ABSTRACT
BAP1 is a powerful tumor suppressor gene characterized by haplo insufficiency. Individuals carrying germline BAP1 mutations often develop mesothelioma, an aggressive malignancy of the serosal layers covering the lungs, pericardium, and abdominal cavity. Intriguingly, mesotheliomas developing in carriers of germline BAP1 mutations are less aggressive, and these patients have significantly improved survival. We investigated the apparent paradox of a tumor suppressor gene that, when mutated, causes less aggressive mesotheliomas. We discovered that mesothelioma biopsies with biallelic BAP1 mutations showed loss of nuclear HIF-1α staining. We demonstrated that during hypoxia, BAP1 binds, deubiquitylates, and stabilizes HIF-1α, the master regulator of the hypoxia response and tumor cell invasion. Moreover, primary cells from individuals carrying germline BAP1 mutations and primary cells in which BAP1 was silenced using siRNA had reduced HIF-1α protein levels in hypoxia. Computational modeling and co-immunoprecipitation experiments revealed that mutations of BAP1 residues I675, F678, I679, and L691 -encompassing the C-terminal domain-nuclear localization signal- to A, abolished the interaction with HIF-1α. We found that BAP1 binds to the N-terminal region of HIF-1α, where HIF-1α binds DNA and dimerizes with HIF-1ß forming the heterodimeric transactivating complex HIF. Our data identify BAP1 as a key positive regulator of HIF-1α in hypoxia. We propose that the significant reduction of HIF-1α activity in mesothelioma cells carrying biallelic BAP1 mutations, accompanied by the significant reduction of HIF-1α activity in hypoxic tissues containing germline BAP1 mutations, contributes to the reduced aggressiveness and improved survival of mesotheliomas developing in carriers of germline BAP1 mutations.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit , Mesothelioma, Malignant , Mesothelioma , Ubiquitin Thiolesterase , Humans , Heterozygote , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Mesothelioma/genetics , Mesothelioma/metabolism , Mesothelioma, Malignant/genetics , Mesothelioma, Malignant/complications , Mutation , Tumor Suppressor Proteins/metabolism , Ubiquitin Thiolesterase/metabolismABSTRACT
Due to the lack of biomarkers predictive of response to atezolizumab-bevacizumab, the standard of care for advanced HCC, we analyzed baseline and early on-treatment variation of peripheral lymphocyte populations of 37 prospective patients treated by atezolizumab-bevacizumab and in 15 prospective patients treated by sorafenib or lenvatinib (TKIs). RNAseq analysis followed by RT-PCR validation on patients-derived PBMC was also performed. At first imaging, re-evaluation 13 patients receiving atezolizumab-bevacizumab, showed an objective response, 17 stable disease, while 7 were nonresponders. Baseline CD8+ and CD8+PD-L1+ peripheral lymphocytes were lower in responders versus nonresponders (T-test, p = 0.012 and 0.004, respectively). At 3 weeks, 28 of 30 responders displayed a rise of CD8+PD1+ lymphocytes with a positive mean fold change of 4.35 (±5.6 SD), whereas 6 of 7 nonresponders displayed a negative fold change of 0.89 (±0.84 SD). These changes were not observed in patients treated by TKIs. TRIM56, TRIM16, TRIM64, and Ki67 mRNAs were validated as upregulated in responders versus nonresponders after 3 weeks after treatment start, providing possible evidence of immune activation. Baseline CD8+ and CD8+PD-L1+ peripheral lymphocytes and early changes in CD8+PD1+ lymphocytes predict response to atezolizumab-bevacizumab providing noninvasive markers to complement clinical practice in the very early phases of treatment of HCC patients.
