Aberrant ERG Expression Research Articles

Abstract 7077: The oncogenic transcription factor ERG has distinct roles and signaling in different prostate cancer cell types

Abstract ERG is the most common oncogene in prostate cancer, as it is aberrantly expressed in about 50% of cases due to the TMPRSS2/ERG gene rearrangement. ERG is not normally expressed in the adult prostate. The normal function of ERG is confined to hematopoietic stem cells and blood vessels. Aberrant expression of ERG in prostate epithelial cells is oncogenic when combined with mutations that activate the PI3K/AKT signaling pathway. The RAS/MAPK pathway can also regulate ERG function through direct phosphorylation of ERG by ERK. The function of these regulatory events and how they can be exploited to develop therapy for ERG-positive prostate cancer remains unclear. Here we find that ERG expression in prostate epithelial cells with low PI3K/AKT pathway activity drives epithelial to mesenchymal transition and stemness properties. This function requires ERG phosphorylation via a positive feedback loop with the TLR4 signaling pathway, and can be repressed by a TLR4 inhibitor. However, activation of the PI3K/AKT pathway rearranges the ERG cistrome and alters ERG function, where ERG now promotes luminal epithelial differentiation. In the presence of activated AKT, new ERG targets include genes involved in vascular development and ERG phosphorylation is regulated through a positive feedback loop with the VEGF signaling pathway. Despite these differences in cellular differentiation, we find that ERG can promote stemness and tumorigenesis in both AKT-high and AKT-low cellular backgrounds. ERG functions in prostate epithelial cells appear to mirror ERG functions in hematopoietic stem cells and blood vessels. These findings suggest strategies of combinatorial inhibition of ERG function through signaling pathway inhibition. Citation Format: Saranya Rajendran, Peter C. Hollenhorst. The oncogenic transcription factor ERG has distinct roles and signaling in different prostate cancer cell types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7077.

Abstract 1246: Erg-driven prostate cancer emerges from a basal subset of cells with luminal transcriptomic features

Abstract Translocations of the ETS family transcription factor ERG are found in nearly 150,000 newly diagnosed prostate cancers (PCas) each year in the United States underscoring the importance of defining the mechanism by which ERG promotes prostate epithelial transformation. A consequence of nearly all ERG translocations is the aberrant expression of ERG in prostate luminal cells under transcriptional control of TMPRSS2. Multiple lines of evidence implicate ERG translocations as an initiating event in prostate cancer. ERG expression is seen in prostatic intraepithelial neoplasia (PIN) and proliferative inflammatory atrophy; expression in cancers is typically uniform; and, in the context of PI3K pathway activation, ERG is sufficient to induce cancers in mice. Yet, despite extensive research the mechanism by which ERG initiates prostate cancer is unclear. To gain insight into how ERG translocations cause prostate cancer, we performed both transcriptional profiling (scRNA-seq) and chromatin accessibility via Assay for Transposase-Accessible Chromatin using sequencing (snATAC-seq) in single cells of an autochthonous mouse model at an early stage of disease initiation. Surprisingly, we observed in prostate epithelial cells broadly expressing ERG enhanced proliferation primarily in a hybrid subpopulation of cells having basal identity but with luminal morphology and cytokeratin expression. Through a series of lineage tracing and primary prostate epithelial transplantation experiments, we confirmed tumor initiating activity resided within a subpopulation of basal cells expressing luminal genes (e.g., Tmprss2 and Nkx3-1). A trajectory analysis from single-cell Chromatin accessibility indicated Erg+ cells began to lose basal (Trp63) accessibility and gained in STAT/NFAT TFs while retaining the ability to rapidly differentiate into luminal cells that reflect the lineage of ERG-positive cancers. These findings help resolve a preexisting debate about the cell of origin of ERG-driven prostate cancer and narrow the focus for future mechanistic studies to a basal-luminal subpopulation of tumor initiating cells. Citation Format: Erik Ladewig, Weiran Feng, Christina Leslie, Charles Sawyers. Erg-driven prostate cancer emerges from a basal subset of cells with luminal transcriptomic features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1246.

