TMPRSS2-ERG Gene Fusion Research Articles

ERG deregulation induces IGF-1R expression in prostate cancer cells and affects sensitivity to anti-IGF-1R agents.

Identifying patients who may benefit from targeted therapy is an urgent clinical issue in prostate cancer (PCa). We investigated the molecular relationship between TMPRSS2-ERG (T2E) fusion gene and insulin-like growth factor receptor (IGF-1R) to optimize the use of IGF-1R inhibitors.IGF-1R was analyzed in cell lines and in radical prostatectomy specimens in relation to T2E status. ERG binding to IGF-1R promoter was evaluated by chromatin immunoprecipitation (ChIP). Sensitivity to anti-IGF-1R agents was evaluated alone or in combination with anti-androgen abiraterone acetate in vitro at basal levels or upon ERG modulation.IGF-1R analysis performed in PCa cells or clinical samples showed that T2E expression correlated with higher IGF-1R expression at mRNA and protein levels. Genetic modulation of ERG directly affected IGF-1R protein levels in vitro. ChIP analysis showed that ERG binds IGF-1R promoter and that promoter occupancy is higher in T2E-positive cells. IGF-1R inhibition was more effective in cell lines expressing the fusion gene and combination of IGF-1R inhibitors with abiraterone acetate produced synergistic effects in T2E-expressing cells.Here, we provide the rationale for use of T2E fusion gene to select PCa patients for anti-IGF-1R treatments. The combination of anti-IGF-1R-HAbs with an anti-androgen therapy is strongly advocated for patients expressing T2E.

Genetic interaction between Tmprss2-ERG gene fusion and Nkx3.1-loss does not enhance prostate tumorigenesis in mouse models.

Gene fusions involving ETS family transcription factors (mainly TMPRSS2-ERG and TMPRSS2-ETV1 fusions) have been found in ~50% of human prostate cancer cases. Although expression of TMPRSS2-ERG or TMPRSS2-ETV1 fusion alone is insufficient to initiate prostate tumorigenesis, they appear to sensitize prostate epithelial cells for cooperation with additional oncogenic mutations to drive frank prostate adenocarcinoma. To search for such ETS-cooperating oncogenic events, we focused on a well-studied prostate tumor suppressor NKX3.1, as loss of NKX3.1 is another common genetic alteration in human prostate cancer. Previous studies have shown that deletions at 8p21 (harboring NKX3.1) and 21q22 (resulting in TMPRSS2-ERG fusion) were both present in a subtype of prostate cancer cases, and that ERG can lead to epigenetic silencing of NKX3.1 in prostate cancer cells, whereas NKX3.1 can in turn negatively regulate TMPRSS2-ERG fusion expression via suppression of the TMPRSS2 promoter activity. We recently generated knockin mouse models for TMPRSS2-ERG and TMPRSS2-ETV1 fusions, utilizing the endogenous Tmprss2 promoter. We crossed these knockin models to an Nkx3.1 knockout mouse model. In Tmprss2-ERG;Nkx3.1+/- (or -/-) male mice, although we observed a slight but significant upregulation of Tmprss2-ERG fusion expression upon Nkx3.1 loss, we did not detect any significant cooperation between these two genetic events to enhance prostate tumorigenesis in vivo. Furthermore, retrospective analysis of a previously published human prostate cancer dataset revealed that within ERG-overexpressing prostate cancer cases, NKX3.1 loss or deletion did not predict biochemical relapse after radical prostatectomy. Collectively, these data suggest that although TMPRSS2-ERG fusion and loss of NKX3.1 are among the most common mutational events found in prostate cancer, and although each of them can sensitize prostate epithelial cells for cooperating with other oncogenic events, these two events themselves do not appear to cooperate at a significant level in vivo to enhance prostate tumorigenesis.

Molecular and Clinicopathological Aspects of Prostate Cancer in Bulgarian Probands.

