Gene expression in prostate tissue according to frequency of ejaculation.

Background: TMPRSS2:ERG, a hormonally regulated gene fusion present in about half of prostate tumors, is the most common somatic event in prostate cancer. There is intriguing evidence to suggest that TMPRSS2:ERG-positive tumors may define a distinct subgroup of prostate cancer. In this study we compared gene expression profiles according to fusion status to identify genes and biological pathways differential expressed by TMPRSS2:ERG. Methods: The study included men with prostate cancer in the Health Professionals Follow-up Study and the Physicians’ Health Study diagnosed between 1982 and 2004 and followed through 2012. Tumor biomarker data was available for archival tumor tissue from 380 cases, 191 of whom also had gene expression data for adjacent normal tissue. Whole genome mRNA expression profiling (20,254 genes) was performed using the Affymetrix 1.0 ST array. TMPRSS2:ERG tumor status was assessed by a genetically validated IHC assay for ERG. To relate expression of individual genes to ERG we used linear regression. Gene Set Enrichment Analysis (GSEA) was used to identify pathways of genes associated with ERG in both tumor and adjacent normal tissue. Data from The Cancer Genome Atlas (TCGA) was used to validate these findings. Results: Among 380 cases, 186 (49%) of tumors were positive for the gene fusion. Genome-wide differential expression analysis identified 492 genes upregulated and 369 genes downregulated in tumor tissue comparing ERG+ to ERG- tumors (FDR p-value < 0.05). No genes were identified in the adjacent normal tissue at this significance level. 772 of the 861 genes (90%) identified in the HPFS/PHS dataset were confirmed in TCGA (448 upregulated and 324 downregulated). The top ten genes included ERG, WNK2, CACNA1D, HES1, SEPT9, PLA2G7, TDRD1, ANTXR2, SORBS2, and MYO6. Enrichment analysis identified a number of metabolic pathways associated with ERG tumor status including pathways involved in amino acid and fatty acid metabolism. Conclusions: We identified significant differences in gene expression profiles in prostate tumor tissue according to TMPRSS2:ERG status, providing supportive evidence of this fusion as a unique subtype. Citation Format: Ericka Ebot, Masis Isikbay, Travis Gerke, Thomas U. Ahearn, Rachel S. Kelly, Svitlana Tyekucheva, Andreas Pettersson, Kathryn L. Penney, Lorelei A. Mucci. Gene expression profiling of prostate tissue identifies biological pathways associated with TMPRSS2:ERG gene fusion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4236. doi:10.1158/1538-7445.AM2017-4236

Radiotherapy is a very effective adjuvant treatment for rectal cancer with little side effects. Its killing effect on tumor cells seems to be more profound than the effect on normal tissue. The molecular events caused by irradiation are mainly analyzed in in vitro and animal models; investigations on human material are rare. In the current study, we analyzed the effects of irradiation on gene expression in normal and tumor tissue of rectal cancer patients. Normal and carcinoma tissue of patients from a randomized clinical trial of the benefits of preoperative radiotherapy were analyzed using the Affymetrix Human Cancer Gene Chip. Preoperative radiotherapy was given within 5 days prior to surgery. Results for normal tissue and tumor were compared to investigate the radiation-related differences between normal and tumor cells. We clustered the differentially expressed genes based on their functional annotation. Results were compared with immunohistochemical and literature data. The majority of the investigated cancer-related genes remained unchanged by irradiation (92% in tumor tissue and 93% in normal tissue). The differentially expressed genes varied between tumor and normal tissue except for maspin and IL-8. Both in tumor and normal tissue, differentially expressed genes were present related to cell signaling and cycle control, apoptosis and cell survival and tissue response and repair. However, the spectrum of affected genes was totally different. Pre-existing differences in gene expression between normal tissue and tumor tissue might explain the differences in their responses to radiation. This change in response may explain the clinical beneficial effect of radiotherapy on tumor cells (low local recurrence rate) and the less severe effects on normal tissue (minor side effects).

