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Metastasis‐associated protein 2 (MTA2) is frequently amplified in many types of cancers; however, the role and underlying molecular mechanism of MTA2 in esophageal squamous cell carcinoma (ESCC) remain unknown. Here, we reported that MTA2 is highly expressed in ESCC tissue and cells, and is closely related to the malignant characteristics and poor prognosis of patients with ESCC. Through in vitro and in vivo experiments, we demonstrated that MTA2 significantly promoted ESCC growth, metastasis, and epithelial‐mesenchymal transition (EMT) progression. This integrative analysis combined with expression microarray showed that MTA2 could interact with eukaryotic initiation factor 4E (EIF4E), which positively regulates the expression of Twist, known as a master regulator of EMT. Moreover, the results of chromatin immunoprecipitation revealed that MTA2 was recruited to the E‐cadherin promoter by Twist, which reduced the acetylation level of the promoter region and thus inhibited expression of E‐cadherin, and subsequently promoted the aggressive progression of ESCC. Collectively, our study provided novel evidence that MTA2 plays an aggressive role in ESCC metastasis by a novel EIF4E‐Twist positive feedback loop, which may provide a potential therapeutic target for the management of ESCC., We demonstrated that the upregulation of MTA2 in ESCC was significantly correlated with malignant characteristics and poor survival probability of patients. The abnormal expression of MTA2 in ESCC contributed to the proliferation, metastasis, and EMT phenotype of ESCC cells through a novel EIF4E‐Twist positive feedback loop.

A substantial fraction of transcripts are known as long noncoding RNAs (lncRNAs), and these transcripts play pivotal roles in the development of cancer. However, little information has been published regarding the functions of lncRNAs in oesophageal squamous cell carcinoma (ESCC) and the underlying mechanisms. In our previous studies, we demonstrated that small nucleolar RNA host gene 5 (SNHG5), a known lncRNA, is dysregulated in gastric cancer (GC). In this study, we explored the expression and function of SNHG5 in development of ESCC. SNHG5 was found to be downregulated in human ESCC tissues and cell lines, and this downregulation was associated with cancer progression, clinical outcomes and survival rates of ESCC patients. Furthermore, we also found that overexpression of SNHG5 significantly inhibited the proliferation, migration and invasion of ESCC cells in vivo and in vitro. Notably, we found that metastasis-associated protein 2 (MTA2) was pulled down by SNHG5 in ESCC cells using RNA pulldown assay. We also found that SNHG5 reversed the epithelial–mesenchymal transition by interacting with MTA2. In addition, overexpression of SNHG5 downregulated the transcription of MTA2 and caused its ubiquitin-mediated degradation. Thus, overexpression of MTA2 partially abrogated the effect of SNHG5 in ESCC cell lines. Furthermore, we found that MTA2 mRNA expression was significantly elevated in ESCC specimens, and a negative correlation between SNHG5 and MTA2 expression was detected. Overall, this study demonstrated, for the first time, that SNHG5-regulated MTA2 functions as an important player in the progression of ESCC and provide a new potential therapeutic strategy for ESCC.

Objective: Angiogenesis plays a vital role in tumor growth and metastasis. Here, we aimed to find novel efficient antiangiogenic molecules targeting vascular endothelial growth factor A (VEGFA ) at the transcriptional level to treat triple-negative breast cancer (TNBC). Methods: We used a cell-based seryl tRNA synthetase (SerRS) promoter-driven dual-luciferase reporter system to screen an in-house library of 384 naturally occurring small molecules and their derivatives to find candidate molecules that could upregulate the expression of SerRS, a potent transcriptional repressor of VEGFA. The levels of SerRS and VEGFA were examined by quantitative RT-PCR (qRT-PCR), western blotting, and/or ELISAs in TNBC cells after candidate molecule administration. Zebrafish, the Matrigel plug angiogenesis assay in mice, the TNBC allograft, and xenograft mouse models were used to evaluate the in vivo anti-angiogenic and anti-cancer activities. Furthermore, the potential direct targets of the candidates were identified by proteomics and biochemical studies. Results: We found the most active compound was 3-(4-methoxyphenyl) quinolin-4(1H)-one (MEQ), an isoflavone derivative. In TNBC cells, MEQ treatment resulted in increased SerRS mRNA (P < 0.001) and protein levels and downregulated VEGFA production. Both the vascular development of zebrafish and Matrigel plug angiogenesis in mice were inhibited by MEQ. MEQ also suppressed the angiogenesis in TNBC allografts and xenografts in mice, resulting in inhibited tumor growth and prolonged overall survival (P < 0.05). Finally, we found that MEQ regulated SerRS transcription by interacting with MTA2 (Metastasis Associated 1 Family Member 2). Conclusions: Our findings suggested that the MTA2/SerRS/VEGFA axis is a drug-treatable anti-angiogenic target, and MEQ is a promising anti-tumor molecule that merits further investigation for clinical applications.

