ABSTRACT
Long noncoding RNAs (lncRNAs) can drive tumorigenesis and are susceptible to therapeutic intervention. Here, we used a large-scale CRISPR interference viability screen to interrogate cell-growth dependency to lncRNA genes in multiple myeloma (MM) and identified a prominent role for the miR-17-92 cluster host gene (MIR17HG). We show that an MIR17HG-derived lncRNA, named lnc-17-92, is the main mediator of cell-growth dependency acting in a microRNA- and DROSHA-independent manner. Lnc-17-92 provides a chromatin scaffold for the functional interaction between c-MYC and WDR82, thus promoting the expression of ACACA, which encodes the rate-limiting enzyme of de novo lipogenesis acetyl-coA carboxylase 1. Targeting MIR17HG pre-RNA with clinically applicable antisense molecules disrupts the transcriptional and functional activities of lnc-17-92, causing potent antitumor effects both in vitro and in vivo in 3 preclinical animal models, including a clinically relevant patient-derived xenograft NSG mouse model. This study establishes a novel oncogenic function of MIR17HG and provides potent inhibitors for translation to clinical trials.
Subject(s)
MicroRNAs , Multiple Myeloma , RNA, Long Noncoding , Humans , Animals , Mice , RNA, Long Noncoding/genetics , Multiple Myeloma/genetics , Chromatin , MicroRNAs/metabolism , Cell Proliferation , Gene Expression Regulation, NeoplasticABSTRACT
BACKGROUND: ANGPTL3 (angiopoietin-like protein 3) is a circulating protein with a key role in maintaining lipoprotein homeostasis. A monoclonal antibody against ANGPTL3 is an approved and well-tolerated treatment to reduce lipoproteins in familial hypercholesterolemia homozygotes. However, the reduction of hepatic ANGPTL3 synthesis using an antisense oligonucleotide unexpectedly resulted in a dose-dependent increase in liver lipid content and circulating transaminases, resulting in the termination of the clinical trial. Meanwhile, the use of silencing RNAs remains an area of active investigation. Our study sought to investigate whether intracellular downregulation of ANGPTL3 may lead to a primary increase in neutral lipids within the hepatocyte. METHODS: We downregulated ANGPTL3 by silencing RNA in primary human hepatocytes 3-dimensional spheroids, HepG2/LX-2 3-dimensional spheroids, and in HepG2, Hep3B2, and Huh7 cultured in 2 dimensions. RESULTS: ANGPTL3 downregulation increased neutral lipids in all models investigated. Interestingly, ANGPTL3 induced lower intracellular deiodinase type 1 protein levels resulting in a reduction in beta-oxidation and causing an increase in triglycerides stored in lipid droplets. CONCLUSIONS: In conclusion, intracellular ANGPTL3 downregulation by silencing RNA led to an increase in triglycerides content due to a reduction in energy substrate utilization resembling a primary intracellular hepatocyte hypothyroidism.
Subject(s)
Angiopoietin-Like Protein 3 , Angiopoietin-like Proteins , Down-Regulation , Energy Metabolism , Hepatocytes , RNA Interference , Triglycerides , Humans , Angiopoietin-Like Protein 3/genetics , Angiopoietin-Like Protein 3/metabolism , Angiopoietin-like Proteins/metabolism , Angiopoietin-like Proteins/genetics , Angiopoietins/metabolism , Angiopoietins/genetics , Energy Metabolism/genetics , Hep G2 Cells , Hepatocytes/metabolism , Lipid Metabolism , Transfection , Triglycerides/metabolismABSTRACT
Exercise may differently affect the expression of key molecular markers, including skeletal muscle and circulating miRNAs, involved in cellular and metabolic pathways' regulation in healthy individuals and in patients suffering from non-communicable diseases (NCDs). Epigenetic factors are emerging as potential therapeutic biomarkers in the prognosis and treatment of NCDs and important epigenetic factors, miRNAs, play a crucial role in cellular pathways. This systematic review aims to underline the potential link between changes in miRNA expression after different types of physical activity/exercise in some populations affected by NCDs. In June 2023, we systematically investigated the following databases: PubMed, MEDLINE, Scopus, and Web of Science, on the basis of our previously established research questions and following the PRISMA guidelines. The risk of bias and quality assessment were, respectively, covered by ROB2 and the Newcastle Ottawa scale. Of the 1047 records extracted from the initial search, only 29 studies were found to be eligible. In these studies, the authors discuss the association between exercise-modulated miRNAs and NCDs. The NCDs included in the review are cancer, cardiovascular diseases (CVDs), chronic obstructive pulmonary disease (COPD), and type 2 diabetes mellitus (T2DM). We evidenced that miR-146, miR-181, miR-133, miR-21, and miRNA-1 are the most reported miRNAs that are modulated by exercise. Their expression is associated with an improvement in health markers and they may be a potential target in terms of the development of future therapeutic tools.
