NF?B and STAT3 synergistically activate the expression of FAT10, a gene counteracting the tumor suppressor p53.
ABSTRACT: Chronic inflammation is one of the main causes of cancer, yet the molecular mechanism underlying this effect is not fully understood. In this study, we identified FAT10 as a potential target gene of STAT3, the expression of which is synergistically induced by NF?B co-stimulation. STAT3 binding stabilizes NF?B on the FAT10 promoter and leads to maximum induction of FAT10 gene expression. Increased FAT10 represses the transcriptional activity of the tumor suppressor p53, a protein that accelerates the protein degradation of FAT10. This FAT10-p53 double-negative regulation is critical in the control of tumorigenesis, as overexpressed FAT10 facilitates the tumor progression in the solid tumor model. In conclusion, transcriptional synergy between STAT3 and NF?B functions to put weight on FAT10 in the mutually inhibitory FAT10-p53 regulatory loop and thus favors tumorigenesis under inflammatory conditions.
Project description:FAT10, also known as diubiquitin, has been implicated in the regulation of diverse cellular processes, including mitosis, immune response, and apoptosis. We seek to identify FAT10-targeted proteins, an essential step in elucidating the physiological function of FAT10. To this end, human FAT10 or its non-conjugatable derivative, FAT10?GG, was overexpressed in HEK293 cells. We observed a number of high molecular weight FAT10 conjugates in cells expressing wild-type FAT10, but not in FAT10?GG. The FAT10 conjugates are inducible by TNF-? and accumulated significantly when cells were treated with proteasome inhibitor, MG132. Among them, tumor suppressor p53 was found to be FATylated. The p53 transcriptional activity was found to be substantially enhanced in FAT10-overexpressing cells. In addition, overexpressing FAT10 in HEK293 cells also reduced the population of p53 which cross reacted with monoclonal anti-p53 antibody, PAB240, known to recognize only the transcriptionally inactive p53. FAT10 in the nucleus was found co-localized with p53 and altered its subcellular compartmentalization. Furthermore, overexpressing FAT10 led to a reduction in the size of promyelocytic leukemia nuclear bodies (PML-NBs) and altered their distribution in the nucleus. Based on these observations, a potential mechanism which correlates FATylation of p53 to its translocation and transcriptional activation is discussed.
Project description:Pleiotropic pro-inflammatory cytokines, TNF-? and IFN-? (TI), play important yet diverse roles in cell survival, proliferation, and death. Recent evidence highlights FAT10 as a downstream molecule in the pathway of inflammation-induced tumorigenesis through mediating the effect of cytokines in causing numerical CIN and protecting cells from cytokines-induced cell death. cDNA microarray analysis of cells treated with TI revealed 493 deregulated genes with FAT10 being the most up-regulated (85.7-fold) gene and NF-?B being the key nodal hub of TI-response genes. Silibinin is reported to be a powerful antioxidant and has anti-C effects against various carcinomas by affecting various signaling molecules/pathways including MAPK, NF-?B and STATs. As NF-?B signaling pathway is a major mediator of the tumor-promoting activities of TI, we thus examine the effects of silibinin on TI-induced FAT10 expression and CIN. Our data showed that silibinin inhibited expression of FAT10, TI-induced chromosome instability (CIN) as well as sensitizes cells to TI-induced apoptosis. Significantly, silibinin suppressed intra-tumorally injected TNF-?-induced tumor growth. This represents the first report associating silibinin with FAT10 and demonstrating that silibinin can modulate TI-induced CIN, apoptosis sensitivity and suppressing TNF-?-induced tumor growth.
