Estrogen receptor (ER)-, progesterone receptor (PR)- and HER2-dependent expression of largely understudied genes with large number of copy number gain events in breast cancer

Estrogen receptor (ER)-, progesterone receptor (PR)- and HER2-dependent expression of largely understudied genes with large number of copy number gain events in breast cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Estrogen receptor (ER)-, progesterone receptor (PR)- and HER2-dependent expression of largely understudied genes with large number of copy number gain events in breast cancer Caglar Berkel This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3863281/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Breast cancer can be classified into several molecular subtypes based on the status of hormone receptors including estrogen receptor (ER) and the progesterone receptor (PR), and of human epidermal growth factor receptor 2 (HER2; ERBB2). Copy number variants (CNVs) cover 5-10% of the human genome, and are responsible for the majority of variability among different individuals’ genomes based on nucleotide coverage. In the present study, I identified largely understudied genes for which copy number gain events (CNV gains) are observed in more than half of breast cancer patients, and whose expression is upregulated in breast tumors compared to non-malignant breast tissue. I found 6 genes meeting these criteria: SLC45A3, RGS7, FCRL4, CSMD4, FAM135B and TRAF7. I then studied these 6 genes in terms of ER, PR and HER2 status in breast cancer patients. I found that SLC45A3 expression is lower in breast cancer patients with ER-positive or PR-positive status. RGS7 mRNA levels are higher in breast cancer patients with ER-positive or PR-positive status but lower in those with HER2-positive status. FCRL4 expression is lower in breast tumors with ER-positive or PR-positive status. CSMD3 transcript levels are lower in breast tumors with HER2-positive status. FAM135B expression is higher in breast cancer patients with ER-positive or PR-positive status but lower in those with HER2-positive status. Combined, our study points that most of the studied genes exhibit receptor status-dependent changes in the expression, and that further mechanistic studies are needed for these genes with large number of copy number gain events in breast cancer. breast cancer copy number variation estrogen receptor progesterone receptor HER2 SLC45A3 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Breast cancer is the most common malignancy and the leading cause of cancer-related mortality in women globally [Sung et al., 2021]. Breast cancer can be classified into several molecular subtypes based on the status of hormone receptors (HRs, including estrogen receptor (ER) and the progesterone receptor (PR)) and of human epidermal growth factor receptor 2 (HER2, also called ERBB2) [Allison et al., 2020]. Approximately 70% of breast tumors are hormone receptor-positive (HR+). Patients with HR+ breast cancer generally have a favorable prognosis, and those with HR-negative (HR-) breast cancer have a poor prognosis. PR is an estrogen-regulated gene; therefore, ER-positive (ER+) tumors are mostly also PR positive (PR+), whereas ER-negative (ER-) tumors are usually PR negative (PR-). Thus, single HR+ (i.e., ER+/PR- or ER-/PR+) tumors represent a minority of breast cancer cases (about 10% of all breast cancers) [Bae et al., 2015; Zhao and Gong, 2021; Li et al., 2020]. It was also reported that single HR+ breast tumors have worse prognosis than ER+, PR+ breast tumors; and that ER-, PR+ subtype has similar prognosis relative to ER-, PR- subtype [Dauphine et al., 2020; Lv et al., 2020]. Therefore, it is of high clinical interest to identify molecular variations (for instance, differences in gene regulation and expression) contributing to these differences in prognosis between breast cancer patients with different receptor status. Copy number variants (CNVs) cover approximately 5-10% of the human genome, and are responsible for the majority of genome variability among individuals based on nucleotide coverage [Abel et al., 2020; Zarrei et al., 2015]. CNVs show considerable variability in terms of both size and frequency, and can disrupt gene function substantially via alterations in gene dosage, coding sequences, and gene regulation [Conrad et al., 2010]. CNVs also alter the diploid status of DNA, and may have no phenotypic effect, explain adaptive traits or can contribute to disease [Zarrei et al., 2015]. CNVs are distributed not regularly in the genome; the pericentromeric and subtelomeric regions of chromosomes exhibit a particularly high rate of variation [Zarrei et al., 2015]. The size of CNVs is in most cases defined as larger than 50 bp, whereas smaller elements are known as insertions or deletions (indels) [MacDonald et al., 2014; Redon et al., 2006; Levy et al., 2007]. In this work, I identified largely understudied genes (i.e. maximum 4 papers in PubMed in the context of breast cancer) for which copy number gains (CNV gain) are observed in more than half of breast cancer patients, and which also show increased expression in breast tumors compared to non-malignant breast tissue. I found 6 genes meeting these criteria: SLC45A3, RGS7, FCRL4, CSMD4, FAM135B and TRAF7. I then studied these 6 genes in terms of ER (estrogen receptor), PR (progesterone receptor) and HER2 (human epidermal growth factor receptor 2; ERBB2) expression in breast cancer patients. Furthermore, I analyzed the expression of these genes among all combinations of receptor status (8 different conditions from triple negative to triple positive) in breast tumors. Methods Datasets In this study, I obtained copy number variation (CNV) data from The National Cancer Institute Genomic Data Commons (GDC) Data Portal (https://portal.gdc.cancer.gov/), which includes CNV data from TCGA-BRCA project in addition to others [Jensen et al., 2017; Zhang et al., 2021; Berger et al., 2018; Cancer Genome Atlas Network, 2012; Ciriello et al., 2015]. For transcriptomics analysis, I used alternatively processed and compiled RNA-Seq and clinical data for samples from The Cancer Genome Atlas (TCGA) project (GSE62944) [Rahman et al., 2015; Cancer Genome Atlas Research Network, 2013; Wilks et al., 2014]. This dataset contains data from 1119 breast cancer patients and 113 women with no breast tumors. Total