Abstract B005: The oncogenic transcription factor ERG has distinct roles and signaling regulation in different prostate cancer cell types

Abstract Background: ERG is the most common oncogene in prostate cancer, as it is aberrantly expressed in about 50% of cases due to the TMPRSS2/ERG gene rearrangement. ERG is not normally expressed in the adult prostate. The normal function of ERG is confined to hematopoietic stem cells and blood vessels. Aberrant expression of ERG in prostate epithelial cells is oncogenic when combined with mutations that activate the PI3K/AKT signaling pathway. The RAS/MAPK pathway can also regulate ERG function through direct phosphorylation of ERG by ERK. The function of these regulatory events and how they can be exploited to develop therapy for ERG-positive prostate cancer remains unclear. Results: ERG expression in prostate basal epithelial cells drives epithelial to mesenchymal transition and stemness properties. This function requires ERG phosphorylation via a positive feedback loop with the TLR4 signaling pathway, and can be repressed by a TLR4 inhibitor. Activation of the PI3K/AKT pathway rearranges the ERG cistrome and alters ERG function, where ERG now promotes luminal epithelial differentiation. In the presence of activated AKT, new ERG targets include genes involved in vascular development and ERG phosphorylation is regulated through a positive feedback loop with the VEGF signaling pathway. Conclusions: ERG function in basal and luminal prostate epithelial cells appear to mirror ERG functions in hematopoietic stem cells and blood vessels, respectively. These findings suggest strategies of combinatorial inhibition of ERG function in both cell types. Citation Format: Peter C. Hollenhorst, Saranya Rajendran, Travis J. Jerde. The oncogenic transcription factor ERG has distinct roles and signaling regulation in different prostate cancer cell types [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B005.

Oncogenic ERG Represses PI3K Signaling through Downregulation of IRS2.

Genomic rearrangements leading to the aberrant expression of ERG are the most common early events in prostate cancer and are significantly enriched for the concomitant loss of PTEN. Genetically engineered mouse models reveal that ERG overexpression alone is not sufficient to induce tumorigenesis, but combined loss of PTEN results in an aggressive invasive phenotype. Here, we show that oncogenic ERG repressed PI3K signaling through direct transcriptional suppression of IRS2, leading to reduced RTK levels and activity. In accordance with this finding, ERG-positive human prostate cancers had a repressed AKT gene signature and transcriptional downregulation of IRS2. Although overexpression of IRS2 activated PI3K signaling, promoting cell migration in a PI3K-dependent manner, this did not fully recapitulate the phenotype seen with loss of PTEN as PI3K signaling is not as robust as observed in the setting of loss of PTEN. Importantly, deletions of the PTEN locus, which promotes active PI3K signaling, were among the most significant copy-number alterations that co-occurred with ERG genomic rearrangements. This work provides insight on how initiating oncogenic events may directly influence the selection of secondary concomitant alterations to promote oncogenic signaling during tumor evolution. SIGNIFICANCE: This work provides insight on how initiating oncogenic events may directly influence the selection of secondary concomitant alterations to promote tumorigenesis.

Aberrant Expression of ERG Promotes Resistance to Combined PI3K and AR Pathway Inhibition through Maintenance of AR Target Genes

On the basis of our previous work defining the molecular rationale for combined targeting of the PI3K and AR pathways in PTEN loss prostate cancer, the first clinical trial was recently reported demonstrating a significant benefit for combination therapy in patients with metastatic prostate cancer. In this phase II trial, loss of PTEN was a biomarker predictive of response to combined AKT and AR inhibition. Given that PTEN loss prostate cancers are significantly enriched for ERG genomic rearrangements, we evaluated how the aberrant expression of ERG may impact response to PI3K/AR-targeted therapy. Here, we show that overexpression of ERG in the setting of Pten loss promotes resistance to combined PI3K and AR pathway inhibition with associated maintenance of AR target gene expression. Importantly, following AR knockout in the setting of ERG overexpression, there is maintenance of a subset of AR lineage-specific target genes, making AR dispensable in this context. This has important clinical implications as even in the setting of the androgen-regulated TMPRSS2:ERG genomic rearrangement, ERG expression is never abolished following AR inhibition and may allow for cell survival following AR (lineage)-targeted therapies.

