Response: 'Antibody crossreactivity between the tumour suppressor PHLPP1 and the proto-oncogene β-catenin'.
Ontology highlight
ABSTRACT:
SUBMITTER: McIntush EW
PROVIDER: S-EPMC3674452 | biostudies-literature | 2013 Jun
REPOSITORIES: biostudies-literature
Publications
Response: 'Antibody crossreactivity between the tumour suppressor PHLPP1 and the proto-oncogene β-catenin'.
EMBO reports 20130517 6
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Project description:Beta-catenin is well-known as a key effector of Wnt signalling and aberrant expression is associated with several human cancers. Stabilisation of and atypical subcellular localisation of beta-catenin, regulated in part through specific protein-protein interactions has been linked to cancer development, however the mechanisms behind these pathologies is yet to be fully elucidated. Affinity purification and mass spectrometry were used to identify potential β-catenin interacting proteins in SW480 colon cancer cells. Recombinant β-catenin constructs were used to co-isolate interacting proteins from stable isotope labelled cells followed by detection using mass spectrometry. Several known and new putative interactors were observed. In particular, we identified interaction with a set of coatomer complex I subunits implicated in retrograde transport at the Golgi, and confirmed endogenous interaction of β-catenin with coatomer subunit COPB using immunoprecipitation assays and immunofluorescence microscopy. These observations suggest a hitherto unrecognised role for β-catenin in the secretory pathway and warrant further functional studies to unravel its activity at this cellular location.
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Project description:STAT5B, a specific member of the STAT family, is intimately associated with prostate tumor progression. While the full form of STAT5B is thought to promote tumor progression, a naturally occurring truncated isoform acts as a tumor suppressor. We previously demonstrated that truncated STAT5 is generated by insertion of an alternatively spliced exon and results in the introduction of an early termination codon. Present approaches targeting STAT proteins based on inhibition of functional domains of STAT's, such as DNA-binding, cooperative binding (protein-protein interaction), dimerization and phosphorylation will halt the action of the entire gene, both the proto-oncogenic and tumor suppressor functions of Stat5B. In this report we develop a new approach aimed at inhibiting the expression of full-length STAT5B (a proto-oncogene) while simultaneously enhancing the expression of STAT5?B (a tumor suppressor). We have demonstrated the feasibility of using steric-blocking splice-switching oligonucleotides (SSOs) with a complimentary sequence to the targeted exon-intron boundary to enhance alternative intron/exon retention (up to 10%). The functional effect of the intron/exon proportional tuning was validated by cell proliferation and clonogenic assays. The new scheme applies specific steric-blocking splice-switching oligonucleotides and opens an opportunity for anti-tumor treatment as well as for the alteration of functional abilities of other STAT proteins.
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Project description:BackgroundMicroRNAs (miRNAs) regulate expression of many cancer-related genes through posttranscriptional repression of their mRNAs. In this study we investigate the proto-oncogene MYCN as a target for miRNA regulation.MethodsA luciferase reporter assay was used to investigate software-predicted miRNA target sites in the 3'-untranslated region (3'UTR) of MYCN. The miRNAs were overexpressed in cell lines by transfection of miRNA mimics or miRNA-expressing plasmids. Mutation of the target sites was used to validate MYCN 3'UTR as a direct target of several miRNAs. To measure miRNA-mediated suppression of endogenous N-myc protein, inhibition of proliferation and inhibition of clonogenic growth, miRNAs were overexpressed in a MYCN-amplified neuroblastoma cell line.ResultsThe results from this study show that MYCN is targeted by several miRNAs. In addition to the previously shown mir-34a/c, we experimentally validate mir-449, mir-19a/b, mir-29a/b/c, mir-101 and let-7e/mir-202 as direct MYCN-targeting miRNAs. These miRNAs were able to suppress endogenous N-myc protein in a MYCN-amplified neuroblastoma cell line. The let-7e and mir-202 were strong negative regulators of MYCN expression. The mir-101 and the let-7 family miRNAs let-7e and mir-202 inhibited proliferation and clonogenic growth when overexpressed in Kelly cells.ConclusionThe tumour-suppressor miRNAs let-7 and mir-101 target MYCN and inhibit proliferation and clonogenic growth of MYCN-amplified neuroblastoma cells.
