EPHA2-dependent outcompetition of KRASG12D mutant cells by wild-type neighbors in the adult pancreas.
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ABSTRACT:
SUBMITTER: Hill W
PROVIDER: S-EPMC8231095 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Plasmodium falciparum purine nucleoside phosphorylase (PfPNP) catalyzes an essential step in purine salvage for parasite growth. 4'-Deaza-1'-Aza-2'-Deoxy-1'-(9-Methylene)-Immucillin-G (DADMe-ImmG) is a transition state analog inhibitor of this enzyme, and P. falciparum infections in an Aotus primate malaria model can be cleared by oral administration of DADMe-ImmG. P. falciparum cultured under increasing DADMe-ImmG drug pressure exhibited PfPNP gene amplification, increased protein expression, and point mutations involved in DADMe-ImmG binding. However, the weak catalytic properties of the M183L resistance mutation (∼17,000-fold decrease in catalytic efficiency) are inconsistent with the essential function of PfPNP. We hypothesized that M183L subunits may form mixed oligomers of native and mutant PfPNP monomers to give hybrid hexameric enzymes with properties conferring DADMe-ImmG resistance. To test this hypothesis, we designed PfPNP constructs that covalently linked native and the catalytically weak M183L mutant subunits. Engineered hybrid PfPNP yielded trimer-of-dimer hexameric protein with alternating native and catalytically weak M183L subunits. This hybrid PfPNP gave near-native Km values for substrate, but the affinity for DADMe-ImmG and catalytic efficiency were both reduced approximately ninefold relative to a similar construct of native subunits. Contact between the relatively inactive M183L and native subunits is responsible for altered properties of the hybrid protein. Thus, gene amplification of PfPNP provides adequate catalytic activity while resistance to DADMe-ImmG occurs in the hybrid oligomer to promote parasite survival. Coupled with the slow development of drug resistance, this resistance mechanism highlights the potential for DADMe-ImmG use in antimalarial combination therapies.
| S-EPMC7949152 | biostudies-literature
Project description:The effect of sampling time in the context of growth conditions on a dynamic metabolic system was investigated in order to assess to what extent a single sampling time may be sufficient for general application, as well as to determine if useful kinetic information could be obtained. A wild type yeast strain (W) was compared to a snf1Delta mutant yeast strain (S) grown in high-glucose medium (R) and in low-glucose medium containing ethanol (DR). Under these growth conditions, different metabolic pathways for utilizing the different carbon sources are expected to be active. Thus, changes in metabolite levels relating to the carbon source in the growth medium were anticipated. Furthermore, the Snf1 protein kinase complex is required to adapt cellular metabolism from fermentative R conditions to oxidative DR conditions. So, differences in intracellular metabolite levels between the W and S yeast strains were also anticipated. Cell extracts were collected at four time points (0.5, 2, 4, 6 h) after shifting half of the cells from R to DR conditions, resulting in 16 sample classes (WR, WDR, SR, SDR) x (0.5, 2, 4, 6 h). The experimental design provided time course data, so temporal dependencies could be monitored in addition to carbon source and strain dependencies. Comprehensive two-dimensional (2D) gas chromatography coupled to time-of-flight mass spectrometry (GC x GC-TOFMS) was used with discovery-based data mining algorithms ( Anal. Chem. 2006, 78, 5068-5075 (ref 1); J. Chromatogr., A 2008, 1186, 401-411 (ref 2)) to locate regions within the 2D chromatograms (i.e., metabolites) that provided chemical selectivity between the 16 sample classes. These regions were mathematically resolved using parallel factor analysis to positively identify the metabolites and to acquire quantitative results. With these tools, 51 unique metabolites were identified and quantified. Various time course patterns emerged from these data, and principal component analysis (PCA) was utilized as a comparison tool to determine the sources of variance between these 51 metabolites. The effect of sampling time was investigated with separate PCA analyses using various subsets of the data. PCA utilizing all of the time course data, averaged time course data, and each individual time point data set independently were performed to discern the differences. For the yeast strains examined in the current study, data collection at either 4 or 6 h provided information comparable to averaged time course data, albeit with a few metabolites missing using a single sampling time point.
