Clement W.K. Chow

Human seminal fluid as a source of prostate cancer-specific microRNA biomarkers

6 1 Dame Roma Mitchell Cancer Research Laboratories and Adelaide Prostate Cancer Research 7 Centre, The University of Adelaide, South Australia, Australia; 2 Freemasons Foundation 8 Centre for Men’s Health, The University of Adelaide, South Australia, Australia; 3 University 9 of Queensland Centre for Clinical Research, Herston, Brisbane, Queensland, Australia; 10 4 Department of Urology, Royal Brisbane and Women’s Hospital, Brisbane, Queensland, 11 Australia; 5 Queensland Institute of Medical Research, Herston, Brisbane, Queensland, 12 Australia, 6 School of Population Health, The University of Queensland, Brisbane, 13 Queensland, Australia, 7 Department of Endocrinology, Princess Alexandra Hospital, 14 Brisbane, Queensland, Australia 15

BMCC1 Is an AP-2 associated endosomal protein in prostate cancer cells

The prostate cancer antigen gene 3 (PCA3) is embedded in an intron of a second gene BMCC1 (Bcl2-/adenovirus E1B nineteen kDa-interacting protein 2 (BNIP-2) and Cdc42GAP homology BCH motif-containing molecule at the carboxyl terminal region 1) which is also upregulated in prostate cancer. BMCC1 was initially annotated as two genes (C9orf65/PRUNE and BNIPXL) on either side of PCA3 but our data suggest that it represents a single gene coding for a high molecular weight protein. Here we demonstrate for the first time the expression of a >300 kDa BMCC1 protein (BMCC1-1) in prostate cancer and melanoma cell lines. This protein was found exclusively in the microsomal fraction and localised to cytoplasmic vesicles. We also observed expression of BMCC1 protein in prostate cancer sections using immunohistology. GST pull down, immunoprecipitation and mass spectrometry protein interaction studies identified multiple members of the Adaptor Related Complex 2 (AP-2) as BMCC1 interactors. Consistent with a role for BMCC1 as an AP-2 interacting endosomal protein, BMCC1 co-localised with β-adaptin at the perinuclear region of the cell. BMCC1 also showed partial co-localisation with the early endosome small GTP-ase Rab-5 as well as strong co-localisation with internalised pulse-chase labelled transferrin (Tf), providing evidence that BMCC1 is localised to functional endocytic vesicles. BMCC1 knockdown did not affect Tf uptake and AP-2 knockdown did not disperse BMCC1 vesicular distribution, excluding an essential role for BMCC1 in canonical AP-2 mediated endocytic uptake. Instead, we posit a novel role for BMCC1 in post-endocytic trafficking. This study provides fundamental characterisation of the BMCC1 complex in prostate cancer cells and for the first time implicates it in vesicle trafficking.

Prostate-based biofluids for the detection of prostate cancer: a comparative study of the diagnostic performance of cell-sourced RNA biomarkers

Background Prostate cancer (PCa) diagnosis requires improvement with the aid of more accurate biomarkers. Postejaculate urethral washings (PEUW) could be a physiological equivalent to urine obtained following rectal prostatic massage, the current basis for the prostate cancer antigen 3 (PCA3) test. The aim of this study was to investigate if PEUW contained prostate-based material, evidenced by the presence of prostate specific antigen (PSA), and to evaluate the diagnostic performance of PEUW-based biomarkers. Methods Male patients referred for elevated serum PSA or abnormal digital rectal examination provided ejaculate and PEUW samples. PSA, PCA3, and β2-microglobulin (β2M) were quantified in ejaculate and PEUW and compared with absolute and clinically significant (according to D’Amico criteria) PCa presence, as determined by biopsies. Diagnostic performance was determined and compared with serum PSA using receiver operating characteristic analysis. Results From 83 patients who provided PEUW samples, paired analysis with ejaculate samples was possible for 38 patients, while analysis in an unpaired, extended cohort was possible for 62 patients. PSA and PCA3 were detected in PEUW, normalized to β2M, and PCA3:PSA was calculated. In predicting absolute PCa status, PCA3:β2M in ejaculate [area under the curve (AUC) 0.717] and PEUW (AUC 0.569) were insignificantly better than PCA3:PSA (AUC 0.668 and 0.431, respectively) and comparable with serum PSA (AUC 0.617) with similar trends observed for the extended cohort. When considering clinically significant PCa presence, serum PSA in the comparison (AUC 0.640) and extended cohorts (AUC 0.665) was comparable with PCA3: β2M (AUC 0.667) and PCA3:PSA (AUC 0.605) in ejaculate, with lower estimates for PEUW in the comparison (PCA3: β2M AUC 0.496; PCA3:PSA AUC 0.342) and extended (PCA3: β2M AUC 0.497; PCA3:PSA AUC 0.469) cohorts. The statistical analysis was limited by sample size. Conclusion PEUW contains prostatic material, but has limited diagnostic accuracy when considering cell-derived DNA analysis. PCA3-based markers in ejaculate are comparable to serum PSA and digital rectal examination–urine markers.

