Circulating microRNAs and isomiRs as biomarkers for the initial insult and epileptogenesis in four experimental epilepsy models: The EPITARGET study.

OBJECTIVE: Structural epilepsies can manifest months or years after the occurrence of an initial epileptogenic insult, making them amenable for secondary prevention. However, development of preventive treatments has been challenged by a lack of biomarkers for identifying the subset of individuals with the highest risk of epilepsy after the epileptogenic insult. METHODS: Four different rat models of epileptogenesis were investigated to identify differentially expressed circulating microRNA (miRNA) and isomiR profiles as biomarkers for epileptogenesis. Plasma samples were collected on day 2 and day 9 during the latency period from animals that did or did not develop epilepsy during long-term video-electroencephalographic monitoring. miRNAs and isomiRs were identified and measured in an unsupervised manner, using a genome-wide small RNA sequencing platform. Receiver operating characteristic analysis was performed to determine the performance of putative biomarkers. RESULTS: Two days after an epileptogenic insult, alterations in the levels of several plasma miRNAs and isomiRs predicted epileptogenesis in a model-specific manner. One miRNA, miR-3085, showed good sensitivity (but low specificity) as a prognostic biomarker for epileptogenesis in all four models (area under the curve = .729, sensitivity = 83%, specificity = 64%, p < .05). SIGNIFICANCE: Identified plasma miRNAs and isomiRs are mostly etiology-specific rather than common prognostic biomarkers of epileptogenesis. These data imply that in preclinical and clinical studies, it may be necessary to identify specific biomarkers for different epilepsy etiologies. Importantly, circulating miRNAs like miR-3085 with high negative predictive value for epileptogenesis in different etiologies could be useful candidates for initial screening purposes of epileptogenesis risk.

Epigenetic age acceleration in hematopoietic stem cell transplantation.

Not available.

MicroRNA dysregulation in ataxia telangiectasia.

Introduction: Ataxia telangiectasia (AT) is a rare disorder characterized by neurodegeneration, combined immunodeficiency, a predisposition to malignancies, and high clinical variability. Profiling of microRNAs (miRNAs) may offer insights into the underlying mechanisms of complex rare human diseases, as miRNAs play a role in various biological functions including proliferation, differentiation, and DNA repair. In this study, we investigate the differential expression of miRNAs in samples from AT patients to identify miRNA patterns and analyze how these patterns are related to the disease. Methods: We enrolled 20 AT patients (mean age 17.7 ± 9.6 years old) and collected clinical and genetic data. We performed short non-coding RNA-seq analysis on peripheral blood mononuclear cells (PBMCs) and fibroblasts to compare the miRNA expression profile between AT patients and controls. Results: We observed 42 differentially expressed (DE)-miRNAs in blood samples and 26 in fibroblast samples. Among these, three DE-miRNAs, miR-342-3p, miR-30a-5p, and miR-195-5p, were further validated in additional AT samples, confirming their dysregulation. Discussion: We identified an AT-related miRNA signature in blood cells and fibroblast samples collected from a group of AT patients. We also predicted several dysregulated pathways, primarily related to cancer, immune system control, or inflammatory processes. The findings suggest that miRNAs may provide insights into the pathophysiology and tumorigenesis of AT and have the potential to serve as useful biomarkers in cancer research.

Combining gemcitabine and MSC delivering soluble TRAIL to target pancreatic adenocarcinoma and its stroma.

Pancreatic ductal adenocarcinoma (PDAC) still has a poor response to therapies, partly due to their cancer-associated fibroblasts (CAFs). Here, we investigate the synergistic impact of a combinatory approach between a known chemotherapy agent, such as gemcitabine (GEM), and gene-modified human mesenchymal stromal/stem cells (MSCs) secreting the pro-apoptotic soluble (s)TRAIL (sTRAIL MSCs) on both PDAC cells and CAFs. The combo significantly impacts on PDAC survival in 2D and 3D models. In orthotopic xenograft models, GEM and sTRAIL MSCs induce tumor architecture shredding with a reduction of CK7- and CK8/18-positive cancer cells and the abrogation of spleen metastases. A cytotoxic effect on primary human CAFs is also observed along with an alteration of their transcriptome and a reduction of the related desmoplasia. Collectively, we demonstrate a promising therapeutic profile of combining GEM and sTRAIL MSCs to target both tumoral and stromal compartments in PDAC.

