Kurtis Eisermann

Evolutionary conservation of zinc finger transcription factor binding sites in promoters of genes co-expressed with WT1 in prostate cancer

BackgroundGene expression analyses have led to a better understanding of growth control of prostate cancer cells. We and others have identified the presence of several zinc finger transcription factors in the neoplastic prostate, suggesting a potential role for these genes in the regulation of the prostate cancer transcriptome. One of the transcription factors (TFs) identified in the prostate cancer epithelial cells was the Wilms tumor gene (WT1). To rapidly identify coordinately expressed prostate cancer growth control genes that may be regulated by WT1, we used an in silico approach.ResultsEvolutionary conserved transcription factor binding sites (TFBS) recognized by WT1, EGR1, SP1, SP2, AP2 and GATA1 were identified in the promoters of 24 differentially expressed prostate cancer genes from eight mammalian species. To test the relationship between sequence conservation and function, chromatin of LNCaP prostate cancer and kidney 293 cells were tested for TF binding using chromatin immunoprecipitation (ChIP). Multiple putative TFBS in gene promoters of placental mammals were found to be shared with those in human gene promoters and some were conserved between genomes that diverged about 170 million years ago (i.e., primates and marsupials), therefore implicating these sites as candidate binding sites. Among those genes coordinately expressed with WT1 was the kallikrein-related peptidase 3 (KLK3) gene commonly known as the prostate specific antigen (PSA) gene. This analysis located several potential WT1 TFBS in the PSA gene promoter and led to the rapid identification of a novel putative binding site confirmed in vivo by ChIP. Conversely for two prostate growth control genes, androgen receptor (AR) and vascular endothelial growth factor (VEGF), known to be transcriptionally regulated by WT1, regulatory sequence conservation was observed and TF binding in vivo was confirmed by ChIP.ConclusionOverall, this targeted approach rapidly identified important candidate WT1-binding elements in genes coordinately expressed with WT1 in prostate cancer cells, thus enabling a more focused functional analysis of the most likely target genes in prostate cancer progression. Identifying these genes will help to better understand how gene regulation is altered in these tumor cells.

The Androgen Receptor and VEGF: Mechanisms of Androgen-Regulated Angiogenesis in Prostate Cancer

Prostate cancer progression is controlled by the androgen receptor and new blood vessel formation, or angiogenesis, which promotes metastatic prostate cancer growth. Angiogenesis is induced by elevated expression of vascular endothelial growth factor (VEGF). VEGF is regulated by many factors in the tumor microenvironment including lowered oxygen levels and elevated androgens. Here we review evidence delineating hormone mediated mechanisms of VEGF regulation, including novel interactions between the androgen receptor (AR), epigenetic and zinc-finger transcription factors, AR variants and the hypoxia factor, HIF-1. The relevance of describing the impact of both hormones and hypoxia on VEGF expression and angiogenesis is revealed in recent reports of clinical therapies targeting both VEGF and AR signaling pathways. A better understanding of the complexities of VEGF expression could lead to improved targeting and increased survival time for a subset of patients with metastatic castration-resistant prostate cancer.