Subject(s)
Antibodies, Monoclonal, Humanized , Carcinoma, Hepatocellular , Liver Neoplasms , Humans , Bevacizumab/therapeutic use , B7-H1 Antigen , Prospective Studies , Leukocytes, Mononuclear , CD8-Positive T-Lymphocytes , Biomarkers, Tumor , Tripartite Motif Proteins , Ubiquitin-Protein LigasesABSTRACT
Gene mutations may affect the fate of many tumors including prostate cancer (PCa); therefore, the research of specific mutations associated with tumor outcomes might help the urologist to identify the best therapy for PCa patients such as surgical resection, adjuvant therapy or active surveillance. Genomic DNA (gDNA) was extracted from 48 paraffin-embedded PCa samples and normal paired tissues. Next, gDNA was amplified and analyzed by next-generation sequencing (NGS) using a specific gene panel for PCa. Raw data were refined to exclude false-positive mutations; thus, variants with coverage and frequency lower than 100× and 5%, respectively were removed. Mutation significance was processed by Genomic Evolutionary Rate Profiling, ClinVar, and Varsome tools. Most of 3000 mutations (80%) were single nucleotide variants and the remaining 20% indels. After raw data elaboration, 312 variants were selected. Most mutated genes were KMT2D (26.45%), FOXA1 (16.13%), ATM (15.81%), ZFHX3 (9.35%), TP53 (8.06%), and APC (5.48%). Hot spot mutations in FOXA1, ATM, ZFHX3, SPOP, and MED12 were also found. Truncating mutations of ATM, lesions lying in hot spot regions of SPOP and FOXA1 as well as mutations of TP53 correlated with poor prognosis. Importantly, we have also found some germline mutations associated with hereditary cancer-predisposing syndrome. gDNA sequencing of 48 cancer tissues by NGS allowed to detect new tumor variants as well as confirmed lesions in genes linked to prostate cancer. Overall, somatic and germline mutations linked to good/poor prognosis could represent new prognostic tools to improve the management of PCa patients.
Subject(s)
High-Throughput Nucleotide Sequencing , Prostatic Neoplasms , Germ-Line Mutation , Humans , Male , Mutation/genetics , Nuclear Proteins/genetics , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Repressor Proteins/geneticsABSTRACT
Liquid biopsy has advantages over tissue biopsy, but also some technical limitations that hinder its wide use in clinical applications. In this study, we aimed to evaluate the usefulness of liquid biopsy for the clinical management of patients with advanced-stage oncogene-addicted non-small-cell lung adenocarcinomas. The investigation was conducted on a series of cases-641 plasma samples from 57 patients-collected in a prospective consecutive manner, which allowed us to assess the benefits and limitations of the approach in a real-world clinical context. Thirteen samples were collected at diagnosis, and the additional samples during the periodic follow-up visits. At diagnosis, we detected mutations in ctDNA in 10 of the 13 cases (77%). During follow-up, 36 patients progressed. In this subset of patients, molecular analyses of plasma DNA/RNA at progression revealed the appearance of mutations in 29 patients (80.6%). Mutations in ctDNA/RNA were typically detected an average of 80 days earlier than disease progression assessed by RECIST or clinical evaluations. Among the cases positive for mutations, we observed 13 de novo mutations, responsible for the development of resistance to therapy. This study allowed us to highlight the advantages and disadvantages of liquid biopsy, which led to suggesting algorithms for the use of liquid biopsy analyses at diagnosis and during monitoring of therapy response.