Subject(s)
Exercise , MicroRNAs , Noncommunicable Diseases , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , Gene Expression Regulation , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/metabolism , Cardiovascular Diseases/genetics , Cardiovascular Diseases/metabolism , Neoplasms/genetics , Neoplasms/metabolismABSTRACT
The identification of biomarkers for predicting inter-individual sorafenib response variability could allow hepatocellular carcinoma (HCC) patient stratification. SNPs in angiogenesis- and drug absorption, distribution, metabolism, and excretion (ADME)-related genes were evaluated to identify new potential predictive biomarkers of sorafenib response in HCC patients. Five known SNPs in angiogenesis-related genes, including VEGF-A, VEGF-C, HIF-1a, ANGPT2, and NOS3, were investigated in 34 HCC patients (9 sorafenib responders and 25 non-responders). A subgroup of 23 patients was genotyped for SNPs in ADME genes. A machine learning classifier method was used to discover classification rules for our dataset. We found that only the VEGF-A (rs2010963) C allele and CC genotype were significantly associated with sorafenib response. ADME-related gene analysis identified 10 polymorphic variants in ADH1A (rs6811453), ADH6 (rs10008281), SULT1A2/CCDC101 (rs11401), CYP26A1 (rs7905939), DPYD (rs2297595 and rs1801265), FMO2 (rs2020863), and SLC22A14 (rs149738, rs171248, and rs183574) significantly associated with sorafenib response. We have identified a genetic signature of predictive response that could permit non-responder/responder patient stratification. Angiogenesis- and ADME-related genes correlation was confirmed by cumulative genetic risk score and network and pathway enrichment analysis. Our findings provide a proof of concept that needs further validation in follow-up studies for HCC patient stratification for sorafenib prescription.
Subject(s)
Antineoplastic Agents , Carcinoma, Hepatocellular , Liver Neoplasms , Humans , Sorafenib/pharmacology , Sorafenib/therapeutic use , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Antineoplastic Agents/therapeutic use , Vascular Endothelial Growth Factor A/metabolism , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Niacinamide/therapeutic use , Phenylurea Compounds/therapeutic use , Genetic MarkersABSTRACT
Diabetes mellitus (DM) is a complex and multifactorial disease characterised by high blood glucose. Type 2 Diabetes (T2D), the most frequent clinical condition accounting for about 90% of all DM cases worldwide, is a chronic disease with slow development usually affecting middle-aged or elderly individuals. T2D represents a significant problem of public health today because its incidence is constantly growing among both children and adults. It is also estimated that underdiagnosis prevalence would strongly further increase the real incidence of the disease, with about half of T2D patients being undiagnosed. Therefore, it is important to increase diagnosis accuracy. The current interest in RNA molecules (both protein- and non-protein-coding) as potential biomarkers for diagnosis, prognosis, and treatment lies in the ease and low cost of isolation and quantification with basic molecular biology techniques. In the present study, we analysed the transcriptome in serum samples collected from T2D patients and unaffected individuals to identify potential RNA-based biomarkers. Microarray-based profiling and subsequent validation using Real-Time PCR identified an uncharacterised long non-coding RNA (lncRNA) transcribed from the ASAP1 locus as a potential diagnostic biomarker. ROC curve analysis showed that a molecular signature including the lncRNA and the clinicopathological parameters of T2D patients as well as unaffected individuals showed a better diagnostic performance compared with the glycated haemoglobin test (HbA1c). This result suggests that the application of this biomarker in clinical practice would help to improve the diagnosis, and therefore the clinical management, of T2D patients. The proposed biomarker would be useful in the context of predictive, preventive, and personalised medicine (3PM/PPPM).