Uncovering Androgen Responsive Regulatory Networks in Prostate Cancer

An important goal for prostate cancer therapy is to identify novel mechanisms of androgen signaling that may provide new targets for androgen blockade therapy. Androgen regulated target genes continue to be identified, and include genes with regulatory regions containing 1) classical dimeric androgen receptor elements, or 2) sites for other transcription factors that tether androgen receptor to a regulatory region lacking androgen receptor binding sites, or 3) non-canonical half-sites. The latter category of half-sites is becoming increasingly important, because up to 80% of potential androgen receptor regulatory regions identified by chromatin immunoprecipitation microarray technology contain these monomeric half-sites [1-3]. Determining which of these predicted target genes and androgen pathways are functional is very important, as they contribute to our understanding of prostate cancer progression. Microarray analyses were used to identify genes expressed in laser captured prostate cancer epithelial cells [4], leading to identification of pathways of co-regulated genes. It is expected that important regulatory regions would be conserved between mammalian genomes, thus, comparative evolutionary analyses were used to identify evolutionary conserved transcription factor binding sites [5]. Notably, non-canonical androgen receptor half-sites were identified in a majority of the gene promoters analyzed, and these sites were adjacent to evolutionary conserved zinc finger transcription factor sites. Subsequent ChIP assays showed that indeed SP1, WT1 and AR proteins all bind to a common regulatory region, indicating potential for interaction between these transcription factors that in turn can modulate hormone responsiveness. Overall, our bioinformatics screening coupled with experimental validation has revealed critical components of regulatory networks important in prostate cancer cells and disease progression.

Abstract B33: The zinc finger transcription factor, WT1, regulates growth control genes in leukemia cells.

The Wilms tumor gene, WT1, encodes multiple splice isoforms with varying functions including both transcriptional and post-transcriptional regulation mediated by DNA, RNA and protein binding domains. Involvement of WT1 in regulation of various growth control genes has been widely studied in many solid tumor types. However, evidence of its role in leukemia has been contradictory; although WT1 is both overexpressed and/or mutated in different subsets of leukemia patients, and these aberrations are linked to poor or intermediate prognosis. Our working hypothesis is that in leukemia WT1 regulates expression of genes that play mitogenic roles, such as Cyclin A1 (CCNA1) and Vascular Endothelial Growth Factor (VEGF). To demonstrate that WT1 regulates these genes we first identified potential WT1 binding sites in both CCNA1 and VEGF promoters, using the MatInspector Software. Three potential WT1 binding sites were located within both the CCNA1 and VEGF gene promoters and their functional significance was validated by chromatin immunoprecipitation (ChIP) assays. ChIP analysis of chromatin from K562 cells revealed WT1 binding at 2 of 3 sites within the CCNA1 promoter; and in chromatin of 293 Kidney cells and LNCaP prostate cancer (PC) cells, WT1 binding was observed in VEGF promoter. To demonstrate that these functional sites are involved in modulating transcription from these promoters, we performed luciferase assays using reporter constructs containing CCNA1 (-1180/+145) and VEGF (-411/+50) promoter regions. Co-transfection of WT1 with these reporter constructs demonstrated that both gene promoters were activated by WT1 over-expression in K562 cells. Transcriptional regulation of the endogenous genes was confirmed by Quantitative PCR in K562 cells transfected with WT1. Although CCNA1 mRNA levels increased as expected, no significant changes in the VEGF mRNA levels were observed. This absence of VEGF mRNA up-regulation in K562 cells differs from that observed in kidney and PC cells, and could be attributed to many factors, including a lack of necessary co-activators. To determine the biological relevance of WT1 mediated regulation, we measured the levels of WT1, CCNA1 and VEGF mRNA levels in pediatric leukemia bone marrow (BM) samples using Quantitative PCR. Overall, WT1 levels were higher in Acute Myelogeneous Leukemia- M3 than in Acute Lymphoblastic Leukemia BM samples and WT1 levels were low or undetected in non-neoplastic BM. The AML-M3s samples with high WT1 levels also had higher expression of CCNA1. Conversely, in ALL samples variation was seen in the expression of WT1 and CCNA1. VEGF transcript levels in leukemia BM were generally near or below those in normal BM. Taken together, these results suggest that WT1 transcriptionally up-regulates CCNA1, but the regulation of VEGF may be cell specific. Since CCNA1 is primarily expressed in leukemias and normal testes, this suggests that WT1 could be a significant factor controlling expression of this proliferative gene. Conversely,VEGF is expressed in many different tissues and under many different conditions, including hypoxia, suggesting that WT1 may be part of the many factors contributing to the complex orchestration of VEGF expression. Nonetheless when these proliferative factors are up-regulated in leukemia, WT1 may contribute to their altered expression levels, and therefore be a leukemogenic factor. Citation Format: Sony Pandey, Shorog Al omair, Mustafa Moazam, Kurtis Eisermann, Steven J. Kuerbitz, Gail C. Fraizer. The zinc finger transcription factor, WT1, regulates growth control genes in leukemia cells. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B33.