Abstract LB-152: EZH2-induced lysine methylation and ERG-EZH2 genomic co-occupancy set the basis for extensive transcriptome reprogramming and prostate cancer progression

Abstract The TMPRSS2-ERG gene fusion is found in about half of prostate tumors and represents one of the most frequent genetic rearrangements in human cancers. The gene fusion provides a mechanism for androgen-stimulated ERG over-expression and broad transcriptional reprogramming in prostate cancer. However, the biological impact of aberrant ERG expression on tumor initiation and progression is still unclear. ERG apparently requires additional co-factors to fully exert its oncogenic effects but the molecular details of these interactions are not defined yet. Understanding these mechanisms could have a huge impact on the clinical management of this disease. Enhancer of zest homolog 2 (EZH2), the catalytic subunit of the Polycomb repressive complex 2 (PRC2) catalyzing histone H3 lysine 27 tri-methylation, is over-expressed in many human cancers and is associated with prostate cancer progression. In primary and metastatic prostate tumors ERG and EZH2 are frequently and concomitantly up-regulated. In this study we tested the hypothesis that EZH2 could act as a co-factor of ERG enhancing its transcriptional and oncogenic activity and identified a novel mechanism driving ERG activation and prostate cancer progression. We found that EZH2 physically interacts with ERG in ERG fusion positive cell lines and human tumors. Moreover, ERG/EZH2 co-occupied multiple genomic sites forming co-activator/co-repressor complexes and enabling massive transcriptional reprogramming. Expression of ERG/EZH2 co-occupied genes reflected the level of ERG activation, was preferentially deregulated in ERG-positive tumors and predicted clinical outcome. Furthermore, EZH2 catalyzed the methylation of ERG at a highly conserved lysine (K362) residue, which resulted in increased chromatin binding and transcriptional activity of ERG. PTEN deficiency and AKT activation promoted ERG methylation and ERG/EZH2 genomic co-occupancy along with a more aggressive and metastatic phenotype in ERG fusion positive cancer cells. Thus, this study identifies the ERG/EZH2 interaction and EZH2-induced ERG methylation as important elements promoting prostate tumorigenesis and at the center of cross-talks between the ERG gene fusion and PTEN deficiency in prostate cancer. Notably, these events were blocked effectively by pharmacological inhibitors of EZH2 providing the rationale for novel context-dependent therapeutic strategies in ERG positive prostate cancer. Citation Format: Giuseppina M. R. Carbone, Laura Curti, Marita Zoma, Abhishek Mitra, Dheeraj Shinde, Domenico Albino, Simona Rossi, Gianluca Civenni, George N. Thalmann, Giovanna Chiorino, Carlo Catapano. EZH2-induced lysine methylation and ERG-EZH2 genomic co-occupancy set the basis for extensive transcriptome reprogramming and prostate cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-152.

Abstract 5117: The expression of ERG oncoprotein and Annexin A2 show a strong inverse correlation in prostate cancers.