To correlate the molecular data to the clinicopathological parameters in Bulgarian prostate cancer patients. PCA3 overexpression, TMPRSS2-ERG gene fusion, GSTP1 promoter hypermethylation, somatic mutations in the AR gene and the IVS1-27G > A polymorphism in the KLF6 gene were studied. A total of 148 patients were analyzed: 16 aggressive PCa, 83 non-aggressive PCa, 25 BPH and 24 chronic inflammatory diseases. Real-time RT-PCR, DNA sequencing, and bisulfite conversion of DNA, were applied. All cases with aggressive PCa before treatment were tested positive for PCA3 overexpression, expression of a T2-ERG gene fusion product and GSTP1 promoter hypermethylation. No somatic mutations were detected in the AR gene and all patients showed normal KLF6-IVS1-27G > A genotype. The TMPRSS2-ERG positive status correlates with moderate to poorly differentiated prostate tumors and it is considered as unfavorable disease predictor. Positive GSTP1 promoter hypermethylation seems to be highly specific and the earliest epigenetic change in the prostate gland, which indicates the beginning of the pathological process. The appearance of positive molecular markers in blood was considered as a predictor of PCa dissemination. GSTP1 promoter hypermethylation was found as the earliest and a long-lasting epigenetic marker in blood samples of PCa patients, which makes it suitable as a marker for treatment follow-up. The molecular profile of prostate cancer needs to be strictly monitored during the course of disease treatment, which is of a great help in determining the patient's individual therapy response.

TMPRSS2-ERG gene fusion in prostate cancer.

The TMPRSS2-ERG gene fusion is one of the most widely spread chromosomal rearrangements in carcinomas. Since its discovery, a number of studies have examined its diagnostic, prognostic and therapeutic implications for prostate cancer where suitable biomarkers are still lacking. The publication data are inconsistent. The aim of this review was to critically evaluate the current clinical impact of this gene fusion. The PubMed online database was used to search relevant reviews and original articles. Although the TMPRSS2-ERG gene fusion appears to be a suitable diagnostic biomarker, the prognostic implications of this gene fusion are still unclear. Several new strategies for therapeutically targeting ETS fusions and their modulators have been identified and are currently being investigated. Due to the heterogeneity of prostate cancer, the combination of several biomarkers is necessary to accurately assess the presence of prostate cancer, predict its potential clinical outcome and decide on appropriate therapy (e.g. PARP inhibitors).

Abstract 2233: Rearrangement of ERG and CHD1 genes in prostate cancer as a marker of tumor heterogeneity

Abstract Efforts to develop targeted therapy based on genomic findings are complicated by the polygenic nature of drug resistance and genetic heterogeneity between separate biopsy cores. These findings have led to the conclusion that analysis of multiple biopsy cores and consideration of multiple targeted agents will be important. Because of our concern that these assessments may be missing an additional layer of heterogeneity within even a single biopsy core, which sequencing alone may not identify, we studied the genetic heterogeneity of cells in focal areas using TMA. Using confocal microscopy, we analyzed the most common and prostate cancer specific gene rearrangement (TMPRSS2-ERG fusion and CHD1 deletion) in neighboring single cells in a tumor focus without changing the stromal microenvironment. We evaluated 56 patients that underwent radical prostatectomy at CINJ with pathological assessment, grading, and ERG rearrangement status. Overall, ERG rearrangements occurred in 24/56 (42.9%) patients. The most frequent mechanism of rearrangements was deletion (58%), while a split event was confirmed in 25% of the ERG-rearranged cases. In 88% of cases the ERG rearrangement was associated with TMPRSS2-ERG gene fusion. A copy number gain of the non-rearranged ERG gene occurred in 10/56 (17.6%) cases. A heterogeneous mix of ERG deletions, translocations, and copy number changes occurred in 8/24 (33%) patients, with a combination of deletions and translocations alone occurring in 3/24 (12.5%) cases. 20/21 (95%) of analyzed cases with ERG-rearrangement were negative for CHD1 gene rearrangement (p< 0.01). Overall, 14/45 (31.1%) of patients had CHD1 deletion, and 13/14 (93%) CHD1- cases correlate with ERG-rearrangement negative status (p< 0.01). 10/45 (22.2%) cases represent CHD1 gene copy number gain. These results support the notion that additional heterogeneity may be present even in a focus of a single biopsy core. These data are extremely important and urgently require further study to understand heterogeneity of genetic changes, because clinical studies have already been launched using genetic changes such as TMPRSS2-ERG fusions found in a single core biopsy to direct a specific therapy, including a trial using ABT-888 only in patients with this fusion now ongoing at CINJ. Citation Format: Irina V. Tereshchenko, Hua Zhong, Marina Chekmareva, Urmila Santanam, Whitney Petrosky, Noriko Kane-Goldsmith, Jay A. Tischfield, Robert DiPaola. Rearrangement of ERG and CHD1 genes in prostate cancer as a marker of tumor heterogeneity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2233. doi:10.1158/1538-7445.AM2014-2233