sample size (n) is 1232. Sample sizes for subgroups are as following: ER-negative = 230; ER-positive = 785; PR-negative = 228, PR-positive = 782; HER2-negative = 162 and HER2-positive = 545. This dataset can also be accessed as a SummarizedExperiment through Bioconductor (in Experiment Packages >> GSE62944) [Arora, 2023; Huber et al., 2015; Gentleman et al., 2004]. Genes in this study were selected for further analysis if (1) for these genes, there are less than or equal to 4 papers in PubMed in the context of breast cancer, (2) their expression are upregulated in breast tumors compared to non-malignant breast tissue, and (3) copy number gains (CNV gains) for these genes are observed in more than half of the patients with breast cancer. In the dataset, ER, PR and HER2 status from patient tumor samples had been determined by IHC (Immunohistochemistry) [Cancer Genome Atlas Research Network, 2013]. Data analysis and visualization This study was performed in R programming environment (R version 4.2.1 (2022-06-23)) using R Studio IDE from posit [R Core Team, 2022]. Following R / Bioconductor packages (https://bioconductor.org/) were used throughout the analysis [Huber et al., 2015; Gentleman et al., 2004]: tidyverse [Wickham et al., 2019; Wickham, 2016; Wickham, 2022], readxl [Wickham and Bryan, 2023], ExperimentHub [Morgan and Shepherd, 2022], SummarizedExperiment [Morgan et al., 2022], ggpubr [Kassambara, 2023], glue [Hester and Bryan, 2022], rmarkdown [Allaire et al., 2023] and knitr [Xie, 2023]. When data is not normally distributed, I performed Wilcoxon test [Kassambara, 2023]. Relative expression values shown in plots are log10 transformation of read counts. Data analysis and visualization was performed as previously reported [Berkel and Cacan, 2020; Berkel and Cacan, 2021]. Results Genes with copy number gains observed in more than half of breast cancer patients First, I identified genes with copy number gain events (CNV gain) observed in more than 50% of breast cancer patients (n = 1058), using CNV (copy number variation) data from Genomic Data Commons (GDC) Data Portal (Figure 1). I found that there are 49 genes with copy number gain events observed in more than half of the patients with breast cancer (Figure 1). I ordered genes from highest to lowest CNV gain percentage in Figure 1. Gene with the highest CNV gain percentage (74.1%) in breast cancer patients is MDM4, followed by SLC45A3 (73.44%) and ELK4 (73.35%) (Figure 1). From this list of genes, I only selected genes indicated by red columns in Figure 1 (namely, SLC45A3, RGS7, FCRL4, CSMD4, FAM135B and TRAF7) for further analysis based on two criteria: (1) the expression of the gene is increased in breast tumors compared to healthy breast tissue (see below), and (2) there are maximum 4 papers in PubMed for the gene in the context of breast cancer, so it is largely understudied in breast cancer research. I analyzed the expression of these 6 selected genes in terms of ER (estrogen receptor), PR (progesterone receptor) and HER2 (human epidermal growth factor receptor 2; ERBB2) expression in breast cancer patients. SLC45A3 expression is lower in breast cancer patients with ER-positive or PR-positive status Initially, I found that SLC45A3 (Solute Carrier Family 45 Member 3) expression is higher in breast tumors compared to normal breast tissue (p = 0.026, our first criteria, Figure 2A). Next, I showed that SLC45A3 expression is lower in ER (estrogen receptor)-positive (p < 2e-16) and PR (progesterone receptor)-positive (p = 2.5e-12) breast cancer patients compared to those with ER-negative and PR-negative tumors, respectively (Figure 2B, 2C). However, there is no difference in SLC45A3 transcript levels between HER2-negative and HER-positive breast cancer patients (p = 0.75, Figure 2D). I also observed that breast cancer patients with triple negative status (ER-, PR-, HER2-; “- - -”) have the highest mean expression of SLC45A3 (Figure 2E). RGS7 expression is higher in breast cancer patients with ER-positive or PR-positive status The percentage of breast cancer patients with RGS7 (Regulator Of G Protein Signaling 7) copy number gains (CNV gain) is around 70% (Figure 1). I found that RGS7 transcript levels are higher in breast tumors than normal breast tissue (p = 2.1e-05, Figure 3A). RGS7 expression is higher in breast tumors with ER-positive (p = 1.6e-09) or PR-positive status (p = 1.4e-09) compared to those with ER-negative or PR-negative status, respectively (Figure 3B, 3C). In contrast, RGS7 mRNA levels are lower in breast tumors with HER2+ status than HER2- status (p = 0.018) (Figure 3D). I also showed that among all receptor status combinations, breast tumors with ER+, PR+ and HER2- status (“+ + -”) have the highest RGS7 expression (Figure 3E). FCRL4 expression is lower in breast tumors with ER-positive or PR-positive status After showing that the percentage of breast cancer patients with FCRL4 (Fc Receptor Like 4) copy number gains is around 68% (Figure 1), and that the expression of FCRL4 is higher in breast tumors than non-malignant breast tissue (p = 2.4e-12, Figure 4A), I found that FCRL4 transcript levels are lower in breast tumors with ER-positive (p = 1.9e-06) or PR-positive status (p = 0.0025) than those with ER-negative or PR-negative status, respectively (Figure 4B, 4C). However, FCRL4 expression does not significantly change depending on HER2 status in breast tumors (p =0.63; Figure 4D). FCRL4 expression based on ER, PR and HER2 status combinations is shown in Figure 4E. CSMD3 transcript levels are lower in breast tumors with HER2-positive status I found that the percentage of breast cancer patients with CSMD3 (CUB And Sushi Multiple Domains 3) copy number gains is around 57% (Figure 1), and that the expression of CSMD3 is higher in breast tumors compared to healthy breast tissue (p = 0.025, Figure 5A). Then, I showed that CSMD3 mRNA levels do not change depending on either ER or PR status in breast tumors (p = 0.18 and 0.53, respectively) (Figure 5B, 5C). However, I observed that CSMD3 expression is lower in HER2+ breast tumors than HER2- breast tumors (p = 0.0011; Figure 5D). In addition, CSMD3 expression is higher in ER+,PR+,HER2- breast tumors compared to those with triple positive status (TNBC) (p = 0.0069; Figure 5E). FAM135B expression is higher in breast cancer patients with ER-positive or PR-positive status but