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.

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, H 2 O 2 and H 2 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, H 2 O 2 , and H 2 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 H 2 O 2 and H 2 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 H 2 O 2 and H 2 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.

On page 829, the legend for Fig 1 was incorrect. The corrected figure and legend are shown here. We regret this error.

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.

Objective Selenium‐containing proteins, or selenoproteins, are likely to play a role in prostate cancer progression and mortality. Selenoprotein F (SELENOF) is a selenium‐containing protein implicated in prostate cancer etiology due to its dramatic reduction in prostate cancer tissue when compared to adjacent benign regions. The reduction in SELENOF levels in immortalized prostate epithelial cells results in phenotypic features associated with cellular transformation, indicating that SELENOF functions as a tumor suppressor in the prostate. The mechanism behind the reduction of SELENOF levels in prostate cancer remains unknown, although it is suspected that it involves events at the level of protein synthesis, based on work done in tissue culture cells. SELENOF synthesis is controlled by sequences within the 3′ untranslated sequences of SELENOF mRNA known as selenocysteine insertion sequences (SECIS) that are required for the recognition of in‐frame UGA codons as the selenocysteine amino acid. Elongation initiation factor 4a3 (eIF4a3) binds SECIS during times of low selenium availability and suppresses the synthesis of several selenoproteins. Moreover, higher levels of eIF4a3 are associated with increased grade of prostate cancer. The objective of this study is to determine if elevated eIF4a3 levels suppress SELENOF synthesis and contribute to prostate cancer progression. Methodology In order to establish a model for SELENOF loss in prostate‐derived cells, SELENOF levels were determined by western blot and mRNA levels by RT‐PCR in PC3 prostate cancer cells and non‐tumorigenic RWPE‐1 cells. Reporter constructs in which the production of light‐emitting luciferase requires SECIS function to recognize a UGA placed in the open reading frame of the luciferase gene were generated by in vitro mutagenesis. An eIF4a3 overexpression construct was used to overexpress that protein in transfected cells to directly determine its impact on SELENOF synthesis. Results SELENOF levels were 18‐fold lower in PC3 cells when compared to RWPE‐1 cells, although mRNA levels were similar between the two cell lines. Using the luciferase reporter constructs described above, it was determined that the readthrough of the UGA in the luciferase gene and supported by the SELENOF SECIS element was more efficient in the RWPE‐1 cells as compared to PC3 cells. In addition, over‐expression of eIF4a3 in RWPE‐1 cells was achieved and the consequences on SELENOF translation and the transformed phenotype are currently under investigation. Conclusion The loss of SELENOF in prostate cancer is likely to occur at the level of translation and mediated through molecular events involving the SECIS element. A likely candidate for how this occurs has been identified as eIF4a3, an RNA helicase whose levels are elevated in prostate cancer. Enhancing SELENOF levels and/or diminishing eIF4a3 are potentially novel approaches to treat prostate cancers that are no longer responsive to existing treatments. Support or Funding Information This work was supported by grant PC17...

Objectives Selenoprotein F (SELENOF) is a selenocysteine-containing protein whose levels are responsive to selenium availability and are often lower in prostate cancer (PCa) compared to benign regions. Lowering SELENOF levels in cultured, non-transformed prostate cells results in these cells gaining a transformed phenotype, indicating that SELENOF may function as a tumor suppressor. The reasons for the loss of SELENOF in PCa remain unknown. SELENOF translation requires a selenocysteine insertion sequence (SECIS), a stem-loop structure within the 3’– untranslated region (UTR) of the SELENOF mRNA. The objective of this study is to determine if SECIS-mediated UGA recoding is impeded in PCa, resulting in lower SELENOF protein levels. Methods In order to establish a cell model for the decrease in SELENOF levels, SELENOF levels were determined by western blot analysis of protein from PC3 PCa cells and RWPE-1 immortalized but not tumorigenic prostate cells. SELENOF mRNA levels in each of these cell lines was subsequently determined by RT-qPCR. In order to determine the efficiency of UGA readthrough in PC3 and RWPE-1 cells, reporter constructs were generated in which an in-frame UGA was introduced into the open reading frame of firefly luciferase by in-vitro mutagenesis and the constructs included portions of the SELENOF 3’-UTR required for UGA recoding. Results SELENOF levels were 18 times lower in PC3 cells compared to the RWPE-1 cells, while mRNA levels were similar between the two. Using the generated reporter constructs, differences in UGA recoding efficiency in PC3 and RWPE-1 cells were apparent, as were unexpected contributions of portions of the 3’UTR sequences to UGA readthrough efficiency. Conclusions Differing levels of SELENOF seen in prostate tissues were recapitulated in a cell culture model, with there being significantly less protein in PC3 cells compared to RWPE-1 cells, without a comparable difference in mRNA. Changes in SELENOF levels due to altered regulation of UGA recoding may be a significant event in PCa progression. Funding Sources This work was supported by the University of Illinois at Chicago Liberal Arts and Sciences Undergraduate Research Initiative (LASURI) to SK and Department of Defense Prostate Cancer Research Program to AMD.

Objectives Selenoprotein F (SELENOF) levels are responsive to available dietary selenium and found in high levels in benign prostate cells. It is implicated in prostate cancer (PCa) mortality due to associations between polymorphisms in the corresponding gene and death from the disease. SELENOF levels are dramatically lower in prostate cancer compared to adjacent benign tissue. The objective of this study was to determine whether reducing SELENOF levels in human, non-transformed RWPE-1 prostate epithelial cells alters their phenotype to implicate SELENOF loss in PCa progression. Methods SELENOF levels were reduced in RWPE-1 cells that express high levels of SELENOF using a SELENOF shRNA construct. Proliferation was determined by quantifying DNA using fluorometric dsDNA quantitation. Growth in soft agar and cell mobility of cells in culture (wound healing assay) were imaged using an Evos FL microscope and quantified using Image J software. The oxygen consumption rate (OCR) was measured using a Seahorse XFe24 Analyzer. Results SELENOF levels were reduced in RWPE-1 and these cells exhibited decreased contact inhibition in culture (n = 3, P < 0.001) when compared to controls. Normal prostate epithelial cells are atypical in that they rely on glycolysis for energy production, have a truncated TCA cycle, and a metabolic shift from glycolysis to oxidative phosphorylation (OXPHOS) occurs in PCa. Reducing SELENOF in RWPE-1 cells resulted in higher OCR compared to controls, indicating that SELENOF can impact the sources and pathways used in cellular energy metabolism. Conclusions Reduced SELENOF levels in RWPE-1 prostate cells resulted in properties consistent with a transformed phenotype and an increase in OCR, and indicating that the reduction in SELENOF may contribute a metabolic shift towards a PCa cancer-like metabolism. Together, these results indicate that SELENOF loss likely contributes to cancer progression. Funding Sources This work was supported by a grant from the Department of Defense to AMD and a Pre-Doctoral Education for Clinical and Translational Scientists Fellowship to LKH.

No abstract