Subject(s)
Adenocarcinoma of Lung , Adenocarcinoma , Carcinoma, Non-Small-Cell Lung , Cell-Free Nucleic Acids , Circulating Tumor DNA , Lung Neoplasms , Adenocarcinoma/genetics , Adenocarcinoma of Lung/genetics , Biomarkers, Tumor/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Cell-Free Nucleic Acids/genetics , Circulating Tumor DNA/genetics , High-Throughput Nucleotide Sequencing , Humans , Lung Neoplasms/pathology , Mutation , Oncogenes , Prospective Studies , RNAABSTRACT
Metagenomic next-generation sequencing (mNGS) allows the monitoring of microbiota composition of murine colonies employed for scientific purposes in a single test by assessing the composition of gut microbiome and the detection of pathogens from fecal pellets. In this study, we tested the potential use of mNGS for monitoring both microbiota composition and the presence of pathogens through Environmental Health Monitoring, by using exhaust dust collection filters derived from individually ventilated cages (IVC) systems.mNGS analysis was performed on nucleic acids isolated from filters collecting air from the exhaust of: (1) cages with mice housed in a non-pathogen free facility; (2) animal-free cages with clean chow and bedding from the same facility; (3) cages housing mice from a specific-pathogen free (SPF) facility. mNGS results revealed correspondence between microbiome composition from fecal pellets and filter, including pathogenic bacteria (Helicobacter hepaticus, Helicobacter typhlonius, Chlamydia muridarum, Rodentibacter pneumotropicus, Citrobacter rodentium), intestinal protozoa (Tritrichomonas muris, Spironucleus muris) nematoda (Aspiculuris tetraptera) and eukaryotic parasites (Myocoptes musculinus), present in the colony. Entamoeba muris and Syphacia obvelata were detected in fecal pellets but not in filter. The animal free exhaust dust filter, exposed to clean cages (no mice) placed in the IVC after removal of all mice, exhibited the presence of the same pathogens due to contaminated connecting pipes, confirming the sensitivity of the approach. Conversely, the filter from SPF colony revealed the absence of pathogens.The current use of exhaust dust collection filters in health surveillance requires multiple molecular tests to identify specific pathogens and does not provide information on the colony microbiome. This work provides the proof-of-principle that assaying exhaust dust collection filters by mNGS for microbiota monitoring of laboratory mice is feasible. In its daily application, results suggest the usefulness of the test in SPF facilities, where pathogenic micro-organisms are expected to be absent. mNGS analysis of exhaust dust collection filters allows the analysis of multiple cages, reducing the number of tests required for pathogen detection and corresponding costs, and avoiding the use of sentinel mice.
Subject(s)
Environmental Health , Metagenomics , Mice , AnimalsABSTRACT
BACKGROUND: The microbiome of the oral cavity is the second-largest and diverse microbiota after the gut, harboring over 700 species of bacteria and including also fungi, viruses, and protozoa. With its diverse niches, the oral cavity is a very complex environment, where different microbes preferentially colonize different habitats. Recent data indicate that the oral microbiome has essential functions in maintaining oral and systemic health, and the emergence of 16S rRNA gene next-generation sequencing (NGS) has greatly contributed to revealing the complexity of its bacterial component. However, a detailed site-specific map of oral microorganisms (including also eukaryotes and viruses) and their relative abundance is still missing. Here, we aimed to obtain a comprehensive view of the healthy oral microbiome (HOM), including its drug-resistance features. RESULTS: The oral microbiome of twenty healthy subjects was analyzed by whole-genome sequencing (WGS) and real-time quantitative PCR microarray. Sampled oral micro-habitat included tongue dorsum, hard palate, buccal mucosa, keratinized gingiva, supragingival and subgingival plaque, and saliva with or without rinsing. Each sampled oral niche evidenced a different microbial community, including bacteria, fungi, and viruses. Alpha-diversity evidenced significant differences among the different sampled sites (p < 0.0001) but not among the enrolled subjects (p = 0.876), strengthening the notion of a recognizable HOM. Of note, oral rinse microbiome was more representative of the whole site-specific microbiomes, compared with that of saliva. Interestingly, HOM resistome included highly prevalent genes conferring resistance to macrolide, lincosamides, streptogramin, and tetracycline. CONCLUSIONS: The data obtained in 20 subjects by WGS and microarray analysis provide for the first time a comprehensive view of HOM and its resistome, contributing to a deeper understanding of the composition of oral microbiome in the healthy subject, and providing an important reference for future studies, allowing to identify microbial signatures related to functional and metabolic alterations associated with diseases, potentially useful for targeted therapies and precision medicine.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteria/classification , Drug Resistance, Microbial , Fungi/classification , Mouth/microbiology , Viruses/classification , Whole Genome Sequencing/methods , Adult , Bacteria/drug effects , Bacteria/genetics , Bacteria/isolation & purification , Female , Fungi/drug effects , Fungi/genetics , Fungi/isolation & purification , Genome, Bacterial , Genome, Fungal , Genome, Viral , Healthy Volunteers , High-Throughput Nucleotide Sequencing , Humans , Lincosamides/pharmacology , Macrolides/pharmacology , Male , Microarray Analysis , Phylogeny , Streptogramins/pharmacology , Tetracycline/pharmacology , Viruses/drug effects , Viruses/genetics , Viruses/isolation & purification , Young AdultABSTRACT
