Recruitment Of HDAC1 Research Articles

GATA4 regulates the transcription of MMP9 to suppress the invasion and migration of breast cancer cells via HDAC1-mediated p65 deacetylation

GATA-binding protein 4 (GATA4) is recognized for its significant roles in embryogenesis and various cancers. Through bioinformatics and clinical data, it appears that GATA4 plays a role in breast cancer development. Yet, the specific roles and mechanisms of GATA4 in breast cancer progression remain elusive. In this study, we identify GATA4 as a tumor suppressor in the invasion and migration of breast cancer. Functionally, GATA4 significantly reduces the transcription of MMP9. On a mechanistic level, GATA4 diminishes MMP9 transcription by interacting with p65 at the NF-κB binding site on the MMP9 promoter. Additionally, GATA4 promotes the recruitment of HDAC1, amplifying the bond between p65 and HDAC1. This leads to decreased acetylation of p65, thus inhibiting p65’s transcriptional activity on the MMP9 promoter. Moreover, GATA4 hampers the metastasis of breast cancer in vivo mouse model. In summary, our research unveils a novel mechanism wherein GATA4 curtails breast cancer cell metastasis by downregulating MMP9 expression, suggesting a potential therapeutic avenue for breast cancer metastasis.

Abstract 6004: HOXC8 epigenetically controls caspse-1 expression and pyroptosis in lung cancer cells

Abstract Homeobox C8 (HOXC8) is a transcription factor preferentially overexpressed in a large percentage of lung adenocarcinoma (LUAD). To investigate the function of HOXC8 in LUAD, we showed that knockdown of HOXC8 led to massive LUAD cell death in a mechanism of pyroptosis because both YVAD, a caspase-1 (CASP1) inhibitor, and disulfiram, which prevents gasdemin D (GSDMD) pore formation, blocked cell death caused by HOXC8 depletion. Intriguingly, ASC and NLRP3, components of canonic inflammasome, were not involved in pyroptosis occurring in HOXC8-depleted cells. Instead, we found that silencing HOXC8 led to a marked increase in CASP1 abundance. As forced expression of CASP1 is sufficient to induce CASP1 activation and pyroptosis, we reason that HOXC8 deters pyroptosis by suppressing the level of CASP1. Moreover, we revealed that knockdown of HOXC8 augmented CASP1 transcription involving in HDAC1/2. In fact, HOXC8 and HDAC1 were in the same immunocomplex and the presence of HOXC8 is required for the recruitment of HDAC1 to CASP1 promoter. Since HOXC8 also binds CASP1 promoter, we conclude that HOXC8 negatively regulates CASP1 expression by drafting HDAC1/2 to CASP1. This study suggests that HOXC8 participates LUAD development by controlling CASP1 expression and pyroptosis. Citation Format: Lei Sun, Ravi Padia, Ozlem Calbay, Cheng Chi, Mahmuda Akter, Lingtao Jin, Shuang Huang. HOXC8 epigenetically controls caspse-1 expression and pyroptosis in lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6004.

Comparative interactome analysis of α-arrestin families in human and Drosophila

The α-arrestins form a large family of evolutionally conserved modulators that control diverse signaling pathways, including both G-protein-coupled receptor (GPCR)-mediated and non-GPCR-mediated pathways, across eukaryotes. However, unlike β-arrestins, only a few α-arrestin targets and functions have been characterized. Here, using affinity purification and mass spectrometry, we constructed interactomes for 6 human and 12 Drosophila α-arrestins. The resulting high-confidence interactomes comprised 307 and 467 prey proteins in human and Drosophila, respectively. A comparative analysis of these interactomes predicted not only conserved binding partners, such as motor proteins, proteases, ubiquitin ligases, RNA splicing factors, and GTPase-activating proteins, but also those specific to mammals, such as histone modifiers and the subunits of V-type ATPase. Given the manifestation of the interaction between the human α-arrestin, TXNIP, and the histone-modifying enzymes, including HDAC2, we undertook a global analysis of transcription signals and chromatin structures that were affected by TXNIP knockdown. We found that TXNIP activated targets by blocking HDAC2 recruitment to targets, a result that was validated by chromatin immunoprecipitation assays. Additionally, the interactome for an uncharacterized human α-arrestin ARRDC5 uncovered multiple components in the V-type ATPase, which plays a key role in bone resorption by osteoclasts. Our study presents conserved and species-specific protein–protein interaction maps for α-arrestins, which provide a valuable resource for interrogating their cellular functions for both basic and clinical research.