Targeting Isocitrate Dehydrogenase (IDH) in Solid Tumors: Current Evidence and Future Perspectives.

The isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) enzymes are involved in key metabolic processes in human cells, regulating differentiation, proliferation, and oxidative damage response. IDH mutations have been associated with tumor development and progression in various solid tumors such as glioma, cholangiocarcinoma, chondrosarcoma, and other tumor types and have become crucial markers in molecular classification and prognostic assessment. The intratumoral and serum levels of D-2-hydroxyglutarate (D-2-HG) could serve as diagnostic biomarkers for identifying IDH mutant (IDHmut) tumors. As a result, an increasing number of clinical trials are evaluating targeted treatments for IDH1/IDH2 mutations. Recent studies have shown that the focus of these new therapeutic strategies is not only the neomorphic activity of the IDHmut enzymes but also the epigenetic shift induced by IDH mutations and the potential role of combination treatments. Here, we provide an overview of the current knowledge about IDH mutations in solid tumors, with a particular focus on available IDH-targeted treatments and emerging results from clinical trials aiming to explore IDHmut tumor-specific features and to identify the clinical benefit of IDH-targeted therapies and their combination strategies. An insight into future perspectives and the emerging roles of circulating biomarkers and radiomic features is also included.

Synergic activity of FGFR2 and MEK inhibitors in the treatment of FGFR2-amplified cancers of unknown primary.

Patients with cancer of unknown primary (CUP) carry the double burden of an aggressive disease and reduced access to therapies. Experimental models are pivotal for CUP biology investigation and drug testing. We derived two CUP cell lines (CUP#55 and #96) and corresponding patient-derived xenografts (PDXs), from ascites tumor cells. CUP cell lines and PDXs underwent histological, immune-phenotypical, molecular, and genomic characterization confirming the features of the original tumor. The tissue-of-origin prediction was obtained from the tumor microRNA expression profile and confirmed by single-cell transcriptomics. Genomic testing and fluorescence in situ hybridization analysis identified FGFR2 gene amplification in both models, in the form of homogeneously staining region (HSR) in CUP#55 and double minutes in CUP#96. FGFR2 was recognized as the main oncogenic driver and therapeutic target. FGFR2-targeting drug BGJ398 (infigratinib) in combination with the MEK inhibitor trametinib proved to be synergic and exceptionally active, both in vitro and in vivo. The effects of the combined treatment by single-cell gene expression analysis revealed a remarkable plasticity of tumor cells and the greater sensitivity of cells with epithelial phenotype. This study brings personalized therapy closer to CUP patients and provides the rationale for FGFR2 and MEK targeting in metastatic tumors with FGFR2 pathway activation.

CAR+ extracellular vesicles predict ICANS in patients with B cell lymphomas treated with CD19-directed CAR T cells.

BACKGROUNDPredicting immune effector cell-associated neurotoxicity syndrome (ICANS) in patients infused with CAR T cells is still a conundrum. This complication, thought to be consequent to CAR T cell activation, arises a few days after infusion, when circulating CAR T cells are scarce and specific CAR T cell-derived biomarkers are lacking.METHODSCAR+ extracellular vesicle (CAR+EV) release was assessed in human CD19.CAR T cells cocultured with CD19+ target cells. A prospective cohort of 100 patients with B cell lymphoma infused with approved CD19.CAR T cell products was assessed for plasma CAR+EVs as biomarkers of in vivo CD19.CAR T cell activation. Human induced pluripotent stem cell-derived (iPSC-derived) neural cells were used as a model for CAR+EV-induced neurotoxicity.RESULTSIn vitro release of CAR+EVs occurs within 1 hour after target engagement. Plasma CAR+EVs are detectable 1 hour after infusion. A concentration greater than 132.8 CAR+EVs/µL at hour +1 or greater than 224.5 CAR+EVs/µL at day +1 predicted ICANS in advance of 4 days, with a sensitivity and a specificity outperforming other ICANS predictors. ENO2+ nanoparticles were released by iPSC-derived neural cells upon CAR+EV exposure and were increased in plasma of patients with ICANS.CONCLUSIONPlasma CAR+EVs are an immediate signal of CD19.CAR T cell activation, are suitable predictors of neurotoxicity, and may be involved in ICANS pathogenesis.TRIAL REGISTRATIONNCT04892433, NCT05807789.FUNDINGLife Science Hub-Advanced Therapies (financed by Health Ministry as part of the National Plan for Complementary Investments to the National Recovery and Resilience Plan [NRRP]: E.3 Innovative health ecosystem for APC fees and immunomonitoring).