Abstract Introduction: Gene fusion of the TMPRSS2 promoter and ERG that gives rise to aberrant ERG expression is detected in over 50% of prostate cancers (CaP). ERG overexpression abrogates cell differentiation, promotes cell invasion and epithelial to mesenchymal transition, and additional Pten loss or AKT up-regulation enhances the transformation into well-differentiated prostate adenocarcinoma. Annexin A2 (ANXA2) is a calcium–dependent phospholipid–binding protein that regulates cell adhesion, cellular junction formation, actin remodeling and genesis of cell polarity. Evidence from a variety of malignancies suggests that ANXA2 is vital for promoting adhesion, migration and metastasis in cancer cells. In CaP, ANXA2 expression is lost or reduced, however, is increased in high grade tumors. Microarray gene expression studies have shown an inverse correlated ERG and ANXA2 expression. We examined the cancer associated functions of ERG and ANXA2 such as cell invasion, cell migration and cell polarity in CaP cell lines. Methods: ANXA2 and ERG cellular localization were monitored using fluorescence microscopy. The prostate specific transcriptional initiation site of ANXA2 was defined by 5’–RACE. Ch-IP Assay was used to show the recruitment of ERG to the ANXA2 promoter. Cases consisting of well differentiated and poorly differentiated CaP with and without TMPRSS2-ERG fusion were stained with ANXA2 and ERG antibodies to determine correlation in expression. Results: The inverse correlation between ERG and ANXA2 protein expression was confirmed by ERG knockdown experiments in VCaP cells. ERG overexpression in prostate adenocarcinoma is correlated with the loss ANXA2 expression in 90% of cases examined. A subset of ERG positive tumors exhibit small clusters of cells with clonal expression of ANXA2. We evaluated the patterns of ERG-ANXA2 expression against available clinical-pathological features. Conclusions: Our findings suggest that the expression of ANXA2 and its function in establishing cell polarity and cellular junctions may be disrupted by ERG overexpression through the transcriptional repression of ANXA2. We hypothesize that the reemergence of ANXA2 expression in advanced tumors reflects the accumulation of genetic hits that resulted in a more aggressive tumor with higher metastatic capacity. An IHC based assay with combined ERG and ANXA2 antibody cocktail could enhance the pathological evaluation of prostate tumors discern the re-expression of ANXA2 in ERG positive tumors and improve the prognosis and treatment of CaPs. This research was in part supported by the National Cancer Institute R01CA162383 grant to S. S. and USU–CPDR funds. Citation Format: Nick E. Griner, Shyh-Han Tan, Denise Young, Albert Dobi, Gyorgy Petrovics, Isabell Sesterhenn, Shiv Srivastava. The expression of ERG oncoprotein and Annexin A2 show a strong inverse correlation in prostate cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5117. doi:10.1158/1538-7445.AM2013-5117

Combined analysis of TMPRSS2–ERG and TERT for improved prognosis of biochemical recurrence in prostate cancer

Prostate cancer (PCa) is a heterogeneous disease with diverse clinical outcomes. TMPRSS2-ERG is the most common gene fusion in PCa, whereas activation of telomerase is a common feature of various malignancies. The aim of our study was to explore the combined utility of these and some other biomarkers in predicting biochemical recurrence after radical prostatectomy. Prostate specimens and urine sediments from 179 previously untreated patients with pT2-pT3 stage PCa were analyzed for expression of telomerase (TERT and TR) and the TMPRSS2-ERG fusion gene by means of reverse transcription PCR. Real-time PCR was used for quantification of ERG and SPINK1 expression. In total, 74% (117/158) of the prostate adenocarcinomas were positive for the TMPRSS2-ERG and/or TERT expression. Noninvasively, these transcripts were identified in 31% (19/61) of catheterized urine specimens. Significantly higher expression of ERG was detected in TMPRSS2-ERG-positive tumors (P<0.0001), whereas more intense expression of SPINK1 was characteristic for the TMPRSS2-ERG-negative tumors (P=0.003). TERT-positive cases also had elevated levels of ERG (P=0.016), suggesting a possible link between aberrant expression of ERG and reactivation of TERT in prostate tumors. The cases negative for both transcripts, TMPRSS2-ERG and TERT, rarely recurred (P=0.014) and showed significantly longer biochemical recurrence-free period (P=0.022) as compared to the TMPRSS2-ERG and/or TERT-positive cases. The results of our study suggest that combined analysis of TMPRSS2-ERG and TERT expression can be a valuable tool for early prediction of biochemical recurrence of PCa after radical prostatectomy.