| S-EPMC3464980 | biostudies-literature
Project description:A reduction in pH is known to induce the disassociation of the tetrameric form of transthyretin and favor the formation of amyloid fibers. Using continuum electrostatic techniques, we calculate the titration curves and the stability of dimer and tetramer formation of transthyretin as a function of pH. We find that the tetramer and the dimer become less stable than the monomer as the pH is lowered. The free energy difference is 13.8 kcal/mol for dimer formation and 27 kcal/mol for tetramer formation, from the monomers, when the pH is lowered from 7 to 3.9. Similar behavior is observed for both the wild-type and the mutant protein. Certain residues (namely Glu-72, His-88, His-90, Glu-92, and Tyr-116), play an important role in the binding process, as seen by the considerable pK(1/2) change of these residues upon dimer formation.
| S-EPMC1302845 | biostudies-literature
Project description:BackgroundSemaphorins act as chemotactic cues for cell movement via their transmembrane receptors, plexins. Somatic missense mutations in the plexinB1 gene coupled with overexpression of the protein frequently occur in prostate tumors, indicating a role for plexinB1 in the pathogenesis of prostate cancer. However, the effect of semaphorin/plexin signaling is highly context dependent and whether plexinB1 acts as an inducer or inhibitor of prostate tumor progression in this context is not known.MethodsThe response of prostate cancer cell lines to plexinB1 activation was assessed in migration, invasion, proliferation and protein phosphorylation assays. Expression was assessed by quantitative RTPCR and immunoblotting.ResultsDifferent prostate cancer cell lines respond to Sema4D (the ligand for plexinB1) in diverse ways. Activation of endogenous plexinB1 enhances migration, invasion and anchorage-independent growth of LNCaP prostate cancer cells via activation of ErbB2 and Akt. In contrast, Sema4D-stimulation decreased the motility and proliferative capacity of PC3 cells. LNCaP has a missense mutation (Thr1697Ala) in the plexinB1 gene while LNCaP-LN3, a derivative of LNCaP, expresses high levels of wild-type plexinB1 only. Sema4D stimulation increases the motility and anchorage independent growth of both cell lines, showing that these responses are not dependent on the presence of the Thr1697Ala form of plexinB1. ErbB2 and plexinB1 are expressed in primary prostate epithelial cells.ConclusionsPlexinB1 signals via ErbB2 to increase the invasive phenotype of prostate cancer cells. Both wild-type and mutant forms of plexinB1 are potential targets for anti-cancer therapy in prostate tumors that express ErbB2.
| S-EPMC4282548 | biostudies-literature
Project description:Niches maintain a finite pool of stem cells via restricted space and short-range signals. Stem cells compete for limited niche resources, but the mechanisms regulating competition are poorly understood. Using the Drosophila testis model, we show that germline stem cells (GSCs) lacking the transcription factor Chinmo gain a competitive advantage for niche access. Surprisingly, chinmo-/- GSCs rely on a new mechanism of competition in which they secrete the extracellular matrix protein Perlecan to selectively evict non-mutant GSCs and then upregulate Perlecan-binding proteins to remain in the altered niche. Over time, the GSC pool can be entirely replaced with chinmo-/- cells. As a consequence, the mutant chinmo allele acts as a gene drive element; the majority of offspring inherit the allele despite the heterozygous genotype of the parent. Our results suggest that the influence of GSC competition may extend beyond individual stem cell niche dynamics to population-level allelic drift and evolution.