Graphical abstract, Establishment of the interferon (IFN)-mediated antiviral state provides a crucial initial line of defense against viral infection. Numerous genes that contribute to this antiviral state remain to be identified. Using a loss-of-function strategy, we screened an original library of 1156 siRNAs targeting 386 individual curated human genes in stimulated microglial cells infected with Zika virus (ZIKV), an emerging RNA virus that belongs to the flavivirus genus. The screen recovered twenty-one potential host proteins that modulate ZIKV replication in an IFN-dependent manner, including the previously known IFITM3 and LY6E. Further characterization contributed to delineate the spectrum of action of these genes towards other pathogenic RNA viruses, including Hepatitis C virus and SARS-CoV-2. Our data revealed that APOL3 acts as a proviral factor for ZIKV and several other related and unrelated RNA viruses. In addition, we showed that MTA2, a chromatin remodeling factor, possesses potent flavivirus-specific antiviral functions induced by IFN. Our work identified previously unrecognized genes that modulate the replication of RNA viruses in an IFN-dependent manner, opening new perspectives to target weakness points in the life cycle of these viruses.

MYC overexpression is considered a driver event in gastric cancer (GC), and is frequently correlated with poor prognosis and metastasis. In this study, we evaluated the prognostic value of genes upregulated by MYC in patients with GC. Metastatic GC cells (AGP01) characterized by MYC amplification, were transfected with siRNAs targeting MYC. RNA-seq was performed in silenced and non-silenced AGP01 cells. Among the differentially expressed genes, CIAPIN1, MTA2, and UXT were validated using qRT-PCR, western blot, and immunohistochemistry in gastric tissues of 213 patients with GC; and their expressions were correlated with clinicopathological and survival data. High mRNA and protein levels of CIAPIN1, MTA2, and UXT were strongly associated with advanced GC stages (P < 0.0001). However, only CIAPIN1 and UXT gene expressions were able to predict distant metastases in patients with early-stage GC (P < 0.0001), with high sensitivity (> 92%) and specificity (> 90%). Overall survival rate of patients with overexpressed CIAPIN1 or UXT was significantly lower (P < 0.0001). In conclusion, CIAPIN1 and UXT may serve as potential molecular markers for GC prognosis.

Rationale: Hypoxia has been proved to contribute to aggressive phenotype of cancers, while functional and regulatory mechanism of long noncoding RNA (lncRNA) in the contribution of hypoxia on pancreatic cancer (PC) tumorigenesis is incompletely understood. The aim of this study was to uncover the regulatory and functional roles for hypoxia-induced lncRNA-MTA2TR (MTA2 transcriptional regulator RNA, AF083120.1) in the regulation of PC tumorigenesis. Methods: A lncRNA microarray confirmed MTA2TR expression in tissues of PC patients. The effects of MTA2TR on proliferation and metastasis of PC cells and xenograft models were determined, and the key mechanisms by which MTA2TR promotes PC were further dissected. Furthermore, the expression and regulation of MTA2TR under hypoxic conditions in PC cells were assessed. We also assessed the correlation between MTA2TR expression and PC patient clinical outcomes. Results: We found that metastasis associated protein 2 (MTA2) transcriptional regulator lncRNA (MTA2TR) was overexpressed in PC patient tissues relative to paired noncancerous tissues. Furthermore, we found that depletion of MTA2TR significantly inhibited PC cell proliferation and invasion both in vitro and in vivo. We further demonstrated that MTA2TR transcriptionally upregulates MTA2 expression by recruiting activating transcription factor 3 (ATF3) to the promoter area of MTA2. Consequentially, MTA2 can stabilize the HIF-1α protein via deacetylation, which further activates HIF-1α transcriptional activity. Interestingly, our results revealed that MTA2TR is transcriptionally regulated by HIF-1α under hypoxic conditions. Our clinical samples further indicated that the overexpression of MTA2TR was correlated with MTA2 upregulation, as well as with reduced overall survival (OS) in PC patients. Conclusions: These results suggest that feedback between MTA2TR and HIF-1α may play a key role in regulating PC tumorigenesis, thus potentially highlighting novel avenues PC treatment.

Triple-negative breast cancer (TNBC) has a poorer outcome compared with that of other subtypes of breast cancer, and the discovery of dysregulated microRNA (miRNA) and their role in tumor progression has provided a new avenue for elucidating the mechanism involved in TNBC. Previous studies have revealed that aberrant expression of miRNA-589 (miR-589) was frequently observed in various types of cancer. However, the expression and function of miR-589 in TNBC has not been well illustrated. In the present study, the expression level of miR-589 was explored in TNBC tissues and TNBC cell lines by quantitative polymerase chain reaction (qPCR). The results revealed that the expression of miR-589 was decreased in TNBC tissues and cell lines compared with that in normal tissues and breast cell lines. Furthermore, miR-589 overexpression decreased the TNBC cell proliferation, migration and invasion, whereas miR-589 silencing generated the opposite results in vitro. Bioinformatic algorithms predicted a direct target site for miR-589 in the 3′-untranslated region of metastasis-associated protein 2 (MTA2), which was confirmed with a dual-luciferase reporter assay and western blot analysis. Results of the qPCR and western blot analysis illustrated that miR-589 negatively regulated MTA2 expression with regard to mRNA and protein levels in the TNBC cell lines. MTA2 silencing reversed the promotion function of miR-589 inhibitor in the TNBC cell line. Finally, miR-589 could inhibit the process of epithelial-mesenchymal transition via MTA2. In summary, the present study revealed the biological function and molecular mechanism of miR-589 in the progression of TNBC. MiR-589 inhibition in the progression of TNBC may be a potential therapeutic target for TNBC.

