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. Please note that the title of this document, 'Deinteraction of NKX3.1 and SELENOP genotype with prostate cancer recurrence', differs slightly from the published version

Selenoproteins in Cancer Etiology

Selenium exerts its functions mainly through its incorporation into selenoproteins, which contain the amino acid selenocysteine. Animal and epidemiological, but not supplementation studies, have provided evidence supporting the role of selenium in cancer etiology. Selenoproteins GPx-1 and Sep15, specifically the polymorphisms in their genes, have been implicated in mediating the chemopreventative effect of selenium in several cancer types. Polymorphisms in MnSOD confer different risk for prostate cancer and poor outcome depending on dietary antioxidant intake and the activity of GPx-1. The gene-gene interaction between MnSOD and GPx-1 was examined to elucidate the molecular mechanisms underlying the differing levels of cancer risk. In order to investigate whether a molecular interaction between polymorphic variants of GPx-1 and MnSOD exists, GPx-1 and MnSOD genotype and levels were manipulated in MCF-7 cells to exclusively express allele specific variants of the anti-oxidant enzymes by transfection. Allele-specific interactions of GPx-1 and MnSOD altered the levels of critical proteins implicated in cancer. Despite these interactions, GPx-1 and MnSOD levels were not associated with prostate cancer recurrence after radical prostatectomy. An association between polymorphisms in Sep15, selenium levels and prostate cancer mortality has been reported. The functional polymorphisms, which are 6-fold more prevalent in African Americans, form a haplotype and exhibit a trend toward an association with prostate cancer mortality. In order to determine whether the levels of Sep15 are associated with prostate cancer tumor grade, tissue samples from a cohort of men who underwent radical prostatectomy were examined. Although Sep15 levels were not associated with prostate tumor grade, its levels were reduced in prostate cancers compared to benign tissues, as well as in prostate tumors of African Americans compared to Caucasians. The studies indicate the importance of subcellular localization of selenoproteins in different locations than traditionally observed, and suggest that the localization of GPx-1 and Sep15 may be important in elucidating their roles in carcinogenesis

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

Rapamycin increases levels of GPx-4, MnSOD and pS6 protein levels in cell lines.

<p>The effect of 1 ng/mL rapamycin on GPx-4 and MnSOD and pS6 protein levels in KU812a, MEG-01, GM10832 and MDA-MB-231 is shown. GPx-4 by 3-day 1 ng/mL rapamycin treatment in KU812a and MEG-01 (<i>P</i> > 0.2), but was increased 3-fold in GM10832 (<i>P</i> = 0.05) and 6-fold in MDA-MB-231 cells (<i>P</i> = 0.02). The disappearance of pS6 signal following rapamycin treatment indicates inhibition of mTOR. Data shown is representative of three independent experiments.</p

Rapamycin enhances GPx-1 protein and enzyme activity levels in all cell lines investigated.

<p>The effect of rapamycin on GPx-1 activity (A) and protein levels (B) in KU812a, MEG-01 GM10832 or MDA-MB-231 is shown. The effect of rapamycin on steady-state GPx-1 transcript levels as determined by RT-qPCR and normalization of GPx-1 Ct values to Ct values for 18s RNA (C). Rapamycin significantly increased protein respectively 3-fold and 1.3-fold in KU812a and MEG-01 (<i>P</i> = 0.05). Steady state transcript levels for GPx-1 were unaffected by treatment with rapamycin in KU812a. Data shown is representative of three independent experiments. * = <i>P</i><0.001, ** = <i>P</i><0.01. † = <i>P</i><0.05. Error bars indicate S.D.</p

GPx-1 levels are enhanced in a dose- and time-dependent manner following imatinib treatment.

<p>The dose- and time-dependent increases in GPx activity following 100 nM and 150 nM imatinib treatment of KU812a cells for 7 days (A) or 150 nM imatinib treatment for 3 or 7 days (B). The effect of imatinib treatment on GPx1 protein levels. Data shown is representative of thee independent experiments were performed (C). * = <i>P</i><0.001, † = <i>P</i><0.05, compared to 100 nM or 3-day imatinib treatment. Error bars indicate S.D.</p

GPx-1 protein and activity levels are decreased by exogenous Bcr-Abl in MDA-MB-231.

<p>MDA-MB-231 cells transfected with a GFP-tagged Bcr-Abl vector and the transfection success was as assessed by fluorescence microscopy (A). The effect of ectopic Bcr-Abl expression on GPx enzyme activity in MDA-MB-231 cells. GPx activity was decreased 0.6-fold by ectopic expression of Bcr-Abl. Data shown is representative of one experiment. Two independent experiments were performed. * = <i>P</i><0.001. Error bars indicate S.D.</p

GPx-1 is increased in MEG-01, but not MDA-MB-231 and GM10832, following imatinib treatment.

<p>The effect of imatinib on GPx enzyme activity (A) and GPx-1 protein (B) in MEG-01, MDA-MB-231 and GM10832 is shown. GPx activity was increased 2-fold, and protein increased 4-fold (<i>P</i><0.01) by 300 nM imatinib treatment in MEG-01. GPx-1 activity and protein levels did not change following imatinib treatment of MDA-MB-231 (500 nM imatinib) and GM10832 (300 nM). Data presented in A is the result of three independent experiments * = <i>P</i><0.001. Error bars indicate S.D.</p

GPx-1 activity and protein, but not steady-state transcript levels are enhanced by treatment with imatinib.

<p>KU812a cells were treated for 7 days with 150 nM imatinib and the effects on GPx enzyme activity (A), GPx-1 protein (B) and GPx-1 transcript levels expressed as the GPx-1 Ct values normalized to the Ct values for TBP mRNA(C) is shown. GPx activity was increased 2.5-fold, and protein increased 7-fold (<i>P</i> = 0.02) by imatinib treatment, while steady-state transcript levels were unaltered. Data shown in A and C are the results of three independent experiments. * = <i>P</i><0.001. Error bars indicate the S.D.</p

The translational efficiency of UGA readthrough by the GPx-1 SECIS is not enhanced by rapamycin.

<p>Diagrammatic representation of the pLNCX-UGA-GPx-1 reporter construct used for quantification of UGA read-through, containing the GPx-1 SECIS element (A). The effect of rapamycin (1 ng/mL), selenium (100 nM), or both on the translational efficiency of UGA readthrough in MDA-MB-231 cells infected with the pLNCX-UGA-GPx1 SECIS reporter construct as assessed by determining luciferase activity; relative luciferase levels were normalized to β-galactosidase levels to calculate translational efficiency (B). SECIS-dependent translational efficiency was not increased following 1 ng/mL rapamycin treatment, but was increased 2.6-fold by incubation of cells containing the reporter construct with selenium. Data shown represents two independent experiments. * = <i>P</i><0.001. Error bars indicate the S.D. The effect of rapamycin on U49C GPx-1 levels was determined by immunoblotting for GPx-1 by western blot analysis (C).</p