Abstract 2193: WT1 mediated regulation of growth control genes in leukemia

Prognosis and therapy of acute leukemia is influenced by leukemia-specific genetic alterations, thus highlighting the importance of identifying novel prognostic markers. One such potential marker, the Wilms’ tumor gene (WT1), a zinc finger transcription factor, is expressed in leukemic blasts and has been found to be mutated in approximately 10 percent of leukemia cases. Work in other tumor systems has shown that WT1 up-regulates expression of genes promoting cancer progression, including the angiogenic and mitogenic factor VEGF. Microarray studies have also correlated expression of JAG1 (Jagged 1) and CCNA1 (Cyclin A) with WT1 in Acute Myeloid Leukemia (AML) samples. We have identified potential WT1 binding sites within the promoters of both JAG1 and CCNA1 and have compared the expression of these genes to that of WT1, using real time quantitative PCR (QRTPCR), in pediatric leukemia and normal bone marrow. Compared to normal bone marrow, we observed lower than normal levels of WT1 in a majority of pediatric ALL samples, associated with lower than normal levels of VEGF, JAG-1 and CCNA-1. Conversely in pediatric AML(M3) we observed elevated levels of WT1 associated with elevated JAG-1 and CCNA-1. Mutations of the WT1 zinc finger (ZF) DNA binding domain have also been described in poor prognosis leukemias. To identify novel ZF mutations we sequenced WT1 in twelve pediatric acute leukemia samples. No ZF domain mutations were identified among these samples with high WT1 expression. However, a well-described SNP (rs 16754, also in exon 7), identified as a good prognostic marker in Cytogenetically Normal AML, was observed either as a homozygous or heterozygous variant of the WT1 gene. Because a majority of WT1 mutations identified in leukemias are frameshift mutations leading to a truncated protein lacking the ZF DNA binding domain, we created a truncation mutant of isoform A-WT1 lacking the ZF domain and assessed its function in K562 cells. Using QRTPCR we quantified the effect of over-expression of wild type and mutant WT1 on VEGF, JAG1 and CCNA1 expression. Compared to the vector control, wild type WT1 upregulated levels of JAG1 and CCNA1 but not VEGF mRNA. Mutant WT1 did not upregulate JAG1 or CCNA1. Our data and other studies have shown that JAG1 is elevated in AML patient samples, implicating the NOTCH 1 pathway that promotes cell proliferation and blocks differentiation. Thus, these results suggest that WT1 may act as an oncogene in K562 cells, in part by upregulating the growth promoting NOTCH-1 ligand, JAG1. Similarly, cell cycle regulation by WT1, demonstrated in other systems, may be mediated by transcriptional regulation of CCNA1. WT1 may contribute to leukemogenesis through transcriptional regulation of genes controlling cell proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2193. doi:1538-7445.AM2012-2193

