Regulation of SELENOF translation by eIF4a3: Possible role in prostate cancer progression.

The levels of the SELENOF selenoprotein are dramatically reduced in prostate cancer compared to adjacent benign tissue and reducing SELENOF in prostate epithelial cells results in the acquisition of features of the transformed phenotype. It was hypothesized that the aberrant increase in the eiF4a3 translation factor, which has an established role in RNA splicing and the regulation of selenoprotein translation, contributes to the lower levels of SELENOF. Using the available databases, eIF4a3 messenger RNA (mRNA) levels are elevated in prostate cancer compared to normal tissue as is the hypomethylation of the corresponding gene. Using a prostate cancer tissue microarray, we established that eiF4a3 levels are higher in prostate cancer tissue. Ectopic expression of eIF4a3 in prostate cancer cells reduced SELENOF levels and attenuated the readthrough of the UGA codon using a specialized reporter construct designed to examine UGA decoding, with the opposite effects observed using eIF4a3 knock-down constructs. Direct binding of eIF4a3 to the regulatory regions of SELENOF mRNA was established with pull-down experiments. Lastly, we show that an eIF4a3 inhibitor, eIF4a3-IN-2, increases SELENOF levels, UGA readthrough, and reduces binding of eIF4a3 to the SELENOF mRNA 3'-UTR in exposed cells. These data establish eIF4a3 as a likely prostate cancer oncogene and a regulator of SELENOF translation.

Reticulocyte mitochondrial retention increases reactive oxygen species and oxygen consumption in mouse models of sickle cell disease and phlebotomy-induced anemia.

Sickle cell disease (SCD) is caused by a mutation of the ß-globin gene that results in the production of hemoglobin S (HbS). People with SCD experience anemia, severe acute pain episodes, persistent chronic pain, multiorgan damage, and a reduced life span. The pathophysiology of SCD caused by the polymerization of HbS on deoxygenation results in red cell deformability and the generation of reactive oxygen species (ROS). These 2 factors lead to red cell fragility and hemolysis. Reticulocytosis is an independent predictor of disease morbidity and mortality in SCD. We previously established that humans and mice with SCD exhibit abnormal mitochondrial retention in erythrocytes increasing ROS-associated hemolysis. Here, we investigated the hypothesis that mitochondrial retention and increased ROS are a consequence of stress erythropoiesis. Our results show clearly that stress erythropoiesis in phlebotomized, anemic AA mice results in mitochondrial retention and increased ROS in reticulocytes. We observed that elevated mitochondrial retention in reticulocytes also alters oxygen consumption and potentially contributes to increased HbS polymerization and red blood cell hemolysis. Therefore, these events occurring due to stress erythropoiesis contribute significantly to the pathology of SCD and suggest new therapeutic targets.

Distinct Roles of SELENOF in Different Human Cancers.

SELENOF, previously known as SEP15, is a selenoprotein that contains selenium in the form of the amino acid selenocysteine. Like other selenoproteins, the role for SELENOF in carcinogenesis has been investigated due to its altered expression compared to the corresponding normal tissue, its molecular function, and the association of genetic variations in the SELENOF gene to cancer risk or outcome. This review summarizes SELENOF's discovery, structure, cellular localization, and expression. SELENOF belongs to a new family of thioredoxin-like proteins. Published data summarized here indicate a likely role for SELENOF in redox protein quality control, and in the regulation of lipids, glucose, and energy metabolism. Current evidence indicates that loss of SELENOF contributes to the development of prostate and breast cancer, while its loss may be protective against colon cancer. Additional investigation into SELENOF's molecular mechanisms and its impact on cancer is warranted.

Associations of selenoprotein expression and gene methylation with the outcome of clear cell renal carcinoma.

