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.

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.

A Multiplex One-Step RT-qPCR Protocol to Detect SARS-CoV-2 in NP/OP Swabs and Saliva.

Since December 2019, SARS-CoV-2 has spread extensively throughout the world, with more than 117 million reported cases and 2.6 million deaths (Johns Hopkins coronavirus resource center, https://coronavirus.jhu.edu/map.html). Detecting the virus is the first step in diagnosing the infection, followed by quarantine to prevent transmission. Nasopharyngeal/oropharyngeal swabs (NP/OP) and saliva are two specimen types that are most often analyzed to detect SARS-CoV-2 by molecular tests that detect viral RNA or by antigen/antibody tests that detect viral proteins and/or the host immune response against the virus. Compared to antigen/antibody tests, molecular tests are highly sensitive and specific for detecting the virus. A significant drawback is that specimen collection requirements are specific to each test and cannot be interchanged with another test. Some tests are qualified to be used on NP swabs or saliva, but not both specimen types. Even with NP swabs, a test may be qualified to detect the virus only with swabs collected in viral transport medium (VTM) but not in other media. These restrictive pre-analytic steps are disadvantageous in that a lab would have to develop and validate different tests for SARS-CoV-2 depending on the specimen type and collection media, with added setup cost, infrastructure, and training requirements. To overcome these problems, we developed and validated a cost-effective multiplex reverse-transcription real-time PCR assay that can be used to detect SARS-CoV-2 in different specimen types. The assay is highly sensitive and specific, can be used to detect the virus in saliva as well as NP swabs collected in different media such as VTM, saline, and commercial preservative fluid, and serves as one test for all applications. The protocol also describes an optimal laboratory setup and unidirectional workflow for detecting SARS-CoV-2 by RT-qPCR. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Manual viral nucleic acid extraction from NP/OP swabs collected in different media, and from saliva Alternate Protocol 1: Low-throughput automated extraction on the Qiagen EZ1 Advanced XL machine (1-14 samples) Alternate Protocol 2: High-throughput automated extraction on the Kingfisher Flex machine (1-96 samples) Basic Protocol 2: Multiplex RT-qPCR protocol to detect SARS-CoV-2 Alternate Protocol 3: Multiplex one-step RT-qPCR protocol to detect SARS-CoV-2 with S and E gene probes labeled with the same fluorochrome.

Development and validation of viral load assays to quantitate SARS-CoV-2.

SARS-CoV-2 has infected more than 30 million persons throughout the world. A subset of patients suffer serious consequences that require hospitalization and ventilator support. Current tests for SARS-CoV-2 generate qualitative results and are vital to make a diagnosis of the infection. However, they are not helpful to follow changes in viral loads after diagnosis. The ability to quantitatively assess viral levels is necessary to determine the effectiveness of therapy with anti-viral or immune agents. Viral load analysis is also necessary to determine the replicative potential of strains with different mutations, emergence of resistance to anti-viral agents and the stability of viral nucleic acid and degree of RT-PCR inhibition in different types of collection media. Quantitative viral load analysis in body fluids, plasma and tissue may be helpful to determine the effects of the infection in various organ systems. To address these needs, we developed two assays to quantitate SARS-CoV-2. The assays target either the S or E genes in the virus, produce comparable viral load results, are highly sensitive and specific and have a wide range of quantitation. We believe that these assays will be helpful to manage the clinical course of infected patients and may also help to better understand the biology of infection with SARS-CoV-2.

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.

Cyst Fluid Biosignature to Predict Intraductal Papillary Mucinous Neoplasms of the Pancreas with High Malignant Potential.

BACKGROUND: Current standard-of-care technologies, such as imaging and cyst fluid analysis, are unable to consistently distinguish intraductal papillary mucinous neoplasms (IPMNs) of the pancreas at high risk of pancreatic cancer from low-risk IPMNs. The objective was to create a single-platform assay to identify IPMNs that are at high risk for malignant progression. STUDY DESIGN: Building on the Verona International Consensus Conference branch duct IPMN biomarker review, additional protein, cytokine, mucin, DNA, and microRNA cyst fluid targets were identified for creation of a quantitative polymerase chain reaction-based assay. This included messenger RNA markers: ERBB2, GNAS, interleukin 1ß, KRAS, MUCs1, 2, 4, 5AC, 7, prostaglandin E2R, PTGER2, prostaglandin E synthase 2, prostaglandin E synthase 1, TP63; microRNA targets: miRs 101, 106b, 10a, 142, 155, 17, 18a, 21, 217, 24, 30a, 342, 532, 92a, and 99b; and GNAS and KRAS mutational analysis. A multi-institutional international collaborative contributed IPMN cyst fluid samples to validate this platform. Cyst fluid gene expression levels were normalized, z-transformed, and used in classification and regression analysis by a support vector machine training algorithm. RESULTS: From cyst fluids of 59 IPMN patients, principal component analysis confirmed no institutional bias/clustering. Lasso (least absolute shrinkage and selection operator)-penalized logistic regression with binary classification and 5-fold cross-validation used area under the curve as the evaluation criterion to create the optimal signature to discriminate IPMNs as low risk (low/moderate dysplasia) or high risk (high-grade dysplasia/invasive cancer). The most predictive signature was achieved with interleukin 1ß, MUC4, and prostaglandin E synthase 2 to accurately discriminate high-risk cysts from low-risk cysts with an area under the curve of up to 0.86 (p = 0.002). CONCLUSIONS: We have identified a single-platform polymerase chain reaction-based assay of cyst fluid to accurately predict IPMNs with high malignant potential for additional studies.

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.