Publications by authors named "Nicolas Wernert"

75 Publications

Co-staining of microRNAs and their target proteins by miRNA in situ hybridization and immunohistofluorescence on prostate cancer tissue microarrays.

Lab Invest 2019 10 11;99(10):1527-1534. Epub 2019 Jun 11.

Department of Urology and Pediatric Urology, University Hospital Erlangen, FAU Erlangen-Nürnberg, Erlangen, Germany.

The co-expression of miRNAs and their target proteins was studied on tissue microarrays from different prostate cancer (PCa) patients. PCa of primary Gleason pattern 4 (GP4), lymph node metastases of GP4, distant metastases, and normal tissue from the transitional and peripheral zones were co-stained by fluorescent miRNA in situ hybridization (miRisH) and protein immunohistofluorescence (IHF). The miRNAs and corresponding target proteins include the pairs miR-145/ERG, miR-143/uPAR, and miR-375/SEC23A. The fluorescence-stained and scanned tissue microarrays (TMAs) were evaluated by experienced uropathologists. The pair miR-145/ERG showed an exclusive staining for miR-145 in the nuclei of stromal cells, both in tumor and normal tissue, and for ERG in the cytoplasm with/without co-expression in the nucleus of tumor cells. The pair miR-143/uPAR revealed a clear distinction between miR-143 in the nuclei of stromal cells and uPAR staining in the cytoplasm of tumor cells. Metastases (lymph node and distant) however, showed tumor cells with cytoplasmic staining for miR-143/uPAR. In normal tissues, beside the nuclei of the stroma cells, gland cells could also express miR-143 and uPAR in the cytoplasm. miR-375 showed particular staining in the nucleoli of GP4 and metastatic samples, suggesting that nucleoli play a special role in sequestering proteins and miRNAs. Combined miRisH/IHF allows for the study of miRNA expression patterns and their target proteins at the single-cell level.
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http://dx.doi.org/10.1038/s41374-019-0251-8DOI Listing
October 2019

Ets-1 drives breast cancer cell angiogenic potential and interactions between breast cancer and endothelial cells.

Int J Oncol 2019 Jan 24;54(1):29-40. Epub 2018 Oct 24.

UMR8161 CNRS/University of Lille/Pasteur Institute of Lille, Biology Institute of Lille, F-59021 Lille CEDEX, France.

Ets-1 transcription factor overexpression in breast cancers is associated with invasive features and is associated with a poor prognosis. Beyond its role in driving carcinoma cell invasion, in this study, we wished to determine whether Ets-1 overexpression in cancer cells promotes angiogenesis by creating a paracrine pro-invasive environment for endothelial cells as well. To address this question, we set up different co-culture models of cancer cells with endothelial cells. Conditioned media from cancer cells induced endothelial cell proliferation, migration and morphogenesis in matrix models. Of note, co-culture assays in three-dimensional matrix models also revealed the reciprocal induction of cancer cell morphogenesis by endothelial cells, in support of an angiocrine action on tumor cells. Ets-1 emerged as a key regulator of the angiogenic potential of breast cancer cells, favoring their ability to induce, in a paracrine manner, the morphogenesis of endothelial cells and also to physically interact with the latter. Nevertheless, Ets-1 overexpression in cancer cells also restrained their chemoattractive potential for endothelial cells both in Boyden chambers and in ex vivo 3D co-cultures. Finally, Ets-1 modulation in breast cancer cells qualitatively altered the angiogenic pattern of experimental in vivo tumors, with a balance between vessel recruitment and intratumoral small capillaries sprouting. Taken together, our data highlight a critical and intriguing role for Ets-1 in the angiogenic potential of breast cancer cells, and reveal another facet of Ets-1 oncogenic activities.
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http://dx.doi.org/10.3892/ijo.2018.4605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6254994PMC
January 2019

Melanoma Adjuvant Treatment: Current Insight and Clinical Features.

Curr Cancer Drug Targets 2018 ;18(5):442-456

Department of Onco-Ematology Medical Oncology, S.G. Moscati Hospital of Taranto, Taranto, Italy.

Melanoma represents 2-3% of all cancers, 95% of them arise from skin, while only 5% are non-cutaneous melanoma. Despite an optimal surgery management, the risk of a local and systemic relapse remains high, particularly in high-risk patients (node-positive or node-negative T3b, T4 a/b). We conducted a systematic review of the main published and ongoing phase I/II/III trials between 2000 and June 2015 on the adjuvant treatment of cutaneous melanoma. The IFN remains the only option currently available for this aim. Ipilimumab represents a possible breakthrough in this setting, considering the positive results of the EORTC 18701 trials in terms of disease free survival (DFS), while data regarding OS are pending. Recent advances in the understanding of the biology of melanoma result in the identification of MAPK pathway role in the melanoma development. Based on these features, B-RAF inhibitors and their combination with immunotherapy could represent the upcoming therapeutic strategy.
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http://dx.doi.org/10.2174/1568009617666170208163714DOI Listing
August 2019

Defective DNA single-strand break repair is responsible for senescence and neoplastic escape of epithelial cells.

Nat Commun 2016 Jan 29;7:10399. Epub 2016 Jan 29.

Univ. Lille, CNRS, Institut Pasteur de Lille, UMR 8161 - M3T - Mechanisms of Tumorigenesis and Targeted Therapies, F-59000 Lille, France.

The main characteristic of senescence is its stability which relies on the persistence of DNA damage. We show that unlike fibroblasts, senescent epithelial cells do not activate an ATM-or ATR-dependent DNA damage response (DDR), but accumulate oxidative-stress-induced DNA single-strand breaks (SSBs). These breaks remain unrepaired because of a decrease in PARP1 expression and activity. This leads to the formation of abnormally large and persistent XRCC1 foci that engage a signalling cascade involving the p38MAPK and leading to p16 upregulation and cell cycle arrest. Importantly, the default in SSB repair also leads to the emergence of post-senescent transformed and mutated precancerous cells. In human-aged skin, XRCC1 foci accumulate in the epidermal cells in correlation with a decline of PARP1, whereas DDR foci accumulate mainly in dermal fibroblasts. These findings point SSBs as a DNA damage encountered by epithelial cells with aging which could fuel the very first steps of carcinogenesis.
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http://dx.doi.org/10.1038/ncomms10399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4740115PMC
January 2016

Prognostic relevance of proliferation markers (Ki-67, PHH3) within the cross-relation of ERG translocation and androgen receptor expression in prostate cancer.