Subject(s)
Diabetes Mellitus, Type 2 , Hyperglycemia , RNA, Long Noncoding , Adult , Aged , Child , Humans , Middle Aged , Diabetes Mellitus, Type 2/genetics , Public Health , RNA, Long Noncoding/geneticsABSTRACT
BACKGROUND: DNA ligases are crucial for DNA repair and cell replication since they catalyze the final steps in which DNA breaks are joined. DNA Ligase III (LIG3) exerts a pivotal role in Alternative-Non-Homologous End Joining Repair (Alt-NHEJ), an error-prone DNA repair pathway often up-regulated in genomically unstable cancer, such as Multiple Myeloma (MM). Based on the three-dimensional (3D) LIG3 structure, we performed a computational screening to identify LIG3-targeting natural compounds as potential candidates to counteract Alt-NHEJ activity in MM. METHODS: Virtual screening was conducted by interrogating the Phenol Explorer database. Validation of binding to LIG3 recombinant protein was performed by Saturation Transfer Difference (STD)-nuclear magnetic resonance (NMR) experiments. Cell viability was analyzed by Cell Titer-Glo assay; apoptosis was evaluated by flow cytometric analysis following Annexin V-7AAD staining. Alt-NHEJ repair modulation was evaluated using plasmid re-joining assay and Cytoscan HD. DNA Damage Response protein levels were analyzed by Western blot of whole and fractionated protein extracts and immunofluorescence analysis. The mitochondrial DNA (mtDNA) copy number was determined by qPCR. In vivo activity was evaluated in NOD-SCID mice subcutaneously engrafted with MM cells. RESULTS: Here, we provide evidence that a natural flavonoid Rhamnetin (RHM), selected by a computational approach, counteracts LIG3 activity and killed Alt-NHEJ-dependent MM cells. Indeed, Nuclear Magnetic Resonance (NMR) showed binding of RHM to LIG3 protein and functional experiments revealed that RHM interferes with LIG3-driven nuclear and mitochondrial DNA repair, leading to significant anti-MM activity in vitro and in vivo. CONCLUSION: Taken together, our findings provide proof of concept that RHM targets LIG3 addiction in MM and may represent therefore a novel promising anti-tumor natural agent to be investigated in an early clinical setting.
Subject(s)
DNA Ligase ATP , DNA Repair , Flavonoids , Multiple Myeloma , Animals , Mice , Annexin A5/genetics , Annexin A5/metabolism , DNA Ligase ATP/genetics , DNA Ligase ATP/metabolism , DNA Ligases/chemistry , DNA Ligases/genetics , DNA Ligases/metabolism , DNA Repair/drug effects , DNA Repair/genetics , DNA, Mitochondrial/drug effects , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , Flavonoids/pharmacology , Flavonoids/therapeutic use , Mice, Inbred NOD , Mice, SCID , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Multiple Myeloma/metabolism , Phenols , Recombinant Proteins/metabolismABSTRACT
Type 2 diabetes and renal damage are strictly linked. The progressive increase in T2D incidence has stimulated the interest in novel biomarkers to improve the diagnostic performance of the commonly utilized markers such as albuminuria and eGFR. Through microarray method, we analyzed the entire transcriptome expressed in 12 serum samples of diabetic patients, six without DKD and six with DKD; the downregulation of the most dysregulated transcripts was validated in a wider cohort of 69 patients by qPCRs. We identified a total of 33 downregulated transcripts. The downregulation of four mitochondrial messenger RNAs (MT-ATP6, MT-ATP8, MT-COX3, MT-ND1) and other two transcripts (seysnoy, skerdo) was validated in patients with eGFR stage G3 versus G2 and G1. The four messenger RNAs correlated with creatinine and eGFR stages, while seysnoy and skerdo were associated with white blood cell values. All transcripts correlated also with Blood Urea Nitrogen. The four mitochondrial messenger RNAs had a high diagnostic performance in G3 versus G2 discrimination, with AUC values above 0.8. The most performant transcript was MT-ATP6, with an AUC of 0.846; sensitivity = 90%, specificity = 76%, p-value = 7.8 × 10-5. This study led to the identification of a specific molecular signature of DKD, proposing the dosage of RNAs, especially mitochondrial RNAs, as noninvasive biomarkers of diabetes complication.
Subject(s)
Diabetes Mellitus, Type 2 , Diabetic Nephropathies , Albuminuria/complications , Biomarkers , Diabetes Mellitus, Type 2/complications , Diabetes Mellitus, Type 2/genetics , Diabetic Nephropathies/diagnosis , Diabetic Nephropathies/genetics , Humans , KidneyABSTRACT
Microtubule-targeting agents (MTAs) are effective drugs for cancer treatment. A novel diaryl [1,2]oxazole class of compounds binding the colchicine site was synthesized as cis-restricted-combretastatin-A-4-analogue and then chemically modified to have improved solubility and a wider therapeutic index as compared to vinca alkaloids and taxanes. On these bases, a new class of tricyclic compounds, containing the [1,2]oxazole ring and an isoindole moiety, has been synthetized, among which SIX2G emerged as improved MTA. Several findings highlighted the ability of some chemotherapeutics to induce immunogenic cell death (ICD), which is defined by the cell surface translocation of Calreticulin (CALR) via dissociation of the PP1/GADD34 complex. In this regard, we computationally predicted the ability of SIX2G to induce CALR exposure by interacting with the PP1 RVxF domain. We then assessed both the potential cytotoxic and immunogenic activity of SIX2G on in vitro models of multiple myeloma (MM), which is an incurable hematological malignancy characterized by an immunosuppressive milieu. We found that the treatment with SIX2G inhibited cell viability by inducing G2/M phase cell cycle arrest and apoptosis. Moreover, we observed the increase of hallmarks of ICD such as CALR exposure, ATP release and phospho-eIF2α protein level. Through co-culture experiments with immune cells, we demonstrated the increase of (i) CD86 maturation marker on dendritic cells, (ii) CD69 activation marker on cytotoxic T cells, and (iii) phagocytosis of tumor cells following treatment with SIX2G, confirming the onset of an immunogenic cascade. In conclusion, our findings provide a framework for further development of SIX2G as a new potential anti-MM agent.
