Gene expression analysis of human prostate cell lines with and without tumor metastasis suppressor CD82

Background Tetraspanin CD82 is a tumor metastasis suppressor that is known to down regulate in various metastatic cancers. However, the exact mechanism by which CD82 prevents cancer metastasis is unclear. This study aims to identify genes that are regulated by CD82 in human prostate cell lines. Methods We used whole human genome microarray to obtain gene expression profiles in a normal prostate epithelial cell line that expressed CD82 (PrEC-31) and a metastatic prostate cell line that does not express CD82 (PC3). Then, siRNA silencing was used to knock down CD82 expression in PrEC-31 while CD82 was re-expressed in PC3 to acquire differentially-expressed genes in the respective cell line. Results Differentially-expressed genes with a P < 0.05 were identified in 3 data sets: PrEC-31 (+CD82) vs PrEC-31(−CD82), PC3–57 (+CD82) vs. PC3-5 V (−CD82), and PC3–29 (+CD82) vs. PC3-5 V (−CD82). Top 25 gene lists did not show overlap within the data sets, except (CALB1) the calcium binding protein calbindin 1 which was significantly up-regulated (2.8 log fold change) in PrEC-31 and PC3–29 cells that expressed CD82. Other most significantly up-regulated genes included serine peptidase inhibitor kazal type 1 (SPINK1) and polypeptide N-acetyl galactosaminyl transferase 14 (GALNT14) and most down-regulated genes included C-X-C motif chemokine ligand 14 (CXCL14), urotensin 2 (UTS2D), and fibroblast growth factor 13 (FGF13). Pathways related with cell proliferation and angiogenesis, migration and invasion, cell death, cell cycle, signal transduction, and metabolism were highly enriched in cells that lack CD82 expression. Expression of two mutually inclusive genes in top 100 gene lists of all data sets, runt-related transcription factor (RUNX3) and trefoil factor 3 (TFF3), could be validated with qRT-PCR. Conclusion Identification of genes and pathways regulated by CD82 in this study may provide additional insights into the role that CD82 plays in prostate tumor progression and metastasis, as well as identify potential targets for therapeutic intervention.

Integrative proteomic and phosphoproteomic profiling of prostate cell lines

ABSTRACTBackgroundProstate cancer is a major public health issue, mainly because patients relapse after androgen deprivation therapy. Proteomic strategies, aiming to reflect the functional activity of cells, are nowadays among the leading approaches to tackle the challenges not only of better diagnosis, but also of unraveling mechanistic details related to disease etiology and progression.MethodsWe conducted here a large SILAC-based Mass Spectrometry experiment to map the proteomes and phosphoproteomes of four widely used prostate cell lines, namely PNT1A, LNCaP, DU145 and PC3, representative of different cancerous and hormonal status.ResultsWe identified more than 3000 proteins and phosphosites, from which we quantified more than 1000 proteins and 500 phosphosites after stringent filtering. Extensive exploration of this proteomics and phosphoproteomics dataset allowed characterizing housekeeping as well as cell-line specific proteins, phosphosites and functional features of each cell line. In addition, by comparing the sensitive and resistant cell lines, we identified protein and phosphosites differentially expressed in the resistance context. Further data integration in a molecular network highlighted the differentially expressed pathways, in particular migration and invasion, RNA splicing, DNA damage repair response and transcription regulation.ConclusionsOverall, this study proposes a valuable resource toward the characterization of proteome and phosphoproteome of four widely used prostate cell lines and reveals candidates to be involved in prostate cancer progression for further experimental validation.

