TRIM59 Promotes the Proliferation and Migration of Non-Small Cell Lung Cancer Cells by Upregulating Cell Cycle Related Proteins

TRIM protein family is an evolutionarily conserved gene family implicated in a number of critical processes including inflammation, immunity, antiviral and cancer. In an effort to profile the expression patterns of TRIM superfamily in several non-small cell lung cancer (NSCLC) cell lines, we found that the expression of 10 TRIM genes including TRIM3, TRIM7, TRIM14, TRIM16, TRIM21, TRIM22, TRIM29, TRIM59, TRIM66 and TRIM70 was significantly upregulated in NSCLC cell lines compared with the normal human bronchial epithelial (HBE) cell line, whereas the expression of 7 other TRIM genes including TRIM4, TRIM9, TRIM36, TRIM46, TRIM54, TRIM67 and TRIM76 was significantly down-regulated in NSCLC cell lines compared with that in HBE cells. As TRIM59 has been reported to act as a proto-oncogene that affects both Ras and RB signal pathways in prostate cancer models, we here focused on the role of TRIM59 in the regulation of NSCLC cell proliferation and migration. We reported that TRIM59 protein was significantly increased in various NSCLC cell lines. SiRNA-induced knocking down of TRIM59 significantly inhibited the proliferation and migration of NSCLC cell lines by arresting cell cycle in G2 phase. Moreover, TRIM59 knocking down affected the expression of a number of cell cycle proteins including CDC25C and CDK1. Finally, we knocked down TRIM59 and found that p53 protein expression levels did not upregulate, so we proposed that TRIM59 may promote NSCLC cell growth through other pathways but not the p53 signaling pathway.     

Role of the histone acetyl transferase Tip60 in the p53 pathway

The histone acetyl transferase Tip60 (HTATIP) shares many properties with the tumor suppressor p53 (TP53). Both proteins are involved in the cellular response to DNA damage, are subjected to proteasomal digestion following Mdm2-mediated ubiquitination, and accumulate after UV irradiation. We found here that knock-down of Tip60 affects the p53-dependent response following actinomycin D treatment, most likely because it inhibits p21 (CDKN1A) accumulation. Moreover, Tip60 is required for p53 to activate the endogenous p21 promoter, suggesting that it functions as a p53 co-activator. However, we also found that knock-down of Tip60 increases the turnover rate of p53 under normal growth conditions. Tip60 interferes with Mdm2-mediated degradation of p53, probably because it affects its subcellular localization. Taken together, our results suggest that Tip60 plays a double role in the p53 pathway: under normal growth conditions, Tip60 contributes to maintain a basal pool of p53 by interfering with its degradation; following DNA damage, Tip60 functions as p53 co-activator. That these two distinct roles are linked during the p53-dependent response is an attractive hypothesis.     

Epigenetic deregulation of miR-29a and miR-1256 by isoflavone contributes to the inhibition of prostate cancer cell growth and invasion

The epigenetic regulation of genes has long been recognized as one of the causes of prostate cancer (PCa) development and progression. Recent studies have shown that a number of microRNAs (miRNAs) are also epigenetically regulated in different types of cancers including PCa. In this study, we found that the DNA sequence of the promoters of miR-29a and miR-1256 are partly methylated in PCa cells, which leads to their lower expression both in PCa cells and in human tumor tissues compared with normal epithelial cells and normal human prostate tissues. By real-time PCR, Western Blot analysis and miRNA mimic and 3′-UTR-Luc transfection, we found that TRIM68 is a direct target of miR-29a and miR-1256 and that the downregulation of miR-29a and miR-1256 in PCa cells leads to increased expression of TRIM68 and PGK-1 in PCa cells and in human tumor tissue specimens. Interestingly, we found that a natural agent, isoflavone, could demethylate the methylation sites in the promoter sequence of miR-29a and miR-1256, leading to the upregulation of miR-29a and miR-1256 expression. The increased levels of miR-29a and miR-1256 by isoflavone treatment resulted in decreased expression of TRIM68 and PGK-1, which is mechanistically linked with inhibition of PCa cell growth and invasion. The selective demethylation activity of isoflavone on miR-29a and miR-1256 leading to the suppression of TRIM68 and PGK-1 expression is an important biological effect of isoflavone, suggesting that isoflavone could be a useful non-toxic demethylating agent for the prevention of PCa development and progression.     