Abstract 3313: 17β-Estradiol-ERβ axis confers proliferation and migration through IGF-1R-dependent expression of TMPRSS2 in castration-resistant PC-3 cells

Abstract Prostate cancer (PCa) is the most common and the second leading cause of cancer-related death among males in the Western world. TMPRSS2, a gene that encodes serine-protease type II transmembrane domain, has been shown to be frequently prevalent as a TMPRSS2-ERG gene fusion during early event in the genesis of PCa. However, TMPRESS2 function remains largely unknown, especially in castration-resistant PCa cells. While TMPRESS2 has previously been shown to be an androgen-regulated gene, TMPRSS2-ERG expression is regulated by a novel ERβ-dependent mechanism. The objectives of this study are to determine the mechanisms of expression and the functional significance of TMPRSS2 in growth and migration of castration-resistant AR-null PC-3 cells. 17β-Estradiol (E2; 10nM) induced TMPRSS2 expression (4-fold higher) in an ERβ-dependent manner as evidenced by shRNA silencing, but not by ERα-specific shRNA, in PC-3 cells. Intriguingly, the E2-induced TMPRSS2 expression was blocked by AG1024 (IGF-1R inhibitor) or knockdown of IGF-1R by siRNA, but not by AG1478 (EGFR inhibitor), suggesting that IGF-1R mediates expression of TMPRESS2 by the E2-ERβ axis in PC-3 cells. Moreover, TMPRSS2-specific siRNA markedly attenuated E2-induced cell growth and migration of PC-3 cells, suggesting that TMPRSS2 may be pivotal to E2-mediated progression of castration-resistant disease in vivo. The effects of TMPRSS2 silencing seem to be mediated through inhibition of E2-induced activation of NF-κB-signaling in these cells. Taken together, our results unraveled the underlying mechanisms that govern the expression and function of TMPRSS2 in mediating E2-induced growth and migration of castration-resistant PC-3 cells. Delineation of such critical players may culminate in identifying therapeutic targets to selectively circumvent castration-resistant outgrowth in PCa patients. Citation Format: Hogyoung Kim, Amrita Datta, Zakaria Y. Abd Elmageed, Asim B. Abdel-Mageed. 17β-Estradiol-ERβ axis confers proliferation and migration through IGF-1R-dependent expression of TMPRSS2 in castration-resistant PC-3 cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3313. doi:10.1158/1538-7445.AM2014-3313

Abstract 2863: TMPRSS2-ERG influences IGF-1R expression and affects its prognostic value in prostate cancer

Abstract INTRODUCTION: Insulin like growth factor 1 receptor (IGF-1R) plays a critical role in different tumors including prostate cancer (PCa). Recently, a fusion gene involving the androgen receptor-regulated gene TMPRSS2 and one of the ETS transcription factors family member, predominantly ERG, has been described in half of PCa patients. TMPRSS2-ERG results in the overproduction of a truncated ERG protein and cooperates in the establishment of a neoplastic phenotype in prostate cells. The aim of this study was to evaluate TMPRSS2-ERG impact on IGF-1R expression and to verify the value of IGF-1R as biomarker of PCa progression. MATERIAL AND METHODS: IGF-1R expression was evaluated in a panel of six prostate cell lines, including malignant VCaP, DU-145, PC-3, LNCaP, 22RV1 and non malignant RWPE-1 prostate cells by qRT-PCR, western blot and immunofluorescence. ERG siRNA was transiently transfected in VCaP cells and ERG down-regulation was assessed by western blot. Total RNA was extracted from a cohort of 270 paraffin-embedded PCa samples with more than 5 years of follow-up and IGF-1R expression was analysed by qRT-PCR. The prognostic value of IGF-1R was evaluated both in the univariate and multivariate analysis. The clinicopathological informations as well as the TMPRSS2-ERG fusion gene status were considered in the statistical analysis. RESULTS: The IGF-1R expression profile analysis was performed in PCa cells and pointed out that VCaP cell line, harboring the TMPRSS2-ERG fusion gene, carries the highest IGF-1R expression levels both considering mRNA, total protein and membrane expression levels. Interestingly, IGF-1R protein levels were found to be down-regulated upon ERG silencing in VCaP cells. PCa tumors did not show any remarkable difference in the expression of IGF-1R when compared with normal tissues (median=1.04) but IGF-1R was directly correlated with TMPRSS2-ERG status (p=0.027) in tumors. Log-rank tests showed an association between higher IGF-1R expression and better biochemical progression free survival (p=0.047).When dividing the population according to the TMPRSS2-ERG status we found higher fold-change in IGF-1R expression in tumors expressing TMPRSS2-ERG (median=1.25) compared to non-expressors (median=0.91) and a statistically stronger association between IGF-1R and prognosis in the TMPRSS2-ERG negative tumors (p=0.012). Moreover, multivariate analysis confirmed that higher expression of IGF-1R is an independent indicator of better prognosis in this subgroup (HR:0.48.IC95% [0.25-0.90].p=0.023). CONCLUSIONS: In this study we demonstrated that TMPRSS2-ERG affects IGF-1R expression both in vitro and in clinical samples. High expression of IGF-1R is significantly associated with a better clinical outcome of PCa patients. (Grants: FIRB RBAP11884M_005 to KS; FIS PI10/01206 and FPI11/00505 from ISCIII) Citation Format: Caterina Mancarella, Irene Casanova-Salas, Josè Antonio Lopez-Guerrero, Katia Scotlandi. TMPRSS2-ERG influences IGF-1R expression and affects its prognostic value in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2863. doi:10.1158/1538-7445.AM2014-2863