Subject(s)
Antineoplastic Agents , Multiple Myeloma , Vinca Alkaloids , Humans , Adenosine Triphosphate/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Calreticulin/metabolism , Cell Line, Tumor , Colchicine/pharmacology , Immunogenic Cell Death , Isoindoles/pharmacology , Microtubules/metabolism , Multiple Myeloma/drug therapy , Oxazoles/pharmacology , Taxoids/pharmacology , Vinca Alkaloids/pharmacology , Pemetrexed/pharmacology , Pemetrexed/therapeutic useABSTRACT
Multiple Myeloma (MM) is a hematologic malignancy strongly characterized by genomic instability, which promotes disease progression and drug resistance. Since we previously demonstrated that LIG3-dependent repair is involved in the genomic instability, drug resistance and survival of MM cells, we here investigated the biological relevance of PARP1, a driver component of Alternative-Non Homologous End Joining (Alt-NHEJ) pathway, in MM. We found a significant correlation between higher PARP1 mRNA expression and poor prognosis of MM patients. PARP1 knockdown or its pharmacological inhibition by Olaparib impaired MM cells viability in vitro and was effective against in vivo xenografts of human MM. Anti-proliferative effects induced by PARP1-inhibition were correlated to increase of DNA double-strand breaks, activation of DNA Damage Response (DDR) and finally apoptosis. Importantly, by comparing a gene expression signature of PARP inhibitors (PARPi) sensitivity to our plasma cell dyscrasia (PC) gene expression profiling (GEP), we identified a subset of MM patients which could benefit from PARP inhibitors. In particular, Gene Set Enrichment Analysis (GSEA) suggested that high MYC expression correlates to PARPi sensitivity in MM. Indeed, we identified MYC as promoter of PARP1-mediated repair in MM and, consistently, we demonstrate that cytotoxic effects induced by PARP inhibition are mostly detectable on MYC-proficient MM cells. Taken together, our findings indicate that MYC-driven MM cells are addicted to PARP1 Alt-NHEJ repair, which represents therefore a druggable target in this still incurable disease.
Subject(s)
Multiple Myeloma , Apoptosis , Cell Line, Tumor , DNA Breaks, Double-Stranded , DNA End-Joining Repair , Genomic Instability , Humans , Multiple Myeloma/drug therapy , Multiple Myeloma/geneticsABSTRACT
The microRNA (miRNA) cluster miR-17-92 is oncogenic and represents a valuable therapeutic target in c-MYC (MYC)-driven malignancies. Here, we developed novel LNA gapmeR antisense oligonucleotides (ASOs) to induce ribonuclease H-mediated degradation of MIR17HG primary transcripts and consequently prevent biogenesis of miR-17-92 miRNAs (miR-17-92s). The leading LNA ASO, MIR17PTi, impaired proliferation of several cancer cell lines (n = 48) established from both solid and hematologic tumors by on-target antisense activity, more effectively as compared with miR-17-92 inhibitors. By focusing on multiple myeloma (MM), we found that MIR17PTi triggers apoptosis via impairment of homeostatic MYC/miR-17-92 feed-forward loops (FFLs) in patient-derived MM cells and induces MYC-dependent synthetic lethality. We show that alteration of a BIM-centered FFL is instrumental for MIR17PTi to induce cytotoxicity in MM cells. MIR17PTi exerts strong in vivo antitumor activity in nonobese diabetic severe combined immunodeficient mice bearing clinically relevant models of MM, with advantageous safety and pharmacokinetic profiles in nonhuman primates. Altogether, MIR17PTi is a novel pharmacological tool to be tested in early-phase clinical trials against MM and other MYC-driven malignancies.