Pathology 2015 Dec;47(7):629-36

1Institute of Pathology, University of Bonn, Germany 2Institute of Pathology, University of Bern, Switzerland 3Institute of Pathology, Section for Prostate Cancer Research, University of Bonn 4Department of Urology, Charité - Universitätsmedizin Berlin, Berlin 5Berlin Institute for Urologic Research, Berlin 6Institute of Pathology, Charité - Universitätsmedizin Berlin, Berlin, Germany.

We evaluated the prognostic value of the mitosis-associated marker phosphorylated histone H3 (PHH3) and Ki-67 in prostate cancer with respect to ERG status and androgen receptor (AR) expression.PHH3 and Ki-67 expression was immunohistochemically detected and digitally quantitated in a radical prostatectomy cohort (n = 640). The results were correlated to clinicopathological parameters including biochemical recurrence times. Prognostic values of PHH3 and Ki-67 were analysed by Cox regression and Kaplan-Meier statistics.In prostate cancer, mean Ki-67 and PHH3 rates were 3.40% (95%CI 3.16-3.63%) and 0.0152% (95%CI 0.0112-0.0191%), respectively.Ki-67 showed a significant correlation with Gleason scores, pT status, margin status, and AR expression, while PHH3 showed a significant correlation with Gleason scores and pT status. Univariate analyses for biochemical recurrence times demonstrated a significant prognostic value for median Ki-67 rate and for the PHH3 rate of the 90th percentile. Of importance, in patient subgroups stratified according to AR expression and ERG translocation, the prognostic power of proliferation markers PHH3 and Ki-67 was markedly enhanced in ERG translocation negative and high-level AR expressing ERG translocation positive prostate cancers.As expected, the proliferation markers PHH3 and Ki-67 predict adverse outcome of prostate cancer and have a particularly pronounced prognostic value in specific molecular subsets of prostate cancer (ERG- or AR+).
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http://dx.doi.org/10.1097/PAT.0000000000000320DOI Listing
December 2015

Ets-1 controls breast cancer cell balance between invasion and growth.

Int J Cancer 2014 Nov 5;135(10):2317-28. Epub 2014 May 5.

UMR-8161 CNRS/Université Lille-1/Université Lille-2/Institut Pasteur de Lille, Institut de Biologie de Lille, BP 447, F-59021, Lille Cedex, France.

Ets-1 overexpression in human breast cancers is associated with invasiveness and poor prognosis. By overexpressing Ets-1 or a dominant negative mutant in MMT breast cancer cells, we previously highlighted the key role of Ets-1 in coordinating multiple invasive features of these cells. Interestingly, we also noticed that Ets-1 decreased the density of breast cancer cells cultured in three-dimensional extracellular matrix gels. The 3D context was instrumental to this phenomenon, as such downregulation was not observed in cells grown on two-dimensional plastic or matrix-coated dishes. Ets-1 overexpression was deleterious to anchorage-independent growth of MMT cells in soft agar, a standard model for in vitro tumorigenicity. The relevance of this mechanism was confirmed in vivo, during primary tumor growth and in a metastatic assay of lung colonization. In these models, Ets-1 was associated with epithelial-to-mesenchymal transition features and modulated the ratio of Ki67-positive cells, while hardly affecting in vivo apoptotic cell death. Finally, siRNA-mediated knockdown of Ets-1 in human breast cancer cell lines also decreased colony growth, both in anchorage-independent assays and 3D extracellular matrix cultures. These in vitro and in vivo observations shed light on an unsuspected facet of Ets-1 in breast tumorigenesis. They show that while promoting malignancy through the acquisition of invasive features, Ets-1 also attenuates breast tumor cell growth and could therefore repress the growth of primary tumors and metastases. This work also demonstrates that 3D models may reveal mechanisms of tumor biology that are cryptic in standard 2D models.
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http://dx.doi.org/10.1002/ijc.28881DOI Listing
November 2014

MED15, encoding a subunit of the mediator complex, is overexpressed at high frequency in castration-resistant prostate cancer.

Int J Cancer 2014 Jul 9;135(1):19-26. Epub 2013 Dec 9.

Department of Prostate Cancer Research, University Hospital of Bonn, Bonn, Germany; Institute of Pathology, University Hospital of Bonn, Bonn, Germany.

The mediator complex is an evolutionary conserved key regulator of transcription of protein-coding genes and an integrative hub for diverse signaling pathways. In this study, we investigated whether the mediator subunit MED15 is implicated in castration-resistant prostate cancer (CRPC). MED15 expression and copy number/rearrangement status were assessed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), respectively on 718 prostate cancer (PCa) specimens and sequenced by Sanger on a subset. Furthermore, SMAD3 phosphorylation, androgen receptor (AR) and proliferation markers were evaluated by IHC. In PCa cells, siRNA/shRNA knockdown of MED15 was followed by proliferation assays with/without dihydrotestosterone (DHT), and treatments with recombinant TGF-β3. Our results show that MED15 is overexpressed in 76% of distant metastatic CRPC (CRPC(MET) ) and 70% of local-recurrent CRPC (CRPC(LOC) ), in contrast to low frequencies in androgen-sensitive PCa, and no expression in benign prostatic tissue. Furthermore, MED15 overexpression correlates with worse clinical outcome thus defining a highly lethal phenotype. Moreover, TGF-β signaling activation associates with MED15 overexpression in PCa tissues, and leads to increased expression of MED15 in PCa cells. MED15 knockdown effects phosphorylation and shuttling of p-SMAD3 to the nucleus as well as TGF-β-enhanced proliferation. In PCa tissues, MED15 overexpression associates with AR overexpression/amplification and correlates with high proliferative activity. MED15 knockdown decreases both androgen-dependent and -independent proliferation in PCa cells. Taken together, these findings implicate MED15 in CRPC, and as MED15 is evolutionary conserved, it is likely to emerge as a lethal phenotype in other therapeutic-resistant diseases, and not restricted to our disease model.
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http://dx.doi.org/10.1002/ijc.28647DOI Listing
July 2014

Somatic copy number alterations by whole-exome sequencing implicates YWHAZ and PTK2 in castration-resistant prostate cancer.

J Pathol 2013 Dec;231(4):505-16

Department of Prostate Cancer Research, University Hospital of Bonn, Germany; Institute of Pathology, University Hospital of Bonn, Germany.