Prevalence and prognostic significance of TMPRSS2-ERG gene fusion in lymph node positive prostate cancers.

TMPRSS2-ERG gene fusion is the most frequent genetic alteration in prostate cancer. However, information about its distribution in lymph node positive prostate cancers and the prognostic significance in these advanced tumors is unknown. Gene fusion status was determined by fluorescence in situ hybridization on a tissue-microarray constructed from 119 hormone-naïve nodal positive, surgically treated prostate cancers containing samples from the primary tumors and corresponding lymph node metastases. Data were correlated with various tumor features (Gleason score, stage, cancer volume, nodal tumor burden) and biochemical recurrence-free, disease-specific, and overall survival. TMPRSS2-ERG fusion was detected in 43.5% of the primary tumors. Conversely, only 29.9% of the metastasizing components showed the fusion. Concordance in TMPRSS2-ERG status between primary tumors and metastases was 70.9% (Kappa 0.39); 20.9% and 8.1% of the patients showed the mutation solely in their primary tumors and metastases, respectively. TMPRSS2-ERG fusion was not correlated with specific histopathological tumor features but predicted favorable biochemical recurrence-free, disease-specific and overall survival independently when present in the primary tumor (P < 0.05 each). TMPRSS2-ERG fusion is more frequent in primary prostate cancer than in corresponding metastases suggesting no selection of fusion-positive cells in the metastatic process. The gene fusion in primary tumors independently predicts favorable outcome.

Pyrrole-imidazole polyamide targeted to break fusion sites in TMPRSS2 and ERG gene fusion represses prostate tumor growth.

Aberrant overexpression of ERG induced by the TMPRSS2-ERG gene fusion is likely involved in the development of prostate cancer. Synthetic pyrrole–imidazole (PI) polyamides recognize and attach to the minor groove of DNA with high affinity and specificity. In the present study, we designed a PI polyamide targeting TMPRSS2-ERG translocation breakpoints and assessed its effect on human prostate cancer cells. Our study identified that this PI polyamide repressed the cell and tumor growth of androgen-sensitive LNCaP prostate cancer cells. Targeting of these breakpoint sequences by PI polyamides could be a novel approach for the treatment of prostate cancer.

Potential utility of cancer-specific biomarkers for assessing response to hormonal treatments in metastatic prostate cancer.

Prostate cancer is the second leading cause of cancer death in men and there is an urgent clinical need to improve its detection and treatment. The introduction of prostate-specific antigen (PSA) as a biomarker for prostate cancer several decades ago represented an important step forward in our ability to diagnose this disease and offers the potential for earlier and more effective treatment. PSA measurements are now routinely conducted alongside digital rectal examination, with raised PSA levels leading to biopsy. PSA is also used to monitor disease and assess therapeutic response. However, there are some important limitations to its use, not least its lack of specificity for prostate cancer, and increased PSA screening may have resulted in overdiagnosis and overtreatment of early, low-risk prostate cancer. Therefore, there is a need for more specific and sensitive biomarkers for the diagnosis and monitoring of prostate cancer and treatment response; in particular, biomarkers of response to hormonal treatments in prostate cancer and predictive biomarkers to identify who is most likely to respond to these treatments. Here we review the current utilization of PSA and data on potentially more specific and sensitive biomarkers for the diagnosis and monitoring of prostate cancer: prostate cancer antigen 3 (PCA3) and the TMPRSS2-ERG fusion gene. A description of the design of an ongoing study of the 6-month extended release formulation of leuprorelin acetate (Eligard(®) 45 mg) will provide preliminary data on the potential utility of these new biomarkers for detecting therapeutic response after hormonal therapy.