The nucleosome remodelling and deacetylase complex (NuRD) is a widely conserved regulator of gene expression. The determination of the subunit composition of the complex and identification of its binding partners are important steps towards understanding its architecture and function. The question of how these properties of the complex vary across different cell types has not been addressed in detail to date. Here, we set up a two-step purification protocol coupled to liquid chromatography-tandem mass spectrometry to assess NuRD composition and interaction partners in three different cancer cell lines, using label-free intensity-based absolute quantification (iBAQ). Our data indicate that the stoichiometry of the NuRD complex is preserved across our three different cancer cell lines. In addition, our interactome data suggest ZNF219 and SLC25A5 as possible interaction partners of the complex. To corroborate this latter finding, in vitro and cell-based pull-down experiments were carried out. These experiments indicated that ZNF219 can interact with RBBP4, GATAD2A/B and chromodomain helicase DNA binding 4, whereas SLC25A5 might interact with MTA2 and GATAD2A.

Highlights • MTA2, as component of the NURD complex, sensitizes gastric cancer cells to PARP inhibitor in vitro and in vivo. • MTA2 renders cancer cells susceptible to olaparib by aggravating replication stress. • MTA2 preferentially binds regions of replication origin-associated DNA sequence, which could be strengthened by olaparib. • Lethality of ATR inhibitor alone and in combination with olaparib is both increased by MTA2., Poly (ADP-ribose) polymerase (PARP) inhibitor olaparib selectively kills cancer cells with BRCA-deficiency and is approved for BRCA-mutated breast, ovarian and pancreatic cancers by FDA. However, phase III study of olaparib failed to show a significant improvement in overall survival in patients with gastric cancer (GC). To discover an effective biomarker for GC patient-selection in olaparib treatment, we analyzed proteomic profiling of 12 GC cell lines. MTA2 was identified to confer sensitivity to olaparib by aggravating olaparib-induced replication stress in cancer cells. Mechanistically, we applied Cleavage Under Targets and Tagmentation assay to find that MTA2 proteins preferentially bind regions of replication origin-associated DNA sequences, which could be enhanced by olaparib treatment. Furthermore, MTA2 was validated here to render cancer cells susceptible to combination of olaparib with ATR inhibitor AZD6738. In general, our study identified MTA2 as a potential biomarker for olaparib sensitivity by aggravating olaparib-induced replication stress.

Metastasis-associated protein 2 (MTA2) is a core subunit of the nucleosome remodeling and deacetylating (NuRD) complex and functions by mediating chromatin remodeling and gene silencing. However, its biological actions and clinical significance in pancreatic ductal adenocarcinoma (PDAC) remain elusive. The aim of this study was to explore the function and regulation mechanism of MTA2 in PDAC. As shown in GEO, ICGC, and TCGA databases, a higher expression of MTA2 was noticed in the PDAC tissues than in the normal pancreatic tissues. Moreover, a higher expression level of MTA2 was associated with a shorter overall survival time in these public PDAC databases. We further investigated the underlying mechanisms of these observations by using a chromatin immunoprecipitation (ChIP)-based deep sequencing, luciferase reporter, and quantitative ChIP assays. We identified the repressive binding of MTA2 to the promoter of phosphatase and tensin homolog (PTEN). We also found that Snail recruited MTA2 and HDAC1 to suppress PTEN expression. Ectopic expression and knockdown of MTA2 were performed to evaluate the effects of this gene on PDAC cell proliferation, migration, and invasion. Using CCK-8, colony formation and transwell assays, and a xenograft tumor model, we revealed that MTA2 promoted PDAC cell proliferation, migration, and invasion in vitro and PDAC tumor growth in vivo by downregulation of PTEN. In benzyl isothiocyanate (BITC)-treated MIA Paca-2 cells and PANC-1 cells, MTA2 level decreased in a dose- and time-dependent manner with concomitant upregulation of PTEN level and downregulation of phosphorylated PI3K and AKT levels, providing evidence of the involvement of MTA2 and PTEN in the regulation of the PI3K/AKT pathway in BITC-mediated PDAC suppression. Collectively, these findings uncover a novel role for MTA2 in the regulation of PDAC progression and help to elucidate the mechanisms involved in this process.

Studies have shown the overexpression of metastasis-associated protein 2 (MTA2) to be associated with hepatocellular carcinoma (HCC) progression. However, the molecular mechanism of MTA2 expression in HCC is unclear. In our study, we found a higher level of MTA2 in HCC tissues than in normal tissues and a significant correlation between tumor grade and overall survival of HCC patients. We also found that MTA2 inhibition reduced the migration and invasion capabilities of HCC cells, independent of cell proliferation. Mechanistic studies have suggested that MTA2 protein and mRNA are more highly expressed in SK-Hep-1 and Huh-7 cells compared with other HCC cells. MTA2 silencing drastically reduced migration and invasion capability and also inhibited matrix metalloproteinase 2 (MMP2) at the transcriptional and translation levels in both cells. In addition, treatment with the MMP2 antibody markedly impaired MTA2-knockdown-mediated inhibition of migration and invasion in SK-Hep-1 cells. Furthermore, MTA2 knockdown reduced the phosphorylation of the p38MAPK protein, whereas the inhibition of p38MAPK (SB203580 or si-p38) confirmed that blocking the p38MAPK pathway mediated MTA2-knockdown-inhibited migration and invasion in SK-Hep-1 cells. We demonstrated the molecular mechanism by which MTA2 inhibits human HCC cell metastasis through the p38MAPK/MMP2 pathways, which might be helpful in determining the diagnostic value of this protein in patients with HCC.