Seminal plasma enables selection and monitoring of active surveillance candidates using nuclear magnetic resonance-based metabolomics: A preliminary investigation

Background Diagnosis and monitoring of localized prostate cancer requires discovery and validation of noninvasive biomarkers. Nuclear magnetic resonance (NMR)-based metabolomics of seminal plasma reportedly improves diagnostic accuracy, but requires validation in a high-risk clinical cohort. Materials and methods Seminal plasma samples of 151 men being investigated for prostate cancer were analyzed with 1H-NMR spectroscopy. After adjustment for buffer (add-to-subtract) and endogenous enzyme influence on metabolites, metabolite profiling was performed with multivariate statistical analysis (principal components analysis, partial least squares) and targeted quantitation. Results Seminal plasma metabolites best predicted low- and intermediate-risk prostate cancer with differences observed between these groups and benign samples. Lipids/lipoproteins dominated spectra of high grade samples with less metabolite contributions. Overall prostate cancer prediction using previously described metabolites was not validated. Conclusion Metabolomics of seminal plasma in vitro may assist urologists with diagnosis and monitoring of either low or intermediate grade prostate cancer. Less clinical benefit may be observed for high-risk patients. Further investigation in active surveillance cohorts, and/or in combination with in vivo magnetic resonance spectroscopic imaging may further optimize localized prostate cancer outcomes.

Can atorvastatin with metformin change the natural history of prostate cancer as characterized by molecular, metabolomic, imaging and pathological variables? A randomized controlled trial protocol

BACKGROUND Atorvastatin and metformin are known energy restricting mimetic agents that act synergistically to produce molecular and metabolic changes in advanced prostate cancer (PCa). This trial seeks to determine whether these drugs favourably alter selected parameters in men with clinically-localized, aggressive PCa. METHODS/DESIGN This prospective phase II randomized, controlled window trial is recruiting men with clinically significant PCa, confirmed by biopsy following multiparametric MRI and intending to undergo radical prostatectomy. Ethical approval was granted by the Royal Brisbane and Womens Hospital Human and The University of Queensland Medical Research Ethics Committees. Participants are being randomized into four groups: metformin with placebo; atorvastatin with placebo; metformin with atorvastatin; or placebo alone. Capsules are consumed for 8weeks, a duration selected as the most appropriate period in which histological and biochemical changes may be observed while allowing prompt treatment with curative intent of clinically significant PCa. At recruitment and prior to RP, participants provide blood, urine and seminal fluid. A subset of participants will undergo 7Tesla magnetic resonance spectroscopy to compare metabolites in-vivo with those in seminal fluid and biopsied tissue. The primary end point is biochemical evolution, defined using biomarkers (serum prostate specific antigen; PCA3 and citrate in seminal fluid and prostatic tissue). Standard pathological assessment will be undertaken. DISCUSSION This study is designed to assess the potential synergistic action of metformin and atorvastatin on PCa tumour biology. The results may determine simple methods of tumour modulation to reduce disease progression.