Introduction. The downregulation of the Spastic Paraplegia-20 (SPG20) gene is correlated with a rare autosomal recessive disorder called Troyer Syndrome. Only in recent years has SPG20 been studied and partially characterized in cancer. SPG20 has been shown to be hypermethylated in colorectal cancer, gastric cancer, non-Hodgkin’s lymphoma and hepatocellular carcinoma. In this study, we analyze the methylation status and the gene expression of SPG20 in different tumors of various histological origins. Methods. We analyzed the data generated through Infinium Human Methylation 450 BeadChip arrays and RNA-seq approaches extrapolated from The Cancer Genome Atlas (TCGA) database. The statistics were performed with R 4.0.4. Results. We aimed to assess whether the hypermethylation of this target gene was a common characteristic among different tumors and if there was a correlation between the m-values and the gene expression in paired tumor versus solid tissue normal. Overall, our analysis highlighted that SPG20 open sea upstream the TSS is altogether hypermethylated, and the tumor tissues display a higher methylation heterogeneity compared to the solid tissue normal. The gene expression evidences a reproducible, higher gene expression in normal tissues. Conclusion. Our research, based on data mining from TCGA, evidences that colon and liver tumors display a consistent methylation heterogeneity compared to their normal counterparts. This parallels a downregulation of SPG20 gene expression in tumor samples and suggests a role for this multifunctional protein in the control of tumor progression.

Background: Epidemiologic studies have consistently shown that men engaged in high levels of physical activity have lower risks of advanced and fatal prostate cancer. The mechanisms underlying this association are not well understood but may include changes in hormonal and anti-inflammatory pathways. Our objective was to investigate the link between prediagnostic physical activity and gene expression alterations in prostate tumor and adjacent normal tissue. Methods: We performed whole-gene expression profiling on tumor (N=248) and adjacent normal tissue (N=117) among a subset of men diagnosed with indolent and lethal prostate cancer between 1986 and 2005 in the prospective Health Professionals Follow-up Study. Men were followed prospectively for prostate cancer outcomes through 2011. Physical activity was ascertained by self-report of the average number of hours per week spent engaged in various forms of recreational physical activity at baseline and every two years thereafter. We examined cumulative average physical activity of vigorous intensity in units of metabolic equivalent of task (MET)-hours/week updated from baseline to the time prostate cancer diagnosis. Gene set enrichment analysis was performed among KEGG and hallmark gene sets to identify pathways with differential expression by vigorous activity. Results: We identified 33 KEGG and four Hallmark gene sets enriched in the adjacent normal tissue comparing men in the highest compared to the lowest quintile of vigorous activity (false-discovery rate < 0.25). Among the significant gene sets identified were cancer-related pathways as well as vascular smooth muscle contraction, angiogenesis, and insulin-signaling pathways. No gene sets were significantly enriched in the tumor tissue. Conclusions: These findings suggest that physical activity may influence prostate cancer development through epigenetic alterations in the prostate tissue. Future studies are needed to confirm these findings and further investigate potential mechanisms linking physical activity to risk of lethal prostate cancer. Citation Format: Claire H. Pernar, Giovanni Parmigiani, Edward L. Giovannucci, Eric B. Rimm, Stephen Finn, Michelangelo Fiorentino, Ericka M. Ebot, Lorelei A. Mucci. Gene expression pathways associated with vigorous physical activity in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B045.

We have previously developed a novel adenovirus vector (Adv) that targeted tumor tissues/vasculatures after systemic administration. The surface of this Adv is conjugated with CGKRK tumor homing peptide by the cross-linking reaction of polyethyleneglycol (PEG). In this study, we showed that the condition of PEG modification was important to minimize the gene expression in normal tissues after systemic treatment. When Adv was modified only with PEG-linked CGKRK, its luciferase expression was enhanced even in the liver tissue, as well as the tumor tissue. However, in the reaction with the mixture of non-cross-linking PEG and PEG-linked CGKRK, we found out that the best modification could suppress its gene expression in the liver, without losing that in the tumor. We also studied the internalization mechanisms of CGKRK-conjugated Adv. Results suggested that there is a specific interaction of the CGKRK peptide with a receptor at the cell surface enabling efficient internalization of CGKRK-conjugated Adv. The presence of cell-surface heparan sulfate is important receptor for the cellular binding and uptake of CGKRK-conjugated Adv. Moreover, macropinocytosis-mediated endocytosis is also important in endocytosis of CGKRK-conjugated Adv, aside from clathrin-mediated and caveolae-mediated endocytosis. These results could help evaluate the potentiality of CGKRK-conjugated Adv as a prototype vector with suitable efficacy and safety for systemic cancer gene therapy.