Epigenetic deregulation of miR-29a and miR-1256 by isoflavone contributes to the inhibition of prostate cancer cell growth and invasion

The epigenetic regulation of genes has long been recognized as one of the causes of prostate cancer (PCa) development and progression. Recent studies have shown that a number of microRNAs (miRNAs) are also epigenetically regulated in different types of cancers including PCa. In this study, we found that the DNA sequence of the promoters of miR-29a and miR-1256 are partly methylated in PCa cells, which leads to their lower expression both in PCa cells and in human tumor tissues compared with normal epithelial cells and normal human prostate tissues. By real-time PCR, Western Blot analysis and miRNA mimic and 3′-UTR-Luc transfection, we found that TRIM68 is a direct target of miR-29a and miR-1256 and that the downregulation of miR-29a and miR-1256 in PCa cells leads to increased expression of TRIM68 and PGK-1 in PCa cells and in human tumor tissue specimens. Interestingly, we found that a natural agent, isoflavone, could demethylate the methylation sites in the promoter sequence of miR-29a and miR-1256, leading to the upregulation of miR-29a and miR-1256 expression. The increased levels of miR-29a and miR-1256 by isoflavone treatment resulted in decreased expression of TRIM68 and PGK-1, which is mechanistically linked with inhibition of PCa cell growth and invasion. The selective demethylation activity of isoflavone on miR-29a and miR-1256 leading to the suppression of TRIM68 and PGK-1 expression is an important biological effect of isoflavone, suggesting that isoflavone could be a useful non-toxic demethylating agent for the prevention of PCa development and progression.     

乙酰基转移酶Tip60(KAT5)的功能研究进展

Tip60(KAT5)属于MYST乙酰基转移酶家族,同时它也是进化上非常保守的Nu A4蛋白质复合体的重要成员.过去十几年的研究证实,Tip60一方面可以作为转录调控因子结合核受体(如雄激素受体,AR)或c-MYC、AICD/Fe65、NCo R、E2F等转录因子来激活或抑制下游基因的表达,另一方面,KAT5可以乙酰化一系列蛋白来调控这些蛋白质的活性及稳定性,进而调控DNA损伤修复反应、细胞周期进程、细胞周期检查点的激活、凋亡、代谢及自噬等重要细胞功能.此外,Tip60在肿瘤的发生发展及转移、胚胎发育等过程中也发挥着至关重要的作用.本文将主要对Tip60近几年的研究进展做一个综述.     

HIV-1 Tat targets Tip60 to impair the apoptotic cell response to genotoxic stresses

HIV-1 transactivator Tat uses cellular acetylation signalling by targeting several cellular histone acetyltransferases (HAT) to optimize its various functions. Although Tip60 was the first HAT identified to interact with Tat, the biological significance of this interaction has remained obscure. We had previously shown that Tat represses Tip60 HAT activity. Here, a new mechanism of Tip60 neutralization by Tat is described, where Tip60 is identified as a substrate for the newly reported p300/CBP-associated E4-type ubiquitin-ligase activity, and Tat uses this mechanism to induce the polyubiquitination and degradation of Tip60. Tip60 targeting by Tat results in a dramatic impairment of the Tip60-dependent apoptotic cell response to DNA damage. These data reveal yet unknown strategies developed by HIV-1 to increase cell resistance to genotoxic stresses and show a role of Tat as a modulator of cellular protein ubiquitination.     