Selenoproteins are a ubiquitous class of proteins defined by having a selenocysteine amino acid residue. While many of the selenoproteins have been well characterized with important roles in oxidation-reduction reactions and hormone synthesis among others, there exist some whose biological roles are not as well understood as denoted by the "SELENO" root. In this study, we explored associations between the reported RNA levels of "SELENO" proteins and clear cell renal cell carcinoma (ccRCC), the most common subtype of renal carcinoma in the US. Utilizing The Cancer Genome Atlas (TCGA) alongside other in silico tools, we discovered higher mRNA expression of Selenoprotein I, T, and P was associated with better overall survival outcomes and differential expression of other selenoproteins based on tumor stage. Additionally, we uncovered relative hypomethylation among selenoproteins in primary ccRCC tumor samples compared to normal tissue. Network and enrichment analysis showed numerous genes through which selenoproteins may modulate cancer progression and outcomes such as DERL1, PNPLA2/3, MIEN1, and FOXO1 which have been well-described in other cancers. In light of our findings highlighting an association of selenoprotein methylation and expression patterns with ccRCC outcome, further wet lab research is warranted.

Role of SELENBP1 and SELENOF in prostate cancer bioenergetics.

The contribution of selenium and selenoproteins in prostate cancer etiology remains elusive, potentially due to insufficient information regarding the biochemical pathways in which they are involved. There are twenty-five human selenocysteine-containing proteins or selenoproteins as well as a smaller class of selenium-containing proteins that do not include selenocysteine, and their cancer-associated aberrations, both genetic and functional, have evoked special interest, although their contribution to the metabolic reprogramming of prostate cancers remains has not been extensively studied. While benign prostate tissue exhibits a glycolytic phenotype, neoplastic events restore the truncated tricarboxylic acid cycle and enhance oxidative phosphorylation. Two selenium-containing proteins, selenium binding protein 1 and selenoprotein F, affect prostate cancer phenotypes by modulating tumor cell metabolic profiles with significant effects on mitochondrial biology, including oxidative phosphorylation and ATP synthesis. One of the pathways affected by both proteins is the activation of adenosine monophosphate kinase and its downstream signaling with concomitant induction of glycolysis. This review focuses on highlighting the role of these two proteins in modulating the bioenergetic profile of prostate cancer and in maintaining the metabolic plasticity of these cells rendering growth advantage and possible therapeutic resistance.

SELENOF is a new tumor suppressor in breast cancer.

Epidemiological evidence has indicated an inverse association between selenium status and various types of cancer, including breast cancer. Selenoproteins are the primary mediators of selenium effects in human health. We have previously reported loss of heterozygosity in breast tumor samples of the gene for one of the selenoproteins, SELENOF. The function of SELENOF remains unclear and whether SELENOF levels impact breast cancer risk or outcome is unknown. The mining of breast cancer patient databases revealed that SELENOF mRNA is significantly lower in late-stage tumor samples and lower levels of SELENOF also predict poor patient outcome from breast cancer. Genetically manipulating SELENOF in human breast cancer cells or in the murine mammary gland by overexpression, silencing or knockout impacted cell viability by affecting both proliferation and cell death. Restoring SELENOF can attenuate a number of aggressive cancer phenotypes in breast cancer cells, including clonogenic survival, and enhance the response to drugs or radiation used in breast cancer therapy. Importantly, enhancing SELENOF expression reduced in vivo tumor growth in a murine xenograft model of breast cancer. These data indicate that SELENOF is a new tumor suppressor in breast cancer.

Loss of SELENOF Induces the Transformed Phenotype in Human Immortalized Prostate Epithelial Cells.

SELENOF is a member of the class of selenoproteins in which the amino acid selenocysteine is co-translationally inserted into the elongating peptide in response to an in-frame UGA codon located in the 3'-untranslated (3'-UTR) region of the SELENOF mRNA. Polymorphisms in the 3'-UTR are associated with an increased risk of dying from prostate cancer and these variations are functional and 10 times more frequent in the genomes of African American men. SELENOF is dramatically reduced in prostate cancer compared to benign adjacent regions. Using a prostate cancer tissue microarray, it was previously established that the reduction of SELENOF in the cancers from African American men was significantly greater than in cancers from Caucasian men. When SELENOF levels in human prostate immortalized epithelial cells were reduced with an shRNA construct, those cells acquired the ability to grow in soft agar, increased the ability to migrate in a scratch assay and acquired features of energy metabolism associated with prostate cancer. These results support a role of SELENOF loss in prostate cancer progression and further indicate that SELENOF loss and genotype may contribute to the disparity in prostate cancer mortality experienced by African American men.