Post-translational modification by covalent attachment of isoprenoid lipids (prenylation) regulates the functions and biological activities of several proteins implicated in the oncogenic transformation and metastatic progression of cancer. The largest group of prenylated proteins contains a CAAX motif at the C-terminal that serves as a substrate for a series of post-translational modifications that convert these otherwise hydrophilic proteins to lipidated proteins, thus facilitating membrane association. C17orf37 (chromosome 17 open reading frame 37), also known as C35/Rdx12/MGC14832, located in the 17q12 amplicon, is overexpressed in human cancer, and its expression correlates with the migratory and invasive phenotype of cancer cells. Here we show that C17orf37 contains a functional CAAX motif and is post-translationally modified by protein geranylgeranyltransferase-I (GGTase-I). Geranylgeranylation of C17orf37 at the CAAX motif facilitates association of the protein to the inner leaflet of plasma membrane, enhances migratory phenotype of cells by inducing increased filopodia formation, and potentiates directional migration. A prenylation-deficient mutant of C17orf37 is functionally inactive and fails to trigger dissemination of tail vein-injected cells in a mouse model of metastasis. These findings demonstrate that prenylation is required for the function of the C17orf37 protein in cancer cells and imply that the post-translational modification may functionally regulate metastatic progression of disease.

Proteolytic Cleavage of the CD44 Adhesion Molecule in Multiple Human Tumors

<p>Supplementary Tables 1-4. Table 1. Prostate cancer risk SNP information and frequency of the risk genotypes in these data (Tumor cohort; n=264) Table 2. The associations of the expression of all genes located within 1 Mb window of each risk SNP in both tumor and normal prostate tissue. Table 3. Significant (False Discovery Rate q<0.1) associations of risk SNPs with the expression of genes in tumor or normal prostate tissue across the genome. Table 4. Top pathways (p<1.0E-03) associated with risk SNPs in tumor and normal tissue.</p>

Simple SummaryFew studies have employed expression data of normal tissues to explore the survival of cancer patients. Hence, we identified aging-related genes and microRNAs in normal tissues from patients with various types of cancer and investigated their effects on survival. We built statistical models to predict the survival of cancer patients using the expression levels of aging-related genes and microRNAs. We found that five aging-related genes (DUSP22, MAPK14, MAPKAPK3, STAT1, and VCP) from kidney cancer patients were significantly related to patient survival. We experimentally showed that there is an increase in the five aging-related genes in the macrophages of aged mice and, among these five genes, the invasion of cancer cells is inhibited by downregulating DUSP22. Using the zebrafish model of tumor cell dissemination, it was also confirmed that in vivo treatment with a small molecule inhibitor of DUSP22 blocked the metastasis of cancer cells in the early stage.The relationship between expression of aging-related genes in normal tissues and cancer patient survival has not been assessed. We developed a genome-wide transcriptomic analysis approach for normal tissues adjacent to the tumor to identify aging-related transcripts associated with survival outcome, and applied it to 12 cancer types. As a result, five aging-related genes (DUSP22, MAPK14, MAPKAPK3, STAT1, and VCP) in normal tissues were found to be significantly associated with a worse survival outcome in patients with renal cell carcinoma (RCC). This computational approach was investigated using nontumorigenic immune cells purified from young and aged mice. Aged immune cells showed upregulated expression of all five aging-related genes and promoted RCC invasion compared to young immune cells. Further studies revealed DUSP22 as a regulator and druggable target of metastasis. DUSP22 gene knockdown reduced RCC invasion and the small molecule inhibitor BML-260 prevented RCC dissemination in a tumor/immune cell xenograft model. Overall, these results demonstrate that deciphering the relationship between aging-related gene expression in normal tissues and cancer patient survival can provide new prognostic markers, regulators of tumorigenesis and novel targets for drug development.