Project description:FAT10, which is also known as diubiquitin, has been implicated to play important roles in immune regulation and tumorigenesis. Its expression is up-regulated in the tumors of Hepatocellular Carcinoma (HCC) and other cancer patients. High levels of FAT10 in cells have been shown to result in increased mitotic non-disjunction and chromosome instability, leading to tumorigenesis. To evaluate whether the aberrant up-regulation of the FAT10 gene in the tumors of HCC patients is due to mutations or the aberrant methylation of CG dinucleotides at the FAT10 promoter, sequencing and methylation-specific sequencing of the promoter of FAT10 was performed. No mutations were found that could explain the differential expression of FAT10 between the tumor and non-tumorous tissues of HCC patients. However, six single nucleotide polymorphisms (SNPs), including one that has not been previously reported, were identified at the promoter of the FAT10 gene. Different haplotypes of these SNPs were found to significantly mediate different FAT10 promoter activities. Consistent with the experimental observation, differential FAT10 expression in the tumors of HCC patients carrying haplotype 1 was generally higher than those carrying haplotype II. Notably, the methylation status of this promoter was found to correlate with FAT10 expression levels. Hence, the aberrant overexpression of the FAT10 gene in the tumors of HCC patients is likely due to aberrant methylation, rather than mutations at the FAT10 promoter.
Project description:NF-kappaB is a central mediator of innate immunity and contributes to the pathogenesis of several renal diseases. FAT10 is a TNF-alpha-inducible ubiquitin-like protein with a putative role in immune response, but whether FAT10 participates in TNF-alpha-induced NF-kappaB activation is unknown. Here, using renal tubular epithelial cells (RTECs) derived from FAT10(-/-) and FAT10(+/+) mice, we observed that FAT10 deficiency abrogated TNF-alpha-induced NF-kappaB activation and reduced the induction of NF-kappaB-regulated genes. Despite normal IkBalpha degradation and polyubiquitination, FAT10 deficiency impaired TNF-alpha-induced IkBalpha degradation and nuclear translocation of p65 in RTECs, suggesting defective proteasomal degradation of polyubiquitinated IkBalpha. In addition, FAT10 deficiency reduced the expression of the proteasomal subunit low molecular mass polypeptide 2 (LMP2). Transduction of FAT10(-/-) RTECs with FAT10 restored LMP2 expression, TNF-alpha-induced IkBalpha degradation, p65 nuclear translocation, and NF-kappaB activation. Furthermore, LMP2 transfection restored IkBalpha degradation in FAT10(-/-) RTECs. In humans, common types of chronic kidney disease associated with tubulointerstitial upregulation of FAT10. These data suggest that FAT10 mediates NF-kappaB activation and may promote tubulointerstitial inflammation in chronic kidney diseases.
Project description:FAT10 is a new member of the ubiquitin-like protein family with yet-to-be defined biological functions in the heart. Our objective was to determine the role of FAT10 in the heart. FAT10 is expressed in the normal human and murine hearts, as detected by qPCR and Western blotting. Expression of FAT10 is increased in the heart at the border zone of myocardial infarction and in cultured neonatal rat cardiac myocytes (NRCM) subjected to hypoxia/reoxygenation (H/R) stress. Lentiviral-mediated overexpression of FAT10 in NRCM reduced p53 (TP53) and its target miR-34a levels, while BCL2 level, a target of miR-34a, was increased and BAX level, a pro-apoptotic protein, was reduced. These changes were associated with reduced apoptosis, detected by FACS analysis of annexin-V expression and TUNEL assay, in response to H/R injury. Knock down of FAT10 by shRNA targeting had the opposite effects. Likewise, lentiviral mediated expression of miR-34a was associated with reduced BCL2 and increased BAX levels in NRCM and also reversed changes in BCL-2 and BAX levels observed upon over-expression of FAT10. Treatment of NRCM with proteasome inhibitor MG132 increased p53 and miR-34a levels and reduced BLC2/BAX ratio. These changes were not reversed upon over-expression of FAT10. Thus, FAT10 is upregulated in the heart and NRCM in response to H/R stress, which protects cardiac myocytes against apoptosis. The anti-apoptotic effects of FAT10 are associated with suppression of p53, probably through fatylation and proteasomal degradation, reduced miR-34a expression, and a shift in the BCL2/BAX proteins against apoptosis. Thus, FAT10 is a cardioprotective protein.
