Renee S. Richards

A Novel Role for hSMG-1 in Stress Granule Formation

ABSTRACT hSMG-1 is a member of the phosphoinositide 3 kinase-like kinase (PIKK) family with established roles in nonsense-mediated decay (NMD) of mRNA containing premature termination codons and in genotoxic stress responses to DNA damage. We report here a novel role for hSMG-1 in cytoplasmic stress granule (SG) formation. Exposure of cells to stress causing agents led to the localization of hSMG-1 to SG, identified by colocalization with TIA-1, G3BP1, and eIF4G. hSMG-1 small interfering RNA and the PIKK inhibitor wortmannin prevented formation of a subset of SG, while specific inhibitors of ATM, DNA-PKcs, or mTOR had no effect. Exposure of cells to H2O2 and sodium arsenite induced (S/T)Q phosphorylation of proteins. While Upf2 and Upf1, an essential substrate for hSMG-1 in NMD, were present in SG, NMD-specific Upf1 phosphorylation was not detected in SG, indicating hSMG-1s role in SG is separate from classical NMD. Thus, SG formation appears more complex than originally envisaged and hSMG-1 plays a central role in this process.

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.

Characterization and structural analysis of a potent anticoagulant phospholipase A2 from Pseudechis australis snake venom.

Pseudechis australis is one of the most venomous and lethal snakes in Australia. Numerous phospholipase A2 (PLA2) isoforms constitute a major portion of its venom, some of which have previously been shown to exhibit not only enzymatic, but also haemolytic, neurotoxic and anticoagulant activities. Here, we have purified a potent anticoagulant PLA2 (identified as PA11) from P. australis venom to investigate its phospholipase, anticoagulant, haemolytic and cytotoxic activities and shown that addition of 11 nM PA11 resulted in a doubling of the clotting time of recalcified whole blood. We have also demonstrated that PA11 has high PLA2 enzymatic activity (10.9 × 10(4) Units/mg), but low haemolytic activity (0.6% of red blood cells hydrolysed in the presence of 1 nM PA11). PA11 at a concentration lower than 600 nM is not cytotoxic towards human cultured cells. Chemical modification experiments using p-bromophenacyl bromide have provided evidence that the catalytic histidine of PA11 is critical for the anticoagulant activity of this PLA2. PA11 that was subjected to trypsin digestion without previous reduction and alkylation of the disulfide bonds maintained enzymatic and anticoagulant activity, suggesting that proteolysis alone cannot abolish these properties. Consistent with these results, administration of PA11 by gavage in a rabbit stasis thrombosis model increased the clotting time of recalcified citrated whole blood by a factor of four. These data suggest that PA11 has potential to be developed as an anticoagulant in a clinical setting.

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.

Design and Clinical Verification of Surface-Enhanced Raman Spectroscopy Diagnostic Technology for Individual Cancer Risk Prediction

The use of emerging nanotechnologies, such as plasmonic nanoparticles in diagnostic applications, potentially offers opportunities to revolutionize disease management and patient healthcare. Despite worldwide research efforts in this area, there is still a dearth of nanodiagnostics which have been successfully translated for real-world patient usage due to the predominant sole focus on assay analytical performance and lack of detailed investigations into clinical performance in human samples. In a bid to address this pressing need, we herein describe a comprehensive clinical verification of a prospective label-free surface-enhanced Raman scattering (SERS) nanodiagnostic assay for prostate cancer (PCa) risk stratification. This contribution depicts a roadmap of (1) designing a SERS assay for robust and accurate detection of clinically validated PCa RNA targets; (2) employing a relevant and proven PCa clinical biomarker model to test our nanodiagnostic assay; and (3) investigating the clinical performance on independent training ( n = 80) and validation ( n = 40) cohorts of PCa human patient samples. By relating the detection outcomes to gold-standard patient biopsy findings, we established a PCa risk scoring system which exhibited a clinical sensitivity and specificity of 0.87 and 0.90, respectively [area-under-curve of 0.84 (95% confidence interval: 0.81-0.87) for differentiating high- and low-risk PCa] in the validation cohort. We envision that our SERS nanodiagnostic design and clinical verification approach may aid in the individualized prediction of PCa presence and risk stratification and may overall serve as an archetypical strategy to encourage comprehensive clinical evaluation of nanodiagnostic innovations.