Castration-resistant prostate cancer (CRPC) is the most aggressive form of prostate cancer (PCa) and remains a significant therapeutic challenge. The key to the development of novel therapeutic targets for CRPC is to decipher the molecular alterations underlying this lethal disease. The aim of our study was to identify therapeutic targets for CRPC by assessing somatic copy number alterations (SCNAs) by whole-exome sequencing on five CRPC/normal paired formalin-fixed paraffin-embedded (FFPE) samples, using the SOLiD4 next-generation sequencing (NGS) platform. Data were validated using fluorescence in situ hybridization (FISH) on a PCa progression cohort. PTK2 and YWHAZ amplification, mRNA and protein expression were determined in selected PCa cell lines. Effects of PTK2 inhibition using TAE226 inhibitor and YWHAZ knock-down on cell proliferation and migration were tested in PC3 cells in vitro. In a larger validation cohort, the amplification frequency of YWHAZ was 3% in localized PCa and 48% in CRPC, whereas PTK2 was amplified in 1% of localized PCa and 35% in CRPC. YWHAZ knock-down and PTK2 inhibition significantly affected cell proliferation and migration in the PC3 cells. Our findings suggest that inhibition of YWHAZ and PTK2 could delay the progression of the disease in CRPC patients harbouring amplification of the latter genes. Furthermore, our validated whole-exome sequencing data show that FFPE tissue could be a promising alternative for SCNA screening using next-generation sequencing technologies.
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http://dx.doi.org/10.1002/path.4274DOI Listing
December 2013

Prognostic significance of phospho-histone H3 in prostate carcinoma.

World J Urol 2014 Jun 26;32(3):703-7. Epub 2013 Jul 26.

Institute of Pathology, University Hospital Bonn, Sigmund-Freud-Strasse 25, 53127, Bonn, Germany.

Purpose: Prostate cancer is the second most common cancer in men and the sixth most common cause of death from cancer in men worldwide. Currently, a sufficient pathological distinction between patients requiring further treatment and those for which active surveillance remains an option is still lacking, which leads to the problem of overtreatment. Cell proliferation is routinely assessed by detecting Ki-67 antigen. While Ki-67 is expressed throughout the interphase of proliferating cells, phosphorylation of the chromatin constituent histone H3 occurs only during the late G2 phase and mitosis thus providing a more strict assessment of the mitotic activity. We undertook this study to test whether expression of the recently introduced proliferation marker phospho-histone H3 (pHH3) in prostate carcinoma tissue sections exhibits prognostic significance in comparison with Ki-67.

Methods: Protein expression of pHH3 and Ki-67 was assessed on TMA consisting of paraffin-embedded tissue from men that had undergone radical prostatectomy. The analysis included triplicate tissue cores of a total of 339 tumor foci. Immunohistochemical staining of pHH3 and Ki-67 was performed and analyzed using Definiens imaging software.

Results: Prostate cancer tissue exhibited a significantly higher frequency of pHH3-positive cells compared to benign prostate tissue. pHH3 expression was significantly correlated with Ki-67 expression. Furthermore, statistical analysis revealed positive correlation between pHH3 expression and PSA levels at diagnosis and in addition negatively correlated with overall survival. In contrast to Ki-67 staining, pHH3 expression did not correlate with Gleason grade.

Conclusion: Our data point to a conceivable role of pHH3 as prognostic biomarker in prostate carcinoma.
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http://dx.doi.org/10.1007/s00345-013-1135-yDOI Listing
June 2014

The imbalance between proliferation and apoptosis contributes to degeneration of aortic valves and bioprostheses.

Cardiol J 2013 ;20(3):268-76

Department of Internal Medicine II - Cardiology, Angiology and Pulmonology, University of Bonn, Germany.

Background: The pathomechanisms underlying aortic valve degeneration are incompletely understood. Therefore, the aim of our work was to assess the quantitative changes of proliferation and apoptosis accompanied by cellular phenotype alternations and matrix secretionin aortic sclerotic and stenotic valves and degenerative bioprostheses, as well as to detect the expression pattern of the rapamycin receptor FKBP12 across these three valve types.

Methods: Mild-to-moderate sclerotic and stenotic valves and degenerative bioprostheses from 30 patients (n = 10 per group) were collected at autopsy or by surgery. Ki67+, FKBP12+, alpha-actin+, HSP47+ and TUNEL+ apoptotic cells were analyzed by immunohistochemistry.

Results: The main finding was the reduced proliferation and increased apoptosis in stenotic valves (ST) compared to the sclerotic ones (SC) (proliferation: ST: 20.8 ± 2.0% vs. SC: 30.1 ±2.2%, apoptosis: ST: 40.7 ± 5.0% vs. SC: 28.0 ± 5.1%, p < 0.05, respectively). Analogical alternations were found in degenerative bioprostheses (BP) (proliferation: 4.8 ± 2.3%; apoptosis: 13.1 ± 6.8%). Corresponding changes were observed in the valve cellularity (ST: 893 ± 168, SC: 1034 ± 284, BP: 385 ± 179 cells/mm2, p < 0.05, respectively). The FKBP12 signaling was reduced in diseased valves and bioprostheses (ST: 28.1 ± 3.6%, SC: 42.2 ± 3.8%, BP: 5.8 ± 1.9%, p < 0.05, respectively). Further, the augmented alpha-actin expressionwas observed as the degenerative process progressed (ST: 30.3 ± 5.0%, SC: 22.6 ± 2.7%, BP:8.7 ± 4.0%, p < 0.05, respectively), followed by the upregulation of HSP47 (ST: 22.6 ± 2.8%,SC: 15.4 ± 2.1%, BP: 3.4 ± 1.0%, p < 0.05, respectively) and consecutive matrix accumulation.

Conclusions: The imbalance between proliferation and apoptosis with cellular phenotypical shift and subsequent matrix secretion may contribute to aortic valve stenosis and bioprosthesis degeneration. The identification of FKBP12 expression may implicate potential therapeutic strategies.
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http://dx.doi.org/10.5603/CJ.2013.0072DOI Listing
August 2014

Senescent fibroblasts enhance early skin carcinogenic events via a paracrine MMP-PAR-1 axis.

PLoS One 2013 10;8(5):e63607. Epub 2013 May 10.

Unité Mixte de Recherche UMR 8161, Centre National de la Recherche Scientifique, CNRS, Institut de Biologie de Lille, Lille, France.