Identification of an alternatively spliced form of the Tat interactive protein (Tip60), Tip60(beta)

Tip60 was originally isolated as a Tat interactive protein. It was subsequently shown that Tip60 had histone acetyltransferase (HAT) activity. In studies to understand gene-expression regulation that might involve HAT activity, we PCR-amplified Tip60 from a human heart marathon-ready cDNA library. As a result, we identified an alternatively spliced form of Tip60, Tip60beta (we refer to the previously cloned Tip60 as Tip60alpha). Tip60beta cDNA is slightly smaller than Tip60alpha, and sequencing indicates that there is a deletion of 156 bp in the coding region of the gene. The predicted Tip60beta protein therefore lacks 52 amino acids when compared with Tip60alpha. The Tip60alpha gene is encoded by 14 exons, and Tip60beta is an alternatively spliced form resulting from the exclusion of exon 5 during the splicing process. Exon 5 encodes a proline-rich region that is known to be important for protein-protein interaction. Tip60beta is expressed in a variety of human tissues and cell lines, and the protein is present in both the nucleus and cytoplasm in contrast to Tip60alpha, which is entirely nuclear. The results suggest that Tip60beta may have functions additional to those of Tip60alpha in cells and tissues.     

Tip60 and p400 are both required for UV-induced apoptosis but play antagonistic roles in cell cycle progression

The histone acetyl transferase Tip60 (HTATIP) belongs to a multimolecular complex involved in the cellular response to DNA damage. Tip60 participates in cell cycle arrest following DNA damage by allowing p53 to activate p21CIP (p21) expression. We show here that Tip60 and the E1A-associated p400 protein (EP400), which belongs to the Tip60 complex, are also required for DNA damage-induced apoptosis. Tip60 favours the expression of some proapoptotic p53 target genes most likely through the stimulation of p53 DNA binding activity. In contrast, p400 represses p21 expression in unstressed cells, thereby allowing cell cycle progression and DNA damage-induced apoptosis. Tip60 and p400 have thus opposite effects on p21 expression in the absence of DNA damage. We further found that this antagonism relies on the inhibition of Tip60 function by p400, a property that is abolished following DNA damage. Therefore, taken together, our results indicate that Tip60 and p400 play distinct roles in DNA damage-induced apoptosis and underline the importance of the Tip60 complex and its regulation in the proper control of cell fate.     

The ATDC (TRIM29) Protein Binds p53 and Antagonizes p53-Mediated Functions

The ataxia telangiectasia group D-complementing (ATDC) gene product, also known as TRIM29, is a member of the tripartite motif (TRIM) protein family. ATDC has been proposed to form homo- or heterodimers and to bind nucleic acids. In cell cultures, ATDC expression leads to rapid growth and resistance to ionizing radiation (IR), whereas silencing of ATDC expression decreases growth rates and increases sensitivity to IR. Although ATDC is overexpressed in many human cancers, the biological significance of ATDC overexpression remains obscure. We report here that ATDC increases cell proliferation via inhibition of p53 nuclear activities. ATDC represses the expression of p53-regulated genes, including p21 and NOXA. Mechanistically, ATDC binds p53, and this interaction is potentially fine-tuned by posttranslational acetylation of lysine 116 on ATDC. The association of p53 and ATDC results in p53 sequestration outside of the nucleus. Together, these results provide novel mechanistic insights into the function of ATDC and offer an explanation for how ATDC promotes cancer cell proliferation.Ataxia telangiectasia (AT) is an autosomal-recessive, complex, multisystem disorder (4, 33). One of the hallmarks for cells derived from AT patients is their unusual sensitivity to ionizing radiation (IR) and their failure to delay the cell cycle in S phase, termed radioresistant DNA synthesis. In addition, AT cells contain atypical cytoskeletal organization. An early attempt to complement the defect in an AT cell line (AT5BIVA) by transfection with a human cosmid library and selection by γIR resulted in the isolation of an AT cell line (1B3) that was partially resistant to IR (22). Subsequent isolation of the human DNA in the region of the integrated cosmid sequences in 1B3 cells resulted in the cloning of the ataxia telangiectasia group D-complementing (ATDC) gene (23).The ATDC gene is located at chromosome 11q23, where it is frequently associated with many different kinds of cancers. Analysis of the ATDC gene product revealed that it is a member of the tripartite motif (TRIM) protein family (also known as the RBCC family). This protein family is characterized by three zinc-binding domains, a RING, a B-box type 1, and a B-box type 2, followed by a coiled-coil region (5, 29, 42, 43, 47). Some TRIM proteins homo-multimerize through their coil-coil region, and the integrity of the TRIM motif is required for proper subcellular localization of TRIM proteins (43). Recently, it was discovered that one of the TRIM proteins is a component of the repressor binding site (RBS) binding complex found in EC and ES cells and functions in restricting retroviral replication (60).The ATDC protein has been shown to interact with a protein kinase C substrate and inhibitor, although the significance of this interaction is not exactly clear (6). Although early studies indicate that ATDC can complement the IR sensitivity of AT fibroblasts, later analysis reveals that ATDC does not affect radioresistant DNA synthesis and is most likely not mutated in any AT patients (29). Rather, the ATDC protein probably induces cell survival or confers cell growth advantage independently of IR. Although ATDC is overexpressed in a wide variety of different cancers (12, 17, 19, 26, 34, 38, 45, 66), its expression is highly cell type and tissue specific (6, 43) (see Fig. S1 and S2 in the supplemental material). Further, expression of ATDC in NIH 3T3 cells leads to more rapid growth and resistance to IR, whereas silencing of ATDC expression in BxPC-3 cells leads to decreased growth rate and increased sensitivity to IR (3).The beginning of a mechanistic understanding for the function of ATDC came recently from a study showing that ATDC promotes cell proliferation in vitro and enhances tumor growth and metastasis in vivo by stabilizing β-catenin via the Disheveled-2 protein (59). This finding is consistent with a previous report by the same group that pancreatic cancer cells overexpress ATDC at an average of 20-fold higher than epithelial cells from normal pancreas. In the present study, we propose an alternative, non-mutually-exclusive pathway by which ATDC increases cell proliferation via inhibition of p53 nuclear activities. ATDC binds p53 and represses expression of p53-regulated genes, including p21 and NOXA. Intriguingly, we found that the ATDC-p53 interaction is regulated by posttranslational acetylation of ATDC. Our results provide novel mechanistic insights into the function of ATDC and further explanation of how ATDC promotes cancer cell proliferation.     