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Project description:Whether TMPRSS2-ERG fusion and TP53 gene alteration coordinately promote prostate cancer (PCa) remains unclear. Here we demonstrate that TMPRSS2-ERG fusion and TP53 mutation / deletion co-occur in PCa patient specimens and this co-occurrence accelerates prostatic oncogenesis. p53 gain-of-function (GOF) mutants are now shown to bind to a unique DNA sequence in the CTNNB1 gene promoter and transactivate its expression. ERG and β-Catenin co-occupy sites at pyrimidine synthesis gene (PSG) loci and promote PSG expression, pyrimidine synthesis and PCa growth. β-Catenin inhibition by small molecule inhibitors or oligonucleotide-based PROTAC suppresses TMPRSS2-ERG- and p53 mutant-positive PCa cell growth in vitro and in mice. Our study identifies a gene transactivation function of GOF mutant p53 and reveals β-Catenin as a transcriptional target gene of p53 GOF mutants and a driver and therapeutic target of TMPRSS2-ERG- and p53 GOF mutant-positive PCa.
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Project description:While there is growing evidence that many epigenetically silenced genes in cancer are tumour suppressor candidates, their significance in cancer biology remains unclear. Here, we identify human Neuralized (NEURL), which acts as a novel tumour suppressor targeting oncogenic Wnt/β-catenin signalling in human cancers. The expression of NEURL is epigenetically regulated and markedly suppressed in human colorectal cancer. We, therefore, considered NEURL to be a bona fide tumour suppressor in colorectal cancer and demonstrate that this tumour suppressive function depends on NEURL-mediated oncogenic β-catenin degradation. We find that NEURL acts as an E3 ubiquitin ligase, interacting directly with oncogenic β-catenin, and reducing its cytoplasmic levels in a GSK3β- and β-TrCP-independent manner, indicating that NEURL-β-catenin interactions can lead to a disruption of the canonical Wnt/β-catenin pathway. This study suggests that NEURL is a therapeutic target against human cancers and that it acts by regulating oncogenic Wnt/β-catenin signalling.
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Project description:BackgroundFAT4, a cadherin-related protein, was shown to function as a tumour suppressor; however, its role in human gastric cancer remains largely unknown. Here, we investigated the role of FAT4 in gastric cancer and examined the underlying molecular mechanisms.MethodsThe expression of FAT4 was evaluated by immunohistochemistry, western blotting, and qRT-PCR in relation to the clinicopathological characteristics of gastric cancer patients. The effects of FAT4 silencing on cell proliferation, migration, and invasion were assessed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) assay, and migration and invasion assays in gastric cancer cell lines in vitro and in a mouse xenograft model in vivo.ResultsDownregulation of FAT4 expression in gastric cancer tissues compared with adjacent normal tissues was correlated with lymph-node metastasis and poor survival. Knockdown of FAT4 promoted the growth and invasion of gastric cancer cells via the activation of Wnt/β-catenin signalling, and induced epithelial-to-mesenchymal transition (EMT) in gastric cancer cells, as demonstrated by the upregulation and downregulation of mesenchymal and epithelial markers. Silencing of FAT4 promoted tumour growth and metastasis in a gastric cancer xenograft model in vivo.ConclusionsFAT4 has a tumour suppressor role mediated by the modulation of Wnt/β-catenin signalling, providing potential novel targets for the treatment of gastric cancer.
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Project description:Nitrilase1 was classified as a tumour suppressor in association with the fragile histidine-triad protein Fhit. However, knowledge about nitrilase1 and its tumour suppressor function is still limited. Whereas nitrilase1 and Fhit are discrete proteins in mammals, they are merged in Drosophila melanogaster and Caenorhabditis elegans. According to the Rosetta-Stone hypothesis, proteins encoded as fusion proteins in one organism and as separate proteins in another organism may act in the same signalling pathway. Although a direct interaction of human nitrilase1 and Fhit has not been shown, our previous finding that Fhit interacts with β-catenin and represses its transcriptional activity in the canonical Wnt pathway suggested that human nitrilase1 also modulates Wnt signalling. In fact, human nitrilase1 forms a complex with β-catenin and LEF-1/TCF-4, represses β-catenin-mediated transcription and shows an additive effect together with Fhit. Knockdown of human nitrilase1 enhances Wnt target gene expression. Moreover, our experiments show that β-catenin competes away human nitrilase1 from LEF-1/TCF and thereby contributes to the activation of Wnt-target gene transcription. Inhibitory activity of human nitrilase1 on vertebrate Wnt signalling was confirmed by repression of Wnt-induced double axis formation in Xenopus embryogenesis. In line with this finding, the Drosophila fusion protein Drosophila NitFhit directly binds to Armadillo and represses the Wingless pathway in reporter gene assays. Genetic experiments confirmed the repressive activity of Drosophila NitFhit on Wingless signalling in the Drosophila wing imaginal disc. In addition, colorectal tumour microarray analysis revealed a significantly reduced expression of human nitrilase1 in poorly differentiated tumours. Taken together, repression of the canonical Wnt pathway represents a new mechanism for the human nitrilase1 tumour suppressor function.
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