Impact of MnSOD and GPx1 Genotype at Different Levels of Enteral Nutrition Exposure on Oxidative Stress and Mortality: A Post hoc Analysis From the FeDOx Trial.

BACKGROUND: Converting nutrition support to energy results in mitochondrial free radical production, possibly increasing oxidative stress. Highly prevalent single nucleotide variants (SNV) exist for the genes encoding antioxidant enzymes responsible for the detoxification of reactive oxygen species. Our objective was to explore the interaction between nutrition support and genetic SNV's for two anti-oxidant proteins (rs4880 SNV for manganese superoxide dismutase and rs1050450 SNV for glutathione peroxidase 1) on oxidative stress and secondarily on intensive care unit (ICU) mortality. METHODS: We performed a post-hoc analysis on 34 mechanically ventilated sepsis patients from a randomized control feeding trial. Participants were dichotomized into those who carried both the rs4880 and the rs1050450 at-risk alleles (Risk Group) versus all others (Nonrisk Group). We explored the interaction between genotype and percent time spent in the upper median of energy exposure on oxidative stress and ICU mortality. RESULTS: Adjusting for confounders, the slope of log F2-isoprostane levels across percentage of days spent in the upper median of daily kilocalories per kilogram (kcal/kg) was 0.01 higher in the Risk Group compared to the Non-Risk Group (p=0.01). Every 1 percent increase in days spent in the upper median of daily kcal/kg was associated with an adjusted 10.3 percent increased odds of ICU mortality amongst participants in the Risk Group (odds ratio [OR]=1.103, p=0.06) but was highly insignificant in the Nonrisk group (OR=0.991, P=0.79). CONCLUSION: Nutrition support may lead to increased oxidative stress and worse clinical outcomes in a large percent of ICU patients with an at-risk genotype.

The Interaction between Dietary Selenium Intake and Genetics in Determining Cancer Risk and Outcome.

There is considerable interest in the trace element selenium as a possible cancer chemopreventive dietary component, but supplementation trials have not indicated a clear benefit. Selenium is a critical component of selenium-containing proteins, or selenoproteins. Members of this protein family contain selenium in the form of selenocysteine. Selenocysteine is encoded by an in-frame UGA codon recognized as a selenocysteine codon by a regulatory element, the selenocysteine insertion sequence (SECIS), in the 3'-untranslated region of selenoprotein mRNAs. Epidemiological studies have implicated several selenoprotein genes in cancer risk or outcome based on associations between allelic variations and disease risk or mortality. These polymorphisms can be found in or near the SECIS or in the selenoprotein coding sequence. These variations both function to control protein synthesis and impact the efficiency of protein synthesis in response to the levels of available selenium. Thus, an individual's genetic makeup and nutritional intake of selenium may interact to predispose them to acquiring cancer or affect cancer progression to lethality.

Selenium-binding protein 1 alters energy metabolism in prostate cancer cells.

OBJECTIVE: The broad goal of the research described in this study was to investigate the contributions of selenium-binding protein 1 (SBP1) loss in prostate cancer development and outcome. METHODS: SBP1 levels were altered in prostate cancer cell lines and the consequences on oxygen consumption, expression of proteins associated with energy metabolism, and cellular transformation and migration were investigated. The effects of exposing cells to the SBP1 reaction products, H2 O2 and H2 S were also assessed. In silico analyses identified potential HNF4α binding sites within the SBP1 promoter region and this was investigated using an inhibitor specific for that transcription factor. RESULTS: Using in silico analyses, it was determined that the promoter region of SBP1 contains putative binding sites for the HNF4α transcription factor. The potential for HNF4α to regulate SBP1 expression was supported by data indicating that HNF4α inhibition resulted in a dose-response increase in the levels of SBP1 messenger RNA and protein, identifying HNF4α as a novel negative regulator of SBP1 expression in prostate cancer cells. The consequences of altering the levels of SBP1 were investigated by ectopically expressing SBP1 in PC-3 prostate cancer cells, where SBP1 expression attenuated anchorage-independent cellular growth and migration in culture, both properties associated with transformation. SBP1 overexpression reduced oxygen consumption in these cells and increased the activation of AMP-activated protein kinase (AMPK), a major regulator of energy homeostasis. In addition, the reaction products of SBP1, H2 O2 , and H2 S also activated AMPK. CONCLUSIONS: Based on the obtained data, it is hypothesized that SBP1 negatively regulates oxidative phosphorylation (OXPHOS) in the healthy prostate cells by the production of H2 O2 and H2 S and consequential activation of AMPK. The reduction of SBP1 levels in prostate cancer can occur due to increased binding of HNF4α, acting as a transcriptional inhibitor to the SBP1 promoter. Consequently, there is a reduction in H2 O2 and H2 S-mediated signaling, inhibition of AMPK, and stimulation of OXPHOS and building blocks of biomolecules needed for tumor growth and progression. Other effects of SBP1 loss in tumor cells remain to be discovered.

