OTUB1 de-ubiquitinating enzyme promotes prostate cancer cell invasion in vitro and tumorigenesis in vivo.

BACKGROUND: Ubiquitination is a highly dynamic and reversible process with a central role in cell homeostasis. Deregulation of several deubiquitinating enzymes has been linked to tumor development but their specific role in prostate cancer progression remains unexplored. METHODS: RNAi screening was used to investigate the role of the ovarian tumor proteases (OTU) family of deubiquitinating enzymes on the proliferation and invasion capacity of prostate cancer cells. RhoA activity was measured in relation with OTUB1 effects on prostate cancer cell invasion. Tumor xenograft mouse model with stable OTUB1 knockdown was used to investigate OTUB1 influence in tumor growth. RESULTS: Our RNAi screening identified OTUB1 as an important regulator of prostate cancer cell invasion through the modulation of RhoA activation. The effect of OTUB1 on RhoA activation is important for androgen-induced repression of p53 expression in prostate cancer cells. In localized prostate cancer tumors OTUB1 was found overexpressed as compared to normal prostatic epithelial cells. Prostate cancer xenografts expressing reduced levels of OTUB1 exhibit reduced tumor growth and reduced metastatic dissemination in vivo. CONCLUSIONS: OTUB1 mediates prostate cancer cell invasion through RhoA activation and promotes tumorigenesis in vivo. Our results suggest that drugs targeting the catalytic activity of OTUB1 could potentially be used as therapeutics for metastatic prostate cancer.

REST mediates androgen receptor actions on gene repression and predicts early recurrence of prostate cancer.

The androgen receptor (AR) is a key regulator of prostate tumorgenesis through actions that are not fully understood. We identified the repressor element (RE)-1 silencing transcription factor (REST) as a mediator of AR actions on gene repression. Chromatin immunoprecipitation showed that AR binds chromatin regions containing well-characterized cis-elements known to mediate REST transcriptional repression, while cell imaging studies confirmed that REST and AR closely co-localize in vivo. Androgen-induced gene repression also involves modulation of REST protein turnover through actions on the ubiquitin ligase ß-TRCP. Androgen deprivation or AR blockage with inhibitor MDV3100 (Enzalutamide) leads to neuroendocrine (NE) differentiation, a phenomenon that is mimicked by REST inactivation. Gene expression profiling revealed that REST not only acts to repress neuronal genes but also genes involved in cell cycle progression, including Aurora Kinase A, that has previously been implicated in the growth of NE-like castration-resistant tumors. The analysis of prostate cancer tissue microarrays revealed that tumors with reduced expression of REST have higher probability of early recurrence, independently of their Gleason score. The demonstration that REST modulates AR actions in prostate epithelia and that REST expression is negatively correlated with disease recurrence after prostatectomy, invite a deeper characterization of its role in prostate carcinogenesis.

SOCS2 mediates the cross talk between androgen and growth hormone signaling in prostate cancer.

UNLABELLED: Anabolic signals such as androgens and the growth hormone/insulin-like growth factor 1 (GH/IGF-1) axis play an essential role in the normal development of the prostate but also in its malignant transformation. In this study, we investigated the role of suppressor of cytokine signaling 2 (SOCS2) as mediator of the cross talk between androgens and GH signals in the prostate and its potential role as tumor suppressor in prostate cancer (PCa). We observed that SOCS2 protein levels assayed by immunohistochemistry are elevated in hormone therapy-naive localized prostatic adenocarcinoma in comparison with benign tissue. In contrast, however, castration-resistant bone metastases exhibit reduced levels of SOCS2 in comparison with localized or hormone naive, untreated metastatic tumors. In PCa cells, SOCS2 expression is induced by androgens through a mechanism that requires signal transducer and activator of transcription 5 protein (STAT5) and androgen receptor-dependent transcription. Consequentially, SOCS2 inhibits GH activation of Janus kinase 2, Src and STAT5 as well as both cell invasion and cell proliferation in vitro. In vivo, SOCS2 limits proliferation and production of IGF-1 in the prostate in response to GH. Our results suggest that the use of GH-signaling inhibitors could be of value as a complementary treatment for castration-resistant PCa. SUMMARY: Androgen induced SOCS2 ubiquitin ligase expression and inhibited GH signaling as well as cell proliferation and invasion in PCa, whereas reduced SOCS2 was present in castration-resistant cases. GH-signaling inhibitors might be a complementary therapeutic option for advanced PCa.

Parkin-mediated ubiquitination regulates phospholipase C-gamma1.

Mutations in parkin cause autosomal recessive forms of Parkinson's disease (PD), with an early age of onset and similar pathological phenotype to the idiopathic disease. Parkin has been identified as an E3 ubiquitin ligase that mediates different types of ubiquitination, which has made the search for substrates an intriguing possibility to identify pathological mechanisms linked to PD. In this study, we present PLCgamma1 as a novel substrate for parkin. This association was found in non-transfected human neuroblastoma SH-SY5Y cells as well as in stable cell lines expressing parkin WT and familial mutants R42P and G328E. Analysis of cortical, striatal and nigral human brain homogenates revealed that the interaction between parkin and PLCgamma1 is consistent throughout these regions, suggesting that the interaction is likely to have a physiological relevance for humans. Unlike many of the previously identified substrates, we could also show that the steady-state levels of PLCgamma1 is significantly higher in parkin KO mice and lower in parkin WT human neuroblastoma cells, suggesting that parkin ubiquitination of PLCgamma1 is required for proteasomal degradation. In line with this idea, we show that the ability to ubiquitinate PLCgamma1 in vitro differs significantly between WT and familial mutant parkin. In this study, we demonstrate that parkin interacts with PLCgamma1, affecting PLCgamma1 steady state protein levels in human and murine models with manipulated parkin function and expression levels. This finding could be of relevance for finding novel pathogenic mechanisms leading to PD.