Although NF-κB is known to play a pivotal role in lung cancer, contributing to tumor growth, microenvironmental changes, and metastasis, the epigenetic regulation of NF-κB in tumor context is largely unknown. Here we report that the IKK2/NF-κB signaling pathway modulates metastasis-associated protein 2 (MTA2), a component of the nucleosome remodeling and deacetylase complex (NuRD). In triple transgenic mice, downregulation of IKK2 (Sftpc-cRaf-IKK2DN) in cRaf-induced tumors in alveolar epithelial type II cells restricted tumor formation, whereas activation of IKK2 (Sftpc-cRaf-IKK2CA) supported tumor growth; both effects were accompanied by altered expression of MTA2. Further studies employing genetic inhibition of MTA2 suggested that in primary tumor growth, independent of IKK2, MTA2/NuRD corepressor complex negatively regulates NF-κB signaling and tumor growth, whereas later dissociation of MTA2/NuRD complex from the promoter of NF-κB target genes and IKK2-dependent positive regulation of MTA2 leads to activation of NF-κB signaling, epithelial–mesenchymal transition, and lung tumor metastasis. These findings reveal a previously unrecognized biphasic role of MTA2 in IKK2/NF-κB-driven primary-to-metastatic lung tumor progression. Addressing the interaction between MTA2 and NF-κB would provide potential targets for intervention of tumor growth and metastasis. Significance: These findings strongly suggest a prominent role of MTA2 in primary tumor growth, lung metastasis, and NF-κB signaling modulatory functions.

The role of post-translational modification (PTM) of proteins in regulating developmental and differentiation processes is understudied, but recently we established that O-GlcNAcylation regulates erythropoiesis. O-GlcNAc regulates numerous cellular functions including stress response, transcription, and cell cycle progression. O-GlcNAc is a single O-linked β-N-acetyl-D-glucosamine moiety added to serine/threonine amino acids of nuclear, cytoplasmic, and mitochondrial proteins. O-GlcNAc transferase (OGT), which adds the modification, and O-GlcNAcase (OGA), which removes the modification, are responsible for the dynamic processing of the PTM. Newly developed erythroid-specific OGT conditional knockout mice show that OGT is essential for fetal definitive erythropoiesis, although during primitive erythropoiesis, erythrocytes exhibit hallmarks of ineffective erythropoiesis. In addition, using G1E-ER cells, we have shown that GATA-1 interacts with OGT and OGA and delivers OGT and OGA to GATA-1 regulated genes. When we perturb this process, we observe erythroid defects, most strikingly, a shift in commitment towards other hematopoietic lineages. We hypothesize that at the onset of erythroid lineage commitment, GATA-1 functions as an adaptor protein to deliver these enzymes to erythroid-specific cis-regulatory DNA elements, where the O-GlcNAc status of bound protein complexes is modified to direct transcriptional networks necessary for normal erythroid development and terminal differentiation. Two key proteins, LRF/ZBTB7A and BCL11A, silence the γ-globin promoter during adult erythropoiesis by recruiting the penultimate NuRD repressor complex to these genes. Loss of either of these proteins leads to partial restoration of γ-globin expression. Our previous work demonstrated that O-GlcNAc plays a role in γ-globin gene transcription. GATA-1 recruits FOG-1 and the NuRD complex to silence γ-globin by binding GATA sites located at -566 or -567 relative to the Aγ-globin or Gγ-globin transcription start sites, respectively. OGT and OGA interact with GATA-1, FOG-1 and CHD4, a key component of the NuRD repressor complex and these three proteins are O-GlcNAcylated. Specifically, O-GlcNAcylation modulates the formation of the multi-protein NuRD repressor complex. OGT adds O-GlcNAc to CHD4 stimulating the formation of this repressor complex, whereas removal of this PTM by OGA results in the activation of γ-globin gene expression. In addition, the GATA-1 binding protein, GATAD2A, interacts with CHD4 and organizes it within the NuRD complex. The domain of GATAD2A that interacts with CHD4 is O-GlcNAcylated. Finally, the NuRD complex proteins RBBP7, HDAC1, and MTA2 are also modified by O-GlcNAc. We hypothesize that O-GlcNAcylation controls NuRD repressor assembly by directing subunit interactions. To ascertain the function of OGT and OGA at GATA-1 regulated genes, we developed novel CRISPR/Cas9 targeting tools in K562 cells. We fused OGT and OGA to catalytically dead Cas9 endonuclease (dCas9). Negative controls were generated by mutating a catalytic residue of OGT (H558F) or OGA (D174A). The CBP/p300 acetyltransferase core was fused to dCas9 as a positive control. As a proof of principle, we targeted the γ-globin promoters. Using the UCSC Genome Browser, single guide RNAs (sgRNAs) that do not disrupt endogenous cis-regulatory elements were selected, synthesized, and subsequently introduced into K562 cells to establish stably expressing monoclonal cell lines. Interestingly, when OGA-dCas9 was targeted to the -1 to -223 bp region relative to the mRNA start site of the γ-globin promoters, we observed a significant increase in γ-globin expression. OGT-dCas9 had a significant repressive effect when targeted to regions around the transcriptional start site. All controls behaved as predicted. To validate these results, we are performing chromatin immunoprecipitation (ChIP) to demonstrate occupancy of the targeted regions by our constructs and the presence or absence of O-GlcNAc. Our findings highlight a new post-translational mechanism regulating gene transcription during erythroid development, which affects γ-globin expression and erythropoiesis. Our data will provide insight into how O-GlcNAc controls erythroid differentiation and reveal O-GlcNAcylated protein targets that may be manipulated for the treatment of hemoglobinopathies. Disclosures No relevant conflicts of interest to declare.