lower in those with HER2-positive status After showing that the percentage of breast cancer patients with FAM135B (Family With Sequence Similarity 135 Member B) copy number gains is around 53% (Figure 1), and that FAM135B mRNA levels are higher in breast tumors than non-malignant breast tissue (p = 0.019; Figure 6A), I found that FAM135B transcript levels are higher in breast cancer patients with ER-positive (p < 2e-16) or PR-positive status (p < 2e-16) compared to those with ER-positive or PR-positive status, respectively (Figure 6B, 6C); but, lower in those with HER2-positive status than those with HER2-negative status (p = 0.0028) (Figure 6D). As expected, FAM135B expression is the highest in ER+, PR+, HER2- breast tumors (Figure 6E). TRAF7 expression does not change depending on ER, PR and HER2 status in breast tumors I first showed that the percentage of patients with breast cancer who have TRAF7 (TNF Receptor Associated Factor 7) copy number gains is 50% (Figure 1), and that TRAF7 transcript levels are higher in breast tumors compared to healthy breast tissue (p < 2e-16, Figure 7A). However, I observed that TRAF7 transcript levels do not change depending on ER, PR and HER2 status in tumors from breast cancer patients (p = 0.1, 0.48 and 0.87, respectively) (Figure 7B, 7C, 7D). Therefore, there is no significant difference in TRAF7 expression between different combinations of these three receptors’ status in breast tumors (Figure 7E). Discussion In this study, I identified largely understudied genes for which copy number gains are observed in more than half of the patients with breast cancer, and which also show increased expression in breast tumors compared to non-malignant breast tissue. I found 6 genes meeting these criteria: SLC45A3, RGS7, FCRL4, CSMD4, FAM135B and TRAF7. I then studied these 6 genes in terms of ER (estrogen receptor), PR (progesterone receptor) and HER2 (human epidermal growth factor receptor 2; ERBB2) expression in breast cancer patients. I observed parallel changes in the expression of these genes in terms of ER and PR status; in other words, for instance, if the expression of one of the genes is increased in ER+ breast tumors; similarly, its expression is increased in PR+ cells. First, I found that more than 70% of breast cancer patients have copy number gains in SLC45A3 gene, and that SLC45A3 expression is lower in breast cancer patients with ER-positive or PR-positive status compared to those with ER-negative or PR-negative status. This gene has been mostly studied in the context of prostate cancer [Kasajima et al., 2020]. The fusion RNA of this gene, SLC45A3-ELK4, generated by cis-splicing between neighboring genes, was found in prostate cancer samples, and it was shown to function as a long non-coding chimeric RNA (lnccRNA) [Qin et al., 2017]. SLC45A3-ELK4 regulates cancer cell proliferation by its transcript, not translated protein; and the level of this chimeric transcript correlates with prostate cancer disease progression [Qin et al., 2017; Zhang et al., 2012]. RGS7 have copy number gains in more than 70% of breast cancer patients, and RGS7 expression is higher in breast cancer patients with ER-positive or PR-positive status but lower in those with HER2-positive status, compared to those with ER-negative, PR-negative, HER2-negative status, respectively. In contrast to our finding that RGS7 expression is increased in breast tumors compared to non-malignant breast tissue, Maity et al. showed that RGS7 expression is downregulated in high-grade human breast tumors [2013]. Here, please note that dataset I analyzed contains data for both low and high grade breast cancer samples. However, it should be noted that there can be some differences in RGS7 levels in tumor initiation (from non-malignant to malignant) and progression (from low to high grade). In melanoma, Qutob et al. identified RGS7, which encodes a GTPase-accelerating protein (GAP), as a tumor-suppressor gene [2018]. The percentage of patients with FCRL4 copy number gains is around 68%, and its expression is lower in breast tumors with ER-positive or PR-positive status. Although FCRL4 has not been studied in the context of breast cancer, FCRL4 was shown to be involved in certain types of lymphomas [Ikeda et al., 2017; Wang and Cook, 2019; Falini et al., 2012]. High expression of FCRL4 was also shown to be associated with a shorter overall survival in colorectal cancer [Sorrentino et al., 2021]. I also found that CSMD3 transcript levels are lower in breast tumors with HER2-positive status. Lu et al. found that CSMD3 is associated with tumor mutation burden and immune infiltration in ovarian cancer patients [2021]. CSMD3 was also shown to be one of the most recurrently mutated genes in prostate adenocarcinoma [Zhao et al., 2019]. Liu et al. identified CSMD3 as the second most frequently mutated gene (next to TP53) in lung cancer in their study, and further demonstrated that loss of CSMD3 results in increased proliferation of airway epithelial cells [2012]. In a very recent study, recurrent somatic mutations in CSMD3 was observed in head and neck angiosarcoma [Loh et al., 2023]. Although CSMD3 has been studied in other cancer types, it remains to be understudied in the context of breast cancer and further research is needed. FAM135B expression is higher in breast cancer patients with ER-positive or PR-positive status but lower in those with HER2-positive status. FAM135B has been mostly studied in the context of esophageal cancer. The expression levels of FAM135B was found to be associated with the tumor behavior characteristics and the progression of esophageal cancer [Wang et al., 2020]. Dong et al. showed that FAM135B protein levels are significantly higher in esophageal squamous cell carcinoma tissues than in precancerous tissues, and high FAM135B expression correlates with poorer clinical prognosis, and that ectopic expression of FAM135B promotes esophageal squamous cell carcinoma cell proliferation both in vitro and in vivo [2021]. Another study reported that esophageal squamous cell carcinoma cells with high levels of FAM135B are resistant to irradiation, and that silencing FAM135B inhibits colony formation capability, and that FAM135B regulates downstream PI3K/Akt/mTOR signaling pathway [Bi et al., 2021]. Others reported that the most common driver mutations are found in FAM135B in addition to