Noninvasive Detection of High Grade Prostate Cancer by DNA Methylation Analysis of Urine Cells Captured by Microfiltration.

PURPOSE: With the aim of developing a noninvasive test to detect clinically significant prostate cancer we investigated the potential of capturing cells from urine by microfiltration coupled with analysis of DNA methylation biomarkers. MATERIALS AND METHODS: In this prospective study urine from men suspected of having prostate cancer who were scheduled for transrectal ultrasound guided biopsy of the prostate was collected before digital rectal examination in 99, after digital rectal examination in 58 and from a urethral catheter in 7. Cells were isolated from whole volume voided urine using a filtration device containing a membrane filter with a pore size of 8 µm. Ten additional men provided 4 or 5 urine cell samples by self-collection prior to biopsy. Cellular DNA was analyzed for 5 methylation biomarkers using ddPCR™ (droplet digital polymerase chain reaction). RESULTS: Prostate cancer was diagnosed in 117 patients (71%). Across all tumor grades the sensitivity of urine DNA testing was 81% and 60% in samples obtained before and after digital rectal examination, respectively. In a prediction model including prostate specific antigen, patient age and the result of urine DNA analysis to detect high grade disease (Gleason score 7 or greater) an AUC of 0.95 (95% CI 0.90-1.00) was obtained for post-digital rectal examination samples. Analysis of repeat samples demonstrated substantial intraindividual variation in the shedding of cancerous cells in urine. CONCLUSIONS: Capturing cells from urine by microfiltration provides a novel tool to detect prostate cancer noninvasively with high sensitivity for high grade disease. Repeat sampling may increase sensitivity, particularly when urine is obtained without prior physical manipulation of the prostate.

Autophagy-mediated control of ribosome homeostasis in oncogene-induced senescence.

Oncogene-induced senescence (OIS) is a persistent anti-proliferative response that acts as a barrier against malignant transformation. During OIS, cells undergo dynamic remodeling, which involves alterations in protein and organelle homeostasis through autophagy. Here, we show that ribosomes are selectively targeted for degradation by autophagy during OIS. By characterizing senescence-dependent alterations in the ribosomal interactome, we find that the deubiquitinase USP10 dissociates from the ribosome during the transition to OIS. This release of USP10 leads to an enhanced ribosome ubiquitination, particularly of small subunit proteins, including lysine 275 on RPS2. Both reinforcement of the USP10-ribosome interaction and mutation of RPS2 K275 abrogate ribosomal delivery to lysosomes without affecting bulk autophagy. We show that the selective recruitment of ubiquitinated ribosomes to autophagosomes is mediated by the p62 receptor. While ribophagy is not required for the establishment of senescence per se, it contributes to senescence-related metabolome alterations and facilitates the senescence-associated secretory phenotype.

Screening of metabolic modulators identifies new strategies to target metabolic reprogramming in melanoma.

The prognosis of metastatic melanoma remains poor due to de novo or acquired resistance to immune and targeted therapies. Previous studies have shown that melanoma cells have perturbed metabolism and that cellular metabolic pathways represent potential therapeutic targets. To support the discovery of new drug candidates for melanoma, we examined 180 metabolic modulators, including phytochemicals and anti-diabetic compounds, for their growth-inhibitory activities against melanoma cells, alone and in combination with the BRAF inhibitor vemurafenib. Two positive hits from this screen, 4-methylumbelliferone (4-MU) and ursolic acid (UA), were subjected to validation and further characterization. Metabolic analysis showed that 4-MU affected cellular metabolism through inhibition of glycolysis and enhanced the effect of vemurafenib to reduce the growth of melanoma cells. In contrast, UA reduced mitochondrial respiration, accompanied by an increase in the glycolytic rate. This metabolic switch potentiated the growth-inhibitory effect of the pyruvate dehydrogenase kinase inhibitor dichloroacetate. Both drug combinations led to increased production of reactive oxygen species, suggesting the involvement of oxidative stress in the cellular response. These results support the potential use of metabolic modulators for combination therapies in cancer and may encourage preclinical validation and clinical testing of such treatment strategies in patients with metastatic melanoma.

Inhibition of retinoic acid receptor ß signaling confers glycolytic dependence and sensitization to dichloroacetate in melanoma cells.

Dysregulation of metabolism during melanoma progression is tightly associated with the acquisition of genetic and epigenetic alterations in regulators of metabolic pathways. Retinoic acid receptor beta (RARß) is epigenetically silenced in a large proportion of melanomas, but a link between RARß and metabolic rewiring of melanoma has not been established. Here, we show that in primary human melanocytes, all-trans retinoic acid (a RARß agonist) induced growth inhibition accompanied by a decrease in both glycolytic and oxidative metabolism, whereas selective inhibition of RARß led to an increase in the basal glycolytic rate and increased sensitivity to inhibition of glycolysis. In melanoma cells, inhibition of RARß promoted lower mitochondrial respiration and higher glycolytic activity, which led to energetic stress and activation of the energy sensor AMP-activated protein kinase. This metabolic shift increased the sensitivity to both glycolytic inhibition and stimulation of mitochondrial metabolism with dichloroacetate, an inhibitor of pyruvate dehydrogenase kinase. In melanoma cells harboring the BRAFV600E mutation, RARß activation antagonized the effect of the BRAF inhibitor PLX4032 (vemurafenib). Collectively, these data suggest that RARß signaling is involved in regulating cellular metabolism in melanoma and may provide a potential target in combination treatment strategies.

A Prospective Blinded Evaluation of Urine-DNA Testing for Detection of Urothelial Bladder Carcinoma in Patients with Gross Hematuria.

Retrospective studies have provided proof of principle that bladder cancer can be detected by testing for the presence of tumor DNA in urine. We have conducted a prospective blinded study to determine whether a urine-based DNA test can replace flexible cystoscopy in the initial assessment of gross hematuria. A total of 475 consecutive patients underwent standard urological examination including flexible cystoscopy and computed tomography urography, and provided urine samples immediately before (n=461) and after (n=444) cystoscopy. Urine cells were collected using a filtration device and tested for eight DNA mutation and methylation biomarkers. Clinical evaluation identified 99 (20.8%) patients with urothelial bladder tumors. With this result as a reference and based on the analysis of all urine samples, the DNA test had a sensitivity of 97.0%, a specificity of 76.9%, a positive predictive value of 52.5%, and a negative predictive value of 99.0%. In three patients with a positive urine-DNA test without clinical evidence of cancer, a tumor was detected at repeat cystoscopy within 16 mo. Our results suggest that urine-DNA testing can be used to identify a large subgroup of patients with gross hematuria in whom cystoscopy is not required. PATIENT SUMMARY: We tested the possibility of using a urine-based DNA test to check for bladder cancer in patients with visible blood in the urine. Our results show that the test efficiently detects bladder cancer and therefore may be used to greatly reduce the number of patients who would need to undergo cystoscopy.