OBJECTIVE: Endometrial carcinoma represents the most common gynaecological cancer and the sixth most frequent cancer among women worldwide. The 5-year survival of patients with stage I endometrial carcinoma is 75%-88% versus 50% for stage III or 15% for stage IV disease. Therefore, early detection could improve survival rates. Specifically, in the most prevalent, type 1 endometrial cancer develops from hyperplastic endometrium. The aim of the study was to evaluate the utility of cancer gene mutations from endometrial biopsies towards predicting synchronous or metachronous development of malignant lesions. The aim of the study was to evaluate whether endometrial biopsies could already carry mutations in cancer genes useful for predicting or anticipating subsequent cancer development. METHODS: Patients with a previous endometrial biopsy negative for cancer, followed by a subsequent biopsy positive for cancer, were included in the study. A fifty cancer genes targeted next-generation sequencing panel were used to investigate mutations in matched non-cancerous and malignant samples. RESULTS: All biopsies from cancer tissues harboured mutations in one or more of the following genes: APC, CTNNB1, FBXW7, HNF1A, KRAS, MTOR, NRAS, PIK3CA, PTEN, RB1 and TP53. Additionally, 50% of the biopsies from matched non-cancerous tissues exhibited mutations in PTEN, KRAS or PIK3CA genes. CONCLUSIONS: These results suggest that detecting pathogenic mutations in oncogenes or tumour suppressor genes in an otherwise benign condition is associated with a risk of developing a malignant disease. Given the identification of mutations several months or years before the appearance of a malignancy, our finding suggests that a closer monitoring of patients who present such molecular alterations in non-cancerous uterine mass is warranted.
Subject(s)
Biomarkers, Tumor/genetics , Carcinoma, Endometrioid/diagnosis , Carcinoma, Endometrioid/genetics , Endometrial Neoplasms/diagnosis , Endometrial Neoplasms/genetics , Aged , Biopsy , DNA Mutational Analysis , Early Detection of Cancer , Female , Genes, Neoplasm/genetics , Humans , Middle Aged , Mutation , Neoplasm Staging , Pilot Projects , Prognosis , Retrospective StudiesABSTRACT
Complex karyotype (CK) is a negative prognostic factor in chronic lymphocytic leukaemia (CLL). However, CK is a heterogeneous cytogenetic category. Unbalanced rearrangements were present in 73·3% of 90 CLL patients with CK (i.e. ≥3 chromosome aberrations in the same clone), and were associated with a shorter overall survival (P = 0·025) and a shorter time to first treatment (P = 0·043) by multivariate analysis. Patients with unbalanced rearrangements presented a distinct mRNA expression profile. In conclusion, CLL patients with unbalanced rearrangements might represent a subset of very high-risk CLL patients with distinct clinical and biological characteristics.
Subject(s)
Chromosome Aberrations , Gene Expression Regulation, Leukemic , Leukemia, Lymphocytic, Chronic, B-Cell , RNA, Messenger , RNA, Neoplasm , Aged , Disease-Free Survival , Female , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/mortality , Male , Middle Aged , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Neoplasm/biosynthesis , RNA, Neoplasm/genetics , Survival RateSubject(s)
Carcinoma, Squamous Cell , Lichen Sclerosus et Atrophicus , MicroRNAs , Vulvar Lichen Sclerosus , Vulvar Neoplasms , Female , Humans , Vulvar Lichen Sclerosus/diagnosis , Vulvar Lichen Sclerosus/genetics , Vulvar Lichen Sclerosus/pathology , MicroRNAs/genetics , Lichen Sclerosus et Atrophicus/pathology , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Vulvar Neoplasms/genetics , Vulvar Neoplasms/pathologyABSTRACT
To clarify whether karyotype aberrations (KA) involving regions not covered by the standard fluorescence in situ hybridization (FISH) panel have independent prognostic relevance, we evaluated KA by conventional cytogenetics in a learning cohort (LC; n = 166) and a validation cohort (VC; n = 250) of untreated chronic lymphocytic leukemia (CLL) patients. In the VC, novel mitogens were used to improve metaphase generation and TP53, NOTCH1, and SF3B1 mutations were assessed. KA undetected by FISH were found in 35 and 35% of the cases in the LC and VC, respectively. In addition to FISH, KA allowed reclassification of 23 and 26% of cases in the LC and VC, respectively, into a higher cytogenetic risk group. By multivariate analysis, both in the LC and VC, KA other than isolated 13q deletion correlated with a shorter time to first treatment (TFT; P < 0.001 and 0.003, respectively), while a complex karyotype predicted a worse overall survival (OS, P = 0.015 and 0.010, respectively). In the VC, where a comprehensive biologic assessment was performed, a shorter TFT was also predicted by stage (P < 0.001), IGHV mutational status (P = 0.05), and del(17p)/TP53 mutations (P = 0.033) while stage (P = 0.023) and del(17p)/TP53 mutations (P = 0.024) independently predicted a shorter OS. FISH results did not independently impact on TFT and OS, in the LC and VC cohorts; this was also the case for NOTCH1 and SF3B1 mutations in the VC. We suggest that in CLL, conventional karyotyping with novel mitogens could be more effective than FISH for the detection of KA allowing for a more precise refinement of prognosis.