some other genes in large-cell lung cancer tumors [Wu et al., 2023], and that FAM135B dysregulation is independent prognostic factors for survival outcomes of patients with colon adenocarcinoma [Wang et al., 2022]. Further research is required to see if similar mechanisms are in place for FAM135B in the case of breast cancer for FAM135B. Lastly, I studied the ER-, PR- and HER2-dependent expression of TRAF7, whose expression is increased in breast tumors, and for which there are copy number gains in half of patients with breast cancer. In contrast to other 5 genes studied, I fail to observe any significant change in the expression of TRAF7 depending on ER, PR and HER2 status in breast cancer patients. Wang et al. showed that TRAF7 expression is downregulated in breast tumors, and identified TRAF7 as an E3 ligase for K48-linked ubiquitination of p53 in vitro . Authors suggested that p53 accumulation is due to the defects of TRAF7-mediated ubiquitination, and the downregulation of TRAF7 correlates with poor prognosis in a breast cancer cohort, stating that TRAF7-mediated ubiquitination of p53 plays a critical role in breast cancer development [2012]. Other studies characterized frequent somatic mutations in TRAF7 in meningiomas [Moussalem et al., 2021; Berghoff et al., 2022]. TRAF7 was reported to increase ubiquitin-degradation of KLF4 to promote hepatocellular carcinoma progression, and was found to contribute to tumor progression by promoting ubiquitin-proteasome mediated degradation of P53 in hepatocellular carcinoma [He et al., 2020; Zhang et al., 2021]. In contrast to the better mechanistic understanding of the function of TRAF7 in other cancer types, mechanistic studies on TRAF7 in the context of breast cancer is highly limited and requires further attention. 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Wickham H, Bryan J (2023). _readxl: Read Excel Files_. R package version 1.4.2, . Wilks C., et al. (2014) The Cancer Genomics Hub (CGHub): overcoming cancer through the power of torrential data. Database, 2014, 1–10. Wu X, Yin J, Deng Y, Zu Y. Whole-genome characterization of large-cell lung carcinoma: A comparative analysis based on the histological classification. Front Genet. 2023 Jan 4;13:1070048. doi: 10.3389/fgene.2022.1070048. PMID: 36685819; PMCID: PMC9845284. Yihui Xie (2023). knitr: A General-Purpose Package for Dynamic Report Generation in R. R package version 1.42. Zhang Y, Gong M, Yuan H, Park HG, Frierson HF, Li H. Chimeric transcript generated by cis-splicing of adjacent genes regulates prostate cancer cell proliferation. Cancer Discov. 2012 Jul;2(7):598-607. doi: 10.1158/2159-8290.CD-12-0042. Epub 2012 Jun 19. PMID: 22719019. Zhang Z, Hernandez K, Savage J, Li S, Miller D, Agrawal S, Ortuno F, Staudt LM, Heath A, Grossman RL. Uniform genomic data analysis in the NCI Genomic Data Commons. Nat Commun. 2021 Feb 22;12(1):1226. doi: 10.1038/s41467-021-21254-9. PMID: 33619257; PMCID: PMC7900240. Zhao H, Gong Y. The Prognosis of Single Hormone Receptor-Positive Breast Cancer Stratified by HER2 Status. Front Oncol. 2021 May 17;11:643956. doi: 10.3389/fonc.2021.643956. PMID: 34079755; PMCID: PMC8165305. Zhao X, Lei Y, Li G, Cheng Y, Yang H, Xie L, Long H, Jiang R. Integrative analysis of cancer driver genes in prostate adenocarcinoma. Mol Med Rep. 2019 Apr;19(4):2707-2715. doi: 10.3892/mmr.2019.9902. Epub 2019 Jan 28. PMID: 30720096; PMCID: PMC6423600. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3863281","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":267357236,"identity":"2a2fb01c-22c9-43aa-b8be-405f78779873","order_by":0,"name":"Caglar Berkel","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCklEQVRIiWNgGAWjYHACxgNAIoGBgY2B4UEFREiCkB6EloQzcC0GRGpJbCNCC3/78QsHfu5hyOOXPpb4IHFerZw5A/PB2zwMf/JxaZE4k1NwsOcZQ7FkX9phg8Rtx40tG9iSrXkYDCwbcGgxYMhJOMBzgCFxwxn2NonEbccSNxzgMZMGasHpMgP+NwkH/wC17D/D3v4jcQ5IC/83/Fok0g8cBtvCw3aMIbGhBmQLG14tEjfeMByWOSBRLHGGLVki4dgBY8tmNmPLOQbGOLXw96c/fPjmgE0efw+b4YcPNXVy5uzND2+8qZDDEzE8IDl4dB9mMGCGBAsewP4AmVdHINpHwSgYBaNgJAIAJTxXRAfiVSMAAAAASUVORK5CYII=","orcid":"","institution":"Gaziosmanpaşa University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Caglar","middleName":"","lastName":"Berkel","suffix":""}],"badges":[],"createdAt":"2024-01-14 12:44:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3863281/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3863281/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49837521,"identity":"b1bef215-92d2-4ba3-bd45-c86e2cb3299a","added_by":"auto","created_at":"2024-01-18 19:38:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":245671,"visible":true,"origin":"","legend":"\u003cp\u003eGenes with copy number gains observed in more than half of breast cancer patients \u003cem\u003eGenes with red bars are genes selected for further analyses.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3863281/v1/4d9134997464ae5243554ebf.png"},{"id":49837523,"identity":"3d2315d6-0c0b-4e01-9036-9f46f91d1138","added_by":"auto","created_at":"2024-01-18 19:38:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":909124,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of SLC45A3 depending on malignancy status (normal vs tumor), ER (Estrogen Receptor) status (ER- vs ER+), PR (Progesterone Status) status (PR- vs PR+), HER2 (ERBB2) (HER2- vs HER2) status and on the combinations of receptor status’.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3863281/v1/ce7c639d3222e1899a74050f.png"},{"id":49837522,"identity":"a8158f6b-0888-459a-bf43-c3260193474f","added_by":"auto","created_at":"2024-01-18 19:38:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":879421,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of RGS7 depending on malignancy status (normal vs tumor), ER (Estrogen Receptor) status (ER- vs ER+), PR (Progesterone Status) status (PR- vs PR+), HER2 (ERBB2) (HER2- vs HER2) status and on the combinations of receptor status’.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3863281/v1/e39f4420dc49ddef0906934c.png"},{"id":49837835,"identity":"ab55c40e-22cf-4245-bad7-9f3923ebe8e8","added_by":"auto","created_at":"2024-01-18 19:46:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":843458,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of FCRL4 depending on malignancy status (normal vs tumor), ER (Estrogen Receptor) status (ER- vs ER+), PR (Progesterone Status) status (PR- vs PR+), HER2 (ERBB2) (HER2- vs HER2) status and on the combinations of receptor status’.