Association of miR-146a-5p and miR-21-5p with Prognostic Features in Melanomas.

BACKGROUND: Cutaneous melanoma (CM) is one of the most lethal tumors among skin cancers and its incidence is rising worldwide. Recent data support the role of microRNAs (miRNAs) in melanoma carcinogenesis and their potential use as disease biomarkers. METHODS: We quantified the expression of miR-146a-5p and miR-21-5p in 170 formalin-fixed paraffin embedded (FFPE) samples of CM, namely 116 superficial spreading melanoma (SSM), 26 nodular melanoma (NM), and 28 lentigo maligna melanoma (LMM). We correlated miRNA expression with specific histopathologic features including Breslow thickness (BT), histological subtype, ulceration and regression status, and mitotic index. RESULTS: miR-146a-5p and miR-21-5p were significantly higher in NM compared to SSM and LMM. The positive correlation between miR-146a-5p and miR-21-5p expression and BT was confirmed for both miRNAs in SSM. Considering the ulceration status, we assessed that individual miR-21-5p expression was significantly higher in ulcerated CMs. The increased combined expression of the two miRNAs was strongly associated with ulceration (p = 0.0093) and higher mitotic rate (≥1/mm2) (p = 0.0005). We demonstrated that the combination of two-miRNA expression and prognostic features (BT and ulceration) can better differentiate cutaneous melanoma prognostic groups, considering overall survival and time-to-relapse clinical outcomes. Specifically, miRNA expression can further stratify prognostic groups among patients with BT ≥ 0.8 mm but without ulceration. Our findings provide further insights into the characterization of CM with specific prognostic features. The graphical abstract was created with BioRender.com.

miRNA patterns in male LUSC patients - the 3-way mirror: Tissue, plasma and exosomes.

Lung cancer remains one of the leading causes of cancer-related deaths worldwide. It is classified into two main histological groups: non-small cell lung cancer (NSCLC) and small cell lung cancer. Improving the outcome of cancer patients could be possible by enhancing the early diagnosis. In the current study, we evaluated the levels of three microRNAs - miR-21-5p, miR-155-5p, and miR-181a-5p in tumor (TT) vs adjacent normal tissue (NT), as well as their expression levels in plasma and extracellular vesicles (EVs) from plasma in lung squamous cell carcinoma (LUSC) male patients vs healthy individuals as means to identify a panel of miRNAs that could serve as novel biomarkers for the diagnosis of LUSC in male patients. Matched paired tissue samples from male LUSC (n=40) patients were used for miRNA expression analysis. MiR-21-5p and miR-155-5p in tumor tissue were overexpressed, while underexpression of miR-181a-5p was observed in LUSC TT vs NT. These results were further validated in the TCGA LUSC dataset, considering 279 male samples. These alterations of miR-21-5p, miR-181a-5p, and miR-155-5p in tumor tissue are also present in plasma and plasma extracellular vesicles in LUSC male patients. In addition, ROC curves were performed to assess the sensitivity and specificity of different combinations of these miRNAs, confirming a high diagnostic accuracy for LUSC of up to 88 % in male subjects. The expression levels in tissue samples and the abundance in plasma and plasma EVs of the three miRNAs combined - miR-21-5p, miR-155-5p and miR-181a-5p - could be considered for further studies on biomarkers for the early detection of LUSC in male subjects.

Non-coding RNAs in disease: from mechanisms to therapeutics.

