Targeting cancer stem cell OXPHOS with tailored ruthenium complexes as a new anti-cancer strategy.

BACKGROUND: Previous studies by our group have shown that oxidative phosphorylation (OXPHOS) is the main pathway by which pancreatic cancer stem cells (CSCs) meet their energetic requirements; therefore, OXPHOS represents an Achille's heel of these highly tumorigenic cells. Unfortunately, therapies that target OXPHOS in CSCs are lacking. METHODS: The safety and anti-CSC activity of a ruthenium complex featuring bipyridine and terpyridine ligands and one coordination labile position (Ru1) were evaluated across primary pancreatic cancer cultures and in vivo, using 8 patient-derived xenografts (PDXs). RNAseq analysis followed by mitochondria-specific molecular assays were used to determine the mechanism of action. RESULTS: We show that Ru1 is capable of inhibiting CSC OXPHOS function in vitro, and more importantly, it presents excellent anti-cancer activity, with low toxicity, across a large panel of human pancreatic PDXs, as well as in colorectal cancer and osteosarcoma PDXs. Mechanistic studies suggest that this activity stems from Ru1 binding to the D-loop region of the mitochondrial DNA of CSCs, inhibiting OXPHOS complex-associated transcription, leading to reduced mitochondrial oxygen consumption, membrane potential, and ATP production, all of which are necessary for CSCs, which heavily depend on mitochondrial respiration. CONCLUSIONS: Overall, the coordination complex Ru1 represents not only an exciting new anti-cancer agent, but also a molecular tool to dissect the role of OXPHOS in CSCs. Results indicating that the compound is safe, non-toxic and highly effective in vivo are extremely exciting, and have allowed us to uncover unprecedented mechanistic possibilities to fight different cancer types based on targeting CSC OXPHOS.

Oleocanthal, an Antioxidant Phenolic Compound in Extra Virgin Olive Oil (EVOO): A Comprehensive Systematic Review of Its Potential in Inflammation and Cancer.

BACKGROUND: The Mediterranean diet is linked to various health benefits, especially the consumption of olive oil as a key component. Multiple studies highlight its advantages, particularly due to its fatty acid composition and additional components like phenolic compounds. A significant antioxidant compound, oleocanthal, known for its antioxidant properties, has gained attention in the pharmaceutical industry for its anti-inflammatory and antiproliferative effects. It shows promise in addressing cardiovascular diseases, metabolic syndrome, and neuroprotection. This systematic review aims to evaluate the existing literature on oleocanthal, examining its role in biological processes and potential impact on conditions like inflammation and cancer. METHODS: We performed several searches in PubMed (MEDLINE), Web of Science (WOS), and Cochrane based on the terms "Oleocanthal", "Cancer", and "Inflammation". The inclusion criteria were as follows: studies whose main topics were oleocanthal and cancer or inflammation. On the other hand, the exclusion criteria were studies that were not focused on oleocanthal, reviews, or editorial material. Given that these findings are explanatory rather than derived from clinical trials, we refrained from employing methods to assess potential bias. This systematic review did not receive any external funding. RESULTS: We found 174 records from these searches, where we discarded reviews and editorial material, duplicated articles, and 1 retracted article. Finally, we had 53 reports assessed for eligibility that were included in this review. DISCUSSION: OC exhibits promising therapeutic potential against both inflammation and cancer. We addressed its ability to target inflammatory genes and pathways, offering potential treatments for conditions like rheumatic diseases by regulating pathways such as NF-kB and MAPK. Additionally, OC's anticancer properties, particularly its notable inhibition of c-Met signaling across various cancers, highlight its efficacy, showcasing promise as a potential treatment.

Metabolomic signature and molecular profile of normal and degenerated human intervertebral disc cells.