Abstract 2169: The effect of WT1 on E-cadherin expression in prostate cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Prostate carcinoma is the most common malignancy and second leading cause of death among American men. The molecular mechanisms that control the progression of this type of cancer are still poorly understood. One of the genes expressed in prostate cancer epithelium is the zinc finger transcription factor Wilms’ Tumor 1, WT1. Previous studies in our lab have shown WT1 protein in a majority of high grade prostate tumor sections with little or no WT1 staining in non-neoplastic or benign prostatic hyperplasia (BPH) tissues. However, a mechanistic role for WT1 in prostate cancer has not been established. Recently, WT1 has been associated with the regulation of cell adhesion molecules such as E-cadherin in NIH 3T3 cells and epicardial cells. Loss of E-cadherin expression is frequently associated with increased cellular motility and tumor invasion. Several regulatory mechanisms controlling E-cadherin gene expression have been proposed in breast cancer cells, however the mechanisms of regulation of E-cadherin gene expression in prostate cancer cells are not yet understood. The objective of this study was to determine whether WT1 might regulate E-cadherin expression and contribute to cancer progression in prostate cancer cells. First, potential WT1 binding sites were identified in the promoter of the E-cadherin gene using a bioinformatics approach. Chromatin Immunoprecipitation (ChIP) showed direct in vivo binding of WT1 to the E-cadherin promoter in the chromatin of LNCaP and PC3 cells. The effect of transfection of prostate cancer cells with a GFP/WT1 expression construct was then tested by quantitative real-time PCR (QRT-PCR). QRT-PCR results showed a decrease in E-cadherin transcripts in GFP/WT1 transfected prostate cancer cells. Conversely, knockdown of WT1 mRNA in siWT1 transfected LNCaP cells, showed increased levels of E-cadherin mRNA. Moreover, co-transfection of WT1 expression construct with an E-cadherin promoter reporter construct showed that WT1 decreased the activity of the proximal E-cadherin promoter in PC3 cells. Overall these results demonstrated that WT1 modulated E-cadherin expression in prostate cancer cells and suggests a novel mechanism whereby WT1 may increase migration by decreasing E-cadherin levels, a novel role for WT1 mediated prostate cancer progression. This work was supported by NIHR15CA11360 and Ohio Board of Regents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2169. doi:10.1158/1538-7445.AM2011-2169

Abstract 1237: WT1, Wnt signaling and E-cadherin in prostate cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Prostate carcinoma is the most common malignancy and second leading cause of death among American men. The molecular mechanisms that control the progression of this type of cancer are still poorly understood. Among the genes proposed to play a role in prostate cancer is the zinc finger transcription factor, WT1. Previous studies in our lab have shown WT1 protein in a majority of high grade prostate tumor sections with little or no WT1 staining in non-neoplastic or benign prostatic hyperplasia (BPH) tissues. However, a mechanistic role for WT1 in prostate cancer has not been established. Recently, WT1 has been associated with the Wnt signaling pathway and Beta catenin, the central molecule in the pathway. The cytoplasmic levels of Beta catenin can be regulated through the canonical Wnt signaling or through the intracellular adhesion complex where it binds to E-cadherin. Importantly, E-cadherin has previously been identified as a WT1 target gene in NIH 3T3 cells. The objective of this study was to determine whether WT1 might regulate genes that, in turn, regulate the levels of Beta catenin in prostate cancer cells. First potential WT1 binding sites were identified in the promoters of two candidate genes, GSK3 Beta and E-cadherin, using a bioinformatics approach. Then the effect of transfection of LNCaP prostate cancer cells with GFP-tagged WT1 expression constructs was tested by quantitative real time PCR (QRT-PCR). In vivo evidence of direct binding of these gene promoters by WT1 protein was obtained by Chromatin immunoprecipitation (ChIP) of GFP/WT1 transfected LNCaP cells. Using evolutionary conservation analysis we identified two WT1 binding sites in the E-cadherin promoter conserved among primate species and one that was conserved among primates, rodents and dog. QRT- PCR results showed a decrease in E-cadherin transcripts in GFP/WT1 transfected LNCaP cells, compared with untransfected cells. ChIP was used to demonstrate that this effect on E-cadherin expression was direct and mediated by DNA binding to the E-cadherin promoter in vivo. Overall these results suggest that WT1 modulates E-cadherin and the Wnt signaling pathway in LNCaP cells. These findings suggest a novel mechanism for proliferation and migration of prostate cancer cells and ultimately may contribute towards an improved cancer therapy. This work was supported by NIHR15CA11360 (GF) and GSS KSU grant(AB) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1237.