Non-coding RNAs (ncRNAs) are a heterogeneous group of transcripts that, by definition, are not translated into proteins. Since their discovery, ncRNAs have emerged as important regulators of multiple biological functions across a range of cell types and tissues, and their dysregulation has been implicated in disease. Notably, much research has focused on the link between microRNAs (miRNAs) and human cancers, although other ncRNAs, such as long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), are also emerging as relevant contributors to human disease. In this Review, we summarize our current understanding of the roles of miRNAs, lncRNAs and circRNAs in cancer and other major human diseases, notably cardiovascular, neurological and infectious diseases. Further, we discuss the potential use of ncRNAs as biomarkers of disease and as therapeutic targets.

MiR-4649-5p acts as a tumor-suppressive microRNA in triple negative breast cancer by direct interaction with PIP5K1C, thereby potentiating growth-inhibitory effects of the AKT inhibitor capivasertib.

BACKGROUND: Triple negative breast cancer (TNBC) is a particularly aggressive and difficult-to-treat subtype of breast cancer that requires the development of novel therapeutic strategies. To pave the way for such developments it is essential to characterize new molecular players in TNBC. MicroRNAs (miRNAs) constitute interesting candidates in this regard as they are frequently deregulated in cancer and contribute to numerous aspects of carcinogenesis. METHODS AND RESULTS: Here, we discovered that miR-4649-5p, a miRNA yet uncharacterized in breast cancer, is associated with better overall survival of TNBC patients. Ectopic upregulation of the otherwise very low endogenous expression levels of miR-4646-5p significantly decreased the growth, proliferation, and migration of TNBC cells. By performing whole transcriptome analysis and physical interaction assays, we were able to identify the phosphatidylinositol phosphate kinase PIP5K1C as a direct target of miR-4649-5p. Downregulation or pharmacologic inhibition of PIP5K1C phenocopied the growth-reducing effects of miR-4649-5p. PIP5K1C is known to play an important role in migration and cell adhesion, and we could furthermore confirm its impact on downstream PI3K/AKT signaling. Combinations of miR-4649-5p upregulation and PIP5K1C or AKT inhibition, using the pharmacologic inhibitors UNC3230 and capivasertib, respectively, showed additive growth-reducing effects in TNBC cells. CONCLUSION: In summary, miR-4649-5p exerts broad tumor-suppressive effects in TNBC and shows potential for combined therapeutic approaches targeting the PIP5K1C/PI3K/AKT signaling axis.

Settling the uncertainty about unconventional circulating tumor cells: Epithelial-to-mesenchymal transition, cell fusion and trogocytosis.

Circulating tumor cells (CTCs) were first described 150 years ago. The so-called "classical" CTC populations (EpCAM+/CK+/CD45-) have been fully characterized and proposed as the most representative CTC subset, with clinical relevance. Nonetheless, other "atypical" or "unconventional" CTCs have also been identified, and their critical role in metastasis formation was demonstrated. In this chapter we illustrate the studies that led to the discovery of unconventional CTCs, defined as CTCs that display both epithelial and mesenchymal markers, or both cancer and immune markers, also in the form of hybrid cancer-immune cells. We also present biological explanations for the origin of these unconventional CTCs: epithelial to mesenchymal transition, cell-cell fusion and trogocytosis. We believe that a deeper knowledge on the biology of CTCs is needed to fully elucidate their role in cancer progression and their use as cancer biomarkers.

MiR-494 induces metabolic changes through G6pc targeting and modulates sorafenib response in hepatocellular carcinoma.