| S-EPMC8752517 | biostudies-literature
Project description:Ferroportin [FPN; Slc40a1 (solute carrier family 40, member 1)] is a transmembrane iron export protein expressed in macrophages and duodenal enterocytes. Heterozygous mutations in the FPN gene result in an autosomal dominant form of iron overload disorder, type-4 haemochromatosis. FPN mutants either have a normal iron export activity but have lost their ability to bind hepcidin, or are defective in their iron export function. The mutant protein has been suggested to act as a dominant negative over the wt (wild-type) protein by multimer formation. Using transiently transfected human epithelial cell lines expressing mouse FPN modified by the addition of a haemagglutinin or c-Myc epitope at the C-terminus, we show that the wtFPN is found at the plasma membrane and in Rab5-containing endosomes, as are the D157G and Q182H mutants. However, the delV162 mutant is mostly intracellular in HK2 cells (human kidney-2 cells) and partially addressed at the cell surface in HEK-293 cells (human embryonic kidney 293 cells). In both cell types, it is partially associated with the endoplasmic reticulum and with Rab5-positive vesicles. However, this mutant is complex-glycosylated like the wt protein. D157G and G323V mutants have a defective iron export capacity as judged by their inability to deplete the intracellular ferritin content, whereas Q182H and delV162 have normal iron export function and probably have lost their capacity to bind hepcidin. In co-transfection experiments, the delV162 mutant does not co-localize with the wtFPN, does not prevent its normal targeting to the plasma membrane and cannot be immunoprecipitated in the same complex, arguing against the formation of FPN hetero-oligomers.
| S-EPMC1462719 | biostudies-literature
Project description:Telomerase reverse transcriptase (TERT) promoter (pTERT) mutation has often been described as a late event in gliomagenesis and it has been suggested as a prognostic biomarker in gliomas other than 1p19q codeleted tumors. However, the characteristics of isocitrate dehydrogenase (IDH) wild type (wt) (IDHwt), pTERTwt glioblastomas are not well known. We recruited 72 adult IDHwt, pTERTwt glioblastomas and performed methylation profiling, targeted sequencing, and fluorescence in situ hybridization (FISH) for TERT structural rearrangement and ALT (alternative lengthening of telomeres). There was no significant difference in overall survival (OS) between our cohort and a the Cancer Genome Atlas (TCGA) cohort of IDHwt, pTERT mutant (mut) glioblastomas, suggesting that pTERT mutation on its own is not a prognostic factor among IDHwt glioblastomas. Epigenetically, the tumors clustered into classic-like (11%), mesenchymal-like (32%), and LGm6-glioblastoma (GBM) (57%), the latter far exceeding the corresponding proportion seen in the TCGA cohort of IDHwt, pTERTmut glioblastomas. LGm6-GBM-clustered tumors were enriched for platelet derived growth factor receptor alpha (PDGFRA) amplification or mutation (p = 0.008), and contained far fewer epidermal growth factor receptor (EGFR) amplification (p < 0.01), 10p loss (p = 0.001) and 10q loss (p < 0.001) compared with cases not clustered to this group. LGm6-GBM cases predominantly showed ALT (p = 0.038). In the whole cohort, only 35% cases showed EGFR amplification and no case showed combined chromosome +7/-10. Since the cases were already pTERTwt, so the three molecular properties of EGFR amplification, +7/-10, and pTERT mutation may not cover all IDHwt glioblastomas. Instead, EGFR and PDGFRA amplifications covered 67% and together with their mutations covered 71% of cases of this cohort. Homozygous deletion of cyclin dependent kinase inhibitor 2A (CDKN2A)/B was associated with a worse OS (p = 0.031) and was an independent prognosticator in multivariate analysis (p = 0.032). In conclusion, adult IDHwt, pTERTwt glioblastomas show epigenetic clustering different from IDHwt, pTERTmut glioblastomas, and IDHwt glioblastomas which are pTERTwt may however not show EGFR amplification or +7/-10 in a significant proportion of cases. CDKN2A/B deletion is a poor prognostic biomarker in this group.