Aberrant ERG expression cooperates with loss of PTEN to promote cancer progression in the prostate

Chromosomal translocations involving the ERG locus are frequent events in human prostate cancer pathogenesis; however, the biological role of aberrant ERG expression is controversial. Here we show that aberrant expression of ERG is a progression event in prostate tumorigenesis. We find that prostate cancer specimens containing the TMPRSS2-ERG rearrangement are significantly enriched for loss of the tumor suppressor PTEN. In concordance with these findings, transgenic overexpression of ERG in mouse prostate tissue promotes marked acceleration and progression of high-grade prostatic intraepithelial neoplasia (HGPIN) to prostatic adenocarcinoma in a Pten heterozygous background. In vitro overexpression of ERG promotes cell migration, a property necessary for tumorigenesis, without affecting proliferation. ADAMTS1 and CXCR4, two candidate genes strongly associated with cell migration, were upregulated in the presence of ERG overexpression. Thus, ERG has a distinct role in prostate cancer progression and cooperates with PTEN haploinsufficiency to promote progression of HGPIN to invasive adenocarcinoma.

Differential Expression of ERG Isoforms in Acute Myeloid and T-Lymphoblastic Leukemia Is Regulated by DNA-Methylation.

The oncogenic ETS transcription factor ERG is involved in various cellular pathways including developmental regulation, proliferation, and differentiation. In hematopoiesis ERG plays a specific role during normal T-cell differentiation showing high expression levels in stem cells and down regulation in the progenitor compartment. In this regard, it is intriguing that aberrant expression of ERG was found in a subset of patients with acute T-lymphoblastic leukemia (T-ALL) and was associated with an inferior outcome. Furthermore, high level ERG expression was of adverse prognostic significance in patients with newly diagnosed acute myeloid leukemia (AML), thus highlighting ERG's potential role in myeloid as well as T-lineage leukemogenesis. ERG3 (NM_182918) and ERG2 (NM_004449) represent the main isoforms and show abundant expression in myeloid and lymphoid hematopoietic progenitor cells. The expression pattern of specific ERG isoforms in acute leukemias has yet to be investigated. To further elucidate the nature of aberrant ERG expression we have determined the existence and transcriptional regulation of ERG isoforms in pretreatment bone marrow samples of adult T-ALL (n=21) and AML (n=20) patients as well as in normal CD34+ hematopoietic cells of healthy volunteers (n=5). 5′RACE revealed the presence of a new ERG isoform (ERG3Δex12) characterized by expression of exon 5 and absence of exon 12. Expression of ERG3Δex12 was verified by RT-PCR in AML, T-ALL, and CD34+ cells. In addition, real-time RT-PCR showed concomitant expression of the two main isoforms ERG2 and ERG3 in AML and normal CD34+ cells. In contrast, T-ALL patients lacked expression of ERG isoforms harboring exon 4 (ERG2). Promoter analyses of ERG2 and ERG3 revealed the presence of two CpG islands in the ERG2 promoter region, whereas no CpG island was predicted in the ERG3 promoter. Bisulfit conversion of genomic DNA and sequencing of cloned PCR products revealed a significantly higher degree of methylation of CpG island 2 in T-ALL samples (median: 86.4%, range: 16.0 – 98.8%) as compared to AML (median: 38.1%, range: 10.9 – 60.7%; P-value=0.0002 - two sided T-test). As for CpG island 1, CD34+ cells had the lowest rate of methylation in CpG island 2 (median: 7.7%, range: 2.4 – 20.7%). Thus, the differential expression of ERG isoforms is mediated by epigenetic silencing of exon 4 containing transcripts in T-ALL. In conclusion, the identification of the new ERG isoform (ERG3Δex12) suggests the association with different partners as the central exons, including exon 12, guide the interaction with different proteins. Furthermore, the distinct expression of specific ERG transcripts controlled by methylation adds to the complexity of ERG directed downstream pathways in different leukemic subtypes.