The dysregulation of gene expression is an enabling hallmark of cancer. Computational analysis of transcriptomics data from human cancer specimens, complemented with exhaustive clinical annotation, provides an opportunity to identify core regulators of the tumorigenic process. Here we exploit well-annotated clinical datasets of prostate cancer for the discovery of transcriptional regulators relevant to prostate cancer. Following this rationale, we identify Microphthalmia-associated transcription factor (MITF) as a prostate tumor suppressor among a subset of transcription factors. Importantly, we further interrogate transcriptomics and clinical data to refine MITF perturbation-based empirical assays and unveil Crystallin Alpha B (CRYAB) as an unprecedented direct target of the transcription factor that is, at least in part, responsible for its tumor-suppressive activity in prostate cancer. This evidence was supported by the enhanced prognostic potential of a signature based on the concomitant alteration of MITF and CRYAB in prostate cancer patients. In sum, our study provides proof-of-concept evidence of the potential of the bioinformatics screen of publicly available cancer patient databases as discovery platforms, and demonstrates that the MITF-CRYAB axis controls prostate cancer biology.

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.

Gene dosage is a key defining factor to understand cancer pathogenesis and progression, which requires the development of experimental models that aid better deconstruction of the disease. Here, we model an aggressive form of prostate cancer and show the unconventional association of LKB1 dosage to prostate tumorigenesis. Whereas loss of Lkb1 alone in the murine prostate epithelium was inconsequential for tumorigenesis, its combination with an oncogenic insult, illustrated by Pten heterozygosity, elicited lethal metastatic prostate cancer. Despite the low frequency of LKB1 deletion in patients, this event was significantly enriched in lung metastasis. Modeling the role of LKB1 in cellular systems revealed that the residual activity retained in a reported kinase-dead form, LKB1K78I, was sufficient to hamper tumor aggressiveness and metastatic dissemination. Our data suggest that prostate cells can function normally with low activity of LKB1, whereas its complete absence influences prostate cancer pathogenesis and dissemination.

Glycine N-Methyltransferase (GNMT) is a metabolic enzyme that integrates metabolism and epigenetic regulation. The product of GNMT, sarcosine, has been proposed as a prostate cancer biomarker. This enzyme is predominantly expressed in the liver, brain, pancreas, and prostate tissue, where it exhibits distinct regulation. Whereas genetic alterations in GNMT have been associated to prostate cancer risk, its causal contribution to the development of this disease is limited to cell line-based studies and correlative human analyses. Here we integrate human studies, genetic mouse modeling, and cellular systems to characterize the regulation and function of GNMT in prostate cancer. We report that this enzyme is repressed upon activation of the oncogenic Phosphoinositide-3-kinase (PI3K) pathway, which adds complexity to its reported dependency on androgen signaling. Importantly, we demonstrate that expression of GNMT is required for the onset of invasive prostate cancer in a genetic mouse model. Altogether, our results provide further support of the heavy oncogenic signal-dependent regulation of GNMT in prostate cancer.

Oncogene addiction postulates that the survival and growth of certain tumor cells is dependent upon the activity of one oncogene, despite their multiple genetic and epigenetic abnormalities. This phenomenon provides a foundation for molecular targeted therapy and a rationale for oncogene-based stratification. We have previously reported that the Promyelocytic Leukemia protein (PML) is upregulated in triple negative breast cancer (TNBC) and it regulates cancer-initiating cell function, thus suggesting that this protein can be therapeutically targeted in combination with PML-based stratification. However, the effects of PML perturbation on the bulk of tumor cells remained poorly understood. Here we demonstrate that TNBC cells are addicted to the expression of this nuclear protein. PML inhibition led to a remarkable growth arrest combined with features of senescence in vitro and in vivo. Mechanistically, the growth arrest and senescence were associated to a decrease in MYC and PIM1 kinase levels, with the subsequent accumulation of CDKN1B (p27), a trigger of senescence. In line with this notion, we found that PML is associated to the promoter regions of MYC and PIM1, consistent with their direct correlation in breast cancer specimens. Altogether, our results provide a feasible explanation for the functional similarities of MYC, PIM1, and PML in TNBC and encourage further study of PML targeting strategies for the treatment of this breast cancer subtype.

Activation of the PTEN-PI3K-mTORC1 pathway consolidates metabolic programs that sustain cancer cell growth and proliferation. Here we show that mechanistic target of rapamycin complex 1 (mTORC1) regulates polyamine dynamics, a metabolic route that is essential for oncogenicity. By using integrative metabolomics in a mouse model and human biopsies of prostate cancer, we identify alterations in tumours affecting 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 is validated in mouse and human cancer specimens. AMD1 is upregulated in human prostate cancer with activated mTORC1. Conversely, samples from a clinical trial with the mTORC1 inhibitor everolimus exhibit a predominant decrease in AMD1 immunoreactivity that is associated with 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.

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.

Prostate cancer is among the most frequent cancers in men, and despite its high rate of cure, the high number of cases results in an elevated mortality worldwide. Importantly, prostate cancer incidence is dramatically increasing in western societies in the past decades, suggesting that this type of tumor is exquisitely sensitive to lifestyle changes. Prostate cancer frequently exhibits alterations in the PTEN gene (inactivating mutations or gene deletions) or at the protein level (reduced protein expression or altered sub-cellular compartmentalization). The relevance of PTEN in this type of cancer is further supported by the fact that the sole deletion of PTEN in the murine prostate epithelium recapitulates many of the features of the human disease. In order to study the molecular alterations in prostate cancer, we need to overcome the methodological challenges that this tissue imposes. In this review we present protocols and methods, using PTEN as proof of concept, to study different molecular characteristics of prostate cancer.