Subject(s)
Chromosome Deletion , Interleukin-2/pharmacology , Karyotyping , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Mitogens/pharmacology , Oligonucleotides/pharmacology , Adult , Aged , Aged, 80 and over , Chromosome Disorders/genetics , Chromosomes, Human, Pair 13/genetics , Chromosomes, Human, Pair 17/genetics , Cytogenetics , Female , Humans , Immunoglobulin Heavy Chains/genetics , In Situ Hybridization, Fluorescence , Male , Middle Aged , Multivariate Analysis , Mutation , Phosphoproteins/genetics , Prognosis , RNA Splicing Factors , Receptor, Notch1/genetics , Recombinant Proteins/pharmacology , Ribonucleoprotein, U2 Small Nuclear/genetics , Time-to-Treatment , Tumor Suppressor Protein p53/geneticsABSTRACT
To evaluate the gene expression changes involved in neoplastic progression of cervical intraepithelial neoplasia. Using microarray analysis, large-scale gene expression profile was carried out on HPV16-CIN2, HPV16-CIN3, and normal cervical keratinocytes derived from two HPV16-CIN2, two HPV-CIN3 lesions, and two corresponding normal cervical tissues, respectively. Differentially expressed genes were analyzed in normal cervical keratinocytes compared with HPV16-CIN2 keratinocytes and in HPV16-CIN2 keratinocytes compared with HPV16-CIN3 keratinocytes; 37 candidate genes with continuously increasing or decreasing expression during CIN progression were identified. One of these genes, phosphoglycerate dehydrogenase, was chosen for further characterization. Quantitative reverse transcription-polymerase chain reaction and immunohistochemical analysis confirmed that expression of phosphoglycerate dehydrogenase consistently increases during progression of CIN toward cancer. Gene expression changes occurring during CIN progression were investigated using microarray analysis, for the first time, in CIN2 and CIN3 keratinocytes naturally infected with HPV16. Phosphoglycerate dehydrogenase is likely to be associated with tumorigenesis and may be a potential prognostic marker for CIN progression.