The incidence of carcinoma increases greatly with aging, but the cellular and molecular mechanisms underlying this correlation are only partly known. It is established that senescent fibroblasts promote the malignant progression of already-transformed cells through secretion of inflammatory mediators. We investigated here whether the senescent fibroblast secretome might have an impact on the very first stages of carcinogenesis. We chose the cultured normal primary human epidermal keratinocyte model, because after these cells reach the senescence plateau, cells with transformed and tumorigenic properties systematically and spontaneously emerge from the plateau. In the presence of medium conditioned by autologous senescent dermal fibroblasts, a higher frequency of post-senescence emergence was observed and the post-senescence emergent cells showed enhanced migratory properties and a more marked epithelial-mesenchymal transition. Using pharmacological inhibitors, siRNAs, and blocking antibodies, we demonstrated that the MMP-1 and MMP-2 matrix metalloproteinases, known to participate in late stages of cancer invasion and metastasis, are responsible for this enhancement of early migratory capacity. We present evidence that MMPs act by activating the protease-activated receptor 1 (PAR-1), whose expression is specifically increased in post-senescence emergent keratinocytes. The physiopathological relevance of these results was tested by analyzing MMP activity and PAR-1 expression in skin sections. Both were higher in skin sections from aged subjects than in ones from young subjects. Altogether, our results suggest that during aging, the dermal and epidermal skin compartments might be activated coordinately for initiation of skin carcinoma, via a paracrine axis in which MMPs secreted by senescent fibroblasts promote very early epithelial-mesenchymal transition of keratinocytes undergoing transformation and oversynthesizing the MMP-activatable receptor PAR-1.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0063607PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3651095PMC
December 2013

Comparison of p40 (ΔNp63) and p63 expression in prostate tissues--which one is the superior diagnostic marker for basal cells?

Histopathology 2013 Jul 13;63(1):50-6. Epub 2013 May 13.

Institute of Pathology, University Hospital of Bonn, Bonn, Germany.

Aims: p63 is one of the standard markers for basal cells of the prostate gland. Recently, it has been suggested that the p63 isoform p40 might be more specific as a basal cell marker. In this study we compare the staining characteristics of p63 and p40 in normal and malignant prostate tissues.

Methods And Results: A prostatectomy cohort (n = 640) in tissue microarray format was evaluated for p63 (clone 4A4) and for p40 (rabbit polyclonal) immunoreactivity in malignant and normal tissues. Immunoreactivity of basal and secretory cells was evaluated in a semiquantitative manner and compared case-wise. In normal tissues, p40 showed highly similar immunoreactivity compared to p63. The staining patterns were absolutely identical in 88% of cases. Additional cytoplasmic p40 staining in tumour cells occurred in 59.6% of cancer cases. Differences were seen in nuclear staining of carcinomas: 1.4% of carcinomas were p63-positive, whereas 0.6% were p40-positive.

Conclusions: In most cases, p40 stains prostatic basal cells as reliably as p63, with only minor differences. Aberrant staining of tumour cells is seen more rarely than with p63 (clone 4A4), establishing its higher specificity. However, additional cytoplasmic immunoreactivity narrows its eligibility for antibody cocktails (e.g. with alpha-methylacyl-CoA racemase).
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http://dx.doi.org/10.1111/his.12116DOI Listing
July 2013

Landscape of chromosome number changes in prostate cancer progression.

World J Urol 2013 Dec 20;31(6):1489-95. Epub 2013 Mar 20.

Institute of Pathology, Department of Prostate Cancer Research, University Hospital of Bonn, Sigmund-Freud-Straße 25, 53127, Bonn, Germany.

Purpose: Both genetic instability resulting in aneuploidy and increased proliferative activity are common features of tumor development and progression. Cytometric evaluation of tumor ploidy status was recently suggested as a prognostic marker. However, in prostate cancer (PCa), a chromosome-specific evaluation is lacking. With the present study, we sought to identify distinct chromosomal changes to complement cytometric results concerning the diagnosis and prognosis of PCa patients.

Methods: We assessed a cohort of 428 PCa specimens (186 localized PCa, 75 lymph node metastasized PCa, 125 lymph node metastases, 42 hormone-refractory distant metastases) for numerical alterations of all 24 chromosomes by using fluorescence in situ hybridization (FISH). Conducting immunohistochemistry with phosphorylated histone H3 (PHH3) and Ki-67, we quantified the proliferation rate. FISH results were fit in a linear model and tested for predictive power.

Results: As expected, we observed a significant increase in aneuploidy with advancing tumor stage. Similarly, an increased expression of the mitotic marker PHH3 was significantly associated with aneuploidy and higher pT-stage. We found aneusomy of chromosomes 4, 6, 20, and X to be indicative of lymph node metastasized PCa. However, with an AUC of 65%, this set of chromosomal changes was poorly suited to distinguish non-metastasized and lymph node metastasized primary tumors.

Conclusion: Our results provide thorough insight into the so far incompletely elucidated chromosomal landscape of PCa. While overall ploidy status and PHH3 expression in primary tumors indicate advanced disease, a FISH-based test for distinct alterations does not seem to be beneficial for diagnostic or prognostic decisions.
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http://dx.doi.org/10.1007/s00345-013-1051-1DOI Listing
December 2013

The heterogeneous Gleason 7 carcinoma of the prostate: analyses of low and high grade (risk) carcinomas with criteria of the International Society of Urological Pathology (ISUP).

Pathol Res Pract 2013 Mar 15;209(3):190-4. Epub 2013 Feb 15.

Department of Pathology, HBH Hospital, Singen, Germany.

Prostate carcinoma (PCa) with Gleason score (GS) 7 has to be examined differentially regarding its prognosis. Using the criteria of ISUP and supplementations, we attempted to analyze the heterogeneity of PCa with GS 7 of biopsy and corresponding specimens of radical prostatectomies (RP). PCa of 530 patients were graded according to Gleason under additional consideration of the state of the nucleoli. Investigating the biopsy specimens, we determined the pattern of Gleason 4 of GS 7, the extension of the tumors in percent, and the number of biopsies containing tumor. In the corresponding specimens of RP, the grading and the state of TNM with margins were assessed. Carcinomas with GS 7 (4+3) in biopsy and RP specimens were unequivocally assigned to the group of high-grade tumors. Carcinomas with GS 7 (3+4) were significantly different from carcinomas with GS 6 when only few and small nucleoli in GS 6 were present (low grade type, p≤0.0001), but were similar to the GS 6 group when nucleoli were prominent (intermediary type, p=0.71). The intermediary group showed an upgrading rate of 36% from GS 6 to GS 7. Furthermore the correlation between organ-confined and non-organ-confined growth showed differences of 63% and 37% in the intermediary group (p=0.0001). The values of grading, staging, margins and metastases indicate that carcinomas of the prostate with the Gleason 3+4 pattern correspond to an intermediary group of carcinomas in contrast to high-grade (high risk) carcinomas with GS 7 and pattern 4+3.
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http://dx.doi.org/10.1016/j.prp.2012.10.016DOI Listing
March 2013

Quantification of protein expression in cells and cellular subcompartments on immunohistochemical sections using a computer supported image analysis system.