Comparative interactome analysis of α-arrestin families in human and Drosophila.

The α-arrestins form a large family of evolutionally conserved modulators that control diverse signaling pathways, including both G-protein-coupled receptor (GPCR)-mediated and non-GPCR-mediated pathways, across eukaryotes. However, unlike β-arrestins, only a few α-arrestin targets and functions have been characterized. Here, using affinity purification and mass spectrometry, we constructed interactomes for 6 human and 12 Drosophila α-arrestins. The resulting high-confidence interactomes comprised 307 and 467 prey proteins in human and Drosophila, respectively. A comparative analysis of these interactomes predicted not only conserved binding partners, such as motor proteins, proteases, ubiquitin ligases, RNA splicing factors, and GTPase-activating proteins, but also those specific to mammals, such as histone modifiers and the subunits of V-type ATPase. Given the manifestation of the interaction between the human α-arrestin, TXNIP, and the histone-modifying enzymes, including HDAC2, we undertook a global analysis of transcription signals and chromatin structures that were affected by TXNIP knockdown. We found that TXNIP activated targets by blocking HDAC2 recruitment to targets, a result that was validated by chromatin immunoprecipitation assays. Additionally, the interactome for an uncharacterized human α-arrestin ARRDC5 uncovered multiple components in the V-type ATPase, which plays a key role in bone resorption by osteoclasts. Our study presents conserved and species-specific protein-protein interaction maps for α-arrestins, which provide a valuable resource for interrogating their cellular functions for both basic and clinical research.

High glucose impairs cognitive function through inducing mitochondrial calcium overload in Treg cells

High glucose has been proved to impair cognitive function in type 2 diabetes, but the underlying mechanisms remain elusive. Here, we found that high glucose increased transcription factors' SP1 O-GlcNAcylation in regulatory T (Treg) cells. Glycosylated SP1 further enhanced HDAC2 recruitment and histone deacetylation on Na+/Ca2+/Li+ exchanger (NCLX) promoter, which downregulated NCLX expression and led to mitochondrial calcium overload and oxidative damage, thereby promoting Treg cell dysfunction, M1 microglia polarization, and diabetes-associated cognitive impairment. Importantly, GLP-1 receptor agonist alleviated these deleterious effects via GLP-1-receptor-mediated upregulation of OGA and inhibition of SP1 O-GlcNAcylation in Treg cells. Our study highlighted a link between high-glucose-mediated SP1 O-GlcNAcylation and HDAC2/NCLX signaling in control of mitochondrial calcium concentrations in Treg cells. It also revealed a mechanism for linking Treg cell dysfunction and cognitive impairment in type 2 diabetes and provides an insight into the mechanism underlying the neuroprotective effects of GLP-1 receptor agonist.