Subject(s)
Gene Expression Regulation, Neoplastic/genetics , Keratinocytes/metabolism , Tissue Array Analysis , Uterine Cervical Neoplasms/genetics , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Carcinoma, Squamous Cell/pathology , Cell Transformation, Neoplastic/genetics , Disease Progression , Female , Human papillomavirus 16/isolation & purification , Humans , Papillomavirus Infections/genetics , Phosphoglycerate Dehydrogenase/genetics , Phosphoglycerate Dehydrogenase/metabolism , Uterine Cervical Neoplasms/pathology , Uterine Cervical Dysplasia/geneticsABSTRACT
BACKGROUND: The anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (moAbs) cetuximab or panitumumab are administered to colorectal cancer (CRC) patients who harbor wild-type RAS proto-oncogenes. However, a percentage of patients do not respond to this treatment. In addition to mutations in the RAS genes, mutations in other genes, such as BRAF, PI3KCA, or PTEN, could be involved in the resistance to anti-EGFR moAb therapy. METHODS: In order to develop a comprehensive approach for the detection of mutations and to eventually identify other genes responsible for resistance to anti-EGFR moAbs, we investigated a panel of 21 genes by parallel sequencing on the Ion Torrent Personal Genome Machine platform. We sequenced 65 CRCs that were treated with cetuximab or panitumumab. Among these, 37 samples were responsive and 28 were resistant. RESULTS: We confirmed that mutations in EGFR-pathway genes (KRAS, NRAS, BRAF, PI3KCA) were relevant for conferring resistance to therapy and could predict response (p = 0.001). After exclusion of KRAS, NRAS, BRAF and PI3KCA combined mutations could still significantly associate to resistant phenotype (p = 0.045, by Fisher exact test). In addition, mutations in FBXW7 and SMAD4 were prevalent in cases that were non-responsive to anti-EGFR moAb. After we combined the mutations of all genes (excluding KRAS), the ability to predict response to therapy improved significantly (p = 0.002, by Fisher exact test). CONCLUSIONS: The combination of mutations at KRAS and at the five gene panel demonstrates the usefulness and feasibility of multigene sequencing to assess response to anti-EGFR moAbs. The application of parallel sequencing technology in clinical practice, in addition to its innate ability to simultaneously examine the genetic status of several cancer genes, proved to be more accurate and sensitive than the presently in use traditional approaches.
Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , Biomarkers, Tumor/genetics , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , ErbB Receptors/antagonists & inhibitors , Adult , Aged , Aged, 80 and over , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Antibodies, Monoclonal, Humanized/pharmacology , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cetuximab/pharmacology , Cetuximab/therapeutic use , Colorectal Neoplasms/diagnosis , Female , Humans , Male , Middle Aged , Panitumumab , Predictive Value of Tests , Treatment OutcomeSubject(s)
Creatinine/blood , Leukemia, Lymphocytic, Chronic, B-Cell/mortality , Severity of Illness Index , Adult , Age Factors , Aged , Aged, 80 and over , Biomarkers , Chromosome Aberrations , Comorbidity , Female , Follow-Up Studies , Genes, p53 , Humans , Immunoglobulin Heavy Chains/genetics , Kaplan-Meier Estimate , Karyotyping , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Male , Middle Aged , Neoplasm Staging , Prognosis , beta 2-Microglobulin/analysisABSTRACT
Hepatocellular carcinoma (HCC) is the most common liver cancer and is among the leading causes of cancer-related death worldwide. There is no reliable biomarker for the early diagnosis of HCC. Circulating microRNAs (miRNAs) have attracted attention as potential biomarkers of disease. By small-RNA next-generation sequencing, the analysis of serum miRNAs led to the identification of molecular signatures able to discriminate advanced HCC from early HCC (n = 246); advanced HCC from CIRRHOSIS (n = 299); advanced HCC from HEALTHY (n = 320); HEALTHY from early HCC (n = 343); and HEALTHY from CIRRHOSIS (n = 414). Cirrhotic patients and early HCC patients exhibited similar serum miRNA profiles, yet a small number of miRNAs (n = 57) were able to distinguish these two classes of patients. A second objective of the study was to identify serum miRNAs capable of predicting the response to therapy in patients with advanced HCC. All patients were treated with sorafenib as first-line therapy: 24 were nonresponsive and 24 responsive. Analysis of circulating miRNAs revealed a 54 miRNAs signature able to separate the two subgroups. This study suggested that circulating miRNAs could be useful biomarkers for monitoring patients with liver diseases ranging from cirrhosis to advanced HCC and possibly predicting susceptibility to first-line treatment based on sorafenib.