TMPRSS2-ERG fusion transcripts expression in patients referred for prostate biopsy: combining detection in urine and needle rinse material.

The objective of this study was to combine urine and prostate biopsy rinse material (BRM) assays to increase sensitivity for fusion gene detection. A total of 194 patients with suspicion of prostate cancer were prospectively included. Urine samples were collected before or after prostate biopsy, as well as BRM. RT-qPCR was used for the detection of fusion transcripts. A microfocal cancer on biopsy was defined by a single core involved with less than 3 mm of Gleason score 3 + 3 cancer. The association between RT-qPCR and biopsy results was statistically assessed. Seven patients were excluded because of insufficient material. Cancer was detected on biopsy in 100 (53%) patients. Urine alone, BRM alone and both samples were obtained in 155, 164 and 132 patients, respectively. In patients with evidence of cancer on biopsy, a fusion transcript was detected in 63, 55 and 73% of the cases on urine alone, BRM alone and paired samples, respectively. Fusion gene detection on BRM was only associated with the amount of cancer on biopsy. Urine fusion score had a larger area under the curve than serum PSA (p = 0.002) and was significantly higher in patients with high Gleason score and significant cancer on biopsy. Assays of paired samples allowed increasing sensitivity in all subgroups of patients. TMPRSS2-ERG fusion gene detection may be performed both in the urine and BRM to increase sensitivity. However, only T-E urine score was associated with adverse pathological features.

Increased association of the ERG oncoprotein expression in advanced stages of prostate cancer in Filipinos.

Filipinos with prostate cancer (CaP) are at increased risk of harboring advanced stages and lower survival rates compared to other Asians. This study aims to investigate prevalence of ETS-related gene (ERG) oncoprotein overexpression in Filipinos as surrogate of TMPRSS2-ERG gene fusions, using a highly specific monoclonal antibody (ERG-MAb), and conduct the first attempt to study the role of genetic alterations in the aggressive tumor biologic behaviour of CaP among Filipinos. This case-matched, case-control retrospective study evaluated ERG expression in Filipino patients diagnosed with CaP and its effect on stage and Gleason grade of their disease. Men who underwent radical prostatectomy for organ-confined disease at the University of the Philippines-Philippine General Hospital (UP-PGH) comprised the organ-confined cohort. Age-matched adults who had trans-rectal ultrasound-guided prostate (TRUSP) biopsy or trans-urethral resection of the prostate (TURP) with bilateral orchiectomy for T4 or stage IV CaP composed the advanced disease cohort. Overall ERG expression frequency of 23.08% (N = 104) was demonstrated, with a higher rate observed in the advanced disease cohort (32.69%) compared to the organ-confined group (13.46%). Furthermore, ERG overexpression was only detected among intermediate and high-risk tumors. A high-specificity (98.08%) of the ERG-MAb for malignant prostatic cells was likewise demonstrated. In contrast to higher ERG frequency in Western countries, it is much lower in Filipino CaP, which is similar to lower rates noted from other Asian countries. The 98.08% specificity of ERG oncoprotein for prostate tumor cells combined with its increased association in advanced disease, suggests for prognostic potential of ERG that may aid clinicians in treatment decisions for Filipino CaP patients.

Increased androgen receptor gene copy number is associated with TMPRSS2-ERG rearrangement in prostatic small cell carcinoma.

Small cell carcinoma of the prostate (PSCC) is a highly aggressive malignancy that often develops in patients previously treated with hormonal therapy for metastatic prostatic acinar adenocarcinoma. The TMPRSS2-ERG gene rearrangement is highly specific for prostate cancer and shared by PSCC; however, the role of androgen receptor (AR) gene alterations and interaction with TMPRSS2-ERG rearrangement are incompletely understood in PSCC. Sixty-one cases of PSCC were examined for AR gene copy number and TMPRSS2-ERG rearrangement by fluorescence in situ hybridization (FISH) and AR protein expression by immunohistochemistry. Of 61 cases of PSCC, 51% (31/61) demonstrated increased AR gene copy number (FISH+), 54% (33/61) were positive for TMPRSS2-ERG gene fusion, and 38% (23/61) showed AR protein expression. Of the 31 AR FISH+ cases, 23 also showed TMPRSS2-ERG gene fusion, and 16 expressed AR protein. Of the 33 cases with TMPRSS2-ERG fusion, 28 were AR FISH+ or expressed AR protein. Statistically significant correlations were observed between AR gene copy number or AR protein expression and TMPRSS2-ERG gene fusion (P = 0.001 and P = 0.03, respectively). In summary, high AR gene copy number emerges during the development of PSCC, often in association with TMPRSS2-ERG rearrangement. This potential mechanism warrants further study. Improvement will come from understanding the biology of the disease and integrating new therapies into the treatment of this rare and aggressive tumor.