DNA methylation at the 5-position of cytosine (5mC) is an epigenetic modification that regulates gene expression and cellular plasticity in development and disease. The ten-eleven translocation (TET) gene family oxidizes 5mC to 5-hydroxymethylcytosine (5hmC), providing an active mechanism for DNA demethylation, and it may also provide its own regulatory function. Here we applied oxidative bisulfite sequencing to generate whole-genome DNA methylation and hydroxymethylation maps at single-base resolution in human normal liver and lung as well as paired tumor tissues. We found that 5hmC is significantly enriched in CpG island (CGI) shores while depleted in CGIs themselves, especially in active genes, which exhibit a bimodal distribution of 5hmC around CGI that corresponds to H3K4me1 modifications. Hydroxymethylation on promoters, gene bodies, and transcription termination regions (TTRs) showed strong positive correlation with gene expression within and across tissues, suggesting that 5hmC is a marker of active genes and could play a role in gene expression mediated by DNA demethylation. Comparative analysis of methylomes and hydroxymethylomes revealed that 5hmC is significantly enriched in both tissue-specific DMRs (t-DMRs) and cancer-specific DMRs (c-DMRs), and 5hmC is negatively correlated with methylation changes, especially in non-CGI-associated DMRs. These findings revealed novel reciprocity between epigenetic markers at CGI shores corresponding to differential gene expression in normal tissues and matching tumors. Overall, our study provided a comprehensive analysis of the interplay between the methylome, hydroxymethylome, and histone modifications during tumorigenesis.

Role of Inflammatory Related Gene Expression in Clear Cell Renal Cell Carcinoma Development and Clinical Outcomes

Glioblastomas are a highly aggressive brain tumor, with one of the highest rates of new blood vessel formation. In this study we used a combined experimental and bioinformatics strategy to determine which genes were highly expressed and specific for glioblastoma endothelial cells (GBM-ECs), compared to gene expression in normal tissue and endothelium. Starting from fresh glioblastomas, several rounds of negative and positive selection were used to isolate GBM-ECs and extract total RNA. Using Serial Analysis of Gene Expression (SAGE), 116,259 transcript tags (35,833 unique tags) were sequenced. From this expression analysis, we found 87 tags that were not expressed in normal brain. Further subtraction of normal endothelium, bone marrow, white blood cell and other normal tissue transcripts resulted in just three gene transcripts, ANAPC10, PLXDC1(TEM7), and CYP27B1, that are highly specific to GBM-ECs. Immunohistochemistry with an antibody for PLXDC1 showed protein expression in GBM microvasculature, but not in the normal brain endothelium tested. Our results suggest that this study succeeded in identifying GBM-EC specific genes. The entire gene expression profile for the GBM-ECs and other tissues used in this study are available at SAGE Genie (http://cgap.nci.nih.gov/SAGE). Functionally, the protein products of the three tags most specific to GBM-ECs have been implicated in processes critical to endothelial cell proliferation and differentiation, and are potential targets for anti-angiogenesis based therapy.

The oncogenic transformation of normal cells into malignant, rapidly proliferating cells requires major alterations in cell physiology. For example, the transformed cells remodel their metabolic processes to supply the additional demand for cellular building blocks. We have recently demonstrated essential metabolic processes in tumor progression through the development of a methodological analysis of gene expression. Here, we present the Metabolic gEne RApid Visualizer (MERAV, http://merav.wi.mit.edu), a web-based tool that can query a database comprising ∼4300 microarrays, representing human gene expression in normal tissues, cancer cell lines and primary tumors. MERAV has been designed as a powerful tool for whole genome analysis which offers multiple advantages: one can search many genes in parallel; compare gene expression among different tissue types as well as between normal and cancer cells; download raw data; and generate heatmaps; and finally, use its internal statistical tool. Most importantly, MERAV has been designed as a unique tool for analyzing metabolic processes as it includes matrixes specifically focused on metabolic genes and is linked to the Kyoto Encyclopedia of Genes and Genomes pathway search.