Subject(s)
Biomarkers, Tumor , Carcinoma, Hepatocellular , Circulating MicroRNA , Disease Progression , Liver Neoplasms , Humans , Liver Neoplasms/blood , Liver Neoplasms/genetics , Liver Neoplasms/drug therapy , Liver Neoplasms/diagnosis , Biomarkers, Tumor/blood , Biomarkers, Tumor/genetics , Circulating MicroRNA/blood , Circulating MicroRNA/genetics , Carcinoma, Hepatocellular/blood , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/diagnosis , Male , Female , Middle Aged , Aged , Liver Cirrhosis/blood , Liver Cirrhosis/diagnosis , Liver Cirrhosis/genetics , Liver Cirrhosis/drug therapy , Sorafenib/therapeutic use , MicroRNAs/blood , MicroRNAs/genetics , AdultABSTRACT
BACKGROUND: The microRNA 125b is a double-faced gene expression regulator described both as a tumor suppressor gene (in solid tumors) and an oncogene (in hematologic malignancies). In human breast cancer, it is one of the most down-regulated miRNAs and is able to modulate ERBB2/3 expression. Here, we investigated its targets in breast cancer cell lines after miRNA-mimic transfection. We examined the interactions of the validated targets with ERBB2 oncogene and the correlation of miR-125b expression with clinical variables. METHODS: MiR-125b possible targets were identified after transfecting a miRNA-mimic in MCF7 cell line and analyzing gene expression modifications with Agilent microarrays and Sylamer bioinformatic tool. Erythropoietin (EPO) and its receptor (EPOR) were validated as targets of miR-125b by luciferase assay and their expression was assessed by RT-qPCR in 42 breast cancers and 13 normal samples. The molecular talk between EPOR and ERBB2 transcripts, through miR-125b, was explored transfecting MDA-MD-453 and MDA-MB-157 with ERBB2 RNA and using RT-qPCR. RESULTS: We identified a panel of genes down-regulated after miR-125b transfection and putative targets of miR-125b. Among them, we validated erythropoietin (EPO) and its receptor (EPOR) - frequently overexpressed in breast cancer--as true targets of miR-125b. Moreover, we explored possible correlations with clinical variables and we found a down-regulation of miR-125b in metastatic breast cancers and a significant positive correlation between EPOR and ERBB2/HER2 levels, that are both targets of miR-125b and function as competing endogenous RNAs (ceRNAs). CONCLUSIONS: Taken together our results show a mechanism for EPO/EPOR and ERBB2 co-regulation in breast cancer and confirm the importance of miR-125b in controlling clinically-relevant cancer features.
Subject(s)
Breast Neoplasms/metabolism , Erythropoietin/genetics , MicroRNAs/genetics , RNA Interference , Receptor, ErbB-2/metabolism , Receptors, Erythropoietin/genetics , 3' Untranslated Regions , Binding Sites , Breast Neoplasms/pathology , Erythropoietin/metabolism , Female , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , HEK293 Cells , Humans , MCF-7 Cells , Molecular Sequence Annotation , Neoplasm Metastasis , Receptor, ErbB-2/genetics , Receptors, Erythropoietin/metabolismABSTRACT
UNLABELLED: MicroRNA-221 (miR-221) is one of the most frequently and consistently up-regulated microRNAs (miRNAs) in human cancer. It has been hypothesized that miR-221 may act as a tumor promoter. To demonstrate this, we developed a transgenic (TG) mouse model that exhibits an inappropriate overexpression of miR-221 in the liver. Immunoblotting and immunostaining confirmed a concomitant down-regulation of miR-221 target proteins. This TG model is characterized by the emergence of spontaneous nodular liver lesions in approximately 50% of male mice and by a strong acceleration of tumor development in 100% of mice treated with diethylnitrosamine. Similarly to human hepatocellular carcinoma, tumors are characterized by a further increase in miR-221 expression and a concomitant inhibition of its target protein-coding genes (i.e., cyclin-dependent kinase inhibitor [Cdkn]1b/p27, Cdkn1c/p57, and B-cell lymphoma 2-modifying factor). To validate the tumor-promoting effect of miR-221, we showed that in vivo delivery of anti-miR-221 oligonucleotides leads to a significant reduction of the number and size of tumor nodules. CONCLUSIONS: This study not only establishes that miR-221 can promote liver tumorigenicity, but it also establishes a valuable animal model to perform preclinical investigations for the use of anti-miRNA approaches aimed at liver cancer therapy.
