Cellular transformation and cancer progression is accompanied by changes in the metabolic landscape. Master co-regulators of metabolism orchestrate the modulation of multiple metabolic pathways through transcriptional programs, and hence constitute a probabilistically parsimonious mechanism for general metabolic rewiring. Here we show that the transcriptional co-activator PGC1α suppresses prostate cancer progression and metastasis. A metabolic co-regulator data mining analysis unveiled that PGC1α is down-regulated in prostate cancer and associated to disease progression. Using genetically engineered mouse models and xenografts, we demonstrated that PGC1α opposes prostate cancer progression and metastasis. Mechanistically, the use of integrative metabolomics and transcriptomics revealed that PGC1α activates an Oestrogen-related receptor alpha (ERRα)-dependent transcriptional program to elicit a catabolic state and metastasis suppression. Importantly, a signature based on the PGC1α-ERRα pathway exhibited prognostic potential in prostate cancer, thus uncovering the relevance of monitoring and manipulating this pathway for prostate cancer stratification and treatment.

Activation of the PTEN-PI3K-mTORC1 pathway consolidates metabolic programs that sustain cancer cell growth and proliferation1,2. Here we show that mTORC1 regulates polyamine dynamics, a metabolic route that is essential for oncogenicity. Through the use of integrative metabolomics in a mouse model3 and human biopsies4 of prostate cancer, we identified alterations in tumours impacting on the production of decarboxylated S-adenosylmethionine (dcSAM) and polyamine synthesis. Mechanistically, this metabolic rewiring stems from mTORC1-dependent regulation of S-adenosylmethionine decarboxylase 1 (AMD1) stability. This novel molecular regulation was validated in murine and human cancer specimens. AMD1 was upregulated in prostate cancer specimens with activated mTORC1. Conversely, samples from a clinical trial with the mTORC1 inhibitor everolimus5 exhibited a predominant decrease in AMD1 immunoreactivity that was associated to a decrease in proliferation, in line with the requirement of dcSAM production for oncogenicity. These findings provide fundamental information about the complex regulatory landscape controlled by mTORC1 to integrate and translate growth signals into an oncogenic metabolic program.

Urine sample analysis is irreplaceable as a non-invasive method for disease diagnosis and follow-up. However, in urine samples, non-degraded protein and RNA may be only found in urinary extracellular vesicles (uEVs). In recent years, various methods of uEV enrichment using low volumes of urine and unsophisticated equipment have been developed, with variable success. We compared the results of the differential ultracentrifugation procedure with 4 of these methods. The methods tested were a lectin-based purification, Exoquick (System Biosciences), Total Exosome Isolation from Invitrogen and an in-house modified procedure employing the Exosomal RNA Kit from Norgen Biotek Corp. The analysis of selected gene transcripts and protein markers of extracellular vesicles (EVs) revealed that each method isolates a different mixture of uEV protein markers. In our conditions, the extraction with Norgen's reagent achieved the best performance in terms of gene transcript and protein detection and reproducibility. By using this method, we were able to detect alterations of EVs protein markers in urine samples from prostate cancer adenoma patients. Taken together, our results show that the isolation of uEVs is feasible from small volumes of urine and avoiding ultracentrifugation, making easier the analysis in a clinical facility. However, caution should be taken in the selection of the enrichment method since they have a differential affinity for protein uEVs markers and by extension for different subpopulation of EVs.

Urine contains extracellular vesicles (EVs) that concentrate molecules and protect them from degradation. Thus, isolation and characterisation of urinary EVs could increase the efficiency of biomarker discovery. We have previously identified proteins and RNAs with differential abundance in urinary EVs from prostate cancer (PCa) patients compared to benign prostate hyperplasia (BPH). Here, we focused on the analysis of the metabolites contained in urinary EVs collected from patients with PCa and BPH. Targeted metabolomics analysis of EVs was performed by ultra-high-performance liquid chromatography–mass spectrometry. The correlation between metabolites and clinical parameters was studied, and metabolites with differential abundance in PCa urinary EVs were detected and mapped into cellular pathways. We detected 248 metabolites belonging to different chemical families including amino acids and various lipid species. Among these metabolites, 76 exhibited significant differential abundance between PCa and BPH. Interestingly, urine EVs recapitulated many of the metabolic alterations reported in PCa, including phosphathidylcholines, acyl carnitines, citrate and kynurenine. Importantly, we found elevated levels of the steroid hormone, 3beta-hydroxyandros-5-en-17-one-3-sulphate (dehydroepiandrosterone sulphate) in PCa urinary EVs, in line with the potential elevation of androgen synthesis in this type of cancer. This work supports urinary EVs as a non-invasive source to infer metabolic changes in PCa.