Selenoproteins of the Human Prostate: Unusual Properties and Role in Cancer Etiology.

The prostate is an important organ for the maintenance of sperm health with prostate cancer being a common disease for which there is a critical need to distinguish indolent from aggressive disease. Several selenium-containing proteins have been implicated in prostate cancer risk or outcome due to either enzyme function, the reduced levels of these proteins being associated with cancer recurrence after prostatectomy or their corresponding genes containing single-nucleotide polymorphisms associated with increased risk. Moreover, experimental data obtained from the manipulation of either cultured cells or animal models have indicated that some of these proteins are contributing mechanistically to prostate cancer incidence or progression. Among these are selenocysteine-containing proteins selenoprotein P (SELENOP), glutathione peroxidase (GPX1), and selenoprotein 15 (SELENOF); and the selenium-associated protein selenium-binding protein 1 (SBP1). Genotyping of some of the genes for these proteins has identified functional single-nucleotide polymorphisms that are associated with prostate cancer risk and the direct quantification of these proteins in human prostate tissues has not only revealed associations to clinical outcomes but have also identified unique properties that are different from what is observed in other tissue types. The location of GPX1 in the nucleus and SELENOF in the plasma membrane of prostate epithelial cells indicates that these proteins may have functions in normal prostate tissue that are distinct from that of the other tissue types.

Interaction of NKX3.1 and SELENOP genotype with prostate cancer recurrence.

BACKGROUND: NKX3.1 is a tumor suppressor frequently lost in prostate cancer. Previous studies by others indicated that the risks associated with reduced NKX3.1 levels can be enhanced by anti-oxidant supplementation. Selenium is an essential component of several proteins with anti-oxidant functions and lower levels of selenium have been associated with greater risk of prostate cancer. In contrast, participants of the select prostate cancer prevention trial were at increased risk of prostate cancer when supplemented with selenium when their baseline selenium levels were high. METHODS: In order to investigate whether there was an interaction between a functional polymorphism in NKX3.1 that results in less protein and selenium status with prostate cancer grade or outcome, plasma selenium levels and the genotypes of NKX3.1 and the selenium carrier protein SELENOP were determined from a cohort of men who underwent radical protatectomy. RESULTS: NKX3.1 and SELENOP genotypes were associated with a more aggressive prostate tumor grade at the time of prostatectomy, but there were no significant interactions of NKX3.1 genotype with either selenium status or SELENOP genotype. There was also a significant association between NKX3.1 genotype and prostate cancer recurrence, however this association was modified by SELENOP genotype, but not with plasma selenium levels. CONCLUSIONS: These data indicate that the impact of selenium status on prostate cancer may be influenced by factors other than the amount of selenium in circulation.

Selenium-Binding Protein 1 in Human Health and Disease.

Selenium-binding protein 1 (SBP1) is a highly conserved protein that covalently binds selenium. SBP1 may play important roles in several fundamental physiological functions, including protein degradation, intra-Golgi transport, cell differentiation, cellular motility, redox modulation, and the metabolism of sulfur-containing molecules. SBP1 expression is often reduced in many cancer types compared to the corresponding normal tissues and low levels of SBP1 are frequently associated with poor clinical outcome. In this review, the transcriptional regulation of SBP1, the different physiological roles reported for SBP1, as well as the implications of SBP1 function in cancer and other diseases are presented.