Subject(s)
Apoptosis/drug effects , MicroRNAs/antagonists & inhibitors , Multiple Myeloma/drug therapy , Oligonucleotides/pharmacology , RNA, Neoplasm/antagonists & inhibitors , Animals , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Mice , Mice, Inbred NOD , Mice, SCID , MicroRNAs/genetics , MicroRNAs/metabolism , Multiple Myeloma/genetics , Multiple Myeloma/metabolism , Oligonucleotides/genetics , RNA, Long Noncoding , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Idiopathic Pulmonary Fibrosis (IPF) is a chronic degenerative disease with a median survival of 2-5 years after diagnosis. Therefore, IPF patient identification represents an important and challenging clinical issue. Current research is still searching for novel reliable non-invasive biomarkers. Therefore, we explored the potential use of long non-coding RNAs (lncRNAs) and mRNAs as biomarkers for IPF. METHODS: We first performed a whole transcriptome analysis using microarray (n = 14: 7 Control, 7 IPF), followed by the validation of selected transcripts through qPCRs in an independent cohort of 95 subjects (n = 95: 45 Control, 50 IPF). Diagnostic performance and transcript correlation with functional/clinical data were also analyzed. RESULTS: 1059 differentially expressed transcripts were identified. We confirmed the downregulation of FOXF1 adjacent non-coding developmental regulatory RNA (FENDRR) lncRNA, hsa_circ_0001924 circularRNA, utrophin (UTRN) and Y-box binding protein 3 (YBX3) mRNAs. The two analyzed non-coding RNAs correlated with Forced Vital Capacity (FVC)% and Diffusing Capacity of the Lung for carbon monoxide (DLCO)% functional data, while coding RNAs correlated with smock exposure. All analyzed transcripts showed excellent performance in IPF identification with Area Under the Curve values above 0.87; the most outstanding one was YBX3: AUROC 0.944, CI 95% = 0.895-0.992, sensitivity = 90%, specificity = 88.9%, p-value = 1.02 × 10-13. CONCLUSIONS: This study has identified specific transcript signatures in IPF suggesting that validated transcripts and microarray data could be useful for the potential future identification of RNA molecules as non-invasive biomarkers for IPF.
Subject(s)
Idiopathic Pulmonary Fibrosis/diagnosis , RNA, Long Noncoding/blood , RNA, Messenger/blood , Aged , Aged, 80 and over , Biomarkers/blood , Case-Control Studies , Chronic Disease , Cohort Studies , Female , Humans , Liquid Biopsy , MaleABSTRACT
Alzheimer's disease (AD) diagnosis is actually based on clinical evaluation and brain-imaging tests, and it can often be confirmed only post-mortem. Therefore, new non-invasive molecular biomarkers are necessary to improve AD diagnosis. As circulating microRNA biomarkers have been proposed for many diseases, including AD, we aimed to identify new diagnostic non-small RNAs in AD. Whole transcriptome analysis was performed on plasma samples of five AD and five unaffected individuals (CTRL) using the Clariom D Pico Assay, followed by validation in real-time PCR on 37 AD patients and 37 CTRL. Six differentially expressed (DE) transcripts were identified: GS1-304P7.3 (upregulated), NONHSAT090268, TC0100011037, TC0400008478, TC1400008125, and UBE2V1 (downregulated). Peripheral blood mononuclear cells (PBMCs) may influence the expression of circulating RNAs and their analysis has been proposed to improve AD clinical management. Accordingly, DE transcript expression was also evaluated in PBMCs, showing no difference between AD and CTRL. ROC (receiver operating characteristic) curve analysis was performed to evaluate the diagnostic accuracy of each DE transcript and a signature including all of them. A correlation between cognitive impairment and GS1-304P7.3, NONHSAT090268, TC0100011037, and TC0400008478 was detected, suggesting a potential association between their extracellular abundance and AD clinical phenotype. Finally, this study identified six transcripts showing altered expression in the plasma of AD patients. Given the need for new, accurate blood biomarkers for AD diagnosis, these transcripts may be considered for further analyses in larger cohorts, also in combination with other biomarkers, aiming to identify specific RNA-based biomarkers to be eventually applied to clinical practice.
Subject(s)
Alzheimer Disease/blood , Cell-Free Nucleic Acids/blood , Cognition , RNA, Untranslated/blood , Aged , Aged, 80 and over , Alzheimer Disease/diagnosis , Biomarkers/blood , Female , Humans , Male , TranscriptomeABSTRACT
Deletion of distal 9p is associated with a rare clinical condition characterized by dysmorphic features, developmental delay, and ambiguous genitalia. The phenotype shows variable expressivity and is related to the size of the deletion. 8q24 duplication has been reported in only few cases to date, all showing dysmorphic features and mild psychomotor developmental delay. A case of chromosomal aberration involving a 9p terminal deletion with an 8q duplication has never been reported. Here, we describe a child with a female phenotype, male karyotype, dysmorphic features, ambiguous genitalia, and developmental delay. In order to assess the cause of the patient's phenotype, conventional karyotyping, FISH, and a chromosomal microarray analysis were performed on the patient and her parents. The cytogenetic and molecular analysis revealed an unbalanced chromosomal aberration with a duplication in the long arm of chromosome 8 at 8q24.11q24.3 associated with a distal deletion in the short arm of chromosome 9 at 9p24.3p24.1, derived from a maternal balanced translocation. We compared the clinical picture of our patient with other similar cases reported in the literature and found that some clinical findings, such as strabismus, symphalangism of the first finger, and cubitus valgus, have never been previously associated with 9p deletion or 8q duplication expanding the phenotypic range of this condition. This study is aimed to better define the clinical history and prognosis of patients with this rare chromosomal aberration.