Patient stratification has been instrumental for the success of targeted therapies in breast cancer. However, the molecular basis of metastatic breast cancer and its therapeutic vulnerabilities remain poorly understood. Here we show that PML is a novel target in aggressive breast cancer. The acquisition of aggressiveness and metastatic features in breast tumours is accompanied by the elevated PML expression and enhanced sensitivity to its inhibition. Interestingly, we find that STAT3 is responsible, at least in part, for the transcriptional upregulation of PML in breast cancer. Moreover, PML targeting hampers breast cancer initiation and metastatic seeding. Mechanistically, this biological activity relies on the regulation of the stem cell gene SOX9 through interaction of PML with its promoter region. Altogether, we identify a novel pathway sustaining breast cancer aggressiveness that can be therapeutically exploited in combination with PML-based stratification.

Extracellular vesicles (EV) are emerging structures with promising properties for intercellular communication. In addition, the characterization of EV in biofluids is an attractive source of non-invasive diagnostic, prognostic and predictive biomarkers. Here we show that urinary EV (uEV) from prostate cancer (PCa) patients exhibit genuine and differential physical and biological properties compared to benign prostate hyperplasia (BPH). Importantly, transcriptomics characterization of uEVs led us to define the decreased abundance of Cadherin 3, type 1 (CDH3) transcript in uEV from PCa patients. Tissue and cell line analysis strongly suggested that the status of CDH3 in uEVs is a distal reflection of changes in the expression of this cadherin in the prostate tumor. CDH3 was negatively regulated at the genomic, transcriptional, and epigenetic level in PCa. Our results reveal that uEVs could represent a non-invasive tool to inform about the molecular alterations in PCa.

Urine extracellular vesicles are a valuable low-invasive source of information, especially for the cells of the genitourinary tract. In the search for biomarkers, different techniques have been developed to isolate and characterize the cargo of these vesicles. In the present work, we compare five of these different isolation methods (three commercial isolation kits, ultracentrifugation, and lectin-based purification) and perform miRNA profiling using a multiplex miRNA assay. The results showed high correlation through all isolation techniques, and 48 out of 68 miRNAs were detected above the detection limit at least 10 times. The results obtained by multiplex assay were validated through Taqman qPCR. In addition, using this technique combined with a clinically friendly extracellular vesicle (uEV)-enrichment method, we performed the analysis of selected miRNAs in urine from patients affected with bladder cancer, benign prostate hyperplasia, or prostate cancer. Importantly, we found that those miRNAs could be detected in almost 100% of the samples, and no significant differences were observed between groups. Our results support the feasibility of analyzing exosomes-associated miRNAs using a methodology that requires a small volume of urine and is compatible with a clinical environment and high-throughput analysis.

Prostate cancer is the second most common tumor and the fifth cause of cancer-related death among men worldwide. PC cells exhibit profound signaling and metabolic reprogramming that account for the acquisition of aggressive features. Although the metabolic understanding of this disease has increased in recent years, the analysis of such alterations through noninvasive methodologies in biofluids remains limited. Here, we used NMR-based metabolomics on a large cohort of urine samples (more than 650) from PC and benign prostate hyperplasia (BPH) patients to investigate the molecular basis of this disease. Multivariate analysis failed to distinguish between the two classes, highlighting the modest impact of prostate alterations on urine composition and the multifactorial nature of PC. However, univariate analysis of urine metabolites unveiled significant changes, discriminating PC from BPH. Metabolites with altered abundance in urine from PC patients revealed changes in pathways related to cancer biology, including glycolysis and the urea cycle. We found out that metabolites from such pathways were diminished in the urine from PC individuals, strongly supporting the notion that PC reduces nitrogen and carbon waste in order to maximize their usage in anabolic processes that support cancer cell growth.