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Introduction/Background Endocrine therapy remains the fundamental treatment for advanced HR+ breast cancer (BC). For those patients who become refractory to endocrine therapy, resistance may be associated with mutations, amplifications, and fusions in the ESR1-encoded estrogen receptor. Here we examine the frequency of ESR1 alterations and the associated genomic landscape. Methods HR+/HER2- BC samples were sequenced with the OncomapTM ExTra assay, which uses whole-exome DNA sequencing with germline subtraction to detect somatic single nucleotide substitutions, indels, and copy number alterations (CNAs), and uses RNA sequencing to detect gene fusions. Tumor mutational burden (TMB) and microsatellite instability (MSI) are also reported. For analysis, BC samples were grouped by site: local (primary breast or regional lymph node) versus metastatic. Prior treatment history was unknown. Testing for a possible association between ESR1 and other biomarkers (genes, MSI, and TMB) was done using Fisher’s Exact Test (p≤0.05). Results A total of 988 HR+HER2- breast cancer patient samples were included in the analysis. Of these, 821 (83.1%) were local samples and the remaining 167 were metastatic samples, with liver (63, 37.7%), bone (20, 12.0%), skin (16, 9.6%) and chest wall (15, 9.0%) being the most common locations. ESR1 alterations were present in 84 tumor samples, 37 local and 47 metastatic, representing 4.5% and 28.1% of samples, respectively. ESR1 alterations included missense mutations (54 samples), fusions (29 samples), and amplifications (8 samples). The most common missense mutations were Y537S (20 samples, 37.0%), D538Q (20 samples, 37.0%), and E380Q (6 samples, 11.1%), which were located in the ligand binding domain and included both clonal and subclonal events. There were 30 ESR1 fusions identified, 17 (2.1%) in local and 13 in metastatic (7.7%) samples. Most fusions (24 samples, 82.8%) involved the same partner, CCDC170, while the other 6 fusions had unique partners. Examination of the 143 other biomarkers altered among ESR1-altered samples revealed 15 genes and MSI-high status that appeared to be over-represented (Table 1). However, none of the associations were statistically significant after correcting for multiple comparisons. Further, there was no evidence that the prevalence of ERBB2, TP53, AKT1 and PIK3CA alterations differed by ESR1 status (Table 1). Conclusions ESR1 alterations were significantly more common in HR+/HER2- metastatic breast cancer samples compared to local samples. Comprehensive genomic profiling with RNA sequencing identified both common and rare ESR1 fusions, which were most frequent in the metastatic samples. No significant difference in the molecular profile of ESR1 altered vs ESR1 wildtype samples was found in this cohort. Clinical trials with novel selective ER degraders (SERDs) to target these ESR1 alterations are ongoing. Table 1. Biomarkers that showed the highest association with ESR1 and other notable breast cancer biomarkers. Citation Format: Gargi Basu, Sameer S. Udhane, Susan Dombrowski, Lenny Hong, Fadel Alyaqoub, Michelle Barbi de Moura, Thiruppavai Chandrasekaran, Turgut Dogruluk, David Driscoll, Aimee Jalkanen, Pawan Noel, Szabolcs Szelinger, Min Wang, David Hall, Jess Hoag, Janine Lobello, Frederick Baehner, Snehal Thakkar, Joyce O’Shaughnessy. ESR1-alterations in HR+HER2- breast cancer patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-14-12.

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, H2O2 and H2S 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, H2O2, and H2S 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 H2O2 and H2S 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 H2O2 and H2S‐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.

