Role of dutasteride in pre‐clinical ETS fusion‐positive prostate cancer models

BACKGROUND Androgens play a crucial role in prostate cancer, hence the androgenic pathway has become an important target of therapeutic intervention. Previously we discovered that gene fusions between the 5′‐untranslated region of androgen regulated gene TMPRSS2 and the ETS transcription factor family members were present in a majority of the prostate cancer cases. The resulting aberrant overexpression of ETS genes drives tumor progression. METHODS Here, we evaluated the expression levels of 5α‐reductase isoenzymes in prostate cancer cell lines and tissues. We tested the effect of dutasteride, a 5α‐reductase inhibitor, in TMPRSS2–ERG fusion‐positive VCaP cell proliferation and cell invasion. We also evaluated the effect of dutasteride on the TMPRSS2–ERG fusion gene expression. Finally, we tested dutasteride alone or in combination with an anti‐androgen in VCaP cell xenografts tumor model. RESULTS Our data showed that 5α‐reductase SRD5A1 and SRD5A3 isoenzymes that are responsible for the conversion of testosterone to DHT, are highly expressed in metastatic prostate cancer compared to benign and localized prostate cancer. Dutasteride treatment attenuated VCaP cell proliferation and invasion. VCaP cells pre‐treated with dutasteride showed a reduction in ERG and PSA expression. In vivo studies demonstrated that dutasteride in combination with the anti‐androgen bicalutamide significantly decreased tumor burden in VCaP cell xenograft model. CONCLUSIONS Our findings suggest that dutasteride can inhibit ERG fusion‐positive cell growth and in combination with anti‐androgen, significantly reduce the tumor burden. Our study suggests that anti‐androgens used in combination with dutasteride could synergistically augment the therapeutic efficacy in the treatment of ETS‐positive prostate cancer. Prostate 72:1542–1549, 2012. © 2012 Wiley Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/93574/1/22509_ftp.pd

“Topological Significance” Analysis of Gene Expression and Proteomic Profiles from Prostate Cancer Cells Reveals Key Mechanisms of Androgen Response

The problem of prostate cancer progression to androgen independence has been extensively studied. Several studies systematically analyzed gene expression profiles in the context of biological networks and pathways, uncovering novel aspects of prostate cancer. Despite significant research efforts, the mechanisms underlying tumor progression are poorly understood. We applied a novel approach to reconstruct system-wide molecular events following stimulation of LNCaP prostate cancer cells with synthetic androgen and to identify potential mechanisms of androgen-independent progression of prostate cancer.We have performed concurrent measurements of gene expression and protein levels following the treatment using microarrays and iTRAQ proteomics. Sets of up-regulated genes and proteins were analyzed using our novel concept of "topological significance". This method combines high-throughput molecular data with the global network of protein interactions to identify nodes which occupy significant network positions with respect to differentially expressed genes or proteins. Our analysis identified the network of growth factor regulation of cell cycle as the main response module for androgen treatment in LNCap cells. We show that the majority of signaling nodes in this network occupy significant positions with respect to the observed gene expression and proteomic profiles elicited by androgen stimulus. Our results further indicate that growth factor signaling probably represents a "second phase" response, not directly dependent on the initial androgen stimulus.We conclude that in prostate cancer cells the proliferative signals are likely to be transmitted from multiple growth factor receptors by a multitude of signaling pathways converging on several key regulators of cell proliferation such as c-Myc, Cyclin D and CREB1. Moreover, these pathways are not isolated but constitute an interconnected network module containing many alternative routes from inputs to outputs. If the whole network is involved, a precisely formulated combination therapy may be required to fight the tumor growth effectively

Proteomic Interrogation of Androgen Action in Prostate Cancer Cells Reveals Roles of Aminoacyl tRNA Synthetases

Prostate cancer remains the most common malignancy among men in United States, and there is no remedy currently available for the advanced stage hormone-refractory cancer. This is partly due to the incomplete understanding of androgen-regulated proteins and their encoded functions. Whole-cell proteomes of androgen-starved and androgen-treated LNCaP cells were analyzed by semi-quantitative MudPIT ESI- ion trap MS/MS and quantitative iTRAQ MALDI- TOF MS/MS platforms, with identification of more than 1300 high-confidence proteins. An enrichment-based pathway mapping of the androgen-regulated proteomic data sets revealed a significant dysregulation of aminoacyl tRNA synthetases, indicating an increase in protein biosynthesis- a hallmark during prostate cancer progression. This observation is supported by immunoblot and transcript data from LNCaP cells, and prostate cancer tissue. Thus, data derived from multiple proteomics platforms and transcript data coupled with informatics analysis provides a deeper insight into the functional consequences of androgen action in prostate cancer