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3863281/v1/42b65fb95d3819b895eb2c19.png"},{"id":49837286,"identity":"a87c6328-d5ef-4bbd-be35-057aea5cc990","added_by":"auto","created_at":"2024-01-18 19:30:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":669364,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of CSMD3 depending on malignancy status (normal vs tumor), ER (Estrogen Receptor) status (ER- vs ER+), PR (Progesterone Status) status (PR- vs PR+), HER2 (ERBB2) (HER2- vs HER2) status and on the combinations of receptor status’.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-3863281/v1/9a87382ee7deebf3b8b946fe.png"},{"id":49837285,"identity":"0747d67c-e54f-4d91-9ca7-0a7702ec751d","added_by":"auto","created_at":"2024-01-18 19:30:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":946534,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of FAM135B depending on malignancy status (normal vs tumor), ER (Estrogen Receptor) status (ER- vs ER+), PR (Progesterone Status) status (PR- vs PR+), HER2 (ERBB2) (HER2- vs HER2) status and on the combinations of receptor status’.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-3863281/v1/39c31ea8f80d6dbdc70eb6e2.png"},{"id":49837289,"identity":"8786e957-b86c-455e-929f-c5a4c0b93ed1","added_by":"auto","created_at":"2024-01-18 19:30:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":897003,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of TRAF7 depending on malignancy status (normal vs tumor), ER (Estrogen Receptor) status (ER- vs ER+), PR (Progesterone Status) status (PR- vs PR+), HER2 (ERBB2) (HER2- vs HER2) status and on the combinations of receptor status’.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-3863281/v1/1340e86d464274f109456ea7.png"},{"id":50599852,"identity":"203f2021-f5fc-4f66-a8b6-961c2b7954be","added_by":"auto","created_at":"2024-02-03 11:07:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1947628,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3863281/v1/4bfa807c-4266-4b8a-9d6f-edcabd637204.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Estrogen receptor (ER)-, progesterone receptor (PR)- and HER2-dependent expression of largely understudied genes with large number of copy number gain events in breast cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer is the most common malignancy and the leading cause of cancer-related mortality in women globally [Sung et al., 2021]. Breast cancer can be classified into several molecular subtypes based on the status of hormone receptors (HRs, including estrogen receptor (ER) and the progesterone receptor (PR)) and of human epidermal growth factor receptor 2 (HER2, also called ERBB2) [Allison et al., 2020]. Approximately 70% of breast tumors are hormone receptor-positive (HR+). Patients with HR+ breast cancer generally have a favorable prognosis, and those with HR-negative (HR-) breast cancer have a poor prognosis. PR is an estrogen-regulated gene; therefore, ER-positive (ER+) tumors are mostly also PR positive (PR+), whereas ER-negative (ER-) tumors are usually PR negative (PR-). Thus, single HR+ (i.e., ER+/PR- or ER-/PR+) tumors represent a minority of breast cancer cases (about 10% of all breast cancers) [Bae et al., 2015; Zhao and Gong, 2021; Li et al., 2020]. It was also reported that single HR+ breast tumors have worse prognosis than ER+, PR+ breast tumors; and that ER-, PR+ subtype has similar prognosis relative to ER-, PR- subtype [Dauphine et al., 2020; Lv et al., 2020]. Therefore, it is of high clinical interest to identify molecular variations (for instance, differences in gene regulation and expression) contributing to these differences in prognosis between breast cancer patients with different receptor status.\u003c/p\u003e\n\u003cp\u003eCopy number variants (CNVs) cover approximately 5-10% of the human genome, and are responsible for the majority of genome variability among individuals based on nucleotide coverage [Abel et al., 2020; Zarrei et al., 2015]. CNVs show considerable variability in terms of both size and frequency, and can disrupt gene function substantially via alterations in gene dosage, coding sequences, and gene regulation [Conrad et al., 2010]. CNVs also alter the diploid status of DNA, and may have no phenotypic effect, explain adaptive traits or can contribute to disease [Zarrei et al., 2015]. CNVs are distributed not regularly in the genome; the pericentromeric and subtelomeric regions of chromosomes exhibit a particularly high rate of variation [Zarrei et al., 2015]. The size of CNVs is in most cases defined as larger than 50 bp, whereas smaller elements are known as insertions or deletions (indels) [MacDonald et al., 2014; Redon et al., 2006; Levy et al., 2007].\u003c/p\u003e\n\u003cp\u003eIn this work, I identified largely understudied genes (i.e. maximum 4 papers in PubMed in the context of breast cancer) for which copy number gains (CNV gain) are observed in more than half of breast cancer patients, and which also show increased expression in breast tumors compared to non-malignant breast tissue. I found 6 genes meeting these criteria: SLC45A3, RGS7, FCRL4, CSMD4, FAM135B and TRAF7. I then studied these 6 genes in terms of ER (estrogen receptor), PR (progesterone receptor) and HER2 (human epidermal growth factor receptor 2; ERBB2) expression in breast cancer patients. Furthermore, I analyzed the expression of these genes among all combinations of receptor status (8 different conditions from triple negative to triple positive) in breast tumors.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eDatasets\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, I obtained copy number variation (CNV) data from The National Cancer Institute Genomic Data Commons (GDC) Data Portal (https://portal.gdc.cancer.gov/), which includes CNV data from TCGA-BRCA project in addition to others [Jensen et al., 2017; Zhang et al., 2021; Berger et al., 2018; Cancer Genome Atlas Network, 2012; Ciriello et al., 2015].