Overexpression of wild type or a Q311E mutant MB21D2 promotes a pro-oncogenic phenotype in HNSCC
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE156327 | GEO | 2020/08/18
REPOSITORIES: GEO
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Project description:Cadherin-mediated cell-cell contacts regulated by intracellular binders play critical roles in tissue homeostasis and tumorigenesis. Here, we screened mutational profiles of 312 annotated genes involved in cadherin binding in human squamous cell carcinomas and found MB21D2 to carry a unique recurrent Q311E mutation. MB21D2 overexpression was also frequently found in head and neck cancer (HNSCC) and was associated with poor clinical outcomes. Cell-based characterizations revealed pro-oncogenic roles for MB21D2 wild-type (WT) and its Q311E mutant (Q311E) in cell proliferation, colony formation, sphere growth, and migration/invasion by promoting epithelial-mesenchymal transition. Conversely, MB21D2 knockdown in MB21D2-overexpressing cells resulted in cell growth arrest and apoptosis. Xenograft tumor models with Q311E-expressing cells formed larger and more aggressive lesions, compared to models with WT-MB21D2-expressing cells or an empty vector. Transcriptome and protein interactome analyses revealed enrichment of KRAS signaling by MB21D2 expression. Immunoblotting confirmed RAS elevation, along with upregulation/phosphorylation of PI3K, AKT, and CREB. Blocking RAS signaling in MB21D2-expressing cells by manumycin significantly reduced cell growth and survival. Our study thus defined RAS signaling-dependent pro-oncogenic roles for MB21D2 overexpression and Q311E MB21D2 expression in HNSCC development.
| S-EPMC7718949 | biostudies-literature
Project description:BackgroundA number of homeobox genes have been implicated in the development of various cancers. However, the role of engrailed 2 (EN2), a member of the homeobox gene superfamily, in esophageal squamous cell carcinoma (ESCC) remains unknown.MethodsThe expression of EN2 was examined using quantitative real-time PCR and immunohistochemistry. A stable cell line was established to express exogenous EN2 using a lentivirus system. The malignant phenotype was analyzed with proliferation, clonogenicity, wound-healing and invasion assays. The CRISPR/Cas9 system was adopted to deplete endogenous EN2. RNA profiling was performed using gene expression microarray. The ShRNA-mediated method was used to knock down the expression of SPARC. The structure-function relationship was determined using site-directed mutagenesis.ResultsEN2 is highly expressed in ESCC. The malignant phenotype of the ESCC cell line was amplified by an overexpression of EN2 but was attenuated by a disruption of EN2. RNA profiling analysis revealed that distinct sets of genes were modulated by the expression of EN2 in various ESCC cell lines and oncogenes were among these. EN2 greatly increased the expression of SPARC in Eca109. Site-directed mutagenesis revealed that the induction of SPARC was closely correlated with the protumor function of EN2. ShRNA-mediated knockdown of SPARC attenuated the malignant phenotype of EN2-infected cells. These data suggest that SPARC is crucial for mediating the protumor function of EN2.DiscussionEN2 has an oncogenic function in ESCC that is mediated by upregulating the expression of pro-oncogenic genes downstream. EN2 may potentially act as a diagnostic marker or therapeutic target for ESCC treatment in the future.
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Project description:BackgroundChronic lymphocytic leukemia (CLL) is the most frequent, and still incurable, form of leukemia in the Western World. It is widely accepted that cancer results from an evolutionary process shaped by the acquisition of driver mutations which confer selective growth advantage to cells that harbor them. Clear examples are missense mutations in classic RAS genes (KRAS, HRAS and NRAS) that underlie the development of approximately 13% of human cancers. Although autonomous B cell antigen receptor (BCR) signaling is involved and mutations in many tumor suppressor genes and oncogenes have been identified, an oncogenic driver gene has not still been identified for CLL.MethodsConditional knock-in mice were generated to overexpress wild type RRAS2 and prove its driver role. RT-qPCR analysis of a human CLL sample cohort was carried out to measure RRAS2 transcriptional expression. Sanger DNA sequencing was used to identify a SNP in the 3'UTR region of RRAS2 in human CLL samples. RNAseq of murine CLL was carried out to identify activated pathways, molecular mechanisms and to pinpoint somatic mutations accompanying RRAS2 overexpression. Flow cytometry was used for phenotypic characterization and shRNA techniques to knockdown RRAS2 expression in human CLL.ResultsRRAS2 mRNA is found overexpressed in its wild type form in 82% of the human CLL samples analyzed (n = 178, mean and median = 5-fold) as well as in the explored metadata. A single nucleotide polymorphism (rs8570) in the 3'UTR of the RRAS2 mRNA has been identified in CLL patients, linking higher expression of RRAS2 with more aggressive disease. Deliberate overexpression of wild type RRAS2 in mice, but not an oncogenic Q72L mutation in the coding sequence, provokes the development of CLL. Overexpression of wild type RRAS2 in mice is accompanied by a strong convergent selection of somatic mutations in genes that have been identified in human CLL. R-RAS2 protein is physically bound to the BCR and mediates BCR signals in CLL.ConclusionsThe results indicate that overexpression of wild type RRAS2 is behind the development of CLL.