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 peroxisome proliferator-activated receptor gamma co-activator 1α (PGC1α) suppresses prostate cancer progression and metastasis. A metabolic co-regulator data mining analysis unveiled that PGC1α is downregulated in prostate cancer and associated with 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.

Salp15, a salivary protein of Ixodes ticks, inhibits the activation of naïve CD4 T cells. Treatment with Salp15 results in the inhibition of early signaling events and the production of the autocrine growth factor, interleukin-2. The fate of the CD4 T cells activated in the presence of Salp15 or its long-term effects are, however, unknown. We now show that Salp15 binding to CD4 is persistent and induces a long-lasting immunomodulatory effect. The activity of Salp15 results in sustained diminished cross-antigenic antibody production even after interruption of the treatment with the protein. Transcriptionally, the salivary protein provokes an acute effect that includes known activation markers, such as Il2 or Cd44, and that fades over time. The long-term effects exerted by Salp15 do not involve the induction of either anergy traits nor increased populations of regulatory T cells. Similarly, the treatment with Salp15 does not result in B cell anergy or the generation of myeloid suppressor cells. However, Salp15 induces the increased expression of the ectoenzyme, CD73, in regulatory T cells and increased production of adenosine. Our study provides a profound characterization of the immunomodulatory activity of Salp15 and suggests that its long-term effects are due to the specific regulation of CD73.

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.

Prostate cancer is diagnosed late in life, when co-morbidities are frequent. Among them, hypertension, hypercholesterolemia, diabetes or metabolic syndrome exhibit an elevated incidence. In turn, prostate cancer patients frequently undergo chronic pharmacological treatments that could alter disease initiation, progression and therapy response. Here we show that treatment with anti-cholesterolemic drugs, statins, at doses achieved in patients, enhance the pro-tumorigenic activity of obesogenic diets. In addition, the use of a mouse model of prostate cancer and human prostate cancer xenografts revealed that in vivo simvastatin administration alone increases prostate cancer aggressiveness. In vitro cell line systems supported the notion that this phenomenon occurs, at least in part, through the direct action on cancer cells of low doses of statins, in range of what is observed in human plasma. In sum, our results reveal a prostate cancer experimental system where statins exhibit an undesirable effect, and warrant further research to address the relevance and implications of this observation in human prostate cancer.

In patients infected by SARS-CoV-2 who experience an exaggerated inflammation leading to pneumonia, monocytes likely play a major role but have received poor attention. Thus, we analyzed peripheral blood monocytes from patients with COVID-19 pneumonia and found that these cells show signs of altered bioenergetics and mitochondrial dysfunction, had a reduced basal and maximal respiration, reduced spare respiratory capacity, and decreased proton leak. Basal extracellular acidification rate was also diminished, suggesting reduced capability to perform aerobic glycolysis. Although COVID-19 monocytes had a reduced ability to perform oxidative burst, they were still capable of producing TNF and IFN-γ in vitro. A significantly high amount of monocytes had depolarized mitochondria and abnormal mitochondrial ultrastructure. A redistribution of monocyte subsets, with a significant expansion of intermediate/pro-inflammatory cells, and high amounts of immature monocytes were found, along with a concomitant compression of classical monocytes, and an increased expression of inhibitory checkpoints like PD-1/PD-L1. High plasma levels of several inflammatory cytokines and chemokines, including GM-CSF, IL-18, CCL2, CXCL10, and osteopontin, finally confirm the importance of monocytes in COVID-19 immunopathogenesis.

Fumarate hydratase (FH) is a mitochondrial enzyme that catalyzes the reversible hydration of fumarate to malate in the tricarboxylic acid (TCA) cycle. Germline mutations of FH lead to hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a cancer syndrome characterized by a highly aggressive form of renal cancer. Although HLRCC tumors metastasize rapidly, FH-deficient mice develop premalignant cysts in the kidneys, rather than carcinomas. How Fh1-deficient cells overcome these tumor-suppressive events during transformation is unknown. Here, we perform a genome-wide CRISPR-Cas9 screen to identify genes that, when ablated, enhance the proliferation of Fh1-deficient cells. We found that the depletion of the histone cell cycle regulator (HIRA) enhances proliferation and invasion of Fh1-deficient cells in vitro and in vivo. Mechanistically, Hira loss activates MYC and its target genes, increasing nucleotide metabolism specifically in Fh1-deficient cells, independent of its histone chaperone activity. These results are instrumental for understanding mechanisms of tumorigenesis in HLRCC and the development of targeted treatments for patients.

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a cancer syndrome caused by inactivating germline mutations in fumarate hydratase (FH) and subsequent accumulation of fumarate. Fumarate accumulation leads to profound epigenetic changes and the activation of an anti-oxidant response via nuclear translocation of the transcription factor NRF2. The extent to which chromatin remodeling shapes this anti-oxidant response is currently unknown. Here, we explored the effects of FH loss on the chromatin landscape to identify transcription factor networks involved in the remodeled chromatin landscape of FH-deficient cells. We identify FOXA2 as a key transcription factor that regulates anti-oxidant response genes and subsequent metabolic rewiring cooperating without direct interaction with the anti-oxidant regulator NRF2. The identification of FOXA2 as an anti-oxidant regulator provides additional insights into the molecular mechanisms behind cell responses to fumarate accumulation and potentially provides further avenues for therapeutic intervention for HLRCC.