Androgen induction of prostate cancer cell invasion is mediated by ezrin.

Ezrin is a key signaling molecule that regulates cell survival, adhesion migration, and invasion. We have previously shown that ezrin is regulated by androgen in rat prostate and that its expression is increased in prostate cancer and in prostate intraepithelial neoplasia. We have used the androgen-sensitive cell line LNCaP-FGC to investigate the role of ezrin in androgen-induced cell invasion. We found that androgen treatment of LNCaP-FGC cells induces ezrin expression, an effect that is inhibited by the androgen receptor antagonist, bicalutamide. In addition, androgen treatment induces the phosphorylation of ezrin in Thr-567 and Tyr-353 in a sequential manner. This is mediated through protein kinase C alpha and Src tyrosine kinase, respectively. Androgen treatment induces the translocation of both protein kinase C alpha and ezrin to the cell membrane and their association. Inhibition of ezrin function using short interference RNA or the overexpression of T567A and Y353F-ezrin mutants significantly reduces androgen-induced Matrigel invasion but does not affect cell proliferation or cell adhesion. Matrigel invasion of the androgen-insensitive prostate cancer cell lines PC-3 and LNCaP-R is also dependent on ezrin. In summary, we have shown that androgens regulate ezrin at transcriptional and posttranscriptional levels. Hormonal regulation of ezrin phosphorylation is required for androgen-induced cell invasion.

Proteomic comparison of prostate cancer cell lines LNCaP-FGC and LNCaP-r reveals heatshock protein 60 as a marker for prostate malignancy.

BACKGROUND: Androgen-sensitive prostate cancer cell-line LNCaP-FGC and androgen-resistant line LNCaP-r constitute a model for development of androgen resistance in prostate cancer. METHODS: Proteins differently expressed in the two cell-lines were identified by two-dimensional (2-D) electrophoresis and mass spectrometry. HSP60, more abundant in LNCaP-r, was studied by RT-PCR and immunohistochemistry in specimens of human prostate cancer. RESULTS: HSP60 was upregulated in LNCaP-r, nm23 in LNCaP-FGC, and titin (two isoforms) in either LNCaP-r or LNCaP-FGC. In non-malignant prostate, HSP60-staining was in the glandular compartment, particularly basal epithelial cells. In prostate cancer, most epithelial cells showed moderate-strong staining without apparent correlation between staining intensity and Gleason grade. CONCLUSIONS: The LNCaP-FGC/LNCaP-r model, characterized by 2-D electrophoresis, reveals distinct proteomic alterations. With HSP60, results from cell-lines correlated with clinical results, indicating that this model can be used for dissection of mechanisms involved in transformation to androgen resistance and assignment of protein markers in prostate cancer.

Identifying patterns of DNA for tumor diagnosis using capillary electrophoresis-amplified fragment length polymorphism (CE-AFLP) screening.

Amplified Fragment Length Polymorphism (AFLP) screening is a genome-wide genotyping strategy that has been widely used in plants and bacteria, but little has been reported concerning its use in humans. We investigated if the AFLP procedure could be coupled with high-throughput capillary electrophoresis (CE) for use in tumor diagnosis and classification. Using CE-AFLP, a series of molecular 'fingerprints' were generated for a set of gastric tumor and normal genomic DNA samples. The CE-AFLP procedure was qualitatively and quantitatively robust, and a variety of clustering tools were used to identify a specific DNA marker 'pattern' of 20 features that classified the tumor and normal samples to reasonable degrees of accuracy (Sensitivity 95%, Specificity 80%). The CE-AFLP-based approach also correctly classified 16 tumor samples, which in a previous study had exhibited no detectable genomic aberrations by comparative genome hybridization (CGH). This is the first reported application of CE-AFLP screening in tumor diagnosis. As the procedure is relatively inexpensive and requires minimal prior sequence knowledge and biological material, we suggest that CE-AFLP-based protocols may represent a promising new approach for DNA-based cancer screening and diagnosis.

Regulation of matrix metalloproteinase 13 expression by androgen in prostate cancer.

The matrix metalloproteinases (MMPs) are members of a family of endopeptidases that are able to degrade extra-cellular matrix. MMPs and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), play a key role in the migration of normal and malignant cell. Interaction of MMPs and TIMPs has been involved in the process of tumor invasion and metastasis. Using cDNA microarray as a screening tool to find androgen regulated gene in prostate cancer, we have found that expression of MMP-13 is regulated by androgen in prostate cancer derived cell line LNCaP. This regulation was further confirmed and quantified by real-time RT-PCR. In addition, the upregulation of MMP-13 mRNA by androgen could be abolished by the androgen antagonist Casodex but not the protein inhibitor cycloheximide. Western blot and immunohistochemistry of MMP-13 confirmed the androgen regulation at the protein level. We have furthermore shown that MMP-13 expression is presented in human prostate cancer obtained from aspiration biopsy. In summary, MMP-13 is androgen regulated and detectable in prostate cancer. Further study of MMP-13 in prostate cancer may help us to understand the progression of the cancer and can lead to new therapeutic options.