807 Background: Activation of the Wnt signaling pathway leads to increases in β-catenin levels, which subsequently increases transcription of various genes, including those involved in cell proliferation. Dysregulation of the Wnt signaling pathway has been observed in numerous gastrointestinal (GI) cancers. Here we examine Wnt pathway activation across all GI cancers by investigating alterations in certain genes of the Wnt pathway, which may identify patients eligible for clinical trials. Methods: The Oncomap ExTra genomic profiling assay utilizes tumor-normal, whole-exome, whole-transcriptome DNA and RNA sequencing to identify somatic alterations in tumors. The assay detects single-nucleotide substitutions, indels, copy number alterations, alternative transcripts, and gene fusions. Alterations in genes that activate Wnt signaling and their frequency across all GI cancers was determined. Results: A total of 1928 patients assayed April 2018 to July 2022 were included. The Wnt signaling pathway was activated in 922 (47.8%) patients. Wnt pathway activation varied substantially across GI cancers, from 81.1% in colorectal cancer (CRC) to 3.4% in anal cancer. APC was the most commonly altered gene in CRC, small bowel and biliary carcinomas. CTNNB1 was predominant in liver (41.2%), MEN1 in neuroendocrine (14.6%), and RNF43 in gastric (9.4%) cancer. Of 921 patients with alterations in the Wnt pathway, 5.2% had a co-mutation in another Wnt pathway-related gene. RSPO2/3 fusions were found in 4 CRC, 1 stomach, 1 biliary, and 1 small bowel cancer and were mutually exclusive with other alterations in the Wnt pathway. Conclusions: Wnt signaling is activated in about half of all GI cancers, and APC alterations are the most frequently observed. Whole transcriptome profiling allowed us to identify RSPO2/3 fusions, which also contribute to activation of the Wnt pathway. Wnt activation appears to be a particularly important in CRC, with 81.1% of CRC having a Wnt pathway alteration. Our results highlight the therapeutic potential of targeting the abnormal Wnt/β-catenin signaling pathway in GI malignancies. [Table: see text]

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

OBJECTIVES: Sickle Cell Disease (SCD) is caused by a single point mutation in the β-globin gene, resulting in the polymerization of the altered hemoglobin β(S) in hypoxic conditions, affecting millions of people worldwide. Previous studies have shown that there are lower selenium levels and reduced activity of the antioxidant selenoprotein GPX1 in SCD patients. The objective of this study was to investigate the consequences of selenium deficiency in a SCD mouse model. METHODS: Humanized SCD (HbSS) mice (Townes model) and wild type (HbAA) mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were fed either a selenium-deficient (

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.

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.

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.

OBJECTIVES: Selenium (Se) is a non-metallic, essential trace element for many organisms, including humans. Se may have potential in cancer prevention, as evidenced by multiple animal and human epidemiological studies. Selenium-binding protein 1 (SBP1) is a highly-conserved protein that covalently binds Se. Prostate cells exhibit cytoplasmic and nuclear SBP1 localization. Reduced levels of nuclear SBP1 were previously shown to be associated with a higher tumor grade and a greater likelihood of prostate cancer recurrence following prostatectomy. The Krebs cycle of the normal prostate is inhibited in favor of the production of citrate energy, therefore distinguishing the energy metabolism of the normal prostate from that of other organs. This inhibition is generally relieved during cancer progression. The objective of this study was to investigate a contribution of SBP1 in this process of metabolic shift. METHODS: Human PC-3 prostate cancer cells that express very low levels of SBP1 were engineered to express the native, nuclear-targeted, or nuclear-excluded SBP1 by transfection of constitutive- or inducible-SBP1 expression constructs. Oxidative phosphorylation (OXPHOS) was examined by quantifying the oxygen consumption rate (OCR) using a Seahorse XF analyzer (Agilent, Inc.). Western blotting with SBP1 and phospho-AMPK specific antibodies was employed to interrogate the activation of AMP-activated protein kinase (AMPK), a key regulator of energy homeostasis. RESULTS: Overexpressing SBP1 in PC-3 cells increased the activating phosphorylation of AMPK at Thr172 by approximately 70%. In addition, overexpressing the native, nuclear-targeted or nuclear-excluded SBP1 significantly reduced OCR and mitochondrial ATP synthesis, as measures of mitochondrial respiration (32–43% reduction in basal respiration and 40–55% reduction in maximal respiration). These changes in energy metabolism occurred without affecting cellular proliferation. CONCLUSIONS: These results indicate that the loss of SBP1 during prostate cancer development may contribute to disease progression by facilitating the transition to an energy metabolism that favors increased energy production as well as the building blocks required to sustain tumor growth and survival. FUNDING SOURCES: National Institute of Health.