GPX1 Localizes to the Nucleus in Prostate Epithelium and its Levels are not Associated with Prostate Cancer Recurrence.

Glutathione peroxidase 1 (GPX1) is an extensively studied selenium-dependent protein that reduces hydrogen and lipid peroxides to water. Because of its antioxidant function and its responsiveness to dietary intakes of selenium, an essential trace element whose levels are inversely associated with prostate cancer risk, GPX1 levels were assessed in a prostate cancer tissue microarray, comparing cases of recurrent prostate cancer following prostatectomy to non-recurrent controls. While GPX1 is generally considered as a protein that resides in both the cytoplasm and mitochondria, we detected strong nuclear staining by immunofluorescence using GPX1-specific antibodies. Nuclear localization of GPX1 was also observed in both primary prostate epithelial cells and the immortalized prostate-derived cell line RWPE-1, but not in LNCaP or PC3 prostate tumor-derived cell lines. Quantification of GPX1 levels in the entire cell, the cytoplasm, and the nucleus did not indicate any association of either its levels or subcellular distribution with prostate cancer recurrence. While GPX1 levels may not have an impact on survival among men with prostate cancer, the data indicates that this extensively characterized protein may have a novel function in the nucleus of prostate epithelial cells.

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

BACKGROUND: 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. METHODS: Tissue microarrays were used to assess SELENOF levels and cellular location in prostatic tissue. Sera and DNA from participants of the Chicago-based Adiposity Study Cohort were used to quantify selenium levels and genotype frequencies of the genes for SELENOF and the selenium-carrier protein selenoprotein P (SELENOP). Logistic regression models for dichotomous patient outcomes and regression models for continuous outcome were employed to identify both clinical, genetic, and biochemical characteristics that are associated with these outcomes. RESULTS: SELENOF is dramatically reduced in prostate cancer and lower in tumors derived from African American men as compared to tumors obtained from Caucasians. Differing frequency of SELENOF polymorphisms and lower selenium levels were observed in African Americans as compared to Caucasians. SELENOF genotypes were associated with higher histological tumor grade. A polymorphism in SELENOP was associated with recurrence and higher serum PSA. CONCLUSIONS: These results indicate an interaction between selenium status and selenoprotein genotypes that may contribute to the disparity in prostate cancer incidence and outcome experienced by African Americans.

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

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.

Pharmacological inhibition of LSD1 and mTOR reduces mitochondrial retention and associated ROS levels in the red blood cells of sickle cell disease.

Sickle cell disease (SCD), an inherited blood disorder caused by a point mutation that renders hemoglobin susceptible to polymerization when deoxygenated, affects millions of people worldwide. Manifestations of SCD include chronic hemolytic anemia, inflammation, painful vaso-occlusive crises, multisystem organ damage, and reduced life expectancy. Part of SCD pathophysiology is the excessive formation of intracellular reactive oxygen species (ROS) in SCD red blood cells (RBCs), which accelerates their hemolysis. Normal RBC precursors eliminate their mitochondria during the terminal differentiation process. Strikingly, we observed an increased percentage of RBCs retaining mitochondria in SCD patient blood samples compared with healthy individuals. In addition, using an experimental SCD mouse model, we demonstrate that excessive levels of ROS in SCD are associated with this abnormal mitochondrial retention. Interestingly, the LSD1 inhibitor, RN-1, and the mitophagy-inducing agent mammalian target of rapamycin (mTOR) inhibitor, sirolimus, increased RBC lifespan and reduced ROS accumulation in parallel with reducing mitochondria-retaining RBCs in the SCD mouse model. Furthermore, gene expression analysis of SCD mice treated with RN-1 showed increased expression of mitophagy genes. Our findings suggest that reduction of mitochondria-retaining RBCs may provide a new therapeutic approach to preventing excessive ROS in SCD.

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.

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.