Therapeutic targeting of BET bromodomain proteins in castration-resistant prostate cancer.

Men who develop metastatic castration-resistant prostate cancer (CRPC) invariably succumb to the disease. The development and progression to CRPC following androgen ablation therapy is predominantly driven by unregulated androgen receptor (AR) signaling1-3. Despite the success of recently approved therapies targeting AR signaling such as abiraterone4-6 and second generation anti-androgens MDV3100 (enzalutamide)7,8, durable responses are limited, presumably due to acquired resistance. Recently JQ1 and I-BET, two selective small molecule inhibitors that target the amino-terminal bromodomains of BRD4, have been shown to exhibit anti-proliferative effects in a range of malignancies9-12. Here we show that AR signaling-competent CRPC cell lines are preferentially sensitive to BET bromodomain inhibition. BRD4 physically interacts with the N-terminal domain of AR and can be disrupted by JQ111,13. Like the direct AR antagonist, MDV3100, JQ1 disrupted AR recruitment to target gene loci. In contrast to MDV3100, JQ1 functions downstream of AR, and more potently abrogated BRD4 localization to AR target loci and AR-mediated gene transcription including induction of TMPRSS2-ERG and its oncogenic activity. In vivo, BET bromodomain inhibition was more efficacious than direct AR antagonism in CRPC xenograft models. Taken together, these studies provide a novel epigenetic approach for the concerted blockade of oncogenic drivers in advanced prostate cancer.

OP1-06 IN MOLECULAR DETECTION OF OCCULT LYMPH NODE METASTASES EPITHELIAL MARKERS ARE HERE TO STAY

You have accessJournal of UrologyOutstanding Posters: Research1 Apr 2014OP1-06 IN MOLECULAR DETECTION OF OCCULT LYMPH NODE METASTASES EPITHELIAL MARKERS ARE HERE TO STAY Antoinette Wetterwald, Achim Fleischmann, Janine Hensel, Berna Özdemir, Irena Klima, Marco G. Cecchini, and George N. Thalmann Antoinette WetterwaldAntoinette Wetterwald More articles by this author , Achim FleischmannAchim Fleischmann More articles by this author , Janine HenselJanine Hensel More articles by this author , Berna ÖzdemirBerna Özdemir More articles by this author , Irena KlimaIrena Klima More articles by this author , Marco G. CecchiniMarco G. Cecchini More articles by this author , and George N. ThalmannGeorge N. Thalmann More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.614AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES In prostate cancer patients the presence of pelvic lymph node metastases is a relevant prognostic factor for disease progression. Thus detection of occult lymph node metastases might be of prognostic value. While most previous studies have focused on the detection of epithelial markers, we hypothesized that micrometastatic cells may express markers of stemness and/or of EMT, or induce a stroma reaction in the lymph node. METHODS Individual lymph nodes (n=1023) collected from 60 prostate cancer patients undergoing radical prostatectomy with extended pelvic lymph node dissection for clinical organ-confined disease were cut in halves, one being processed for histology, the second stored in RNALater prior to RNA extraction and real-time PCR analysis. The mRNA expression of epithelial markers, markers of stemness and EMT, as well as markers of stromal activation was tested by real-time PCR and compared to histopathological results for each lymph node and compared to negative controls. Immunohistochemical controls were performed. RESULTS The suitability of the different markers for detecting micrometastases was established by comparing their expression in lymph nodes with overt metastases and negative controls. The epithelial markers PSA, EpCAM, PSCA, PSMA, NKX3-1 and AGR2 were differentially expressed in positive lymph nodes as compared to controls. In contrast, the levels of expression of the EMT markers SNAIL, TWIST and CXCR4 were either higher in control patients or no difference was observed. Similar results were obtained for the markers of reactive stroma (ASPN, POSTN, SPARCL1 and MCAM). The following proven or putative stem cell/self-renewal markers ALDH1A1, NANOG, SOX2, OCT4, KLF4, EGR1, BMI1, LGR5, LGR6, LRIG1, TSPAN7, TSPAN13, and TROP2 were tested. With the exception of TROP2, all those markers were expressed in lymph nodes from control patients to similar or even higher levels than in prostate cancer patients. TROP2 expression was barely detectable in lymph nodes from controls but could be measured in lymph nodes from prostate cancer patients. TMPRSS2-ERG fusion gene was detected in lymph node metastases of a few patients only. Detectable expression of epithelial markers in histopathologically negative lymph nodes was predictable of biological recurrence in several patients in spite of a relatively short follow up. CONCLUSIONS In lymph nodes epithelial markers are the only markers indicative of lymph node metastasis, as other potential markers are expressed by the different lymph node cells as well. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e164 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Antoinette Wetterwald More articles by this author Achim Fleischmann More articles by this author Janine Hensel More articles by this author Berna Özdemir More articles by this author Irena Klima More articles by this author Marco G. Cecchini More articles by this author George N. Thalmann More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Antibody-independent targeted quantification of TMPRSS2-ERG fusion protein products in prostate cancer