BACKGROUND: Metabolic reprogramming is a well-known marker of cancer, and it represents an early event during hepatocellular carcinoma (HCC) development. The recent approval of several molecular targeted agents has revolutionized the management of advanced HCC patients. Nevertheless, the lack of circulating biomarkers still affects patient stratification to tailored treatments. In this context, there is an urgent need for biomarkers to aid treatment choice and for novel and more effective therapeutic combinations to avoid the development of drug-resistant phenotypes. This study aims to prove the involvement of miR-494 in metabolic reprogramming of HCC, to identify novel miRNA-based therapeutic combinations and to evaluate miR-494 potential as a circulating biomarker. METHODS: Bioinformatics analysis identified miR-494 metabolic targets. QPCR analysis of glucose 6-phosphatase catalytic subunit (G6pc) was performed in HCC patients and preclinical models. Functional analysis and metabolic assays assessed G6pc targeting and miR-494 involvement in metabolic changes, mitochondrial dysfunction, and ROS production in HCC cells. Live-imaging analysis evaluated the effects of miR-494/G6pc axis in cell growth of HCC cells under stressful conditions. Circulating miR-494 levels were assayed in sorafenib-treated HCC patients and DEN-HCC rats. RESULTS: MiR-494 induced the metabolic shift of HCC cells toward a glycolytic phenotype through G6pc targeting and HIF-1A pathway activation. MiR-494/G6pc axis played an active role in metabolic plasticity of cancer cells, leading to glycogen and lipid droplets accumulation that favored cell survival under harsh environmental conditions. High miR-494 serum levels associated with sorafenib resistance in preclinical models and in a preliminary cohort of HCC patients. An enhanced anticancer effect was observed for treatment combinations between antagomiR-494 and sorafenib or 2-deoxy-glucose in HCC cells. CONCLUSIONS: MiR-494/G6pc axis is critical for the metabolic rewiring of cancer cells and associates with poor prognosis. MiR-494 deserves attention as a candidate biomarker of likelihood of response to sorafenib to be tested in future validation studies. MiR-494 represents a promising therapeutic target for combination strategies with sorafenib or metabolic interference molecules for the treatment of HCC patients who are ineligible for immunotherapy.

Anti-miR-93-5p therapy prolongs sepsis survival by restoring the peripheral immune response.

Sepsis remains a leading cause of death for humans and currently has no pathogenesis-specific therapy. Hampered progress is partly due to a lack of insight into deep mechanistic processes. In the past decade, deciphering the functions of small noncoding miRNAs in sepsis pathogenesis became a dynamic research topic. To screen for new miRNA targets for sepsis therapeutics, we used samples for miRNA array analysis of PBMCs from patients with sepsis and control individuals, blood samples from 2 cohorts of patients with sepsis, and multiple animal models: mouse cecum ligation puncture-induced (CLP-induced) sepsis, mouse viral miRNA challenge, and baboon Gram+ and Gram- sepsis models. miR-93-5p met the criteria for a therapeutic target, as it was overexpressed in baboons that died early after induction of sepsis, was downregulated in patients who survived after sepsis, and correlated with negative clinical prognosticators for sepsis. Therapeutically, inhibition of miR-93-5p prolonged the overall survival of mice with CLP-induced sepsis, with a stronger effect in older mice. Mechanistically, anti-miR-93-5p therapy reduced inflammatory monocytes and increased circulating effector memory T cells, especially the CD4+ subset. AGO2 IP in miR-93-KO T cells identified important regulatory receptors, such as CD28, as direct miR-93-5p target genes. In conclusion, miR-93-5p is a potential therapeutic target in sepsis through the regulation of both innate and adaptive immunity, with possibly a greater benefit for elderly patients than for young patients.

A Systematic Review of Diagnostic and Prognostic Biomarkers for Head and Neck Cancer of Unknown Primary: An Unmet Clinical Need.

Head and neck cancer of unknown primary (HNCUP) is defined as cervical lymph node metastases without a detectable primary tumor. The management of these patients presents a challenge to clinicians since guidelines in the diagnosis and treatment of HNCUP remain controversial. An accurate diagnostic workup is fundamental for the search for the hidden primary tumor to allow the best adequate treatment strategy. The purpose of this systematic review is to present the currently available data about the diagnostic and prognostic molecular biomarkers for HNCUP. Systematic research in an electronic database was conducted using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) protocol and identified 704 articles, of which 23 studies were selected and included in the analysis. Fourteen studies investigated HNCUP diagnostic biomarkers and focused on the human papilloma virus (HPV) and the Epstein-Barr virus (EBV) due to the strong associations with oropharyngeal cancer and nasopharyngeal cancer, respectively. HPV status was shown to possess prognostic value, correlating with longer disease-free survival and overall survival. HPV and EBV are the only available HNCUP biomarkers, and they are already used in clinical practice. A better characterization of the molecular profiling and the development of tissue-of-origin classifiers are necessary to improve the diagnosis, staging, and therapeutic management of patients with HNCUP.