Subject(s)
Chromosomes, Human, Pair 9/genetics , Developmental Disabilities/genetics , Gonadal Dysgenesis, 46,XY/genetics , Trisomy/genetics , Chromosome Deletion , Chromosome Mapping , Chromosomes, Human, Pair 8/genetics , Female , HumansABSTRACT
BACKGROUND & AIMS: In patients with non-alcoholic fatty liver disease (NAFLD), liver biopsy is the gold standard to detect non-alcoholic steatohepatitis (NASH) and stage liver fibrosis. We aimed to identify differentially expressed mRNAs and non-coding RNAs in serum samples of biopsy-diagnosed mild and severe NAFLD patients with respect to controls and to each other. METHODS: We first performed a whole transcriptome analysis through microarray (n = 12: four Control: CTRL; four mild NAFLD: NAS ≤ 4 F0; four severe NAFLD NAS ≥ 5 F3), followed by validation of selected transcripts through real-time PCRs in an independent internal cohort of 88 subjects (63 NAFLD, 25 CTRL) and in an external cohort of 50 NAFLD patients. A similar analysis was also performed on liver biopsies and HepG2 cells exposed to oleate:palmitate or only palmitate (cellular model of NAFL/NASH) at intracellular/extracellular levels. Transcript correlation with histological/clinical data was also analysed. RESULTS: We identified several differentially expressed coding/non-coding RNAs in each group of the study cohort. We validated the up-regulation of UBE2V1, BNIP3L mRNAs, RP11-128N14.5 lncRNA, TGFB2/TGFB2-OT1 coding/lncRNA in patients with NAS ≥ 5 (vs NAS ≤ 4) and the up-regulation of HBA2 mRNA, TGFB2/TGFB2-OT1 coding/lncRNA in patients with Fibrosis stages = 3-4 (vs F = 0-2). In in vitro models: UBE2V1, RP11-128N14.5 and TGFB2/TGFB2-OT1 had an increasing expression trend ranging from CTRL to oleate:palmitate or only palmitate-treated cells both at intracellular and extracellular level, while BNIP3L was up-regulated only at extracellular level. UBE2V1, RP11-128N14.5, TGFB2/TGFB2-OT1 and HBA2 up-regulation was also observed at histological level. UBE2V1, RP11-128N14.5, BNIP3L and TGFB2/TGFB2-OT1 correlated with histological/biochemical data. Combinations of TGFB2/TGFB2-OT1 + Fibrosis Index based on the four factors (FIB-4) showed an Area Under the Curve (AUC) of 0.891 (P = 3.00E-06) or TGFB2/TGFB2-OT1 + Fibroscan (AUC = 0.892, P = 2.00E-06) improved the detection of F = 3-4 with respect to F = 0-2 fibrosis stages. CONCLUSIONS: We identified specific serum coding/non-coding RNA profiles in severe and mild NAFLD patients that possibly mirror the molecular mechanisms underlying NAFLD progression towards NASH/fibrosis. TGFB2/TGFB2-OT1 detection improves FIB-4/Fibroscan diagnostic performance for advanced fibrosis discrimination.
Subject(s)
Liver Cirrhosis/diagnosis , Liver Cirrhosis/pathology , Non-alcoholic Fatty Liver Disease/diagnosis , Non-alcoholic Fatty Liver Disease/pathology , RNA, Untranslated/blood , Adult , Biomarkers/blood , Biopsy , Cohort Studies , Disease Progression , Female , Gene Expression Profiling , Humans , Liver/enzymology , Liver/pathology , Liver Cirrhosis/blood , Male , Middle Aged , Non-alcoholic Fatty Liver Disease/blood , Predictive Value of Tests , ROC Curve , Severity of Illness IndexABSTRACT
Over the past 10 years, the biological role of lipid droplets (LDs) has gained significant attention in the context of both physiological and pathological conditions. Considerable progress has been made in elucidating key aspects of these organelles, yet much remains to be accomplished to fully comprehend the myriad functions they serve in the progression of hepatic tumors. Our current perception is that LDs are complex and active structures managed by a distinct set of cellular processes. This understanding represents a significant paradigm shift from earlier perspectives. In this review, we aim to recapitulate the function of LDs within the liver, highlighting their pivotal role in the pathogenesis of metabolic dysfunction-associated steatotic liver disease (MASLD) (Hsu and Loomba, 2024) and their contribution to the progression towards more advanced pathological stages up to hepatocellular carcinoma (HC) (Farese and Walther, 2009). We are aware of the molecular complexity and changes occurring in the neoplastic evolution of the liver. Our attempt, however, is to summarize the most important and recent roles of LDs across both healthy and all pathological liver states, up to hepatocarcinoma. For more detailed insights, we direct readers to some of the many excellent reviews already available in the literature (Gluchowski et al., 2017; Hu et al., 2020; Seebacher et al., 2020; Paul et al., 2022).