Fusions between the transmembrane protease serine 2 (TMPRSS2) and ETS related gene (ERG) represent one of the most specific biomarkers that define a distinct molecular subtype of prostate cancer. Studies of TMPRSS2-ERG gene fusions have seldom been performed at the protein level, primarily due to the lack of high-quality antibodies suitable for quantitative studies. Herein, we applied a recently developed PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing)-SRM (selected reaction monitoring) strategy for quantifying ERG protein in prostate cancer cell lines and tumors. The highly sensitive PRISM-SRM assays provided confident detection of 6 unique ERG peptides in both TMPRSS2-ERG positive cell lines and tissues, but not in cell lines or tissues lacking the TMPRSS2-ERG rearrangement, clearly indicating that ERG protein expression is significantly increased in the presence of the TMPRSS2-ERG gene fusion. Significantly, our results provide evidence that two distinct ERG protein isoforms are simultaneously expressed in TMPRSS2-ERG positive samples as evidenced by the concomitant detection of two mutually exclusive peptides in two patient tumors and in the VCaP prostate cancer cell line. Three peptides, shared across almost all fusion protein products, were determined to be the most abundant peptides, providing “signature” peptides for detection of ERG over-expression resulting from TMPRSS2-ERG gene fusion. The PRISM-SRM assays provide valuable tools for studying TMPRSS2-ERG gene fusion protein products in prostate cancer.

TMPRSS2-ERG Expression Predicts Prostate Cancer Survival and Associates with Stromal Biomarkers

The TMPRSS2-ERG gene fusion is found in approximately half of all prostate cancers. The functional and prognostic significance of TMPRSS2-ERG is, however, not fully understood. Based on a historical watchful waiting cohort, an association between TMPRSS2-ERG, evaluated as positive immune staining, and shorter survival of prostate cancer patients was identified. Expression of ERG was also associated with clinical markers such as advanced tumor stage, high Gleason score, presence of metastasis and prognostic tumor cell markers such as high Ki67, pEGFR and pAkt. Novel associations between TMPRSS2-ERG and alterations in the tumor stroma, for example, increased vascular density, hyaluronan and PDGFRβ and decreased Caveolin-1, all known to be associated with an aggressive disease, were found. The present study suggests that the TMPRSS2-ERG fusion gene is associated with a more aggressive prostate cancer phenotype, supported by changes in the tumor stroma.

ERG rearrangement and protein expression in the progression to castration-resistant prostate cancer