Histol Histopathol 2013 05 30;28(5):605-10. Epub 2013 Jan 30.

Institute of Pathology, and Department of Prostate Cancer Research, University Hospital of Bonn, Bonn, Germany.

Quantification of protein expression based on immunohistochemistry (IHC) is an important step for translational research and clinical routine. Several manual ('eyeballing') scoring systems are used in order to semi-quantify protein expression based on chromogenic intensities and distribution patterns. However, manual scoring systems are time-consuming and subject to significant intra- and interobserver variability. The aim of our study was to explore, whether new image analysis software proves to be sufficient as an alternative tool to quantify protein expression. For IHC experiments, one nucleus specific marker (i.e., ERG antibody), one cytoplasmic specific marker (i.e., SLC45A3 antibody), and one marker expressed in both compartments (i.e., TMPRSS2 antibody) were chosen. Stainings were applied on TMAs, containing tumor material of 630 prostate cancer patients. A pathologist visually quantified all IHC stainings in a blinded manner, applying a four-step scoring system. For digital quantification, image analysis software (Tissue Studio v.2.1, Definiens AG, Munich, Germany) was applied to obtain a continuous spectrum of average staining intensity. For each of the three antibodies we found a strong correlation of the manual protein expression score and the score of the image analysis software. Spearman's rank correlation coefficient was 0.94, 0.92, and 0.90 for ERG, SLC45A3, and TMPRSS2, respectively (p⟨0.01). Our data suggest that the image analysis software Tissue Studio is a powerful tool for quantification of protein expression in IHC stainings. Further, since the digital analysis is precise and reproducible, computer supported protein quantification might help to overcome intra- and interobserver variability and increase objectivity of IHC based protein assessment.
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http://dx.doi.org/10.14670/HH-28.605DOI Listing
May 2013

Development and clinical validation of a real-time PCR assay for PITX2 DNA methylation to predict prostate-specific antigen recurrence in prostate cancer patients following radical prostatectomy.

J Mol Diagn 2013 Mar 22;15(2):270-9. Epub 2012 Dec 22.

Institute of Pathology, University Hospital Bonn, Bonn, Germany.

Prostate cancer is the most common cancer among men. The prospective discrimination of aggressive and clinically insignificant tumors still poses a significant and, as yet, unsolved problem. PITX2 DNA methylation is a strong prognostic biomarker in prostate cancer. Recently, a diagnostic microarray for prostate cancer prognosis based on PITX2 methylation has been developed and validated. Because this microarray requires nonstandard laboratory equipment, its use in a diagnostic setting is limited. This study aimed to develop and validate an alternative quantitative real-time PCR assay for measuring PITX2 methylation that can easily be established in clinical laboratories, thereby facilitating the implementation of this biomarker in clinical practice. A methylation cut-off for patient stratification was established in a training cohort (n = 157) and validated in an independent test set (n = 523) of men treated with radical prostatectomy. In univariate Cox proportional hazards analysis, PITX2 hypermethylation was a significant predictor for biochemical recurrence (P < 0.001, hazard ratio = 2.614). Moreover, PITX2 hypermethylation added significant prognostic information (P = 0.003, hazard ratio = 1.814) to the Gleason score, pathological T stage, prostate-specific antigen, and surgical margins in a multivariate analysis. The clinical performance was particularly high in patients at intermediate risk (Gleason score of 7) and in samples containing high tumor cell content. This assay might aid in risk stratification and support the decision-making process when determining whether a patient might benefit from adjuvant treatment after radical prostatectomy.
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http://dx.doi.org/10.1016/j.jmoldx.2012.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5707187PMC
March 2013

Epigenetics-related genes in prostate cancer: expression profile in prostate cancer tissues, androgen-sensitive and -insensitive cell lines.

Int J Mol Med 2013 Jan 6;31(1):21-5. Epub 2012 Nov 6.

Institute of Pathology, University Hospital Bonn, D-53127 Bonn, Germany.

Epigenetic changes have been suggested to drive prostate cancer (PCa) development and progression. Therefore, in this study, we aimed to identify novel epigenetics-related genes in PCa tissues, and to examine their expression in metastatic PCa cell lines. We analyzed the expression of epigenetics-related genes via a clustering analysis based on gene function in moderately and poorly differentiated PCa glands compared to normal glands of the peripheral zone (prostate proper) from PCa patients using Whole Human Genome Oligo Microarrays. Our analysis identified 12 epigenetics-related genes with a more than 2-fold increase or decrease in expression and a p-value <0.01. In modera-tely differentiated tumors compared to normal glands of the peripheral zone, we found the genes, TDRD1, IGF2, DICER1, ADARB1, HILS1, GLMN and TRIM27, to be upregulated, whereas TNRC6A and DGCR8 were found to be downregulated. In poorly differentiated tumors, we found TDRD1, ADARB and RBM3 to be upregulated, whereas DGCR8, PIWIL2 and BC069781 were downregulated. Our analysis of the expression level for each gene in the metastatic androgen-sensitive VCaP and LNCaP, and -insensitive PC3 and DU-145 PCa cell lines revealed differences in expression among the cell lines which may reflect the different biological properties of each cell line, and the potential role of each gene at different metastatic sites. The novel epigenetics-related genes that we identified in primary PCa tissues may provide further insight into the role that epigenetic changes play in PCa. Moreover, some of the genes that we identified may play important roles in primary PCa and metastasis, in primary PCa only, or in metastasis only. Follow-up studies are required to investigate the functional role and the role that the expression of these genes play in the outcome and progression of PCa using tissue microarrays.
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http://dx.doi.org/10.3892/ijmm.2012.1173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3573772PMC
January 2013

Significance of Gleason grading of low-grade carcinoma of the prostate with therapeutic option of active surveillance.

Urol Int 2013 24;90(1):17-23. Epub 2012 Oct 24.

Institute of Pathology, HB Hospital Singen, Singen, Germany. burkhard.helpap @ hbh-kliniken.de

Introduction: Active surveillance needs a precise grading diagnosis of a low-grade carcinoma of the prostate (Gleason score (GS) 6) within a small organ-confined tumor. However, how accurate is the gold standard of GS 6 in predicting a small pT2 carcinoma? To answer this question, we have analyzed grading systems in this study.

