Dede N. Ekoue

MnSOD upregulation sustains the Warburg effect via mitochondrial ROS and AMPK-dependent signalling in cancer

Manganese superoxide dismutase (MnSOD/SOD2) is a mitochondria-resident enzyme that governs the types of reactive oxygen species egressing from the organelle to affect cellular signaling. Here, we demonstrate that MnSOD upregulation in cancer cells establishes a steady flow of H2O2 originating from mitochondria that sustains AMP-activated kinase (AMPK) activation and the metabolic shift to glycolysis. Restricting MnSOD expression or inhibiting AMPK suppress the metabolic switch and dampens the viability of transformed cells indicating that the MnSOD/AMPK axis is critical in support cancer cell bioenergetics. Recapitulating in vitro findings, clinical and epidemiologic analyses of MnSOD expression and AMPK activation indicated that the MnSOD/AMPK pathway is most active in advanced stage and aggressive breast cancer subtypes. Taken together, our results indicate that MnSOD serves as a biomarker of cancer progression and acts as critical regulator of tumor cell metabolism.

Natural Allelic Variations in Glutathione Peroxidase-1 Affect Its Subcellular Localization and Function

Glutathione peroxidase 1 (GPx-1) has been implicated in the etiology of several common diseases due to the association between specific allelic variations and cancer risk. The most common among these variations are the codon 198 polymorphism that results in either a leucine or proline and the number of alanine repeat codons in the coding sequence. The molecular and biologic consequences of these variations remain to be characterized. Toward achieving this goal, we have examined the cellular location of GPx-1 encoded by allelic variants by ectopically expressing these genes in MCF-7 human breast carcinoma cells that produce undetectable levels of GPx-1, thus achieving exclusive expression in the same cellular environment. A differential distribution between the cytoplasm and mitochondria was observed, with the allele expressing the leucine-198 polymorphism and 7 alanine repeats being more cytoplasmically located than the other alleles examined. To assess whether the distribution of GPx-1 between the cytoplasm and mitochondria had a biologic consequence, we engineered derivative GPx-1 proteins that were targeted to the mitochondria by the addition of a mitochondria targeting sequence and expressed these proteins in MCF-7 cells. These cells were examined for their response to oxidative stress, energy metabolism, and impact on cancer-associated signaling molecules. The results obtained indicated that both primary GPx-1 sequence and cellular location have a profound impact on cellular biology and offer feasible hypotheses about how expression of distinct GPx-1 alleles can affect cancer risk. Cancer Res; 74(18); 5118-26. ©2014 AACR.

Evidence that selenium binding protein 1 is a tumor suppressor in prostate cancer.

Selenium-Binding Protein 1 (SBP1, SELENBP1, hSP56) is a selenium-associated protein shown to be at lower levels in tumors, and its lower levels are frequently predictive of a poor clinical outcome. Distinguishing indolent from aggressive prostate cancer is a major challenge in disease management. Associations between SBP1 levels, tumor grade, and disease recurrence following prostatectomy were investigated by duplex immunofluorescence imaging using a tissue microarray containing tissue from 202 prostate cancer patients who experienced biochemical (PSA) recurrence after prostatectomy and 202 matched control patients whose cancer did not recur. Samples were matched by age, ethnicity, pathological stage and Gleason grade, and images were quantified using the Vectra multispectral imaging system. Fluorescent labels were targeted for SBP1 and cytokeratins 8/18 to restrict scoring to tumor cells, and cell-by-cell quantification of SBP1 in the nucleus and cytoplasm was performed. Nuclear SBP1 levels and the nuclear to cytoplasm ratio were inversely associated with tumor grade using linear regression analysis. Following classification of samples into quartiles based on the SBP1 levels among controls, tumors in the lowest quartile were more than twice as likely to recur compared to those in any other quartile. Inducible ectopic SBP1 expression reduced the ability of HCT-116 human tumor cells to grow in soft agar, a measure of transformation, without affecting proliferation. Cells expressing SBP1 also demonstrated a robust induction in the phosphorylation of the p53 tumor suppressor at serine 15. These data indicate that loss of SBP1 may play an independent contributing role in prostate cancer progression and its levels might be useful in distinguishing indolent from aggressive disease.

