNA (Figure 2a). Likewise, proliferation of the human PC3 prostate cancer cell line was diminished by siGLS (Figure 2a), indicating that GLS is necessary for cell proliferation. Because glutamine is converted by GLS to glutamate for further catabolism by the TCA cycle and previous studies indicate that overexpression of Myc sensitizes human cells to glutamine withdrawal induced apoptosis11, we determined the metabolic responses of P493-6 or PC3 cells to glutamine deprivation (Figure 2b). The growth of both cell lines was diminished significantly by glutamine withdrawal and moderately with glucose withdrawal. Glutamine withdrawal also resulted in a decrease in ATP levels (Figure 2c) associated with a diminished cellular oxygen consumption rate (Figures S3a and S3b). Reduction of GLS by RNAi also reduced ATP levels (Figure 2d). Because glutamine is a precursor for glutathione20, glutathione levels were measured by flow cytometry and were found diminished with glutamine withdrawal or RNAi mediated reduction of GLS (Figure S4 and Table S2) that is also associated with an increase in ROS levels (Figure S3c) and cell death in the P493-6 cells (Figures 2e and S5). It is notable shortly after the MYC proto-oncogene was discovered, that the MC29 retrovirus which bears the v-myc oncogene was found to enhance glutamine catabolism and mitochondrial respiration in transplantable avian liver tumor cells21. Thus, our findings functionally link historical observations with Myc, glutaminase and glutamine metabolism. Since GLS catabolizes glutamine for ATP and glutathione synthesis, its reduction affects proliferation and cell death presumably through depletion of ATP and augmentation of ROS, respectively. Hence, we sought to rescue the P493-6 cells with the TCA cycle metabolite oxaloacetate (OAA) and the oxygen radical scavenger N-acetylcysteine (NAC)11. Both OAA and NAC partially rescued the decreased proliferation and death of P493-6 cells deprived of GLS (Figures 2e and S6). Likewise, OAA and NAC both partially rescued glutamine-deprived P493-6 cells (Figures S5 and S6). These findings support the notion that glutamine catabolism through GLS is critical for cell proliferation induced by Myc and protection against ROS generated by enhanced mitochondrial function in response to Myc11,20. Given that GLS is critical for cell proliferation and is induced by Myc, we determined the mechanism by which Myc regulates GLS. Because Myc is a transcription factor9, we hypothesized that Myc transactivates GLS directly as a target gene. Despite the presence of a canonical Myc binding site (5′-CACGTG-3′) in the GLS gene intron 1, GLS mRNA levels do not respond to alterations in Myc levels in the P493-6 cells, suggesting that GLS is regulated at the post-transcriptional level (Figure 3a). As such, we hypothesized that GLS could be regulated by miRNAs that are in turn directly regulated by Myc. The TargetScan algorithm predicts that miR-23a and miR-23b could target the GLS 3′UTR seed sequence. Intriguingly, our earlier studies uncovered that both miR-23a and miR-23b are suppressed by Myc in P493-6 cells7, and both miR-23s are decreased in human prostate cancers22, which are associated with elevated Myc expression15. To verify that miR-23a and miR-23b (herein referred to as miR-23) are suppressed by Myc and could be diminished by antisense miR-23 locked nucleic acid (LNA) oligomers, northern analysis was performed and miR-23 was suppressed by Myc and profoundly diminished by antisense miR-23 LNAs (Figure 3b). Using quantitative real-time PCR, we document (Figure S7) that miR-23 levels increased with diminished Myc expression and then decreased upon Myc re-induction in a manner that is compatible with the GLS protein levels seen in Figure 1c. We also found an inverse relationship between Myc and the levels of miR-23a and miR-23b in the CB33 human lymphoid cells and PC3 prostate cancer cell line (Figure S8). Furthermore, Myc directly bound the transcriptional unit, C9orf3, encompassing miR-23b, as demonstrated for other Myc miRNA targets7 by chromatin immunoprecipitation (Figure 3c). Because the transcriptional unit involving miR-23a has not been mapped, we did not study miR-23a in this context. These observations document that Myc represses miR-23a and miR23b, which appear to be directly regulated by Myc. We determined next whether miR-23 targets and inhibits the expression of GLS through the 3′UTR. In this regard, we cloned the 3′UTR sequence of GLS including the predicted