Metastasis-associated protein 2 (MTA2) was previously known as a requirement to maintain malignant potentials in several human cancers. However, the role of MTA2 in the progression of renal cell carcinoma (RCC) has not yet been delineated. In this study, MTA2 expression was significantly increased in RCC tissues and cell lines. Increased MTA2 expression was significantly associated with tumour grade (p = 0.002) and was an independent prognostic factor for overall survival with a high RCC tumour grade. MTA2 knockdown inhibited the migration, invasion, and in vivo metastasis of RCC cells without effects on cell proliferation. Regarding molecular mechanisms, MTA2 knockdown reduced the activity, protein level, and mRNA expression of matrix metalloproteinase-9 (MMP-9) in RCC cells. Further analyses demonstrated that patients with lower miR-133b expression had poorer survival rates than those with higher expression from The Cancer Genome Atlas database. Moreover, miR-133b modulated the 3&prime, untranslated region (UTR) of MMP-9 promoter activities and subsequently the migratory and invasive abilities of these dysregulated expressions of MTA2 in RCC cells. The inhibition of MTA2 could contribute to human RCC metastasis by regulating the expression of miR-133b targeting MMP-9 expression.

Minhua Wu,1,2,* Xiaoxia Ye,2,* Xubin Deng,3,* Yanxia Wu,4 Xiaofang Li,4 Lin Zhang11Department of Histology and Embryology, Southern Medical University, Guangzhou, 2Department of Histology and Embryology, Guangdong Medical University, Zhanjiang, 3Affiliated Cancer Hospital of Guangzhou Medical University, Cancer Center of Guangzhou Medical University, Guangzhou, 4Pathological Diagnosis and Research Center, Affiliated Hospital of Guangdong Medical University, Zhanjiang, People’sRepublic of China*These authors contributed equally tothis workAims: Metastasis-associated gene 2 (MTA2) is reported to play an important role in tumor progression, but little is known about the role of MTA2 in nasopharyngeal carcinoma (NPC). The aim of the study was to explore the expression and function of MTA2 in NPC.Methods: Expression of MTA2 in NPC tissues and cell lines was detected by immunohistochemistry and Western blotting. Relationship between MTA2 expression and clinicopathological features was analyzed. Stable MTA2-overexpressing and MTA2-siliencing NPC cells were established by transfection with plasmids encoding MTA2 cDNA and lentivirus-mediated short hairpin RNA, respectively. Cell viability was determined by Cell Counting Kit-8 and colony formation assay. Cell migration ability was evaluated by wound healing and transwell invasion assay. The impact of MTA2 knockdown on growth and metastasis of CNE2 cells in vivo was determined by nude mouse xenograft models. Expression of several Akt pathway proteins was detected by Western blotting.Results: MTA2 was upregulated in NPC tissues and three NPC cell lines detected (CNE1, CNE2, and HNE1). MTA2 expression was related to clinical stage and lymph node metastasis of patients with NPC. MTA2 upregulation promoted proliferation and invasion of CNE1 cells, while MTA2 depletion had opposite effects on CNE2 cells. Moreover, MTA2 depletion suppressed growth and metastasis of CNE2 cells in vivo. MTA2 overexpression activated Akt and upregulated the expression of matrix metalloproteinase 7 and cyclin D1.Conclusion: We conclude that MTA2 acts as an oncogene in tumorigenesis of NPC. MTA2 may be a potential target for gene therapy in NPC.Keywords: MTA2, nasopharyngeal carcinoma, proliferation, invasion, Akt pathway

Multiprotein chromatin remodelling complexes show remarkable conservation of function amongst metazoans, even though components present in invertebrates are often found as multiple paralogous proteins in vertebrate complexes. In some cases, these paralogues specify distinct biochemical and/or functional activities in vertebrate cells. Here, we set out to define the biochemical and functional diversity encoded by one such group of proteins within the mammalian Nucleosome Remodelling and Deacetylation (NuRD) complex: Mta1, Mta2 and Mta3. We find that, in contrast to what has been described in somatic cells, MTA proteins are not mutually exclusive within embryonic stem (ES) cell NuRD and, despite subtle differences in chromatin binding and biochemical interactions, serve largely redundant functions. ES cells lacking all three MTA proteins exhibit complete NuRD loss of function and are viable, allowing us to identify a previously unreported function for NuRD in reducing transcriptional noise, which is essential for maintaining a proper differentiation trajectory during early stages of lineage commitment.