BACKGROUND CONTEXT: Intervertebral disc degeneration (IVDD) is an incurable, specific treatment-orphan disease with an increasing burden worldwide. Although great efforts have been made to develop new regenerative therapies, their clinical success is limited. PURPOSE: Characterize the metabolomic and gene expression changes underpinning human disc degeneration. This study also aimed to disclose new molecular targets for developing and optimizing novel biological approaches for IVDD. STUDY DESIGN: Intervertebral disc cells were obtained from IVDD patients undergoing circumferential arthrodesis surgery or from healthy subjects. Mimicking the harmful microenvironment of degenerated discs, cells isolated from the nucleus pulposus (NP) and annulus fibrosus (AF) were exposed to the proinflammatory cytokine IL-1ß and the adipokine leptin. The metabolomic signature and molecular profile of human disc cells were unraveled for the first time. METHODS: The metabolomic and lipidomic profiles of IVDD and healthy disc cells were analyzed by high-performance liquid chromatography-mass spectrometry (UHPLC-MS). Gene expression was investigated by SYBR green-based quantitative real-time RT-PCR. Altered metabolites and gene expression were documented. RESULTS: Lipidomic analysis revealed decreased levels of triacylglycerols (TG), diacylglycerol (DG), fatty acids (FA), phosphatidylcholine (PC), lysophosphatidylinositols (LPI) and sphingomyelin (SM), and increased levels of bile acids (BA) and ceramides, likely promoting disc cell metabolism changing from glycolysis to fatty acid oxidation and following cell death. The gene expression profile of disc cells suggests LCN2 and LEAP2/GHRL as promising molecular therapeutic targets for disc degeneration and demonstrates the expression of genes related to inflammation (NOS2, COX2, IL-6, IL-8, IL-1ß, and TNF-α) or encoding adipokines (PGRN, NAMPT, NUCB2, SERPINE2, and RARRES2), matrix metalloproteinases (MMP9 and MMP13), and vascular adhesion molecules (VCAM1). CONCLUSIONS: Altogether, the presented results disclose the NP and AF cell biology changes from healthy to degenerated discs, allowing the identification of promising molecular therapeutic targets for intervertebral disc degeneration. CLINICAL SIGNIFICANCE: Our results are relevant to improving current biological-based strategies aiming to repair IVD by restoring cellular lipid metabolites as well as adipokines homeostasis. Ultimately, our results will be valuable for successful, long-lasting relief of painful IVDD.

Monomeric CRP regulates inflammatory responses in human intervertebral disc cells.

CRP is an acute-phase protein that is used as a biomarker to follow severity and progression in infectious and inflammatory diseases. Its pathophysiological mechanisms of action are still poorly defined. CRP in its pentameric form exhibits weak anti-inflammatory activity. The monomeric isoform (mCRP) exerts potent proinflammatory properties in chondrocytes, endothelial cells, and leucocytes. No data exist regarding mCRP effects in human intervertebral disc (IVD) cells. This work aimed to verify the pathophysiological relevance of mCRP in the aetiology and/or progression of IVD degeneration. We investigated the effects of mCRP and the signalling pathways that are involved in cultured human primary annulus fibrosus (AF) cells and in the human nucleus pulposus (NP) immortalized cell line HNPSV-1. We determined messenger RNA (mRNA) and protein levels of relevant factors involved in inflammatory responses, by quantitative real-time polymerase chain reaction (RT-qPCR) and western blot. We also studied the presence of mCRP in human AF and NP tissues by immunohistochemistry. We demonstrated that mCRP increases nitric oxide synthase 2 (NOS2), cyclooxygenase 2 (COX2), matrix metalloproteinase 13 (MMP13), vascular cell adhesion molecule 1 (VCAM1), interleukin (IL)-6, IL-8, and Lipocalin 2 (LCN2) expression in human AF and NP cells. We also showed that nuclear factor-κß (NF-κß), extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphoinositide 3-kinase (PI3K) are at play in the intracellular signalling of mCRP. Finally, we demonstrated the presence of mCRP in human AF and NP tissues. Our results indicate, for the first time, that mCRP can be localized in IVD tissues, where it triggers a proinflammatory and catabolic state in degenerative and healthy IVD cells, and that NF-κß signalling may be implicated in the mediation of this mCRP-induced state.

Progranulin in Musculoskeletal Inflammatory and Degenerative Disorders, Focus on Rheumatoid Arthritis, Lupus and Intervertebral Disc Disease: A Systematic Review.