Manganese superoxide dismutase and glutathione peroxidase-1 contribute to the rise and fall of mitochondrial reactive oxygen species which drive oncogenesis ☆

Reactive oxygen species (ROS) largely originating in the mitochondria play essential roles in the metabolic and (epi)genetic reprogramming of cancer cell evolution towards more aggressive phenotypes. Recent studies have indicated that the activity of superoxide dismutase (SOD2) may promote tumor progression by serving as a source of hydrogen peroxide (H2O2). H2O2 is a form of ROS that is particularly active as a redox agent affecting cell signaling due to its ability to freely diffuse out of the mitochondria and alter redox active amino acid residues on regulatory proteins. Therefore, there is likely a dichotomy whereas SOD2 can be considered a protective antioxidant, as well as a pro-oxidant during cancer progression, with these effects depending on the accumulation and detoxification of H2O2. Glutathione peroxidase-1 GPX1, is a selenium-dependent scavenger of H2O2 which partitions between the mitochondria and the cytosol. Epidemiologic studies indicated that allelic variations in the SOD2 and GPX1 genes alter the distribution and relative concentrations of SOD2 and GPX1 in mitochondria, thereby affecting the dynamic between the production and elimination of H2O2. Experimental and epidemiological evidence supporting a conflicting role of SOD2 in tumor biology, and epidemiological evidence that SOD2 and GPX1 can interact to affect cancer risk and progression indicated that it is the net accumulation of mitochondrial H2O2 (mtH2O2) resulting from of the balance between the activities SOD2 and anti-oxidants such as GPX1 that determines whether SOD2 prevents or promotes oncogenesis. In this review, research supporting the idea that GPX1 is a gatekeeper restraining the oncogenic power of mitochondrial ROS generated by SOD2 is presented. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux.

Translational Regulation of GPx-1 and GPx-4 by the mTOR Pathway

Glutathione peroxidase activity was previously determined to be elevated in lymphocytes obtained from patients treated with the Bcr-Abl kinase inhibitor imatinib mesylate. In order to expand upon this observation, the established chronic myelogenous leukemia cell lines KU812 and MEG-01 were treated with imatinib and the effect on several anti-oxidant proteins was determined. The levels of GPx-1 were significantly increased following treatment with imatinib. This increase was not due to altered steady-state mRNA levels, and appeared to be dependent on the expression of Bcr-Abl, as no increases were observed following imatinib treatment of cells that did not express the fusion protein. The nutrient-sensing signaling protein, mammalian target of rapamycin (mTOR), can be activated by Bcr-Abl and its activity regulates the translation of many different proteins. Treatment of those same cells used in the imatinib studies with rapamycin, an inhibitor of mTOR, resulted in elevated GPx-1 and GPx-4 protein levels independent of Bcr-Abl expression. These proteins all belong to the selenoprotein family of peptides that contain the UGA-encoded amino acid selenocysteine. Collectively, these data provide evidence of a novel means of regulating anti-oxidants of the selenoprotein family via the mTOR pathway.

Selenium levels in human breast carcinoma tissue are associated with a common polymorphism in the gene for SELENOP (Selenoprotein P)

Selenium supplementation of the diets of rodents has consistently been shown to suppress mammary carcinogenesis and some, albeit not all, human epidemiological studies have indicated an inverse association between selenium and breast cancer risk. In order to better understand the role selenium plays in breast cancer, 30 samples of tumor tissue were obtained from women with breast cancer and analyzed for selenium concentration, the levels of several selenium-containing proteins and the levels of the MnSOD anti-oxidant protein. Polymorphisms within the genes for these same proteins were determined from DNA isolated from the tissue samples. There was a wide range of selenium in these tissues, ranging from 24 to 854ng/gm. The selenium levels in the tissues were correlated to the genotype of the SELENOP selenium carrier protein, but not to other proteins whose levels have been reported to be responsive to selenium availability, including GPX1, SELENOF and SBP1. There was an association between a polymorphism in the gene for MnSOD and the levels of the encoded protein. These studies were the first to examine the relationship between selenium levels, genotypes and protein levels in human tissues. Furthermore, the obtained data provide evidence for the need to obtain data about the effects of selenium in breast cancer by examining samples from that particular tissue type.