Transcriptional repressor GATA binding 1 (TRPS1), an atypical GATA transcription factor, functions as a transcriptional repressor and is also implicated in human cancers. However, the underlying mechanism of TRPS1 contributing to malignancy remains obscure. In the current study, we report that TRPS1 recognizes both gene proximal and distal transcription start site (TSS) sequences to repress gene expression. Co-IP mass spectrometry and biochemical studies showed that TRPS1 binds to CHD4/NuRD(MTA2). Genome-wide and molecular studies revealed that CHD4/NuRD(MTA2) is required for TRPS1 transcriptional repression. Mechanically, TRPS1 and CHD4/NuRD(MTA2) form precision-guided transcriptional repression machinery in which TRPS1 guides the machinery to specific target sites by recognizing GATA elements, and CHD4/NuRD(MTA2) represses the transcription of target genes. Furthermore, TP63 was identified and validated to be a direct target of TRPS1-CHD4/NuRD(MTA2) complex, which represses TP63 expression by involving decommission of TP63 enhancer in the described precision-guided manner, leading to a reduction of the ΔNp63 level and contributing to migration and invasion of cancer cells.

Multiprotein chromatin remodelling complexes show remarkable conservation of function amongst metazoans, even though components present in invertebrates are often present as multiple paralogous proteins in vertebrate complexes. In some cases these paralogues specify distinct biochemical and/or functional activities in vertebrate cells. Here we set out to define the biochemical and functional diversity encoded by one such group of proteins within the mammalian Nucleosome Remodelling and Deacetylation (NuRD) complex: Mta1, Mta2 and Mta3. We find that, in contrast to what has been described in somatic cells, MTA proteins are not mutually exclusive within ES cell NuRD and, despite subtle differences in chromatin binding and biochemical interactions, serve largely redundant functions. Nevertheless, ES cells lacking all three MTA proteins represent a complete NuRD null and are viable, allowing us to identify a previously undetected function for NuRD in maintaining differentiation trajectory during early stages of lineage commitment.

Background MicroRNAs deregulation are common in human tumor progression. miR-1236-3p has been reported to function as tumor suppressor microRNA in various malignancies. The aim of this study was to demonstrate the downregulated expression of miR-1236-3p in gastric cancer (GC) tissues and cell lines, and clarify its biological function in GC. Methods Real-time polymerase chain reaction was used to measure the mRNA level of miR-1236-3p in GC. Dual luciferase assay was used to demonstrate that MTA2 was one of the candidate target genes of miR-1236-3p. Western blots were utilized to detect the protein levels. Cell function assays were also performed to determine the function of miR-1236-3p in GC. Results miR-1236-3p expression, which was associated with lymph node metastasis, differentiation and clinical stage, was significantly reduced in GC tissues and cell lines. miR-1236-3p over-expression could inhibit GC cell proliferation, migration and invasion, and inhibition of miR-1236-3p expression had opposite effects. Furthermore, we demonstrated that MTA2 was a candidate target of miR-1236-3p, and miR-1236-3p over-expression significantly inhibited the process of epithelial–mesenchymal transition. We also found that miR-1236-3p could suppress the PI3K/Akt signaling pathway in GC cells. Conclusions Our results suggest that miR-1236-3p functions as a tumor suppressor in GC and could be a promising therapeutic target for GC.

SUMMARY The NuRD complex contains both chromatin remodeling and histone deacetylase activities. Mice lacking the MTA2 subunit of NuRD show developmental defects in pro-B, pre-B, immature B, and marginal zone B cells, and abnormal germinal center B cell differentiation during immune responses. Mta2 inactivation also causes a derepression of Igll1 and VpreB1 genes in pre-B cells. Furthermore, MTA2/NuRD interacts directly with AIOLOS/IKAROS and shows a striking overlap with AIOLOS/IKAROS target genes in human pre-B cells, suggesting a functional interdependence between MTA2/NuRD and AIOLOS. Mechanistically, MTA2 deficiency in mice leads to increased H3K27 acetylation at both Igll1 and VpreB1 promoters. Gene profiling analyses also identify distinct MTA2-dependent transcription programs in pro-B and pre-B cells. In addition, we find a strong synergy between MTA2 and OCA-B in repressing Igll1 and VpreB1 at the pre-B cell stage, and in regulating both the pre-B to immature B transition and splenic B cell development., Graphical Abstract, In Brief Lu et al. examine B cell developmental defects in MTA2-deficient mice. MTA2 interacts with AIOLOS/IKAROS, represses Igll1 expression, co-binds to most AIOLOS/IKAROS target genes in pre-B cells, and cooperates with OCA-B in the pre-B to immature B transition. These data suggest that AIOLOS/IKAROS functions through MTA2/NuRD during B cell development.

The aim of our study was to assess the differential gene expression of Parkinson protein 7 (PARK7) interactome in malignant pleural mesothelioma (MPM) using data mining techniques to identify novel candidate genes that may play a role in the pathogenicity of MPM. We constructed the PARK7 interactome using the ConsensusPathDB database. We then interrogated the Oncomine Cancer Microarray database using the Gordon Mesothelioma Study, for differential gene expression of the PARK7 interactome. In ConsensusPathDB, 38 protein interactors of PARK7 were identified. In the Gordon Mesothelioma Study, 34 of them were assessed out of which SUMO1, UBC3, KIAA0101, HDAC2, DAXX, RBBP4, BBS1, NONO, RBBP7, HTRA2, and STUB1 were significantly overexpressed whereas TRAF6 and MTA2 were significantly underexpressed in MPM patients ( network 2). Furthermore, Kaplan-Meier analysis revealed that MPM patients with high BBS1 expression had a median overall survival of 16.5 vs. 8.7 mo of those that had low expression. For validation purposes, we performed a meta-analysis in Oncomine database in five sarcoma datasets. Eight network 2 genes (KIAA0101, HDAC2, SUMO1, RBBP4, NONO, RBBP7, HTRA2, and MTA2) were significantly differentially expressed in an array of 18 different sarcoma types. Finally, Gene Ontology annotation enrichment analysis revealed significant roles of the PARK7 interactome in NuRD, CHD, and SWI/SNF protein complexes. In conclusion, we identified 13 novel genes differentially expressed in MPM, never reported before. Among them, BBS1 emerged as a novel predictor of overall survival in MPM. Finally, we identified that PARK7 interactome is involved in novel pathways pertinent in MPM disease.