Progranulin (PGRN) is a glycoprotein formed by 593 amino acids encoded by the GRN gene. It has an important role in immunity and inflammatory response, as well as in tissue recovery. Its role in musculoskeletal inflammatory diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and intervertebral disc degeneration disease (IVDD), is, nowadays, an important target to investigate. The objective of this review is to systematically sum up all the recent findings concerning PGRN as a target in the development and resolution of the inflammatory diseases. PubMed was examined with the terms combinations (Progranulin) AND (Lupus Erythematosus, Systemic), (Progranulin) AND (Arthritis, Rheumatoid), and (Progranulin) AND (Intervertebral Disc Degeneration). PubMed was examined with the terms combinations (Atsttrin) AND (Lupus Erythematosus, Systemic), (Atsttrin) AND (Arthritis, Rheumatoid), and (Atsttrin) AND (Intervertebral Disc Degeneration). Moreover, research through Web of Science was performed searching the same items. The inclusion criteria were: studies whose main topic were progranulin, or atsttrin, with emphasis on the three selected diseases. On the other hand, the exclusion criteria were studies that only focused on diseases not related to RA, lupus or IVDD, in addition to the previous published literature reviews. Since few results were obtained, we did not filter by year. The records assessed for eligibility were 23, including all the studies with the information in state of art of progranulin and its capability to be a potential target or treatment for each one of the selected diseases. As these results are descriptive and not clinical trials, we did not perform risk of bias methods. Within these results, many studies have shown an anti-inflammatory activity of PGRN in RA. PGRN levels in serum and synovial fluids in RA patients were reported higher than controls. On the other hand, serum levels were directly correlated with SLE disease activity index, suggesting an important role of PGRN as a player in the progression of inflammatory diseases and a therapeutical approach for the recovery. This review has some limitations due to the small number of studies in this regard; therefore, we highlight the importance and the necessity of further investigation. No external funding was implicated in this systematical review.

Adipokines as targets in musculoskeletal immune and inflammatory diseases.

Adipokines are the principal mediators in adipose signaling. Nevertheless, besides their role in energy storage, these molecules can be produced by other cells, such as immune cells or chondrocytes. Given their pleiotropic effects, research over the past few years has also focused on musculoskeletal diseases, showing that these adipokines might have relevant roles in worsening the disease or improving the treatment response. In this review, we summarize recent advances in our understanding of adipokines and their role in the most prevalent musculoskeletal immune and inflammatory disorders.

WISP-2 modulates the induction of inflammatory mediators and cartilage catabolism in chondrocytes.

Wnt-1 inducible signaling pathway protein 2 (WISP-2/CCN5) is a recently identified adipokine that has been described as an important mediator of canonical Wnt activation in adipogenic precursor cells. In osteoarthritis (OA), the most common form of arthritis, chondrocytes exhibit aberrant and increased production of pro-inflammatory mediators and matrix degrading enzymes such as IL-1ß and MMP-13. Although recent evidence suggests a role for Wnt signaling in OA physiopathology, little is known about the involvement of WISP-2 in cartilage degradation. In the present study, we determined the expression of WISP-2 in healthy and OA human chondrocytes. WISP-2 expression is modulated along chondrocyte differentiation and downregulated at the onset of hypertrophy by inflammatory mediators. We also investigated the effect of WISP-2 on cartilage catabolism and performed WISP-2 loss-of-function experiments using RNA interference technology in human T/C-28a2 immortalized chondrocytes. We demonstrated that recombinant human WISP-2 protein reduced IL-1ß-mediated chondrocyte catabolism, that IL-1ß and WNT/b-catenin signaling pathways are involved in rhWISP-2 protein and IL-1ß effects in human chondrocytes, and that WISP-2 has a regulatory role in attenuating the catabolic effects of IL-1ß in chondrocytes. Gene silencing of WISP-2 increased the induction of the catabolic markers MMP-13 and ADAMTS-5 and the inflammatory mediators IL-6 and IL-8 triggered by IL-1ß in human primary OA chondrocytes in a Wnt/ß-catenin dependent manner. In conclusion, here we have shown for the first time that WISP-2 may have relevant roles in modulating the turnover of extracellular matrix in the cartilage and that its downregulation may detrimentally alter the inflammatory environment in OA cartilage. We also proved the participation of Wnt/ß-catenin signaling pathway in these processes. Thus, targeting WISP-2 might represent a potential therapeutical approach for degenerative and/or inflammatory diseases of musculoskeletal system, such as osteoarthritis.

Leptin in Osteoarthritis and Rheumatoid Arthritis: Player or Bystander?