Allele-specific interaction between glutathione peroxidase 1 and manganese superoxide dismutase affects the levels of Bcl-2, Sirt3 and E-cadherin

Abstract Manganese superoxide dismutase (MnSOD) is a mitochondrial-resident enzyme that reduces superoxide to hydrogen peroxide (H2O2), which can be further reduced to water by glutathione peroxidase (GPX1). Data from human studies have indicated that common polymorphisms in both of these proteins are associated with the risk of several cancers, including breast cancer. Moreover, polymorphisms in MnSOD and GPX1 were shown to interact to increase the risk of breast cancer. To gain an understanding of the molecular mechanisms behind these observations, we engineered human MCF-7 breast cancer cells to exclusively express GPX1 and/or MnSOD alleles and investigated the consequences on the expression of several proteins associated with cancer aetiology. Little or no effect was observed on the ectopic expression of these genes on the phosphorylation of Akt, although allele-specific effects and interactions were observed for the impact on the levels of Bcl-2, E-cadherin and Sirt3. The patterns observed were not consistent with the steady-state levels of H2O2 determined in the transfected cells. These results indicate plausible contributing factors to the effects of allelic variations on cancer risk observed in human epidemiological studies.

Correlations of SELENOF and SELENOP genotypes with serum selenium levels and prostate cancer.

Selenium status is inversely associated with the incidence of prostate cancer. However, supplementation trials have not indicated a benefit of selenium supplementation in reducing cancer risk. Polymorphisms in the gene encoding selenoprotein 15 (SELENOF) are associated with cancer incidence/mortality and present disproportionately in African Americans. Relationships among the genotype of selenoproteins implicated in increased cancer risk, selenium status, and race with prostate cancer were investigated.

Abstract 225: Allelic variations in MnSOD and GPx-1 affect metabolism, mitochondrial membrane potential and expression of signaling proteins

MnSOD detoxifies superoxide and impacts tumor biology by generating H 2 O 2 which can diffuse through the mitochondrial membrane and affect metabolism and apoptosis. The selenium-dependent enzyme GPx-1 localizes to both the cytoplasm and the mitochondria and reduces H 2 O 2 to water and may therefore modulate the impact of MnSOD on carcinogenesis. A val to ala polymorphism in codon 16 of the MnSOD gene has been shown to be associated with increased prostate cancer risk in men with the lowest level of dietary antioxidant intake and individuals who consumed less dietary antioxidants, including selenium, had the greatest risk of prostate cancer (Li et al, 2005). A polymorphism in the GPx-1 gene resulting in a leucine (L) instead of a proline (P) at position 198 has frequently been reported to be associated with elevated cancer risk. To examine the molecular mechanism behind the epidemiological observation that genotypes in GPx-1 gene modify elevated risk of cancer associated with MnSOD genotypes, MCF-7 human breast cancer cells null for GPx-1 and having negligible levels of endogenous MnSOD were transfected with GPx-1 and MnSOD allele-specific expression constructs to determine outcomes related to cellular signaling and metabolism. MnSOD and GPx-1 alleles differentially interacted to modulate the expression of the anti-oxidant stress response regulator Nrf2, the cell adhesion protein E-Cadherin, the cell signaling protein pAkt, the anti-apoptotic protein Bcl-2 and the mitochondria located deacetylase Sirt3. Also, co-expression of the GPx-1 A7L and either MnSOD val or MnSOD ala increased oxidative phosphorylation as measured by O 2 uptake using the Seahorse XF24 XF analyzer, which simultaneously determines relative mitochondrial respiration and glycolysis. In order to assess whether GPx-1 and MnSOD allelic variants modulate mitochondrial membrane potential, CMX-ROS fluorescence was measured. Independent and irrespective of which allele was evaluated, MnSOD and GPx-1 individually decreased mitochondrial potential as compared to that seen using MCF-7 control cells. In addition, co-expression of GPx-1 A7L with either MnSOD val or MnSOD ala further decreased membrane potential above that observed for either MnSOD and GPx-1 alone. In order to determine if MnSOD and GPx-1 levels are associated with a higher risk for biochemical recurrence of prostate cancer after radical prostatectomy, immunohistochemistry of human prostate tissue cores will be performed. Preliminary results indicate that a high MnSOD/ GPx-1 ratio was observed in prostate cancer tissue compared to adjacent normal tissue. Citation Format: Dede N. Ekoue, Soumen Bera, Emmanuel Ansong, Peter Hart, Virgilia Macias, Andre Kajdacsy-Balla, Marcelo Bonini, Alan M. Diamond. Allelic variations in MnSOD and GPx-1 affect metabolism, mitochondrial membrane potential and expression of signaling proteins. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 225.