Approximately half of the prostate carcinomas are characterized by a chromosomal rearrangement fusing the androgen-regulated gene TMPRSS2 to the oncogenic ETS transcription factor ERG. Aim of this study was to comprehensively analyze the role and impact of the ERG rearrangement and protein expression on the progression to castration-resistant (CR) disease. We used a tissue microarray (TMA) constructed from 114 hormone naive (HN) and 117 CR PCs. We analyzed the ERG rearrangement status by fluorescence in situ hybridization and the expression profiles of ERG, androgen receptor (AR) and the proliferation marker Ki67 by immunohistochemistry. Nearly half of the PC tissue specimens (HN: 38%, CR: 46%) harbored a TMPRSS2-ERG gene fusion. HN PCs with positive translocation status showed increased tumor cell proliferation (P<0.05). As expected, TMPRSS2-ERG gene fusion was strongly associated with increased ERG protein expression in HN and CR PCs (both P<0.0001). Remarkably, the study revealed a subgroup (26%) of CR PCs with ERG rearrangement but without any detectable ERG protein expression. This subgroup showed significantly lower levels of AR protein expression and androgen-regulated serum PSA (both P<0.05). In this study, we identified a subgroup of ERG-rearranged CR PCs without detectable ERG protein expression. Our results suggest that this subgroup could represent CR PCs with a dispensed AR pathway. These tumors might represent a thus far unrecognized subset of patients with AR-independent CR PC who may not benefit from conventional therapy directed against the AR pathway.

Loss of the NKX3.1 tumorsuppressor promotes the TMPRSS2-ERG fusion gene expression in prostate cancer

BackgroundIn normal prostate epithelium the TMPRSS2 gene encoding a type II serine protease is directly regulated by male hormones through the androgen receptor. In prostate cancer ERG protooncogene frequently gains hormonal control by seizing gene regulatory elements of TMPRSS2 through genomic fusion events. Although, the androgenic activation of TMPRSS2 gene has been established, little is known about other elements that may interact with TMPRSS2 promoter sequences to modulate ERG expression in TMPRSS2-ERG gene fusion context.MethodsComparative genomic analyses of the TMPRSS2 promoter upstream sequences and pathway analyses were performed by the Genomatix Software. NKX3.1 and ERG genes expressions were evaluated by immunoblot or by quantitative Real-Time PCR (qRT-PCR) assays in response to siRNA knockdown or heterologous expression. QRT-PCR assay was used for monitoring the gene expression levels of NKX3.1-regulated genes. Transcriptional regulatory function of NKX3.1 was assessed by luciferase assay. Recruitment of NKX3.1 to its cognate elements was monitored by Chromatin Immunoprecipitation assay.ResultsComparative analysis of the TMPRSS2 promoter upstream sequences among different species revealed the conservation of binding sites for the androgen inducible NKX3.1 tumor suppressor. Defects of NKX3.1, such as, allelic loss, haploinsufficiency, attenuated expression or decreased protein stability represent established pathways in prostate tumorigenesis. We found that NKX3.1 directly binds to TMPRSS2 upstream sequences and negatively regulates the expression of the ERG protooncogene through the TMPRSS2-ERG gene fusion.ConclusionsThese observations imply that the frequently noted loss-of-function of NKX3.1 cooperates with the activation of TMPRSS2-ERG fusions in prostate tumorigenesis.

Relation of ETS transcription factor family member ERG, androgen receptor and topoisomerase 2β expression to TMPRSS2-ERG fusion status in prostate cancer

Fusion of TMPRSS2 with ERG in prostate cells is determined by double-strand DNA breaks induced by androgen signaling and transcription stress. The enzyme topoisomerase 2β (TOP2B) mediating DNA processing, plays an important role in DNA cleavage. The aim of this study was to analyse expression of AR, TOP2B and ERG in relation to TMPRSS2-ERG gene rearrangement and relevant clinicopathological characteristics in prostate cancer (CaP). Immunohistochemical staining and FISH were used for investigation. ERG expression in prostate cell lesions positively correlated with levels of TMPRSS2-ERG fusion gene (p<0.0001). The most significant co-expression of ERG was found with AR in CaP (p=0.001). Significantly more frequent co-expression of ERG was also revealed with TOP2B (p=0.028). ERG protein expression did not correlate with CaP differentiation status as we found no significant differences in ERG expression for different Gleason categories. We demonstrated a statistically significant positive correlation between the percentage of cells with fusion gene TMPRSS2-ERG in CaP and metastatic potential of tumors (p=0.011). Besides these positive corelations of AR with ERG (p=0.001) and TOP2B with ERG (p=0.028), we also demonstrated a significant co-expression of AR with TOP2B (p=0.007) in CaP. There was a statistically significant increase in the TOP2B H-index in locally advanced CaP in comparison with localized tumors (p=0.046). ERG expression correlates with occurrence of TMPRSS2-ERG fusion and with AR-driven malignant transformation. The results indicate that detection of the TMPRSS2-ERG fusion gene and parallel immunohistochemical examination of AR, TOP2B and ERG has diagnostic significance and may be useful in assessing the biological character of the prostate cancer as well as selecting the best treatment.