Role of dutasteride in pre‐clinical ETS fusion‐positive prostate cancer models

BACKGROUND Androgens play a crucial role in prostate cancer, hence the androgenic pathway has become an important target of therapeutic intervention. Previously we discovered that gene fusions between the 5′‐untranslated region of androgen regulated gene TMPRSS2 and the ETS transcription factor family members were present in a majority of the prostate cancer cases. The resulting aberrant overexpression of ETS genes drives tumor progression. METHODS Here, we evaluated the expression levels of 5α‐reductase isoenzymes in prostate cancer cell lines and tissues. We tested the effect of dutasteride, a 5α‐reductase inhibitor, in TMPRSS2–ERG fusion‐positive VCaP cell proliferation and cell invasion. We also evaluated the effect of dutasteride on the TMPRSS2–ERG fusion gene expression. Finally, we tested dutasteride alone or in combination with an anti‐androgen in VCaP cell xenografts tumor model. RESULTS Our data showed that 5α‐reductase SRD5A1 and SRD5A3 isoenzymes that are responsible for the conversion of testosterone to DHT, are highly expressed in metastatic prostate cancer compared to benign and localized prostate cancer. Dutasteride treatment attenuated VCaP cell proliferation and invasion. VCaP cells pre‐treated with dutasteride showed a reduction in ERG and PSA expression. In vivo studies demonstrated that dutasteride in combination with the anti‐androgen bicalutamide significantly decreased tumor burden in VCaP cell xenograft model. CONCLUSIONS Our findings suggest that dutasteride can inhibit ERG fusion‐positive cell growth and in combination with anti‐androgen, significantly reduce the tumor burden. Our study suggests that anti‐androgens used in combination with dutasteride could synergistically augment the therapeutic efficacy in the treatment of ETS‐positive prostate cancer. Prostate 72:1542–1549, 2012. © 2012 Wiley Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/93574/1/22509_ftp.pd

Golgi Protein GOLM1 Is a Tissue and Urine Biomarker of Prostate Cancer

Prostate cancer is the most common type of tumor found in American men and is the second leading cause of cancer death in males. To identify biomarkers that distinguish prostate cancer from normal, we compared multiple gene expression profiling studies. Through meta-analysis of expression array data from multiple prostate cancer studies, we identified GOLM1 (Golgi membrane protein 1, Golm 1) as consistently up-regulated in clinically localized prostate cancer. This observation was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and validated at the protein level by immunoblot assay and immunohistochemistry. Prostate epithelial cells were identified as the cellular source of GOLM1 expression using laser capture microdissection. Immunohistochemical staining localized the GOLM1 signal to the subapical cytoplasmic region, typical of a Golgi distribution. Surprisingly, GOLM1 immunoreactivity was detected in the supernatants of prostate cell lines and in the urine of patients with prostate cancer. The mechanism by which intact GOLM1 might be released from cells has not yet been elucidated. GOLM1 transcript levels were measured in urine sediments using quantitative PCR on a cohort of patients presenting for biopsy or radical prostatectomy. We found that urinary GOLM1 mRNA levels were a significant predictor of prostate cancer. Further, GOLM1 outperformed serum prostate-specific antigen (PSA) in detecting prostate cancer. The area under the receiver-operating characteristic curve was 0.622 for GOLM1 (P = .0009) versus 0.495 for serum PSA (P = .902). Our data indicating the up-regulation of GOLM1 expression and its appearance in patients' urine suggest GOLM1 as a potential novel biomarker for clinically localized prostate cancer

Mapping intact protein isoforms in discovery mode using top-down proteomics.

A full description of the human proteome relies on the challenging task of detecting mature and changing forms of protein molecules in the body. Large-scale proteome analysis has routinely involved digesting intact proteins followed by inferred protein identification using mass spectrometry. This 'bottom-up' process affords a high number of identifications (not always unique to a single gene). However, complications arise from incomplete or ambiguous characterization of alternative splice forms, diverse modifications (for example, acetylation and methylation) and endogenous protein cleavages, especially when combinations of these create complex patterns of intact protein isoforms and species. 'Top-down' interrogation of whole proteins can overcome these problems for individual proteins, but has not been achieved on a proteome scale owing to the lack of intact protein fractionation methods that are well integrated with tandem mass spectrometry. Here we show, using a new four-dimensional separation system, identification of 1,043 gene products from human cells that are dispersed into more than 3,000 protein species created by post-translational modification (PTM), RNA splicing and proteolysis. The overall system produced greater than 20-fold increases in both separation power and proteome coverage, enabling the identification of proteins up to 105 kDa and those with up to 11 transmembrane helices. Many previously undetected isoforms of endogenous human proteins were mapped, including changes in multiply modified species in response to accelerated cellular ageing (senescence) induced by DNA damage. Integrated with the latest version of the Swiss-Prot database, the data provide precise correlations to individual genes and proof-of-concept for large-scale interrogation of whole protein molecules. The technology promises to improve the link between proteomics data and complex phenotypes in basic biology and disease research