ABSTRACT
BACKGROUND: Long-term survival induced by anticancer treatments discloses emerging frailty among breast cancer (BC) survivors. Trastuzumab-induced cardiotoxicity (TIC) is reported in at least 5% of HER2+BC patients. However, TIC mechanism remains unclear and predictive genetic biomarkers are still lacking. Interaction between systemic inflammation, cytokine release and ADME genes in cancer patients might contribute to explain mechanisms underlying individual susceptibility to TIC and drug response variability. We present a single institution case series to investigate the potential role of genetic variants in ADME genes in HER2+BC patients TIC experienced. METHODS: We selected data related to 40 HER2+ BC patients undergone to DMET genotyping of ADME constitutive variant profiling, with the aim to prospectively explore their potential role in developing TIC. Only 3 patients ("case series"), who experienced TIC, were compared to 37 "control group" matched patients cardiotoxicity-sparing. All patients underwent to left ventricular ejection fraction (LVEF) evaluation at diagnosis and during anti-HER2 therapy. Each single probe was clustered to detect SNPs related to cardiotoxicity. RESULTS: In this retrospective analysis, our 3 cases were homogeneous in terms of clinical-pathological characteristics, trastuzumab-based treatment and LVEF decline. We identified 9 polymorphic variants in 8 ADME genes (UGT1A1, UGT1A6, UGT1A7, UGT2B15, SLC22A1, CYP3A5, ABCC4, CYP2D6) potentially associated with TIC. CONCLUSION: Real-world TIC incidence is higher compared to randomized clinical trials and biomarkers with potential predictive value aren't available. Our preliminary data, as proof of concept, could suggest a predictive role of pharmacogenomic approach in the identification of cardiotoxicity risk biomarkers for anti-HER2 treatment.
Subject(s)
Breast Neoplasms , Cardiotoxicity , Polymorphism, Single Nucleotide , Trastuzumab , Humans , Female , Trastuzumab/adverse effects , Trastuzumab/pharmacokinetics , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Cardiotoxicity/genetics , Middle Aged , Retrospective Studies , Antineoplastic Agents, Immunological/adverse effects , Antineoplastic Agents, Immunological/pharmacokinetics , Aged , Receptor, ErbB-2/genetics , Receptor, ErbB-2/metabolism , AdultABSTRACT
G protein-coupled estrogen receptor 1 (GPER1) activation is emerging as a promising therapeutic strategy against several cancer types. While GPER targeting has been widely studied in the context of solid tumors, its effect on hematological malignancies remains to be fully understood. Here, we show that GPER1 mRNA is down-regulated in plasma cells from overt multiple myeloma (MM) and plasma cell leukemia patients as compared to normal donors or pre-malignant conditions (monoclonal gammopathy of undetermined significance and smoldering MM); moreover, lower GPER1 expression associates with worse overall survival of MM patients. Using the clinically applicable GPER1-selective agonist G-1, we demonstrate that the pharmacological activation of GPER1 triggered in vitro anti-MM activity through apoptosis induction, also overcoming the protective effects exerted by bone marrow stromal cells. Noteworthy, G-1 treatment reduced in vivo MM growth in two distinct xenograft models, even bearing bortezomib-resistant MM cells. Mechanistically, G-1 upregulated the miR-29b oncosuppressive network, blunting an established miR-29b-Sp1 feedback loop operative in MM cells. Overall, this study highlights the druggability of GPER1 in MM, providing the first preclinical framework for further development of GPER1 agonists to treat this malignancy.