\u003c/p\u003e\n\u003cp\u003eFor transcriptomics analysis, I used alternatively processed and compiled RNA-Seq and clinical data for samples from The Cancer Genome Atlas (TCGA) project (GSE62944) [Rahman et al., 2015; Cancer Genome Atlas Research Network, 2013; Wilks et al., 2014]. This dataset contains data from 1119 breast cancer patients and 113 women with no breast tumors. Total sample size (n) is 1232. Sample sizes for subgroups are as following: ER-negative = 230; ER-positive = 785; PR-negative = 228, PR-positive = 782; HER2-negative = 162 and HER2-positive = 545. This dataset can also be accessed as a SummarizedExperiment through Bioconductor (in Experiment Packages \u0026gt;\u0026gt; GSE62944) [Arora, 2023; Huber et al., 2015; Gentleman et al., 2004].\u003c/p\u003e\n\u003cp\u003eGenes in this study were selected for further analysis if (1) for these genes, there are less than or equal to 4 papers in PubMed in the context of breast cancer, (2) their expression are upregulated in breast tumors compared to non-malignant breast tissue, and (3) copy number gains (CNV gains) for these genes are observed in more than half of the patients with breast cancer.\u003c/p\u003e\n\u003cp\u003eIn the dataset, ER, PR and HER2 status from patient tumor samples had been determined by IHC (Immunohistochemistry) [Cancer Genome Atlas Research Network, 2013].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData analysis and visualization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in R programming environment (R version 4.2.1 (2022-06-23)) using R Studio IDE from posit [R Core Team, 2022]. Following R / Bioconductor packages (https://bioconductor.org/) were used throughout the analysis [Huber et al., 2015; Gentleman et al., 2004]: tidyverse [Wickham et al., 2019; Wickham, 2016; Wickham, 2022], readxl [Wickham and Bryan, 2023], ExperimentHub [Morgan and Shepherd, 2022], SummarizedExperiment [Morgan et al., 2022], ggpubr [Kassambara, 2023], glue [Hester and Bryan, 2022], rmarkdown [Allaire et al., 2023] and knitr [Xie, 2023].\u003c/p\u003e\n\u003cp\u003eWhen data is not normally distributed, I performed Wilcoxon test [Kassambara, 2023]. Relative expression values shown in plots are log10 transformation of read counts. Data analysis and visualization was performed as previously reported [Berkel and Cacan, 2020; Berkel and Cacan, 2021].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eGenes with copy number gains observed in more than half of breast cancer patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirst, I identified genes with copy number gain events (CNV gain) observed in more than 50% of breast cancer patients (n = 1058), using CNV (copy number variation) data from Genomic Data Commons (GDC) Data Portal (Figure 1). I found that there are 49 genes with copy number gain events observed in more than half of the patients with breast cancer (Figure 1). I ordered genes from highest to lowest CNV gain percentage in Figure 1. Gene with the highest CNV gain percentage (74.1%) in breast cancer patients is MDM4, followed by SLC45A3 (73.44%) and ELK4 (73.35%) (Figure 1). From this list of genes, I only selected genes indicated by red columns in Figure 1 (namely, SLC45A3, RGS7, FCRL4, CSMD4, FAM135B and TRAF7) for further analysis based on two criteria: (1) the expression of the gene is increased in breast tumors compared to healthy breast tissue (see below), and (2) there are maximum 4 papers in PubMed for the gene in the context of breast cancer, so it is largely understudied in breast cancer research. I analyzed the expression of these 6 selected genes in terms of ER (estrogen receptor), PR (progesterone receptor) and HER2 (human epidermal growth factor receptor 2; ERBB2) expression in breast cancer patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSLC45A3 expression is lower in breast cancer patients with ER-positive or PR-positive status\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInitially, I found that SLC45A3 (Solute Carrier Family 45 Member 3) expression is higher in breast tumors compared to normal breast tissue (p = 0.026, our first criteria, Figure 2A). Next, I showed that SLC45A3 expression is lower in ER (estrogen receptor)-positive (p \u0026lt; 2e-16) and PR (progesterone receptor)-positive (p = 2.5e-12) breast cancer patients compared to those with ER-negative and PR-negative tumors, respectively (Figure 2B, 2C). However, there is no difference in SLC45A3 transcript levels between HER2-negative and HER-positive breast cancer patients (p = 0.75, Figure 2D). I also observed that breast cancer patients with triple negative status (ER-, PR-, HER2-; “- - -”) have the highest mean expression of SLC45A3 (Figure 2E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRGS7 expression is higher in breast cancer patients with ER-positive or PR-positive status\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe percentage of breast cancer patients with RGS7 (Regulator Of G Protein Signaling 7) copy number gains (CNV gain) is around 70% (Figure 1). I found that RGS7 transcript levels are higher in breast tumors than normal breast tissue (p = 2.1e-05, Figure 3A). RGS7 expression is higher in breast tumors with ER-positive (p = 1.6e-09) or PR-positive status (p = 1.4e-09) compared to those with ER-negative or PR-negative status, respectively (Figure 3B, 3C). In contrast, RGS7 mRNA levels are lower in breast tumors with HER2+ status than HER2- status (p = 0.018) (Figure 3D). I also showed that among all receptor status combinations, breast tumors with ER+, PR+ and HER2- status (“+ + -”) have the highest RGS7 expression (Figure 3E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFCRL4 expression is lower in breast tumors with ER-positive or PR-positive status\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter showing that the percentage of breast cancer patients with FCRL4 (Fc Receptor Like 4) copy number gains is around 68% (Figure 1), and that the expression of FCRL4 is higher in breast tumors than non-malignant breast tissue (p = 2.4e-12, Figure 4A), I found that FCRL4 transcript levels are lower in breast tumors with ER-positive (p = 1.9e-06) or PR-positive status (p = 0.0025) than those with ER-negative or PR-negative status, respectively (Figure 4B, 4C). However, FCRL4 expression does not significantly change depending on HER2 status in breast tumors (p =0.63; Figure 4D). FCRL4 expression based on ER, PR and HER2 status combinations is shown in Figure 4E.