Mammalian circadian rhythms are generated by a negative feedback loop in which PERIOD (PER) proteins accumulate, form a large nuclear complex (PER complex), and bind the transcription factor CLOCK-BMAL1, repressing their own expression. We found that mouse PER complexes include the Mi-2/nucleosome remodelling and deacetylase (NuRD) transcriptional corepressor. Unexpectedly, two NuRD subunits, CHD4 and MTA2, constitutively associate with CLOCK-BMAL1, with CHD4 functioning to promote CLOCK-BMAL1 transcriptional activity. At the onset of negative feedback, the PER complex delivers the remaining complementary NuRD subunits to DNA-bound CLOCK-BMAL1, thereby reconstituting a NuRD corepressor that is important for circadian transcriptional feedback and clock function. The PER complex thus acquires full repressor activity only upon successful targeting of CLOCK-BMAL1. Our results show how specificity is generated in the clock despite its dependence on generic transcriptional regulators and reveal the existence of active communication between the positive and negative limbs of the circadian feedback loop.

Metastasis remains a major clinical problem in breast cancer. One family of genes previously linked with metastasis is the metastasis tumor associated (MTA) family, with members MTA1 enhancing and MTA3 inhibiting cancer metastasis. We have previously found that MTA2 enhances anchorage-independent growth in estrogen receptor α (ERα) breast cancers, and, in combination with other genes, performed as a predictive biomarker in ERα-positive breast cancer. We therefore hypothesized that MTA2 enhances breast cancer progression. To test this, cell growth, soft-agar colony formation, migration, and in vivo metastasis were examined in MTA2-overexpressing and vector control transfected ERα-negative breast cancer cells. Pathways regulating cell-cell interaction, adhesion, and signaling through the Rho pathway were also investigated. Effects of the inhibition of the Rho pathway using a Rho Kinase (ROCK) inhibitor were assessed in soft agar colony formation and motility assays in MTA2-overexpressing cells. MTA2 expression was associated with poor prognostic markers, and levels of MTA2 were associated with increased risk of early recurrence in retrospective analyses. MTA2 overexpression was associated with enhanced metastasis, and pathways regulating cell-cell interactions in vitro and in vivo. Most critically, MTA2-enhanced motility could be blocked by inhibiting Rho pathway signaling. We present the novel finding that MTA2 defined a subset of ERα-negative patients with a particularly poor outcome.

Minhua Wu,1,2,* Xiaoxia Ye,2,* Xubin Deng,3,* Yanxia Wu,4 Xiaofang Li,4 Lin Zhang11Department of Histology and Embryology, Southern Medical University, Guangzhou, 2Department of Histology and Embryology, Guangdong Medical University, Zhanjiang, 3Affiliated Cancer Hospital of Guangzhou Medical University, Cancer Center of Guangzhou Medical University, Guangzhou, 4Pathological Diagnosis and Research Center, Affiliated Hospital of Guangdong Medical University, Zhanjiang, People’sRepublic of China*These authors contributed equally tothis workAims: Metastasis-associated gene 2 (MTA2) is reported to play an important role in tumor progression, but little is known about the role of MTA2 in nasopharyngeal carcinoma (NPC). The aim of the study was to explore the expression and function of MTA2 in NPC.Methods: Expression of MTA2 in NPC tissues and cell lines was detected by immunohistochemistry and Western blotting. Relationship between MTA2 expression and clinicopathological features was analyzed. Stable MTA2-overexpressing and MTA2-siliencing NPC cells were established by transfection with plasmids encoding MTA2 cDNA and lentivirus-mediated short hairpin RNA, respectively. Cell viability was determined by Cell Counting Kit-8 and colony formation assay. Cell migration ability was evaluated by wound healing and transwell invasion assay. The impact of MTA2 knockdown on growth and metastasis of CNE2 cells in vivo was determined by nude mouse xenograft models. Expression of several Akt pathway proteins was detected by Western blotting.Results: MTA2 was upregulated in NPC tissues and three NPC cell lines detected (CNE1, CNE2, and HNE1). MTA2 expression was related to clinical stage and lymph node metastasis of patients with NPC. MTA2 upregulation promoted proliferation and invasion of CNE1 cells, while MTA2 depletion had opposite effects on CNE2 cells. Moreover, MTA2 depletion suppressed growth and metastasis of CNE2 cells in vivo. MTA2 overexpression activated Akt and upregulated the expression of matrix metalloproteinase 7 and cyclin D1.Conclusion: We conclude that MTA2 acts as an oncogene in tumorigenesis of NPC. MTA2 may be a potential target for gene therapy in NPC.Keywords: MTA2, nasopharyngeal carcinoma, proliferation, invasion, Akt pathway