Subject(s)
Hematologic Neoplasms , MicroRNAs , Multiple Myeloma , Smoldering Multiple Myeloma , Humans , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Plasma CellsABSTRACT
BACKGROUND: Multiple myeloma (MM) is a hematologic malignancy characterized by high genomic instability, and telomere dysfunction is an important cause of acquired genomic alterations. Telomeric repeat-containing RNA (TERRA) transcripts are long non-coding RNAs involved in telomere stability through the interaction with shelterin complex. Dysregulation of TERRAs has been reported across several cancer types. We recently identified a small molecule, hit 17, which stabilizes the secondary structure of TERRA. In this study, we investigated in vitro and in vivo anti-MM activities of hit 17. METHODS: Anti-proliferative activity of hit 17 was evaluated in different MM cell lines by cell proliferation assay, and the apoptotic process was analyzed by flow cytometry. Gene and protein expressions were detected by RT-qPCR and western blotting, respectively. Microarray analysis was used to analyze the transcriptome profile. The effect of hit 17 on telomeric structure was evaluated by chromatin immunoprecipitation. Further evaluation in vivo was proceeded upon NCI-H929 and AMO-1 xenograft models. RESULTS: TERRA G4 stabilization induced in vitro dissociation of telomeric repeat-binding factor 2 (TRF2) from telomeres leading to the activation of ATM-dependent DNA damage response, cell cycle arrest, proliferation block, and apoptotic death in MM cell lines. In addition, up-regulation of TERRA transcription was observed upon DNA damage and TRF2 loss. Transcriptome analysis followed by gene set enrichment analysis (GSEA) confirmed the involvement of the above-mentioned processes and other pathways such as E2F, MYC, oxidative phosphorylation, and DNA repair genes as early events following hit 17-induced TERRA stabilization. Moreover, hit 17 exerted anti-tumor activity against MM xenograft models. CONCLUSION: Our findings provide evidence that targeting TERRA by hit 17 could represent a promising strategy for a novel therapeutic approach to MM.
Subject(s)
Multiple Myeloma , Humans , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Telomere , Transcription, Genetic , Apoptosis , TranscriptomeABSTRACT
BACKGROUND: We developed a 13-mer locked nucleic acid (LNA) inhibitor of miR-221 (LNA-i-miR-221) with a full phosphorothioate (PS)-modified backbone. This agent downregulated miR-221, demonstrated anti-tumor activity against human xenografts in mice, and favorable toxicokinetics in rats and monkeys. Allometric interspecies scaling allowed us to define the first-in-class LNA-i-miR-221 safe starting dose for the clinical translation. METHODS: In this first-in-human, open-label, dose-escalation phase 1 trial, we enrolled progressive cancer patients (aged ≥ 18 years) with ECOG 0-2 into 5 cohorts. The treatment cycle was based on a 30-min IV infusion of LNA-i-miR-221 on 4 consecutive days. Three patients within the first cohort were treated with 2 cycles (8 infusions), while 14 patients were treated with a single course (4 infusions); all patients were evaluated for phase 1 primary endpoint. The study was approved by the Ethics Committee and Regulatory Authorities (EudraCT 2017-002615-33). RESULTS: Seventeen patients received the investigational treatment, and 16 were evaluable for response. LNA-i-miR-221 was well tolerated, with no grade 3-4 toxicity, and the MTD was not reached. We recorded stable disease (SD) in 8 (50.0%) patients and partial response (PR) in 1 (6.3%) colorectal cancer case (total SD + PR: 56.3%). Pharmacokinetics indicated non-linear drug concentration increase across the dose range. Pharmacodynamics demonstrated concentration-dependent downregulation of miR-221 and upregulation of its CDKN1B/p27 and PTEN canonical targets. Five mg/kg was defined as the recommended phase II dose. CONCLUSIONS: The excellent safety profile, the promising bio-modulator, and the anti-tumor activity offer the rationale for further clinical investigation of LNA-i-miR-221 (ClinTrials.Gov: NCT04811898).
Subject(s)
MicroRNAs , Neoplasms , Humans , MicroRNAs/genetics , MicroRNAs/therapeutic use , Neoplasms/drug therapy , Oligonucleotides/therapeutic useABSTRACT
The understanding of the biological differences which underlie the inter-individual variability in drug response improved the efficacy of cancer therapy in the era of precision medicine. In fact molecularly targeted drugs and immunotherapy represent a revolution in cancer treatment. The identification of genetic predictive and/or prognostic biomarkers linked to drug pharmacokinetics (PK) and pharmacodynamics (PD) is allowed by the development of high-throughput omics tools for detecting and understanding biological differences among individuals, in order to improve drug efficacy and minimize risk of toxicity. Personalized medicine in cancer treatment reduces costs of the healthcare system. Unfortunately, pharmacogenomics biomarkers discovery is influenced by complexity, need of high-quality evidence, and a validation process for regulatory purposes. This chapter is focused on the critic analysis of presently available pharmacogenomics tools for discovering or testing genetic polymorphic variants in drug metabolizing enzyme to be introduced in clinical practice for the prospective stratification of cancer patients.