High NRBP1 expression in prostate cancer is linked with poor clinical outcomes and increased cancer cell growth.

BACKGROUND: We recently established the rationale that NRBP1 (nuclear receptor binding protein 1) has a potential growth-promoting role in cell biology. NRBP1 interacts directly with TSC-22, a potential tumor suppressor gene that is differently expressed in prostate cancer. Consequently, we analyzed the role of NRBP1 expression in prostate cancer cell lines and its expression on prostate cancer tissue microarrays (TMA). METHODS: The effect of NRBP1 expression on tumor cell growth was analyzed by using RNAi. NRBP1 protein expression was evaluated on two TMAs containing prostate samples from more than 1,000 patients. Associations with clinico-pathological features, the proliferation marker Ki67 and survival data were analyzed. RESULTS: RNAi mediated silencing of NRBP1 expression in prostate cancer cell lines resulted in reduced cell growth (P < 0.05). TMA analysis revealed NRBP1 protein expression in benign prostate hyperplasia in 6% as compared to 60% in both, high-grade intraepithelial neoplasia and prostate cancer samples. Strong NRBP1 protein expression was restricted to prostate cancer and correlated with higher expression of the proliferation marker Ki67 (P < 0.05). Further, patients with strong NRBP1 protein expression showed poor clinical outcomes (P < 0.05). Analysis of matched localized cancer tissues before and after castration revealed that post-therapy-related repression of NRBP1 expression was significantly associated with better overall survival. CONCLUSIONS: We demonstrate that expression of NRBP1 is up-regulated during the progression of prostate cancer and that high NRBP1 expression is linked with poor prognosis and enhanced tumor cell growth.

Amplification and overexpression of vinculin are associated with increased tumour cell proliferation and progression in advanced prostate cancer.

Androgen withdrawal is the standard treatment for advanced prostate cancer. Although this therapy is initially effective, nearly all prostate cancers become refractory to it. Approximately 15% of these castration-resistant prostate cancers harbour a genomic amplification at 10q22. The aim of this study was to explore the structure of the 10q22 amplicon and to determine the major driving genes. Application of high-resolution array-CGH using the 244k Agilent microarrays to cell lines with 10q22 amplification allowed us to narrow down the common amplified region to a region of 5.8 megabases. We silenced each of the genes of this region by an RNAi screen in the prostate cancer cell lines PC-3 and 22Rv1. We selected genes with a significant growth reduction in the 10q22 amplified cell line PC-3, but not in the non-amplified 22Rv1 cells, as putative target genes of this amplicon. Immunohistochemical analysis of the protein expression of these candidate genes on a tissue microarray enriched for 10q22 amplified prostate cancers revealed vinculin as the most promising target of this amplicon. We found a strong association between vinculin gene amplification and overexpression (p < 0.001). Further analysis of 443 specimens from across all stages of prostate cancer progression showed that vinculin expression was highest in castration-resistant prostate cancers, but negative or very low in benign prostatic hyperplasia (p < 0.0001). Additionally, high tumour cell proliferation measured by Ki67 expression was significantly associated with high vinculin expression in prostate cancer (p < 0.0001). Our data suggest that vinculin is a major driving gene of the 10q22 amplification in prostate cancer and that vinculin overexpression might contribute to prostate cancer progression by enhancing tumour cell proliferation.

Culture and Drug Profiling of Patient Derived Malignant Pleural Effusions for Personalized Cancer Medicine.

INTRODUCTION: The use of patients' own cancer cells for in vitro selection of the most promising treatment is an attractive concept in personalized medicine. Human carcinoma cells from malignant pleural effusions (MPEs) are suited for this purpose since they have already adapted to the liquid environment in the patient and do not depend on a stromal cell compartment. Aim of this study was to develop a systematic approach for the in-vitro culture of MPEs to analyze the effect of chemotherapeutic as well as targeted drugs. METHODS: MPEs from patients with solid tumors were selected for this study. After morphological and molecular characterization, they were cultured in medium supplemented with patient-derived sterile-filtered effusion supernatant. Growth characteristics were monitored in real-time using the xCELLigence system. MPEs were treated with a targeted therapeutic (erlotinib) according to the mutational status or chemotherapeutics based on the recommendation of the oncologists. RESULTS: We have established a robust system for the ex-vivo culture of MPEs and the application of drug tests in-vitro. The use of an antibody based magnetic cell separation system for epithelial cells before culture allowed treatment of effusions with only moderate tumor cell proportion. Experiments using drugs and drug-combinations revealed dose-dependent and specific growth inhibitory effects of targeted drugs. CONCLUSIONS: We developed a new approach for the ex-vivo culture of MPEs and the application of drug tests in-vitro using real-time measuring of cell growth, which precisely reproduced the effect of clinically established treatments by standard chemotherapy and targeted drugs. This sets the stage for future studies testing agents against specific targets from genomic profiling of metastatic tumor cells and multiple drug-combinations in a personalized manner.

E2F3 amplification and overexpression is associated with invasive tumor growth and rapid tumor cell proliferation in urinary bladder cancer.