White adipose tissue (WAT) is a specialized tissue whose main function is lipid synthesis and triglyceride storage. It is now considered as an active organ secreting a plethora of hormones and cytokines namely adipokines. Discovered in 1994, leptin has emerged as a key molecule with pleiotropic functions. It is primarily recognized for its role in regulating energy homeostasis and food intake. Currently, further evidence suggests its potent role in reproduction, glucose metabolism, hematopoiesis, and interaction with the immune system. It is implicated in both innate and adaptive immunity, and it is reported to contribute, with other adipokines, in the cross-talking networks involved in the pathogenesis of chronic inflammation and immune-related diseases of the musculo-skeletal system such as osteoarthritis (OA) and rheumatoid arthritis (RA). In this review, we summarize the most recent findings concerning the involvement of leptin in immunity and inflammatory responses in OA and RA.

Analgesic and antiinflammatory effects of Nigella orientalis L. seeds fixed oil: Pharmacological potentials and molecular mechanisms.

Nigella species have been widely used in traditional medicine. The aim of this study was to evaluate the antiinflammatory and analgesic potentials of Nigella orientalis L. seeds fixed oil (NOO). The acetic acid writhing test and the formaldehyde-induced licking paw were performed to assess the analgesic activity of the oil. The antiinflammatory activity was first evaluated in vitro by the erythrocyte membrane stabilization then in vivo by xylene- and carrageenan-induced ear and paw edema, respectively. To further understand the molecular mechanism of action of the Nigella extract, lipopolysaccharide-activated RAW 264.7 macrophages were used. Nitric oxide (NO) production was measured by Griess reaction and cell viability by MTT assay. The gene and protein expression of inflammatory mediators were assessed by RT-PCR and western blot, respectively. NOO exerted a potent analgesic effect in in vivo models of writhing test and induced edema. The analyzed molecular mechanisms revealed a role for NO and prostaglandins as molecules mediating the pharmacological effects of the extract through a mechanism involving nuclear factor-κB and mitogen-activated protein kinases. This study demonstrates, for the first time, that the fixed oil of N. orientalis has strong antinociceptive and antiinflammatory properties and might be a promising agent for the treatment of certain inflammation-related diseases.

Asprosin in health and disease, a new glucose sensor with central and peripheral metabolic effects.

Adipose tissue malfunction leads to altered adipokine secretion which might consequently contribute to an array of metabolic diseases spectrum including obesity, diabetes mellitus, and cardiovascular disorders. Asprosin is a novel diabetogenic adipokine classified as a caudamin hormone protein. This adipokine is released from white adipose tissue during fasting and elicits glucogenic and orexigenic effects. Although white adipose tissue is the dominant source for this multitask adipokine, other tissues also may produce asprosin such as salivary glands, pancreatic B-cells, and cartilage. Significantly, plasma asprosin levels link to glucose metabolism, lipid profile, insulin resistance (IR), and ß-cell function. Indeed, asprosin exhibits a potent role in the metabolic process, induces hepatic glucose production, and influences appetite behavior. Clinical and preclinical research showed dysregulated levels of circulating asprosin in several metabolic diseases including obesity, type 2 diabetes mellitus (T2DM), polycystic ovarian syndrome (PCOS), non-alcoholic fatty liver (NAFLD), and several types of cancer. This review provides a comprehensive overview of the asprosin role in the etiology and pathophysiological manifestations of these conditions. Asprosin could be a promising candidate for both novel pharmacological treatment strategies and diagnostic tools, although developing a better understanding of its function and signaling pathways is still needed.

An Update on the Role of Leptin in the Immuno-Metabolism of Cartilage.

Since its discovery in 1994, leptin has been considered as an adipokine with pleiotropic effects. In this review, we summarize the actual information about the impact of this hormone on cartilage metabolism and pathology. Leptin signalling depends on the interaction with leptin receptor LEPR, being the long isoform of the receptor (LEPRb) the one with more efficient intracellular signalling. Chondrocytes express the long isoform of the leptin receptor and in these cells, leptin signalling, alone or in combination with other molecules, induces the expression of pro-inflammatory molecules and cartilage degenerative enzymes. Leptin has been shown to increase the proliferation and activation of immune cells, increasing the severity of immune degenerative cartilage diseases. Leptin expression in serum and synovial fluid are related to degenerative diseases such as osteoarthritis (OA), rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Inhibition of leptin signalling showed to have protective effects in these diseases showing the key role of leptin in cartilage degeneration.

Evaluation of Virola oleifera activity in musculoskeletal pathologies: Inhibition of human multiple myeloma cells proliferation and combination therapy with dexamethasone or bortezomib.