TRIM36 interacts with the kinetochore protein CENP-H and delays cell cycle progression

The tripartite motif-containing protein (TRIM) family is defined by the presence of a common domain structure composed of a RING finger, a B-box, and a coiled-coil motif. TRIM family proteins are involved in a broad range of biological processes and, consistently, their alterations result in diverse pathological conditions such as genetic diseases, viral infection, and cancer development. In this study, we found by using yeast two-hybrid screening that TRIM36 has a ubiquitin ligase activity and interacts with centromere protein-H, one of the kinetochore proteins. We also found by immunofluorescence analysis that TRIM36 colocalizes with α-tubulin, one of the microtubule proteins. Moreover, we found that overexpression of TRIM36 decelerates the cell cycle and attenuates cell growth. These results indicate that TRIM36 is potentially associated with chromosome segregation and that an excess of TRIM36 may cause chromosomal instability.     

TRIM16 inhibits neuroblastoma cell proliferation through cell cycle regulation and dynamic nuclear localization

Neuroblastoma is the most common solid tumor in childhood and represents 15% of all children’s cancer deaths. We have previously demonstrated that tripartite motif 16 (TRIM16), a member of the RING B-box coiled-coil (RBCC)/tripartite totif (TRIM) protein family, has significant effects on neuroblastoma proliferation and migration in vitro and tumorigenicity in vivo. However, the mechanism by which this putative tumor suppressor influences cell proliferation and tumorigenicity was undetermined. Here we show, for the first time, TRIM16’s striking pattern of expression and dynamic localization during cell cycle progression and neuroblastoma tumor development. In a tyrosine hydroxylase MYCN (TH-MYCN) neuroblastoma mouse model, immunohistochemical staining revealed strong nuclear TRIM16 expression in differentiating ganglia cells but not in the tumor-initiating cells. Furthermore in vitro studies clearly demonstrated that during G₁ cell cycle phase, TRIM16 protein expression is upregulated and shifts to the nucleus of cells. TRIM16 also plays a role in cell cycle progression through changes in Cyclin D1 and p27 expression. Importantly, using TRIM16 deletion mutants, an uncharacterized protein domain of TRIM16 was found to be required for both TRIM16’s growth inhibitory effects and its nuclear localization. Taken together, our data suggest that TRIM16 acts as a novel regulator of both neuroblastoma G₁/S progression and cell differentiation.