E2F3 is located in the 6p22 bladder amplicon and encodes a transcription factor important for cell cycle regulation and DNA replication. To further investigate the role of E2F3 in bladder cancer, a tissue microarray containing samples from 2317 bladder tumors was used for gene copy number and expression analysis by means of fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). E2F3 amplification was strongly associated with invasive tumor phenotype and high tumor grade (P < 0.0001 each). None of 272 pTaG1/G2 tumors, but 35 of 311 pT1-4 carcinomas (11.3%), had E2F3 amplification. A high E2F3 expression level was associated with high grade, advanced stage, and E2F3 gene amplification (P < 0.0001 each). To evaluate whether E2F3 expression correlates with tumor proliferation, the Ki67 labeling index (LI) was analysed for each tumor. There was a strong association between a high Ki67 LI and E2F3 expression (P < 0.0001), which was independent of grade and stage. We conclude that E2F3 is frequently amplified and overexpressed in invasively growing bladder cancer (stage pT1-4). E2F3 expression appears to provide a growth advantage to tumor cells by activating cell proliferation in a subset of bladder tumors.

Ceramic foam plates: a new tool for processing fresh radical prostatectomy specimens.

Procurement of fresh tissue of prostate cancer is critical for biobanking and generation of xenograft models as an important preclinical step towards new therapeutic strategies in advanced prostate cancer. However, handling of fresh radical prostatectomy specimens has been notoriously challenging given the distinctive physical properties of prostate tissue and the difficulty to identify cancer foci on gross examination. Here, we have developed a novel approach using ceramic foam plates for processing freshly cut whole mount sections from radical prostatectomy specimens without compromising further diagnostic assessment. Forty-nine radical prostatectomy specimens were processed and sectioned from the apex to the base in whole mount slices. Putative carcinoma foci were morphologically verified by frozen section analysis. The fresh whole mount slices were then laid between two ceramic foam plates and fixed overnight. To test tissue preservation after this procedure, formalin-fixed and paraffin-embedded whole mount sections were stained with hematoxylin and eosin (H&E) and analyzed by immunohistochemistry, fluorescence, and silver in situ hybridization (FISH and SISH, respectively). There were no morphological artifacts on H&E stained whole mount sections from slices that had been fixed between two plates of ceramic foam, and the histological architecture was fully retained. The quality of immunohistochemistry, FISH, and SISH was excellent. Fixing whole mount tissue slices between ceramic foam plates after frozen section examination is an excellent method for processing fresh radical prostatectomy specimens, allowing for a precise identification and collection of fresh tumor tissue without compromising further diagnostic analysis.

Role of KCNMA1 in breast cancer.

KCNMA1 encodes the α-subunit of the large conductance, voltage and Ca(2+)-activated (BK) potassium channel and has been reported as a target gene of genomic amplification at 10q22 in prostate cancer. To investigate the prevalence of the amplification in other human cancers, the copy number of KCNMA1 was analyzed by fluorescence-in-situ-hybridization (FISH) in 2,445 tumors across 118 different tumor types. Amplification of KCNMA1 was restricted to a small but distinct fraction of breast, ovarian and endometrial cancer with the highest prevalence in invasive ductal breast cancers and serous carcinoma of ovary and endometrium (3-7%). We performed an extensive analysis on breast cancer tissue microarrays (TMA) of 1,200 tumors linked to prognosis. KCNMA1 amplification was significantly associated with high tumor stage, high grade, high tumor cell proliferation, and poor prognosis. Immunofluorescence revealed moderate or strong KCNMA1 protein expression in 8 out of 9 human breast cancers and in the breast cancer cell line MFM223. KCNMA1-function in breast cancer cell lines was confirmed by whole-cell patch clamp recordings and proliferation assays, using siRNA-knockdown, BK channel activators such as 17ß-estradiol and the BK-channel blocker paxilline. Our findings revealed that enhanced expression of KCNMA1 correlates with and contributes to high proliferation rate and malignancy of breast cancer.

High-throughput tissue microarray analysis of CMYC amplificationin urinary bladder cancer.

Alterations of chromosome 8, preferentially deletions of 8p and gains of 8q, belong to the most frequent cytogenetic changes in bladder cancer. CMYC on 8q24 is a candidate oncogene in this region. Little is known about the clinical significance of CMYC copy number changes in urinary bladder cancer because its frequency is low and a limited numbers of tumors were analyzed so far. To investigate the impact of CMYC alterations on tumor progression and patient prognosis in bladder cancer, we applied FISH to a tissue microarray containing 2317 bladder cancer samples. Presence of CMYC copy number increase was associated with advanced stage and high grade. CMYC amplifications were seen in 3 of 467 pTa (0.6%), 10 of 247 pT1 (4%) and 11 of 201 pT2-4 urothelial carcinomas (5.5%; p < 0.0001), as well as in 1 of 123 G1 (0.8%), 8 of 470 G2 (1.7%) and 17 of 365 G3 urothelial carcinomas (4.7%; p < 0.0001). CMYC gains were present in 49 of 467 pTa (10.5%), 39 of 247 pT1 (15.8%) and 43 of 201 pT2-4 urothelial carcinoma (21.4%; p < 0.0001), as well as in 7 of 123 G1 (5.7%), 56 of 470 G2 (11.9%) and 72 of 365 G3 urothelial carcinomas (19.7%; p < 0.0001). CMYC copy number changes were unrelated to prognosis of bladder cancer patients. We conclude that alterations of the CMYC gene, including copy number gains and amplifications, are linked to genetically unstable bladder cancers that are characterized by a high histologic grade and/or invasive growth. Patient prognosis was not affected by CMYC gene copy number changes.