Mi-2/nucleosome remodeling and deacetylase (NuRD) chromatin remodeling complexes are important regulators of chromatin structure and DNA accessibility. We examined requirements for individual domains of chromodomain helicase DNA-binding protein 4 (CHD4), a core catalytic component of NuRD complexes, as well as the NuRD subunit methyl-binding domain protein 2 (MBD2) and methylated DNA, for NuRD function in the context of tissue-specific transcription. By itself, loss of NuRD activity is not sufficient for transcriptional activation. However, NuRD complexes greatly reduce activation of the B cell-specific mb-1 (Cd79a) gene by the transcription factors EBF1 and Pax5. Using our B cell model system, we determined that the two chromodomains and ATPase/helicase and C-terminal domains (CTD) of CHD4 are all necessary for repression of mb-1 promoters by NuRD. All of these domains except the CTD are required for efficient association of CHD4 with mb-1 promoter chromatin. Loss of MBD2 expression or of DNA methylation impaired association of CHD4 with mb-1 promoter chromatin and enhanced its transcription. We conclude that repressive functions of MBD2-containing NuRD complexes are dependent on cooperative interactions between the major domains of CHD4 with histones and DNA and on binding of methylated DNA by MBD2.

Thymomas of the same histological subtype sometimes manifest different biological behaviors. Metastasis Tumor Antigen-2 (MTA2) is targeted by the transcriptional repressor Kaiso, the distribution which is thought to be modulated by p120catenin (p120ctn). It is currently unclear if expression of p120ctn, Kaiso, and MTA2 relates to the biological behavior of thymoma. P120ctn, Kaiso, and MTA2 expression were examined in 137 cases of thymoma, three cases of thymic carcinoma, and 18 paired autologous normal thymic tissues using immunohistochemistry, and correlation of these proteins with histological subtypes and clinical stages were analyzed. In normal thymic epithelial cells, p120ctn was expressed on the cell membrane but Kaiso and MTA2 were not detected. Membranous p120ctn expression was reduced in thymoma epithelial cells, while ectopic cytoplasmic expression was observed in 76.6 % (105/137) of the cases. Cytoplasmic Kaiso was detected in 69.3 % (95/137) and nuclear MTA2 was detected in 70.8 % (97/137) of the thymomas. There were good consistencies (Kappa = 0.559, 0.512, 0.652; all P < 0.001) and correlations (r = 0.733, 0.652, 0.708; all P < 0.001) between cytoplasmic p120ctn, cytoplasmic Kaiso, and nuclear MTA2 expression in thymomas. All three protein factors correlated with histological type and clinical stage in thymoma (P < 0.05). Specifically, cytoplasmic p120ctn and Kaiso expression and nuclear MTA2 expression were higher in high-risk (types B2 and B3) thymomas and Masaoka stage III/IV thymomas than low-risk (types A, AB, and B1) and stage I/II thymomas (both P < 0.001), respectively. Cytoplasmic p120ctn, cytoplasmic Kaiso, and nuclear MTA2 expression correlated directly with histological type and Masaoka stage and may thus be used as potential biomarkers to predict biological behavior of thymoma.

The aim of this study was to clarify the clinicopathological outcome and prognostic significance of metastasis-associated gene 2 (MTA2) in esophageal squamous cell carcinoma (ESCC). Immunohistochemical staining for MTA2 was performed on surgical specimens obtained from 162 patients with ESCC. Relationships between MTA2 expression and clinicopathological factors, including prognosis, were analyzed. Significant correlations were noted among MTA2 overexpression and TNM clinical classification staging, depth of invasion, presence of regional lymph node metastasis, presence of distant metastasis, lymphatic invasion, blood-vessel invasion and the 5-year survival rates. The expression of MTA2 protein was correlated with tumour progression. Patients with MTA2 overexpression tended to have poor prognosis compared to patients with MTA2 underexpression.

Several key transcription factors and coregulators important to peripheral nerve myelination have been identified, but the contributions of specific chromatin remodeling complexes to peripheral nerve myelination have not been analyzed. Chromodomain helicase DNA-binding protein 4 (Chd4) is the core catalytic subunit of the nucleosome remodeling and deacetylase (NuRD) chromatin remodeling complex. Previous studies have shown Chd4 interacts with Nab (NGFI-A/Egr-binding) corepressors, which are required for early growth response 2 (Egr2/Krox20), to direct peripheral nerve myelination by Schwann cells. In this study, we examined the developmental importance of the NuRD complex in peripheral nerve myelination through the generation of conditional Chd4 knock-out mice in Schwann cells (Chd4loxP/loxP; P0-cre). Chd4 conditional null mice were found to have delayed myelination, radial sorting defects, hypomyelination, and the persistence of promyelinating Schwann cells. Loss of Chd4 leads to elevated expression of immature Schwann cell genes (Id2, c-Jun, and p75), and sustained expression of the promyelinating Schwann cell gene, Oct6/Scip, without affecting the levels of Egr2/Krox20. Furthermore, Schwann cell proliferation is upregulated in Chd4-null sciatic nerve.In vivochromatin immunoprecipitation studies reveal recruitment of Chd4 and another NuRD component, Mta2, to genes that are positively and negatively regulated by Egr2 during myelination. Together, these results underscore the necessity of Chd4 function to guide proper terminal differentiation of Schwann cells and implicate the NuRD chromatin remodeling complex as a requisite factor in timely and stable peripheral nerve myelination.