binding site for miR-23 to the pGL3 luciferase reporter vector and transfected MCF-7 cells, which is known to express miR-2323. The GLS 3′UTR inhibited luciferase activity in a fashion that was blocked by co-transfection with the antisense miR-23 LNAs, but not with control LNAs (Figure 3d). We next mutated the predicted binding site by a site-directed mutagenesis strategy8 and observed that mutant 3′UTR did not inhibit luciferase activity as the wild-type sequence did. Using these reporters in PC3 cells, we observed that siRNA-mediated diminished Myc expression resulted in decreased luciferase activity with wild-type but not with the mutant 3′UTR reporter (Figure S9). Most importantly, diminished GLS protein level, which follows decreased Myc expression (Figure 1c), was rescued by antisense miR-23 LNAs (Figure 3e). The antisense miR-23 LNAs also partially rescued the diminished GLS level associated with RNAi-mediated reduction of Myc expression in the PC3 cells (Figure 3e). We also examined events upstream and downstream of GLS24 and found that the glutamine transporter SLC7A5 is induced by Myc in P493-6 cells at the transcriptional level (5-fold by nuclear run-on, unpublished data) with a >7-fold induction of its mRNA level. The glutamine transporter ASCT2 is induced by Myc at the mRNA level by 2-fold, whereas glutamate dehydrogenase mRNA levels appear unaltered (unpublished data). Furthermore, we found that elevated levels of Myc protein in human prostate cancer correspond to levels of GLS, which was not increased in the accompanying normal tissue from the same patients (Figure S10). Intriguingly, miR-23a and miR-23b are significantly decreased in human prostate cancer as compared with normal prostate tissue22. It is notable that loss of GLS function by antisense suppression significantly inhibited the tumorigenesis of Ehrlich ascites tumor cells in vivo25. Our findings here uncover a pathway by which Myc suppression of miR-23, which targets GLS, enhances glutamine catabolism through increased mitochondrial glutaminase expression. Taken together, these observations provide a regulatory mechanism involving Myc and miRNAs for elevated expression of glutaminase and glutamine metabolism in human cancers. Methods Summary Human cell lines were cultured under standard conditions. Isolation of mitochondria, enrichment for mitochondrial proteins, and proteomic analysis were performed as described26-29. RNA interference experiments and luciferase reporter analysis of miRNA activity were as reported8,30. Flow cytometric analyses of reactive oxygen species, cell death and glutathione level were performed as described11,30. Human samples were acquired with the approval of the Johns Hopkins University School of Medicine Institutional Review Board. Supplementary Material 1 2

            Author and article information

            H. M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois, United States of America
            University of Pennsylvania Medical School, United States of America
            Author notes

            The authors have declared that no competing interests exist.

            Conceived and designed the experiments: MVV BC. Performed the experiments: MVV DD VB CB OG NSW SD. Analyzed the data: MVV BC. Wrote the paper: MVV BC.

            Role: Editor
            PLoS Pathog
            PLoS Pathog
            PLoS Pathogens
            Public Library of Science (San Francisco, USA )
            October 2014
            9 October 2014
            : 10
            : 10
            25299066 4192595 PPATHOGENS-D-14-00383 10.1371/journal.ppat.1004389

            This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

            Pages: 19
            This study was supported in part by the Public Health Service grants CA 075911 and CA 168472, and the Rosalind Franklin University of Medicine and Science H.M. Bligh Cancer Research Fund to BC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
            Research Article
            Biology and Life Sciences
            Cell Biology
            Cell Processes
            Cell Growth
            Molecular Cell Biology
            Medicine and health sciences
            Infectious Diseases
            Viral Diseases
            Cancers and neoplasms
            AIDS-related cancers
            Cancer Treatment
            Antiangiogenesis Therapy
            Oncology Agents

            Infectious disease & Microbiology


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