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Project description:Protein aggregates are a neuropathological feature of Huntington's disease and Parkinson's disease. Mutant huntingtin exon 1 with 72 CAG repeats fused to enhanced green fluorescent protein (EGFP) forms hyperfluorescent inclusions in PC12 cells. Inclusion formation is enhanced in cells co-transfected with EGFP-huntingtin-(CAG)(72) and alpha-synuclein, a major component of Parkinson's disease aggregates. However, alpha-synuclein does not form aggregates by itself, nor does it appear in huntingtin inclusions in vitro.
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Project description:The metabolic inflammation (meta-inflammation) of obesity is characterized by proinflammatory macrophage infiltration into adipose tissue. Catalysis by deoxyhypusine synthase (DHPS) modifies the translation factor eIF5A to generate a hypusine (Hyp) residue. Hypusinated eIF5A (eIF5AHyp) controls the translation of mRNAs involved in inflammation, but its role in meta-inflammation has not been elucidated. Levels of eIF5AHyp were found to be increased in adipose tissue macrophages from obese mice and in murine macrophages activated to a proinflammatory M1-like state. Global proteomics and transcriptomics revealed that DHPS deficiency in macrophages altered the abundance of proteins involved in NF-κB signaling, likely through translational control of their respective mRNAs. DHPS deficiency in myeloid cells of obese mice suppressed M1 macrophage accumulation in adipose tissue and improved glucose tolerance. These findings indicate that DHPS promotes the post-transcriptional regulation of a subset of mRNAs governing inflammation and chemotaxis in macrophages and contributes to a proinflammatory M1-like phenotype.
| S-EPMC8432737 | biostudies-literature
Project description:NF-Y is the CCAAT-binding trimer formed by the histone fold domain (HFD), NF-YB/NF-YC and NF-YA. The CCAAT box is generally prevalent in promoters of "cancer" genes. We reported the overexpression of NF-YA in BRCA, LUAD and LUSC, and of all subunits in HCC. Altered splicing of NF-YA was found in breast and lung cancer. We analyzed RNA-seq datasets of TCGA and cell lines of head and neck squamous cell carcinomas (HNSCC). We partitioned all TCGA data into four subtypes, deconvoluted single-cell RNA-seq of tumors and derived survival curves. The CCAAT box was enriched in the promoters of overexpressed genes. The "short" NF-YAs was overexpressed in all subtypes and the "long" NF-YAl in Mesenchymal. The HFD subunits are overexpressed, except Basal (NF-YB) and Atypical (NF-YC); NF-YAl is increased in p53 mutated tumors. In HPV-positive tumors, high levels of NF-YAs, p16 and ΔNp63 correlate with better prognosis. Deconvolution of single cell RNA-seq (scRNA-seq) found a correlation of NF-YAl with Cancer Associated Fibroblasts (CAFs) and p-EMT cells, a population endowed with metastatic potential. We conclude that overexpression of HFD subunits and NF-YAs is protective in HPV-positive tumors; expression of NF-YAl is largely confined to mutp53 tumors and malignant p-EMT cells.
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Project description:Aberrant activation of the RAS family of guanosine triphosphatases (GTPases) is prevalent in lung adenocarcinoma, with somatic mutation of KRAS occurring in ~30% of tumors. We previously identified somatic mutations and amplifications of the gene encoding RAS family GTPase RIT1 in lung adenocarcinomas. To explore the biological pathways regulated by RIT1 and how they relate to the oncogenic KRAS network, we performed quantitative proteomic, phosphoproteomic, and transcriptomic profiling of isogenic lung epithelial cells in which we ectopically expressed wild-type or cancer-associated variants of RIT1 and KRAS. We found that both mutant KRAS and mutant RIT1 promoted canonical RAS signaling and that overexpression of wild-type RIT1 partially phenocopied oncogenic RIT1 and KRAS, including induction of epithelial-to-mesenchymal transition. Our findings suggest that RIT1 protein abundance is a factor in its pathogenic function. Therefore, chromosomal amplification of wild-type RIT1 in lung and other cancers may be tumorigenic.