ETHNOPHARMACOLOGICAL RELEVANCE: Virola oleifera (Schott) A.C. Smith, Myristicaceae, has been widely used in traditional medicine in Brazil to treat rheumatic pain, joint tumours, skin diseases, halitosis, bronchial asthma, haemorrhoids, and intestinal worms. Recently, research data showed the antioxidant properties in several oxidative stress-related models. However, there is no experimental evidence supporting its potential use in managing rheumatic diseases and bone malignancies. AIMS OF THE STUDY: To evaluate the therapeutic potential of the resin from Virola oleifera in joint and bone diseases, namely arthritis, osteosarcoma, chondrosarcoma, and multiple myeloma. MATERIALS AND METHODS: To determine Virola oleifera resin (VO) effects on arthritis-associated inflammation and cartilage degradation, the LPS-induced NO production, and mRNA and protein expression of ADAMTS5, MMP13, COL2, and ACAN, were evaluated in chondrocytes (ATDC5 and TC28 cell lines). The cytotoxic effects of VO (0.05-50 µg/ml) on multiple myeloma (ARH-77), osteosarcoma (SAOS-2), and chondrosarcoma (SW-1353) cell lines were analysed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The VO effects, combined with dexamethasone or bortezomib, were evaluated in a multiple myeloma cell line. The mechanisms of VO, alone or in combination with bortezomib, were determined by cell cycle analysis through flow cytometry, while expression levels of p-Akt/Akt, p-ERK/ERK, p-p38/p38 MAPK, Bax, Bcl-2, and cleaved-caspase-3/caspase-3 proteins by Western blot. RESULTS: VO had no significant effect on LPS-induced NO production in chondrocytes at non-cytotoxic concentrations. VO treatment diminished the mRNA levels of metalloproteinases and ECM components; however, any significant effect was observed on the protein expression levels. The cell viability of a multiple myeloma cell line was strongly reduced by VO treatment in a dose- and time-dependent manner, while osteosarcoma and chondrosarcoma cell lines viability was significantly affected only by the highest dose assessed. In multiple myeloma cells, VO leads to G2/M cell cycle arrest. Furthermore, it synergizes with dexamethasone by increasing cell toxicity. Finally, VO reverts bortezomib activity by counteracting ERK1/2, Bax, and caspase-3 activation. CONCLUSIONS: The current work supports the ethnopharmacological use of Virola oleifera (Schott) A.C. Smith in bone and joint diseases, but there is no evidence for the amelioration of arthritis-associated inflammatory or catabolic processes. Our data also supports the potential use of Virola oleifera as adjuvant therapy to optimize the pharmacologic effects of current chemotherapeutic drugs. However, possible herb-drug interactions should be considered before clinical application.

Pharmacological Extracts and Molecules from Virola Species: Traditional Uses, Phytochemistry, and Biological Activity.

Virola is the largest genus of Myristicaceae in America, comprising about 60 species of medium-sized trees geographically spread from Mexico to southern Brazil. The plant species of this genus have been widely used in folk medicine for the treatment of several ailments, such as rheumatic pain, bronchial asthma, tumors in the joints, intestinal worms, halitosis, ulcers, and multiple infections, due to their pharmacological activity. This review presents an updated and comprehensive summary of Virola species, particularly their ethnomedicinal uses, phytochemistry, and biological activity, to support the safe medicinal use of plant extracts and provide guidance for future research. The Virola spp.'s ethnopharmacology, including in the treatment of stomach pain and gastric ulcers, as well as antimicrobial and tryponosomicidal activities, is attributable to the presence of a myriad of phytoconstituents, such as flavonoids, tannins, phenolic acids, lignans, arylalkanones, and sitosterol. Hence, such species yield potential leads or molecular scaffolds for the development of new pharmaceutical formulations, encouraging the elucidation of not-yet-understood action mechanisms and ascertaining their safety for humans.

CD133 Expression in Medullary Thyroid Cancer Cells Identifies Patients with Poor Prognosis.