Project description:FAT10 (HLA-F-adjacent transcript 10) is a ubiquitin-like modifier that is commonly overexpressed in various tumors. It was found to play a role in mitotic regulation through its interaction with mitotic arrest-deficient 2 (MAD2). Overexpression of FAT10 promotes tumor growth and malignancy. Here, we identified the MAD2-binding interface of FAT10 to be located on its first ubiquitin-like domain whose NMR structure thus was determined. We further proceeded to demonstrate that disruption of the FAT10-MAD2 interaction through mutation of specific MAD2-binding residues did not interfere with the interaction of FAT10 with its other known interacting partners. Significantly, ablation of the FAT10-MAD2 interaction dramatically limited the promalignant capacity of FAT10, including promoting tumor growth in vivo and inducing aneuploidy, proliferation, migration, invasion, and resistance to apoptosis in vitro. Our results strongly suggest that the interaction of FAT10 with MAD2 is a key mechanism underlying the promalignant property of FAT10 and offer prospects for the development of anticancer strategies.
Project description:Human HLA-F adjacent transcript 10 (FAT10) is a member of the ubiquitin-like-modifier family of proteins, which have been implicated in cancer development. In addition, the Survivin protein promotes proliferation in bladder cancer (BC). In this study, we explored the link between FAT10 and Survivin. FAT10 expression was dramatically up-regulated in BC tissue samples, and Kaplan-Meier survival analysis revealed that BC patients with high FAT10 expression had shorter overall survival than those with low FAT10 expression. Moreover, RNAi-mediated FAT10 knockdown decreased Survivin protein levels and inhibited BC proliferation both in vitro and in vivo. FAT10 directly bound to and stabilized Survivin protein, thereby promoting cancer cell proliferation by inhibiting ubiquitin-mediated degradation. These results reveal a novel mechanism by which FAT10 promotes tumor proliferation by directly stabilizing Survivin protein in BC.
Project description:FAT10 interacts with MAD2 and caused tumor progression. The hypothesis tested in the present study was FAT10 induces tumor progression through its binding to MAD2 and Disruption of this binding restored its global gene expression Overall design: Total RNA obtained from wild-type HCT116, stable FAT overexpressed HCT116, and FAT10 mutants, where its binding with MAD2 was disrupted or abolished.
Project description:FAT10 is a ubiquitin-like protein modifier that is induced in vertebrates following certain inflammatory stimuli. Its functions and the repertoire of its target substrates have remained elusive. In contrast to ubiquitin, its cellular abundance is tightly controlled by both transcriptional and posttranslational regulation, and it was reported to be rapidly degraded by the proteasome. Here we provide data to indicate that the degradation of FAT10 requires ubiquitination: degradation was inhibited in cells expressing a ubiquitin mutant that cannot be polymerized and in a mutant cell harboring a thermolabile ubiquitin-activating enzyme, E1. Of importance, FAT10 can serve as a degradation signal for otherwise stable proteins, and in this case, too, the targeting to the proteasome requires ubiquitination. Degradation of FAT10 is accelerated after induction of apoptosis, suggesting that it plays a role in prosurvival pathways.
Project description:RIG-I is a key cytosolic RNA sensor that mediates innate immune defense against RNA virus. Aberrant RIG-I activity leads to severe pathological states such as autosomal dominant multi-system disorder, inflammatory myophathies and dermatomyositis. Therefore, identification of regulators that ensure efficient defense without harmful immune-pathology is particularly critical to deal with RIG-I-associated diseases. Here, we presented the inflammatory inducible FAT10 as a novel negative regulator of RIG-I-mediated inflammatory response. In various cell lines, FAT10 protein is undetectable unless it is induced by pro-inflammatory cytokines. FAT10 non-covalently associated with the 2CARD domain of RIG-I, and inhibited viral RNA-induced IRF3 and NF-kB activation through modulating the RIG-I protein solubility. We further demonstrated that FAT10 was recruited to RIG-I-TRIM25 to form an inhibitory complex where FAT10 was stabilized by E3 ligase TRIM25. As the result, FAT10 inhibited the antiviral stress granules formation contains RIG-I and sequestered the active RIG-I away from the mitochondria. Our study presented a novel mechanism to dampen RIG-I activity. Highly accumulated FAT10 is observed in various cancers with pro-inflammatory environment, therefore, our finding which uncovered the suppressive effect of the accumulated FAT10 during virus-mediated inflammatory response may also provide molecular clue to understand the carcinogenesis related with infection and inflammation.