Methods: Prostatic carcinomas in biopsy and corresponding radical prostatectomy (RP) specimens of 960 patients were graded by the Gleason system in which glandular fusions and nucleolar stage (prominence and location) were considered.

Results: Using the modified Gleason grading, a high upgrading rate from the biopsy to RP specimens (GS 6-7) and in even 30% a non-organ-confined growth pattern (pT3) of GS 6 carcinoma in RP was found. When considering glandular fusion and the incorporation of the state of nucleoli within the Gleason grading, the agreement of score 6 between biopsy and RP specimens as well as the prediction of a pT2a tumor increased from about 80 to 90%.

Conclusion: The combination of Gleason grading and grading of the nuclear and nucleolar features may help to identify patients eligible for active surveillance.
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http://dx.doi.org/10.1159/000342810DOI Listing
July 2013

ERG is specifically associated with ETS-2 and ETV-4, but not with ETS-1, in prostate cancer.

Int J Mol Med 2012 Nov 20;30(5):1029-33. Epub 2012 Aug 20.

Institute of Pathology, University Hospital of Bonn, D-53127 Bonn, Germany.

The erythroblast transformation-specific (ETS) family of transcription factors plays important roles in both physiological and pathological conditions. Even though many studies have focused on single ETS factors within a single tissue and within the context of specific promoters, the functional impact of multiple ETS members present within a specific cell type has not yet been investigated, especially in prostate cancer (PCa). As the most prominent gene rearrangement in PCa leads to the overexpression of the ETS-related gene (ERG), the aim of this study was to investigate whether ERG is part of a complex integrated transcriptional network that involves other ETS factors. More specifically, as the ETS family consists of 27 members, we focused our efforts initially on investigating whether ERG is associated with the three family members, ETS-1, ETS-2 and ETS variant gene‑4 (ETV‑4), in PCa as a proof of principle. Using western blot analysis, we show that ERG, ETS-1, ETS-2 and ETV-4 are expressed in PC3 cell nuclear extracts and in protein lysates prepared from human PCa prostatectomy specimens. Immunoprecipitations using an anti-ERG antibody were used with PC3 cell nuclear extracts as well as with a pooled protein lysate sample prepared from the PCa tissue samples of five patients. Importantly, our results revealed that ERG is specifically associated with ETS-2 and ETV-4, but not with ETS-1, in PC3 cell nuclear extracts and PCa tissue protein lysates. Our findings strongly support the notion that ERG is part of a complex integrated transcriptional network that involves other ETS factors, which are likely to cooperate or influence the activity of ERG in PCa. The functional impact of multiple ETS factors being associated with ERG in PCa requires further study, as it may provide insights into the mechanism by which ERG exerts its influence in PCa and may subsequently contribute to our understanding of the molecular basis of PCa.
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http://dx.doi.org/10.3892/ijmm.2012.1097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3572757PMC
November 2012

Loss of SLC45A3 protein (prostein) expression in prostate cancer is associated with SLC45A3-ERG gene rearrangement and an unfavorable clinical course.

Int J Cancer 2013 Feb 30;132(4):807-12. Epub 2012 Aug 30.

Institute of Pathology, University Hospital of Bonn, Bonn, Germany.

The majority of prostate cancer harbors recurrent gene fusions involving ETS transcription factors, most commonly ERG. The second most common 5' fusion partner after TMPRSS2 is SLC45A3. The aim of our study was to quantify the protein expression of ERG, TMPRSS2 and SLC45A3 in prostate cancer to assess for diagnostic or prognostic utility. Six hundred and forty consecutive prostate cancer cases in tissue microarray format were immunohistochemically analyzed for ERG, TMPRSS2 and SLC45A3 protein. Resultant protein expression data was correlated to the respective gene rearrangement status and clinico-pathological parameters including PSA follow up times. ERG showed no expression in benign prostate glands. In cancer tissue, ERG protein expression showed a high rate of concordance with an underlying ERG rearrangement (91.5%). SLC45A3 showed a weaker expression in cancer as compared to benign tissue, which was pronounced in cases with SLC45A3-ERG fusion. Importantly, SLC45A3 down regulation was significantly associated with shorter PSA-free survival times. In contrast, TMPRSS2 was neither differentially expressed nor did it show a correlation between protein expression and rearrangement status. This study provides first evidence that the expression of SLC45A3 protein is down regulated through SLC45A3-ERG fusion in prostate cancer. Moreover, these cases may represent a distinct molecular subclass of ERG rearranged prostate cancer with distinct clinical features. This study also confirms that ERG protein expression is predominantly found in prostate carcinomas with ERG gene rearrangement and does not occur in benign glands.
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http://dx.doi.org/10.1002/ijc.27733DOI Listing
February 2013

ERG rearrangement in local recurrences compared to distant metastases of castration-resistant prostate cancer.

Virchows Arch 2012 Aug 6;461(2):157-62. Epub 2012 Jul 6.

Institute of Pathology, University Hospital Tuebingen, Tuebingen, Germany.

Castration-resistant prostate cancer is the second most common cause of cancer death and results in a median survival of less than 2 years. In prostate cancer, fusions between TMPRSS2 and ERG are common. The ERG rearrangement prevalence in local recurrent castration-resistant prostate cancer compared to distant metastatic prostate cancer is unknown. We investigated the frequency of ERG rearrangement in local recurrent castration-resistant prostate cancer compared to distant metastatic prostate cancer, and assessed for associations between androgen receptor (AR) amplification and ERG rearrangement status. Samples from 134 patients diagnosed with prostate cancer (84 local recurrent castration resistant prostate cancer, 55 distant metastatic prostate cancer) were assessed for their ERG rearrangement and AR amplification status by fluorescence in situ hybridization. Statistical analysis was performed using the χ (2) test. We found that the ERG rearrangement occurs at a significantly lower frequency in distant metastatic prostate cancer (25 %) than in local recurrent castration-resistant prostate cancer (45 %). The AR amplification frequencies were 45 and 35 % in local recurrent castration-resistant prostate cancer and distant metastatic prostate cancer, respectively. The ERG rearrangement occurred at a lower frequency in distant metastatic prostate cancer compared to local recurrent castration-resistant prostate cancer.
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http://dx.doi.org/10.1007/s00428-012-1270-7DOI Listing
August 2012

Regulation of prostate cancer immunity-related genes in PC3 prostate cancer cells by ETS-1.

Oncol Lett 2012 Mar 2;3(3):513-516. Epub 2011 Dec 2.