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Project description:Autosomal recessive homozygous or compound heterozygous mutations in FKRP result in forms of muscular dystrophy-dystroglycanopathy varying in age of onset, clinical presentation, and disease progression, ranging from the severe Walker-Warburg, type A,5 (MDDGA5), muscle-eye-brain (MDDGB5) with or without cognitive deficit, to limb-girdle type 2I (MDDGC5). Phenotypic variation indicates degrees of functionality of individual FKRP mutation, which has been supported by the presence of residual expression of functionally glycosylated ?-dystroglycan (DG) in muscles of both animal models and patients. However, direct evidence showing enhancement in glycosylation of ?-DG by mutant FKRP is lacking. Using AAV9-mediated overexpression of mutant human FKRP bearing the P448L mutation (mhFKRP-P448L) associated with severe congenital muscular dystrophy (CMD), we demonstrate the restoration of functional glycosylation of ?-DG and reduction in markers of disease progression. Expression of mhFKRP-P448L also corrects dystrophic phenotypes in the models of L276I mutation with mild disease phenotype and causes no obvious histological or biomarker alteration in C57BL/6 normal mice. Our results confirm the existing function of mutant FKRP. The results also suggest that mutant FKRP could be an alternative approach for potential gene therapy should normal FKRP gene products be immunogenic.
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Project description:Pathological conditions associated with dysfunctional wound healing are characterized by impaired remodelling of extracellular matrix (ECM), increased macrophage infiltration, and chronic inflammation. Macrophages also play an important role in wound healing as they drive wound closure by secretion of molecules like transforming growth factor beta-1 (TGF-β). As the functions of macrophages are regulated by their metabolism, local administration of small molecules that alter this might be a novel approach for treatment of wound-healing disorders. Itaconate is a tricarboxylic acid (TCA) cycle-derived metabolite that has been associated with resolution of macrophage-mediated inflammation. However, its effects on macrophage wound healing functions are unknown. In this study, we investigated the effects of the membrane-permeable 4-octyl itaconate (4-OI) derivative on ECM scavenging by cultured human blood monocyte-derived macrophages (hMDM). We found that 4-OI reduced signalling of p38 mitogen-activated protein kinase (MAPK) induced by the canonical immune stimulus lipopolysaccharide (LPS). Likely as a consequence of this, the production of the inflammatory mediators like tumor necrosis factor (TNF)-α and cyclooxygenase (COX)-2 were also reduced. On the transcriptional level, 4-OI increased expression of the gene coding for TGF-β (TGFB1), whereas expression of the collagenase matrix metalloprotease-8 (MMP8) was reduced. Furthermore, surface levels of the anti-inflammatory marker CD36, but not CD206 and CD11c, were increased in these cells. To directly investigate the effect of 4-OI on scavenging of ECM by macrophages, we developed an assay to measure uptake of fibrous collagen. We observed that LPS promoted collagen uptake and that this was reversed by 4-OI-induced signaling of nuclear factor erythroid 2-related factor 2 (NRF2), a regulator of cellular resistance to oxidative stress and the reduced glycolytic capacity of the macrophage. These results indicate that 4-OI lowers macrophage inflammation, likely promoting a more wound-resolving phenotype.
| S-EPMC9800195 | biostudies-literature
Project description:ObjectivesForkhead box D1, the core transcription factor member of FOX family, has gradually seen as a key cancerous regulatory. However, its expression and carcinogenicity in head and neck squamous cell carcinoma (HNSCC) have not been reported yet. This study was to investigate its expression pattern, clinicopathological significance and biological roles in HNSCC.MethodsHNSCC data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) was used to indicate the detailed expression pattern and outcome association of FOXD1, while Western Blot assay to detect FOXD1 level in a panel of HNSCC cell lines as well as immunocytochemistry to explore FOXD1 protein abundance and sublocation. Series of siRNA-mediated FOXD1 knock-down experiments to assess the proliferation, migration, invasion and anti- apoptosis ability after FOXD1 down-regulation. Bioinformatic analysis to find out which biological function and cancer-related pathways of FOXD1 associated genes involved in.ResultsFOXD1 mRNA was significantly overexpressed in TCGA-HNSCC, GSE6631, GSE12452, GSE25099 and GSE30784. Besides, IHC results shown that nuclear location FOXD1 protein was significantly higher in primary HNSCC specimens from cohort involved in this study. Also, FOXD1 abundance was significantly correlated with cervical node metastasis and poor over-all/disease-free survival after combination analysis with patient pathological information. siRNA-mediated FOXD1 knock-down significantly inhibited cell proliferation, migration and invasion and induced apoptosis in HNSCC cells. Further analysis of GSEA, GO and KEGG showed that FOXD1 expression was significantly associated with oncological function and cancer-related pathways.ConclusionsTaken together, our study implies that the potential oncogene, FOXD1, facilitates oncological behavior who can be identified as a brand-new HNSCC biomarker with diagnostic and prognostic significance.
| S-EPMC8588630 | biostudies-literature