FAT10 Plays a Role in the Regulation of Chromosomal Stability

Abstract

Aneuploidy is a key process in tumorigenesis. Dysfunction of the mitotic spindle checkpoint proteins has been implicated as a cause of aneuploidy in cells. We have previously reported that FAT10, a member of the ubiquitin-like modifier family of proteins, is overexpressed in several gastrointestinal and gynecological cancers. Here we show that FAT10 interacts with MAD2, a spindle checkpoint protein, during mitosis. Notably, we show that localization of MAD2 at the kinetochore during the prometaphase stage of the cell cycle was greatly reduced in FAT10-overexpressing cells. Furthermore, compared with parental HCT116 cells, fewer mitotic cells were observed after double thymidine-synchronized FAT10-overexpressing cells were released into nocodazole for more than 4 h. Nonetheless, when these double thymidine-treated cells were released into media, a similar number of G 1 parental and FAT10-overexpressing HCT116 cells was observed throughout the 10-h time course. Additionally, more nocodazole-treated FAT10-overexpressing cells escape mitotic controls and are multinucleate compared with parental cells. Significantly, we observed a higher degree of variability in chromosome number in cells overexpressing FAT10. Hence, our data suggest that high levels of FAT10 protein in cells lead to increased mitotic nondisjunction and chromosome instability, and this effect is mediated by an abbreviated mitotic phase and the reduction in the kinetochore localization of MAD2 during the prometaphase stage of the cell cycle. Genetic instability is an important phenomenon that underlies tumorigenesis. Chromosome instability (CIN)2 involving gains and loss of chromosomes has been found to occur in most malignancies, whereas microsatellite instability, which occurs at the nucleotide level, is less commonly observed in cancers (1). Two forms of CIN, namely structural instability and numerical instability (aneuploidy), can be observed in various tumors. Genes responsible for CIN in human cancers include those involved in the condensation of chromosomes, cohesion of sister chromatids, formation of microtubules, and kinetochore structure and function as well as mitotic "checkpoint" genes that monitor the proper progression through the cell cycle (1, 2).MAD2 (mitotic arrest-deficient 2) is a key mitotic spindle checkpoint protein whose primary role is to ensure that all of the chromosomes are properly attached to the mitotic spindle before the onset of anaphase (3). It is activated by associating with unattached kinetochores. Activated MAD2 binds to Cdc20 and prevents the anaphase-promoting complex from ubiquitylating securin. As a result, anaphase is delayed until all of the kinetochores are attached by microtubules and the chromosomes are properly aligned along the metaphase plate (4 -6). MAD2 is an essential gene, and MAD2 Ϫ/Ϫ mice die in utero (7). Loss of one allele of MAD2 has been reported to result in premature anaphase and CIN in mammalian cells (8). Dysregulation of MAD2 has been implicated in various cancers. Reduced expression of ...

The ubiquitin-like modifier FAT10 in cancer development

“…In FAT10 overexpressing HCT116 cells as well as in TNF-␣-treated HCT116 cells, the localization of MAD2 at the kinetochores was much reduced in the prometaphase of the cell cycle, indicating that FAT10 interferes with the binding of MAD2 to kinetochores (Fig. 2B) (Ren et al, 2006(Ren et al, , 2011. Moreover, after release from G1/S arrest, FAT10 overexpressing cells showed a delayed entry into mitosis.…”

“…Concurrent with these findings, FAT10 overexpression induced the formation of multinucleated cells with abnormal nuclear morphology. A constitutive overexpression of FAT10 as well as constitutive treatment with TNF-␣ resulted in an increased chromosome number as compared to the parental HCT116 cell line (Ren et al, 2006(Ren et al, , 2011. However, when the five putative interaction sites of MAD2 within the N-terminal ubiquitin-like domain of FAT10 were mutated (H11D, R13Q, H75D, T77D and K79Q), binding of MAD2 to FAT10 was greatly diminished.…”

“…Upregulation of endogenous FAT10 expression by TNF-␣ as well as overexpression of FAT10 has directly been linked to chromosomal instability in several reports (Gao et al, 2015;Ren et al, 2006Ren et al, , 2011Theng et al, 2014). It was shown that about 60% of HCT116 and HepG2 cells which had been treated for about ten passages with IFN-␥ and TNF-␣ displayed abnormally high chromosome numbers in a karyotype analysis and inhibition of FAT10 expression with silibinin, a potent inhibitor of the NF-B signaling pathway, was able to abolish this effect (Gao et al, 2015).…”

“…FAT10 was shown to be cell-cycle regulated with a peak of expression in S-phase but low expression levels during G2/M phase (Lim et al, 2006;Liu et al, 2014) and a proteomics based study provided evidence for a role of FAT10 in mitotic regulation (Merbl et al, 2013). Already 17 years ago MAD2 (Mitotic arrest deficient 2), a mitotic spindle checkpoint protein, was identified as a non-covalent interaction partner of FAT10 in a yeast two hybrid screen using a human B cell cDNA library (Liu et al, 1999) and this interaction was shown to prevail during mitosis (Ren et al, 2006). MAD2 binds to the kinetochores of sister chromatids in the metaphase of the cell cycle.…”

Molecular Mechanisms of Liver Carcinogenesis in the Mdr2-Knockout Mice

“…The most prominent among them being up-regulated-Axl, Cbr3, Cd36, Lgals1, Lgals3, Jun, and Ubd; down-regulated-Ahcy, Cbs, Gnmt (three genes encoding enzymes that control Sadenosylmethionine catabolism), C6, C8a, C8b (encoding complement components), and cytochrome P450 genes Cyp1a2, Cyp2e1, Cyp4v3, Cyp8b1 (8,(25)(26)(27)(28)(29). Overexpression of the Ubd protein may cause chromosomal instability (30). We confirmed the up-regulation of its transcript in Mdr2-KO livers by real-time RT-PCR: Ubd was highly up-regulated in nontumorous liver tissues (6-to 23-fold compared with Mdr2-heterozygotes) and progressively up-regulated in most tumors (Table 1 and Supplementary Tables S2 and S3).…”