MicroRNAs as regulators of tumor metabolism.

Cancer cells reprogram their metabolism to support their growth. Since the discovery of the Warburg effect, several other metabolic alterations and metabolites have been described in cancer cells, including lactate, glutamine, and lipid metabolism reprogramming. Together these alterations provide rapidly dividing tumor cells with metabolic intermediates needed for nucleotide, protein, and fatty acid biosynthesis. MicroRNAs are a class of small non-coding RNAs involved in the regulation of virtually all biological pathways. Altered microRNA expression patterns are associated with the onset and development of several diseases, including cancer. Tumor suppressor microRNAs targeting molecules involved in tumor metabolism are frequently downregulated in cancers. Therefore, microRNAs can serve as potential tumor biomarkers and also represent interesting therapeutic targets. This review summarizes recent findings about microRNAs involved in the regulation of tumor metabolism.

ARID1A in cancer: Friend or foe?

ARID1A belongs to a class of chromatin regulatory proteins that function by maintaining accessibility at most promoters and enhancers, thereby regulating gene expression. The high frequency of ARID1A alterations in human cancers has highlighted its significance in tumorigenesis. The precise role of ARID1A in cancer is highly variable since ARID1A alterations can have a tumor suppressive or oncogenic role, depending on the tumor type and context. ARID1A is mutated in about 10% of all tumor types including endometrial, bladder, gastric, liver, biliopancreatic cancer, some ovarian cancer subtypes, and the extremely aggressive cancers of unknown primary. Its loss is generally associated with disease progression more often than onset. In some cancers, ARID1A loss is associated with worse prognostic features, thus supporting a major tumor suppressive role. However, some exceptions have been reported. Thus, the association of ARID1A genetic alterations with patient prognosis is controversial. However, ARID1A loss of function is considered conducive for the use of inhibitory drugs which are based on synthetic lethality mechanisms. In this review we summarize the current knowledge on the role of ARID1A as tumor suppressor or oncogene in different tumor types and discuss the strategies for treating ARID1A mutated cancers.

Disclosing quantitative RT-PCR raw data during manuscript submission: a call for action.

Accuracy and transparency of scientific data are becoming more and more relevant with the increasing concern regarding the evaluation of data reproducibility in many research areas. This concern is also true for quantifying coding and noncoding RNAs, with the remarkable increase in publications reporting RNA profiling and sequencing studies. To address the problem, we propose the following recommendations: (a) accurate documentation of experimental procedures in Materials and methods (and not only in the supplementary information, as many journals have a strict mandate for making Materials and methods as visible as possible in the main text); (b) submission of RT-qPCR raw data for all experiments reported; and (c) adoption of a unified, simple format for submitted RT-qPCR raw data. The Real-time PCR Data Essential Spreadsheet Format (RDES) was created for this purpose.

Diagnostic and Prognostic Value of microRNAs in Patients with Laryngeal Cancer: A Systematic Review.

Laryngeal squamous cell cancer (LSCC) is one of the most common malignant tumors of the head and neck region, with a poor survival rate (5-year overall survival 50-80%) as a consequence of an advanced-stage diagnosis and high recurrence rate. Tobacco smoking and alcohol abuse are the main risk factors of LSCC development. An early diagnosis of LSCC, a prompt detection of recurrence and a more precise monitoring of the efficacy of different treatment modalities are currently needed to reduce the mortality. Therefore, the identification of effective diagnostic and prognostic biomarkers for LSCC is crucial to guide disease management and improve clinical outcomes. In the past years, a dysregulated expression of small non-coding RNAs, including microRNAs (miRNAs), has been reported in many human cancers, including LSCC, and many miRNAs have been explored for their diagnostic and prognostic potential and proposed as biomarkers. We searched electronic databases for original papers that were focused on miRNAs and LSCC, using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) protocol. According to the outcome, 566 articles were initially screened, of which 177 studies were selected and included in the analysis. In this systematic review, we provide an overview of the current literature on the function and the potential diagnostic and prognostic role of tissue and circulating miRNAs in LSCC.