| S-EPMC9616088 | biostudies-literature
Project description:Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer, and is associated with a poor prognosis due to frequent distant metastasis and lack of effective targeted therapies. Previously, we identified maternal embryonic leucine zipper kinase (MELK) to be highly expressed in TNBCs as compared with ER-positive breast cancers. Here we determined the molecular mechanism by which MELK is overexpressed in TNBCs. Analysis of publicly available data sets revealed that MELK mRNA is elevated in p53-mutant breast cancers. Consistent with this observation, MELK protein levels are higher in p53-mutant vs. p53 wild-type breast cancer cells. Furthermore, inactivation of wild-type p53, by loss or mutation of the p53 gene, increases MELK expression, whereas overexpression of wild-type p53 in p53-null cells reduces MELK promoter activity and MELK expression. We further analyzed MELK expression in breast cancer data sets and compared that with known wild-type p53 target genes. This analysis revealed that MELK expression strongly correlates with genes known to be suppressed by wild-type p53. Promoter deletion studies identified a p53-responsive region within the MELK promoter that did not map to the p53 consensus response elements, but to a region containing a FOXM1-binding site. Consistent with this result, knockdown of FOXM1 reduced MELK expression in p53-mutant TNBC cells and expression of wild-type p53 reduced FOXM1 expression. ChIP assays demonstrated that expression of wild-type p53 reduces binding of E2F1 (a critical transcription factor controlling FOXM1 expression) to the FOXM1 promoter, thereby, reducing FOXM1 expression. These results show that wild-type p53 suppresses FOXM1 expression, and thus MELK expression, through indirect mechanisms. Overall, these studies demonstrate that wild-type p53 represses MELK expression by inhibiting E2F1A-dependent transcription of FOXM1 and that mutation-driven loss of wild-type p53, which frequently occurs in TNBCs, induces MELK expression by suppressing FOXM1 expression and activity in p53-mutant breast cancers.
| S-EPMC6941974 | biostudies-literature
Project description:Tumor protein p53 is a key regulator of several cellular pathways, including DNA repair, cell cycle and angiogenesis. Kevetrin exhibits p53‑dependent as well as‑independent activity in solid tumors, while its effects on leukemic cells remain unknown. The aim of the present study was to analyze the response of acute myeloid leukemia (AML) cell lines (TP53 wild‑type: OCI‑AML3 and MOLM‑13; and TP53‑mutant: KASUMI‑1 and NOMO‑1) to kevetrin at a concentration range of 85‑340 µM. The cellular and molecular effects of the treatment were analyzed in terms of cell growth, viability [Annexin V‑propidium iodide (PI) staining] and cell cycle alterations (PI staining). Gene expression profiling, western blotting and immunofluorescence were performed to elucidate the pathways underlying kevetrin activity. Pulsed exposure exerted no effect on the wild‑type cells, but was effective on mutant cells. After continuous treatment, significant cell growth arrest and apoptosis were observed in all cell lines, with TP53‑mutant models displaying a higher sensitivity and p53 induction. Kevetrin also displayed efficacy against TP53 wild‑type and mutant primary AML, with a preferential cytotoxic activity against blast cells. Gene expression profiling revealed a common core transcriptional program altered by drug exposure and the downregulation of glycolysis, DNA repair and unfolded protein response signatures. These findings suggest that kevetrin may be a promising therapeutic option for patients with both wild‑type and TP53‑mutant AML.
| S-EPMC7448420 | biostudies-literature
Project description:Stem cells are undifferentiated cells that can self-renew and generate specialized (functional) cell types. The remarkable ability of stem cells to differentiate towards functional cells makes them suitable modalities in cellular therapy (which means treating diseases with the body's own cells). Potential targets for cellular therapy include diabetes and liver failure. However, in order for stem cells to be clinically useful, we must learn to identify them and to regulate their differentiation. We will use the intestine as a classical example of a stem cell compartment, and then examine the evidence for the existence of adult stem cells in two endodermally derived organs: pancreas and liver. We will review the characteristics of the putative stem cells in these tissues and the transcription factors controlling their differentiation towards functional cell types.
| S-EPMC2715288 | biostudies-literature