CONTEXT: The identification of markers able to determine medullary thyroid cancer (MTC) patients at high-risk of disease progression is critical to improve their clinical management and outcome. Previous studies have suggested that expression of the stem cell marker CD133 is associated with MTC aggressiveness. OBJECTIVE: To evaluate CD133 impact on disease progression in MTC and explore the regulatory mechanisms leading to the upregulation of this protein in aggressive tumors. PATIENTS: We compiled a series of 74 MTCs with associated clinical data and characterized them for mutations in RET and RAS proto-oncogenes, presumed to be related with disease clinical behavior. RESULTS: We found that CD133 immunohistochemical expression was associated with adverse clinicopathological features and predicted a reduction in time to disease progression even when only RET-mutated cases were considered in the analysis (log-rank test P < 0.003). Univariate analysis for progression-free survival revealed CD133 expression and presence of tumor emboli in peritumoral blood vessels as the most significant prognostic covariates among others such as age, gender, and prognostic stage. Multivariate analysis identified both variables as independent factors of poor prognosis (hazard ratio = 16.6 and 2; P = 0.001 and 0.010, respectively). Finally, we defined hsa-miR-30a-5p, a miRNA downregulated in aggressive MTCs, as a CD133 expression regulator. Ectopic expression of hsa-miR-30a-5p in MZ-CRC-1 (RETM918T) cells significantly reduced CD133 mRNA expression. CONCLUSIONS: Our results suggest that CD133 expression may be a useful tool to identify MTC patients with poor prognosis, who may benefit from a more extensive primary surgical management and follow-up.

Hsa-miR-139-5p is a prognostic thyroid cancer marker involved in HNRNPF-mediated alternative splicing.

It is critical to identify biomarkers and functional networks associated with aggressive thyroid cancer to anticipate disease progression and facilitate personalized patient management. We performed miRNome sequencing of 46 thyroid tumors enriched with advanced disease patients with a median follow-up of 96 months. MiRNome profiles correlated with tumor-specific histopathological and molecular features, such as stromal cell infiltration and tumor driver mutation. Differential expression analysis revealed a consistent hsa-miR-139-5p downexpression in primary carcinomas from patients with recurrent/metastatic disease compared to disease-free patients, sustained in paired local metastases and validated in publicly available thyroid cancer series. Exogenous expression of hsa-miR-139-5p significantly reduced migration and proliferation of anaplastic thyroid cancer cells. Proteomic analysis indicated RICTOR, SMAD2/3 and HNRNPF as putative hsa-miR-139-5p targets in our cell system. Abundance of HNRNPF mRNA, encoding an alternative splicing factor involved in cryptic exon inclusion/exclusion, inversely correlated with hsa-miR-139-5p expression in human tumors. RNA sequencing analysis revealed 174 splicing events differentially regulated upon HNRNPF repression in our cell system, affecting genes involved in RTK/RAS/MAPK and PI3K/AKT/MTOR signaling cascades among others. These results point at the hsa-miR-139-5p/HNRNPF axis as a novel regulatory mechanism associated with the modulation of major thyroid cancer signaling pathways and tumor virulence.

Integrative multi-omics analysis identifies a prognostic miRNA signature and a targetable miR-21-3p/TSC2/mTOR axis in metastatic pheochromocytoma/paraganglioma.

Rationale: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors that present variable outcomes. To date, no effective therapies or reliable prognostic markers are available for patients who develop metastatic PPGL (mPPGL). Our aim was to discover robust prognostic markers validated through in vitro models, and define specific therapeutic options according to tumor genomic features. Methods: We analyzed three PPGL miRNome datasets (n=443), validated candidate markers and assessed them in serum samples (n=36) to find a metastatic miRNA signature. An integrative study of miRNome, transcriptome and proteome was performed to find miRNA targets, which were further characterized in vitro. Results: A signature of six miRNAs (miR-21-3p, miR-183-5p, miR-182-5p, miR-96-5p, miR-551b-3p, and miR-202-5p) was associated with metastatic risk and time to progression. A higher expression of five of these miRNAs was also detected in PPGL patients' liquid biopsies compared with controls. The combined expression of miR-21-3p/miR-183-5p showed the best power to predict metastasis (AUC=0.804, P=4.67·10-18), and was found associated in vitro with pro-metastatic features, such as neuroendocrine-mesenchymal transition phenotype, and increased cell migration rate. A pan-cancer multi-omic integrative study correlated miR-21-3p levels with TSC2 expression, mTOR pathway activation, and a predictive signature for mTOR inhibitor-sensitivity in PPGLs and other cancers. Likewise, we demonstrated in vitro a TSC2 repression and an enhanced rapamycin sensitivity upon miR-21-3p expression. Conclusions: Our findings support the assessment of miR-21-3p/miR-183-5p, in tumors and liquid biopsies, as biomarkers for risk stratification to improve the PPGL patients' management. We propose miR-21-3p to select mPPGL patients who may benefit from mTOR inhibitors.