Specific and reliable detection of Myosin 1C isoform A by RTqPCR in prostate cancer cells

Background Prostate cancer (PC) diagnostics and treatment often present a challenging task due to cancer subtype heterogeneity and differential disease progression in patient subgroups. Hence, the critical issue is finding a reliable and sensitive diagnostic and prognostic PC marker, especially for cases of biopsies with low percentages of cancer cells. Isoform A of myosin 1C was shown to be expressed in PC cells and responsible for their invasive properties, however, its feasibility for diagnostic purposes remains to be elucidated. Methods To verify the role of myosin 1C isoform A mRNA expression as a putative prostate cancer marker we performed RT qPCR normalized by three reference genes (GAPDH, YWHAZ, HPRT1) on PC3, RWPE-1, LNCaP and 22Rv1 cell lines. Myosin 1C isoform A detection specificity was confirmed by immunofluorescence staining, cancer and non-cancer prostate cell lines were immunophenotyped by flow cytometry. Results Median normalized mRNA expression level of myosin 1C isoform A in PC cells (PC3 and 22Rv1) is two orders of magnitude higher compared to RWPE-1 cells, which functionally correspond to benign prostate cells. Myosin 1C isoform A expression allows PC cell detection even at a dilution ratio of 1:1000 cancer to non-cancer cells. At the protein level, the mean fluorescence intensity of myosin 1C isoform A staining in PC3 nuclei was only twice as high as in RWPE-1, while the immunophenotypes of both cell lines were similar (CD44+/CD90-/CD133-/CD57-/CD24+-). Conclusions We report a distinct difference in myosin 1C isoform A mRNA levels in malignant (PC3) and benign (RWPE-1) prostate cell lines and suggest a combination of three reference genes for accurate data normalization. For the first time we provide an immunophenotype comparison of RWPE-1 and PC3 cells and demonstrate that RT qPCR analysis of MYO 1C A using appropriate reference genes is sufficient for PC detection even in low-abundance cancer specimens.

Characterisation of CCS1477: A novel small molecule inhibitor of p300/CBP for the treatment of castration resistant prostate cancer.

11590 Background: Targeted degradation of androgen receptor (AR) and AR variants (ARV) remains an attractive therapeutic opportunity for patients with castrate resistant prostate cancer (CRPC). E1A binding protein (p300) and CREB binding protein (CBP) are two closely related transcriptional activators of AR. We have developed CCS1477 which is a potent, selective and orally active small molecule inhibitor of the bromodomain of p300/CBP and investigated its role in regulating androgen receptor expression and function. Methods: Binding of CCS1477 to p300, CBP and BRD4, was measured in a surface plasmon resonance (SPR) assay. Potency and functional activity (proliferation and biomarker knockdown) was demonstrated in prostate cell lines in vitro (22Rv1, VCaP). Cross species in vivo pharmacokinetic (PK) properties were assessed, and in vivo efficacy, linked to inhibition of biomarkers, was determined in 22Rv1 and LNCaP xenograft models. Results: CCS1477 binds to p300 and CBP with high affinity (Kd = 1.3/1.7nM) and selectivity (Kd = 222nM; BRD4). It is a potent inhibitor of cell proliferation in prostate cell lines (IC50 = 96nM,22Rv1; 49nM,VCaP) with minimal effect in AR-ve lines. In 22Rv1 cells, p300/CBP inhibition down-regulates AR-FL, AR-V7 and c-Myc protein by Western, an effect not seen with the BET inhibitor, JQ1 at equivalent proliferation IC50s. Inhibition of p300/CBP also reduces c-Myc, KLK3 and TMPRSS2 gene expression (qPCR) in 22Rv1 cells in vitro. The in vivo PK properties of CCS1477 are consistent with qd or qod oral dosing in mouse. CCS1477 dosed at 10mg, 20mg/kg qd or 30mg/kg qod, caused complete tumour growth inhibition over 28 days in a 22RV1 xenograft model, including extended duration in the absence of the drug for a further 24 days. This was accompanied by complete inhibition of plasma PSA and significant knockdown of tumour AR-FL, AR-V7, and C-Myc protein as well as C-Myc and TMPRSS2 mRNA expression. Conclusions: Taken together these data support the clinical testing of CCS1477 in castrate resistant prostate cancer by down-regulation of AR, AR-SV and c-MYC expression and function.