Subject(s)
Liver Neoplasms/genetics , Liver Neoplasms/pathology , MicroRNAs/physiology , Animals , Male , Mice , Mice, TransgenicABSTRACT
Metagenomic Next Generation Sequencing (mNGS) allows the evaluation of complex microbial communities, avoiding isolation and cultivation of each microbial species, and does not require prior knowledge of the microbial sequences present in the sample. Applications of mNGS include virome characterization, new virus discovery and full-length viral genome reconstruction, either from virus preparations enriched in culture or directly from clinical and environmental specimens. Here, we systematically reviewed studies that describe novel virus identification through mNGS from samples of different origin (plant, animal and environment). Without imposing time limits to the search, 379 publications were identified that met the search parameters. Sample types, geographical origin, enrichment and nucleic acid extraction methods, sequencing platforms, bioinformatic analytical steps and identified viral families were described. The review highlights mNGS as a feasible method for novel virus discovery from samples of different origins, describes which kind of heterogeneous experimental and analytical protocols are currently used and provides useful information such as the different commercial kits used for the purification of nucleic acids and bioinformatics analytical pipelines.
ABSTRACT
Palbociclib is in early-stage clinical testing in advanced hepatocellular carcinoma (HCC). Here, we investigated whether the anti-tumor activity of palbociclib, which prevents the CDK4/6-mediated phosphorylation of RB1 but simultaneously activates AKT signaling, could be improved by its combination with a PI3K/AKT/mTOR inhibitor in liver cancer models. The selective pan-AKT inhibitor, MK-2206, or the microRNA-199a-3p were tested in combination with palbociclib in HCC cell lines and in the TG221 HCC transgenic mouse model. The combination palbociclib/MK-2206 was highly effective, but too toxic to be tolerated by mice. Conversely, the combination miR-199a-3p mimics/palbociclib not only induced a complete or partial regression of tumor lesions, but was also well tolerated. After 3 weeks of treatment, the combination produced a significant reduction in number and size of tumor nodules in comparison with palbociclib or miR-199a-3p mimics used as single agents. Moreover, we also reported the efficacy of this combination against sorafenib-resistant cells in vitro and in vivo. At the molecular level, the combination caused the simultaneous decrease of the phosphorylation of both RB1 and of AKT. Our findings provide pre-clinical evidence for the efficacy of the combination miR-199a-3p/palbociclib as anti-HCC treatment or as a new approach to overcome sorafenib resistance.
ABSTRACT
[This corrects the article DOI: 10.1016/j.omtn.2022.07.015.].
ABSTRACT
Despite the significant improvements in advanced melanoma therapy, there is still a pressing need for biomarkers that can predict patient response and prognosis, and therefore support rational treatment decisions. Here, we investigated whether circulating miRNAs could be biomarkers of clinical outcomes in patients treated with targeted therapy. Using next-generation sequencing, we profiled plasma miRNAs at baseline and at progression in patients treated with BRAF inhibitors (BRAFi) or BRAFi + MEKi. Selected miRNAs associated with response to therapy were subjected to validation by real-time quantitative RT-PCR. Receiver Operating Characteristics (ROC), Kaplan-Meier and univariate and multivariate Cox regression analyses were performed on the validated miR-1246 and miR-485-3p baseline levels. The median baseline levels of miR-1246 and miR-485-3p were significantly higher and lower, respectively, in the group of patients not responding to therapy (NRs) as compared with the group of responding patients (Rs). In Rs, a trend toward an increase in miR-1246 and a decrease in miR-485-3p was observed at progression. Baseline miR-1246 level and the miR-1246/miR-485-3p ratio showed a good ability to discriminate between Rs and NRs. Poorer PFS and OS were observed in patients with unfavorable levels of at least one miRNA. In multivariate analysis, a low level of miR-485-3p and a high miR-1246/miR-485-3p ratio remained independent negative prognostic factors for PFS, while a high miR-1246/miR-485-3p ratio was associated with an increased risk of mortality, although statistical significance was not reached. Evaluation of miR-1246 and miR-485-3p baseline plasma levels might help clinicians to identify melanoma patients most likely to be unresponsive to targeted therapy or at higher risk for short-term PFS and mortality, thus improving their management.