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCSMD3 transcript levels are lower in breast tumors with HER2-positive status\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI found that the percentage of breast cancer patients with CSMD3 (CUB And Sushi Multiple Domains 3) copy number gains is around 57% (Figure 1), and that the expression of CSMD3 is higher in breast tumors compared to healthy breast tissue (p = 0.025, Figure 5A). Then, I showed that CSMD3 mRNA levels do not change depending on either ER or PR status in breast tumors (p = 0.18 and 0.53, respectively) (Figure 5B, 5C). However, I observed that CSMD3 expression is lower in HER2+ breast tumors than HER2- breast tumors (p = 0.0011; Figure 5D). In addition, CSMD3 expression is higher in ER+,PR+,HER2- breast tumors compared to those with triple positive status (TNBC) (p = 0.0069; Figure 5E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFAM135B expression is higher in breast cancer patients with ER-positive or PR-positive status but lower in those with HER2-positive status\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter showing that the percentage of breast cancer patients with FAM135B (Family With Sequence Similarity 135 Member B) copy number gains is around 53% (Figure 1), and that FAM135B mRNA levels are higher in breast tumors than non-malignant breast tissue (p = 0.019; Figure 6A), I found that FAM135B transcript levels are higher in breast cancer patients with ER-positive (p \u0026lt; 2e-16) or PR-positive status (p \u0026lt; 2e-16) compared to those with ER-positive or PR-positive status, respectively (Figure 6B, 6C); but, lower in those with HER2-positive status than those with HER2-negative status (p = 0.0028) (Figure 6D). As expected, \u0026nbsp;FAM135B expression is the highest in ER+, PR+, HER2- breast tumors (Figure 6E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTRAF7 expression does not change depending on ER, PR and HER2 status in breast tumors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI first showed that the percentage of patients with breast cancer who have TRAF7 (TNF Receptor Associated Factor 7) copy number gains is 50% (Figure 1), and that TRAF7 transcript levels are higher in breast tumors compared to healthy breast tissue (p \u0026lt; 2e-16, Figure 7A). However, I observed that TRAF7 transcript levels do not change depending on ER, PR and HER2 status in tumors from breast cancer patients (p = 0.1, 0.48 and 0.87, respectively) (Figure 7B, 7C, 7D). Therefore, there is no significant difference in TRAF7 expression between different combinations of these three receptors’ status in breast tumors (Figure 7E).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, I identified largely understudied genes for which copy number gains are observed in more than half of the patients with breast cancer, and which also show increased expression in breast tumors compared to non-malignant breast tissue. I found 6 genes meeting these criteria: SLC45A3, RGS7, FCRL4, CSMD4, FAM135B and TRAF7. I then studied these 6 genes in terms of ER (estrogen receptor), PR (progesterone receptor) and HER2 (human epidermal growth factor receptor 2; ERBB2) expression in breast cancer patients. I observed parallel changes in the expression of these genes in terms of ER and PR status; in other words, for instance, if the expression of one of the genes is increased in ER+ breast tumors; similarly, its expression is increased in PR+ cells.\u003c/p\u003e\n\u003cp\u003eFirst, I found that more than 70% of breast cancer patients have copy number gains in SLC45A3 gene, and that SLC45A3 expression is lower in breast cancer patients with ER-positive or PR-positive status compared to those with ER-negative or PR-negative status. This gene has been mostly studied in the context of prostate cancer [Kasajima et al., 2020]. The fusion RNA of this gene, SLC45A3-ELK4, generated by cis-splicing between neighboring genes, was found in prostate cancer samples, and it was shown to function as a long non-coding chimeric RNA (lnccRNA) [Qin et al., 2017]. SLC45A3-ELK4 regulates cancer cell proliferation by its transcript, not translated protein; and the level of this chimeric transcript correlates with prostate cancer disease progression [Qin et al., 2017; Zhang et al., 2012].\u003c/p\u003e\n\u003cp\u003eRGS7 have copy number gains in more than 70% of breast cancer patients, and RGS7 expression is higher in breast cancer patients with ER-positive or PR-positive status but lower in those with HER2-positive status, compared to those with ER-negative, PR-negative, HER2-negative status, respectively. In contrast to our finding that RGS7 expression is increased in breast tumors compared to non-malignant breast tissue, Maity et al. showed that RGS7 expression is downregulated in high-grade human breast tumors [2013]. Here, please note that dataset I analyzed contains data for both low and high grade breast cancer samples. However, it should be noted that there can be some differences in RGS7 levels in tumor initiation (from non-malignant to malignant) and progression (from low to high grade). In melanoma, Qutob et al. identified RGS7, which encodes a GTPase-accelerating protein (GAP), as a tumor-suppressor gene [2018].\u003c/p\u003e\n\u003cp\u003eThe percentage of patients with FCRL4 copy number gains is around 68%, and its expression is lower in breast tumors with ER-positive or PR-positive status. Although FCRL4 has not been studied in the context of breast cancer, FCRL4 was shown to be involved in certain types of lymphomas [Ikeda et al., 2017; Wang and Cook, 2019; Falini et al., 2012]. High expression of FCRL4 was also shown to be associated with a shorter overall survival in colorectal cancer [Sorrentino et al., 2021].\u003c/p\u003e\n\u003cp\u003eI also found that CSMD3 transcript levels are lower in breast tumors with HER2-positive status. Lu et al. found that CSMD3 is associated with tumor mutation burden and immune infiltration in ovarian cancer patients [2021]. CSMD3 was also shown to be one of the most recurrently mutated genes in prostate adenocarcinoma [Zhao et al., 2019]. Liu et al. identified CSMD3 as the second most frequently mutated gene (next to TP53) in lung cancer in their study, and further demonstrated that loss of CSMD3 results in increased proliferation of airway epithelial cells [2012]. In a very recent study, \u0026nbsp;recurrent somatic mutations in CSMD3 was observed in head and neck angiosarcoma [Loh et al., 2023]. Although CSMD3 has been studied in other cancer types, it remains to be understudied in the context of breast cancer and further research is needed.