Institute of Pathology, University Hospital Bonn, D-53127 Bonn, Germany.

Prostate cancer (PCa) is one of the most prevalent forms of cancer affecting males worldwide, and knowledge of the immune defenses involved in PCa remains incomplete. Since the identification of immunity-related genes may have enormous implications for the understanding of PCa immunology, we recently reported the identification of immunity-related genes in PCa tissues and found potential binding sites for the ETS family prototype, ETS-1, in the majority of genes identified. Therefore, as a continuation of our previous study, we investigated whether ETS-1 regulates these genes in an in vitro PCa cell line model, PC3 cells. We specifically blocked ETS-1 in PC3 cells by transfection with an ETS-1 inverse antisense expression vector or a mock control vector. We then assessed the effect of the blockade on the expression of the recently identified PCa immunity-related genes using a comprehensive oligo gene expression microarray analysis. The results showed that ETS-1 is involved in the activation or repression of the recently identified immunity-related genes in PCa. These findings provide insights into the regulation of immunity-related genes in PCa, and emphasize the importance of ETS-1 in prostate cancer immunology.
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http://dx.doi.org/10.3892/ol.2011.509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3362418PMC
March 2012

Improved method of detecting the ERG gene rearrangement in prostate cancer using combined dual-color chromogenic and silver in situ hybridization.

J Mol Diagn 2012 Jul 27;14(4):322-7. Epub 2012 May 27.

Institute of Pathology, University Hospital of Bonn, Sigmund-Freud-Straße 25, Bonn, Germany.

The recently detected TMPRSS2-ERG fusion gene was revealed as a recurrent and prevalent prostate cancer (PCa)-specific event, potentially qualifying it for clinical use. To detect this alteration, fluorescence in situ hybridization (FISH) is the method of choice. However, FISH has some disadvantages for widespread adoption in clinical practice. Subsequently, chromogenic in situ hybridization, which uses organic chromogens, and enzymatic metallography silver in situ hybridization have emerged as promising bright-field alternatives. Compared with chromogenic in situ hybridization, silver in situ hybridization signals are very distinct and superior with regard to signal clarity and resolution, but the method excludes multicolor protocols. Based on the ERG break-apart FISH assay, we established a dual-color ERG break-apart assay using combined chromogenic in situ hybridization and silver in situ hybridization (CS-ISH) and compared these results with those obtained by FISH. We assessed 178 PCa and 10 benign specimens for their ERG rearrangement status by applying dual-color FISH and CS-ISH ERG break-apart assays to consecutive sections. We observed a highly significant concordance (97.7%) between FISH- and CS-ISH-based results (Pearson's correlation coefficient = 0.955, P < 0.001). Our findings demonstrate that the ERG rearrangement status can reliably be assessed by CS-ISH. Further, the CS-ISH technique combines the accuracy and precision of FISH with the ease of bright-field microscopy. This tool allows a much broader spectrum of applications in which to study the biological role and clinical use of ERG rearrangements in PCa.
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http://dx.doi.org/10.1016/j.jmoldx.2012.01.017DOI Listing
July 2012

Rearrangement of the ETS genes ETV-1, ETV-4, ETV-5, and ELK-4 is a clonal event during prostate cancer progression.

Hum Pathol 2012 Nov 7;43(11):1910-6. Epub 2012 May 7.

Institute of Pathology, University Hospital Bonn, 53127 Bonn, Germany.

ETS gene rearrangements are frequently found in prostate cancer. Several studies have assessed the rearrangement status of the most commonly found ETS rearranged gene ERG, and the less frequent genes, ETV-1, ETV-4, ETV-5, and ELK-4 in primary prostate cancer. However, frequency in metastatic disease is not well investigated. Recently, we have assessed the ERG rearrangement status in both primary and corresponding lymph node metastases and observed that ERG rearrangement in primary prostate cancer transfers into lymph node metastases, suggesting it to be a clonal expansion event during prostate cancer progression. As a continuation, we investigated in this study whether this observation is valid for the less frequent ETS rearranged genes. Using dual-color break-apart fluorescent in situ hybridization assays, we evaluated the status of all less frequent ETS gene rearrangements for the first time on tissue microarrays constructed from a large cohort of 86 patients with prostate cancer and composed of primary and corresponding lymph node metastases, as well as in a second cohort composed of 43 distant metastases. ETV-1, ETV-4, ETV-5, and ELK-4 rearrangements were found in 8 (10%) of 81, 5 (6%) of 85, 1 (1%) of 85, and 2 (2%) of 86 of primary prostate cancer, respectively, and in 6 (8%) of 73, 4 (6%) of 72, 1 (1%) of 75, and 1 (1%) of 78 of corresponding lymph node metastases, respectively. ETV-1 and ETV-5 rearrangements were not found in the distant metastases cases, whereas ETV-4 and ELK-4 rearrangements were found in 1 (4%) of 25 and 1 (4%) of 24, respectively. Our findings suggest that rearrangement of the less frequent ETS genes is a clonal event during prostate cancer progression.
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http://dx.doi.org/10.1016/j.humpath.2012.01.018DOI Listing
November 2012

Classifying prostate cancer malignancy by quantitative histomorphometry.

J Urol 2012 May 16;187(5):1867-75. Epub 2012 Mar 16.

Institute for Medical Informatics, Statistics, and Epidemiology, University of Leipzig, Leipzig, Germany.

Purpose: Prostate cancer is routinely graded according to the Gleason grading scheme. This scheme is predominantly based on the textural appearance of aberrant glandular structures. Gleason grade is difficult to standardize and often leads to discussion due to interrater and intrarater disagreement. Thus, we investigated whether digital image based automated quantitative histomorphometry could be used to achieve a more standardized, reproducible classification outcome.

Materials And Methods: In a proof of principle study we developed a method to evaluate digitized histological images of single prostate cancer regions in hematoxylin and eosin stained sections. Preprocessed color images were subjected to color deconvolution, followed by the binarization of obtained hematoxylin related image channels. Highlighted neoplastic epithelial gland related objects were morphometrically assessed by a classifier based on 2 calculated quantitative and objective geometric measures, that is inverse solidity and inverse compactness. The procedure was then applied to the prostate cancer probes of 125 patients. Each probe was independently classified for Gleason grade 3, 4 or 5 by an experienced pathologist blinded to image analysis outcome.

Results: Together inverse compactness and inverse solidity were adequate discriminatory features for a powerful classifier that distinguished Gleason grade 3 from grade 4/5 histology. The classifier was robust on sensitivity analysis.