In the past years a number of target antigens recognized by cytotoxic T-lymphocytes (CTL) have been identified in human malignancies. In most cases, the CTL-recognized antigens did not arise from mutations, but were instead encoded by genes that were identical in normal and tumor cells. Gene expression in normal tissues may result in tolerance of high avidity CTL, leaving behind low avidity CTL that cannot provide effective immunity against tumors expressing the relevant target antigens. In this chapter we describe a strategy to circumvent immunological tolerance that can be used to generate high avidity CTL against self-proteins, including human tumor-associated antigens.

Introduction: Obesity is a risk factor for breast cancer, including the more aggressive triple-negative (TN) subtype, but the relevance of obesity to prognosis remains unclear. Unlike other subtypes, triple-negative breast cancer (TNBC) is not hormonally responsive and is characterized by a lack of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2 expression. Thus, obesity-related development of TN tumors is unlikely to rely on the peripheral aromatization of androgens to estrogens in fat. Chronic, low-grade inflammation elicited by obesity could increase risk of TNBC. Here we sought to assess which mechanisms mediate obesity’s effect on normal tissue and TN tumors using a gene expression approach. Because African American women are more likely to be obese and twice as likely to develop TNBC compared to white women, understanding the mechanisms of obesity-related risk of TNBC is critical for this high-risk group. Methods: We performed whole-genome expression profiling on formalin-fixed, paraffin-embedded samples from 104 African American women with TNBC (59 tumor, 45 adjacent normal). Body mass index was obtained by medical record abstraction, and women with BMI at least 30 kg/m2 were classified as obese. Gene Ontology (GO) was used to identify biologic processes enriched in differentially expressed genes by obesity status in tumors and adjacent normal tissue (p-value<0.05). CIBERSORT was used to estimate proportions of 22 immune cell subtypes using expression profiles, and differences in median immune cell proportions by obesity status were evaluated. Results: 28 (47%) TNBC tumor and 29 (64%) adjacent normal samples were from obese women. Among genes more highly expressed in tumors from obese compared to nonobese women, GO analysis revealed enrichment for proinflammatory processes involving regulation of immune system, activation of immune responses, leukocyte activation, and neutrophil activation (FDR-corrected p-value <0.05). In contrast, GO analysis showed that genes in pathways related to B and T cell activation, cytokine production, regulation of immune system, and immune response (all FDR-corrected p <0.05) were downregulated in adjacent normal tissue from obese compared to nonobese women. Tumors from obese women exhibited a proinflammatory environment with increased levels of CD4 T memory resting cells, resting mast cells, and neutrophils, and reduced levels of regulatory T cells, resting dendritic cells, and activated mast cells (p < 0.1). We also found reduced proportions of naïve B cells, CD4 T memory resting cells, and M1 macrophages but an increased proportion of resting mast cells in the adjacent normal tissue of obese women compared to nonobese women (p< 0.1). Conclusions: Obesity modifies the immune cell environment present not only in TN tumors but also in the surrounding normal tissue. Obesity promotes a more proinflammatory setting within TN tumors, and in contrast, an anti-inflammatory setting in normal tissue adjacent. These findings may have important implications for understanding how obesity influences TNBC risk and prognosis, particularly among African American women who are at high risk for both. Citation Format: Asra N. Shaik, Julie L. Boerner, Michele L. Cote, Gregory Dyson, Rouba Ali-Fehmi, Sudeshna Bandyopadhyay, Kristen S. Purrington. Immune cell infiltrates differ by obesity in tumor and adjacent normal breast tissue in African American women with triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B01.