\u003c/p\u003e\n\u003cp\u003eFAM135B expression is higher in breast cancer patients with ER-positive or PR-positive status but lower in those with HER2-positive status. FAM135B has been mostly studied in the context of esophageal cancer. The expression levels of FAM135B was found to be associated with the tumor behavior characteristics and the progression of esophageal cancer [Wang et al., 2020]. Dong et al. showed that FAM135B protein levels are significantly higher in esophageal squamous cell carcinoma tissues than in precancerous tissues, and high FAM135B expression correlates with poorer clinical prognosis, and that ectopic expression of FAM135B promotes esophageal squamous cell carcinoma cell proliferation both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e [2021]. Another study reported that esophageal squamous cell carcinoma cells with high levels of FAM135B are resistant to irradiation, and that silencing FAM135B inhibits colony formation capability, and that FAM135B regulates downstream PI3K/Akt/mTOR signaling pathway [Bi et al., 2021]. Others reported that the most common driver mutations are found in FAM135B in addition to some other genes in large-cell lung cancer tumors [Wu et al., 2023], and that FAM135B dysregulation is independent prognostic factors for survival outcomes of patients with colon adenocarcinoma [Wang et al., 2022]. Further research is required to see if similar mechanisms are in place for FAM135B in the case of breast cancer for FAM135B.\u003c/p\u003e\n\u003cp\u003eLastly, I studied the ER-, PR- and HER2-dependent expression of TRAF7, whose expression is increased in breast tumors, and for which there are copy number gains in half of patients with breast cancer. In contrast to other 5 genes studied, I fail to observe any significant change in the expression of TRAF7 depending on ER, PR and HER2 status in breast cancer patients. Wang et al. showed that TRAF7 expression is downregulated in breast tumors, and identified TRAF7 as an E3 ligase for K48-linked ubiquitination of p53 \u003cem\u003ein vitro\u003c/em\u003e. Authors suggested that p53 accumulation is due to the defects of TRAF7-mediated ubiquitination, and the downregulation of TRAF7 correlates with poor prognosis in a breast cancer cohort, stating that TRAF7-mediated ubiquitination of p53 plays a critical role in breast cancer development [2012]. Other studies characterized frequent somatic mutations in TRAF7 in meningiomas [Moussalem et al., 2021; Berghoff et al., 2022]. TRAF7 was reported to increase ubiquitin-degradation of KLF4 to promote hepatocellular carcinoma progression, and was found to contribute to tumor progression by promoting ubiquitin-proteasome mediated degradation of P53 in hepatocellular carcinoma [He et al., 2020; Zhang et al., 2021]. In contrast to the better mechanistic understanding of the function of TRAF7 in other cancer types, mechanistic studies on TRAF7 in the context of breast cancer is highly limited and requires further attention.\u003c/p\u003e\n\u003cp\u003eCombined, I here studied ER-, PR-, HER2-dependent expression of understudied genes with large percentage of copy number gain events in breast cancer patients. Although most of these genes show differential expression depending on receptor status in breast cancer, further research is needed to obtain functional and mechanistic details on the role of these genes in breast cancer initiation and progression.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthor declares no conflicts of interest. 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PMID: 30720096; PMCID: PMC6423600.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"breast cancer, copy number variation, estrogen receptor, progesterone receptor, HER2, SLC45A3","lastPublishedDoi":"10.21203/rs.3.rs-3863281/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3863281/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Breast cancer can be classified into several molecular subtypes based on the status of hormone receptors including estrogen receptor (ER) and the progesterone receptor (PR), and of human epidermal growth factor receptor 2 (HER2; ERBB2). Copy number variants (CNVs) cover 5-10% of the human genome, and are responsible for the majority of variability among different individuals’ genomes based on nucleotide coverage. In the present study, I identified largely understudied genes for which copy number gain events (CNV gains) are observed in more than half of breast cancer patients, and whose expression is upregulated in breast tumors compared to non-malignant breast tissue. I found 6 genes meeting these criteria: SLC45A3, RGS7, FCRL4, CSMD4, FAM135B and TRAF7. I then studied these 6 genes in terms of ER, PR and HER2 status in breast cancer patients. I found that SLC45A3 expression is lower in breast cancer patients with ER-positive or PR-positive status. RGS7 mRNA levels are higher in breast cancer patients with ER-positive or PR-positive status but lower in those with HER2-positive status. FCRL4 expression is lower in breast tumors with ER-positive or PR-positive status. CSMD3 transcript levels are lower in breast tumors with HER2-positive status. FAM135B expression is higher in breast cancer patients with ER-positive or PR-positive status but lower in those with HER2-positive status. Combined, our study points that most of the studied genes exhibit receptor status-dependent changes in the expression, and that further mechanistic studies are needed for these genes with large number of copy number gain events in breast cancer.","manuscriptTitle":"Estrogen receptor (ER)-, progesterone receptor (PR)- and HER2-dependent expression of largely understudied genes with large number of copy number gain events in breast cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-18 19:30:13","doi":"10.21203/rs.3.rs-3863281/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7a0e95b1-9956-43f6-9190-7c530bd8a89b","owner":[],"postedDate":"January 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-03T10:59:27+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-18 19:30:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3863281","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3863281","identity":"rs-3863281","version":["v1"]},"buildId":"zQwnuV7TCBrMSSSToR1PI","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}