Conclusions: Results suggest that quantitative and interpretable measures can be obtained from image based analysis, permitting algorithmic differentiation of prostate Gleason grades. The method must be validated in a large independent series of specimens.
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http://dx.doi.org/10.1016/j.juro.2011.12.054DOI Listing
May 2012

ETS transcription factors and prostate cancer: the role of the family prototype ETS-1 (review).

Int J Oncol 2012 Jun 21;40(6):1748-54. Epub 2012 Feb 21.

Institute of Pathology, University Hospital Bonn, D-53127 Bonn, Germany.

The ETS family of transcription factors is known to play important roles in various biological processes such as development, differentiation, proliferation, apoptosis, migration, tissue remodeling, invasion and angiogenesis in various cell types including B cells, endothelial cells, fibroblasts as well as diverse neoplastic cells. In prostate cancer, recurrent gene fusions involving members of the ETS family are frequently reported. ETS-1, the prototype of the ETS family, is expressed in different cell types and is known to play various roles during both physiological and pathological conditions. In this review, we focus on studies investigating the role of ETS-1 in prostate cancer.
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http://dx.doi.org/10.3892/ijo.2012.1380DOI Listing
June 2012

Novel identification of the ETS-1 splice variants p42 and p27 in prostate cancer cell lines.

Oncol Rep 2012 May 1;27(5):1321-4. Epub 2012 Feb 1.

Institute of Pathology, University Hospital Bonn, D-53127 Bonn, Germany.

ETS-1 is involved in cellular functions such as proliferation, migration, invasion, apoptosis and angiogenesis. The ETS-1 gene encodes three distinct proteins, ETS-1 p51 encoded by a full-length mRNA, ETS-1 p42 and ETS-1 p27 encoded by an alternatively spliced mRNA lacking exon VII and exons III-VI, respectively. ETS-1 p51, commonly considered to be the active form, has been studied in prostate cancer (PCa). However, the ETS-1 p42 and p27 variants have not yet been identified in PCa. Therefore, we aimed in this study at investigating whether the splice variants p42 and p27 are expressed in the androgen-dependent VCaP and LNCaP and the androgen-independent PC3 and DU-145 PCa cell lines. Using RT-PCR, we found the expression of both splice variants p42 and p27 at the mRNA level in the VCaP, LNCaP, PC3 and DU-145 PCa cell lines. We then confirmed the expression of ETS-1 p51 and its splice variants p42 and p27 at the protein level using an anti-ETS-1 antibody directed against the DNA-binding domain (DBD) in lysates prepared from the latter-mentioned cell lines, as well as in PC3 cell nuclear extract. Moreover, differences in the expression ratios of the ETS-1 splice variants within each cell line were also found. In conclusion, we have demonstrated for the first time the novel identification of the ETS-1 splice variants p42 and p27 in PCa cell lines. It is very likely that the role of ETS-1 p51 in PCa is significantly influenced by the presence of its splice variants ETS-1 p42 and p27 as competition, abundance, affinity, interactions and cross-talk among them will eventually determine the genes that will be targeted and subsequently affect cellular functions. Follow-up studies will need to address in functional terms, the roles of these splice variants in PCa cell lines, as well as their expression in PCa tissues and its correlation with clinical outcome.
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http://dx.doi.org/10.3892/or.2012.1667DOI Listing
May 2012

The peripheral zone of the prostate is more prone to tumor development than the transitional zone: is the ETS family the key?

Mol Med Rep 2012 Feb 27;5(2):313-6. Epub 2011 Oct 27.

Institute of Pathology, University Hospital Bonn, 53127 Bonn, Germany.

Predisposition to develop prostate cancer (PCA) varies among the prostate zones, with the peripheral zone (PZ) more prone to tumor development than the transitional zone (TZ). In view of the fact that molecular differences between the zones may explain this difference, combined with the findings that translocations between TMPRSS2 and several ETS members are frequently observed in PCA, we hypothesized that the ETS family may be crucial to explaining this difference. Normal tissues from the PZ and the TZ of 20 PCA patients were laser microdissected to separate glands from stroma. Two oligo microarrays were performed in order to investigate the variation in ETS family gene expression between the glands and the stroma of the two zones. The ETS members, ELF-3, ELF-5, ERG, ETV-1, ETV-4, ETV-5, ETV-7 and FEV, were found to be differentially expressed. A striking observation was that ERG and ETV-1 were found to be up-regulated in the glands of the PZ compared to the TZ, particularly when considering that ERG and ETV-1 fusions account for 50-80% and 20% of PCA occurrences, respectively. These results indicate that the glands and stroma of the two zones display distinct molecular differences and zonal-specific expression of ETS members. Furthermore, ETS members up-regulated in PCA are already overexpressed in the normal PZ, suggesting that these members play a role in the development and progression of PCA.
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http://dx.doi.org/10.3892/mmr.2011.647DOI Listing
February 2012

Overexpression of hepatoma-derived growth factor in melanocytes does not lead to oncogenic transformation.

BMC Cancer 2011 Oct 20;11:457. Epub 2011 Oct 20.

Institute of Biochemistry and Molecular Biology, University of Bonn, Nussallee 11, 53115 Bonn, Germany.

Background: HDGF is a growth factor which is overexpressed in a wide range of tumors. Importantly, expression levels were identified as a prognostic marker in some types of cancer such as melanoma.

Methods: To investigate the presumed oncogenic/transforming capacity of HDGF, we generated transgenic mice overexpressing HDGF in melanocytes. These mice were bred with mice heterozygous for a defective copy of the Ink4a tumor suppressor gene and were exposed to UV light to increase the risk for tumor development both genetically and physiochemically. Mice were analyzed by immunohistochemistry and Western blotting. Furthermore, primary melanocytes were isolated from different strains created.

Results: Transgenic animals overexpressed HDGF in hair follicle melanocytes. Interestingly, primary melanocytes isolated from transgenic animals were not able to differentiate in vitro whereas cells isolated from wild type and HDGF-deficient animals were. Although, HDGF-/-/Ink4a+/- mice displayed an increased number of epidermoid cysts after exposure to UV light, no melanomas or premelanocytic alterations could be detected in this mouse model.

Conclusions: The results therefore provide no evidence that HDGF has a transforming capacity in tumor development. Our results in combination with previous findings point to a possible role in cell differentiation and suggest that HDGF promotes tumor progression after secondary upregulation and may represent another protein fitting into the concept of non-oncogene addiction of tumor tissue.
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http://dx.doi.org/10.1186/1471-2407-11-457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3213223PMC
October 2011
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