GATA-1 and BCL11A Collaboratively Regulate Erythroid Gene Expression through HDAC1 Dependent Manner

Transcription factor GATA-1 plays important roles in hematopoiesis through interaction with other key transcription factors and epigenetic modulators. Previously, we established that GATA-1 can physically interact with HDAC1 at two arginine residues of the linker region between two zinc fingers. Arginine to alanine mutation at these sites blocks the HDAC1 interaction and results in constitutive GATA-1 acetylation, and reduced GATA-1 chromatin binding at the gene regulatory regions. In addition, the loss of HDAC1 interaction causes the blockage of erythroid and megakaryocytic differentiation in mice. Having established the importance of GATA-1 and HDAC1 interaction in GATA-1 function, next we aim to further investigate whether HDAC1-associated proteins are involved in regulating the function of GATA-1. We performed LC-MS/MS analysis using GATA-1 wild type and mutant expressing cells and identified that BCL11A was one of the major proteins that associates with GATA-1 through HDAC1. BCL11A is a master regulator of γ-globin gene silencing through association with the NuRD complex and blocking of NFYA recruitment to γ-globin gene promoters. The role of BCL11A in regulating gene expression during erythroid differentiation is not well studied. HDAC1 mediated GATA-1 and BCL11A association was validated by pull down and immunoprecipitation assay. Next, the BCL11A and HDAC1 interacting site was determined. The N-terminal region of BCL11A is found to be important for HDAC1 interaction by pull down assay. Through analysis of the secondary structure of BCL11A N-terminal, L12/K14 are predicted to be important for protein-protein interaction. Indeed, L12A/K14A mutation significantly reduced HDAC1 interaction as well as HDAC1 mediated GATA-1 and NuRD complex association. To assess the effects of the L12A/K14A mutation of BCL11A on gene expression, we performed RNA-seq analysis using BCL11A knock down (KD) or L12A/K14A mutant overexpressed in HUDEP-2 cells. The result shows that the L12A/K14A mutation de-repressed γ-globin expression and positively affects genes for erythrocyte development that is similar to the expression profile of BCL11A KD. Therefore, L12/K14 residues of BCL11A might have an important role in γ-globin silencing and erythropoiesis by mediating HDAC1 associated GATA-1 and NuRD complex. The Cut&Run assay was performed to study the recruitment of BCL11A, GATA-1, HDAC1, NuRD complex, and NFYA in WT, BCL11A KD, and Flag-BCL11A L12A/K14A mutant cells. There are over 7000 GATA-1 and BCL11A co-binding peaks detected in wild type cells. Within these peaks, about 2000 peaks are at BCL11A binding motif site. GATA-1, HDAC1 and NuRD binding were abolished at these sites in BCL11A KD or L12A/K14A mutant cells, while BCL11A binding was not affected. There are about 3000 peaks at GATA-1 binding motif sites. While GATA-1 binding is not affected at these sites in BCL11A KD or L12A/K14A mutant cells, BCL11A, HDAC1 and NuRD complex binding were significantly reduced. This study demonstrates that there are two modes of BCL11A and GATA-1 co-recruitment: BCL11A can recruit GATA-1 and GATA-1 can recruit BCL11A to their specific binding motifs, respectively. Interestingly, these two modes regulate differential gene targets. BCL11A recruits GATA-1 at genes mainly involved in chromatin organization and cell cycle, while GATA-1 recruits BCL11A to genes mainly involved in transcription, erythrocyte and megakaryocyte differentiation. At the β-globin locus, BCL11A KD or L12A/K14A mutation reduced GATA-1 and NuRD binding and enhanced NFYA binding at the γ-globin promoter, suggesting that BCL11A cooperates with GATA-1, HDAC1, and NuRD complex and prevents NFYA binding at the distal binding region to suppress γ-globin expression. At LCR hypersensitive site 2 (HS2) region, we also observed a reduction of GATA-1 and NuRD recruitment with BCL11A KD or L12A/K14A mutation while GATA-1 recruitment at other promoters or LCR regions were not changed, suggesting that the HS2 region may be involved in reactivating γ-globin in BCL11A KD or L12A/K14A mutation cells. Future study will continue to analyze how BCL11A KD or mutation affects BCL11A and GATA-1 co-binding and target gene expression, and NFYA or other factors recruitment. In summary, our study shows that BCL11A interacts with GATA-1 in a HDAC1 dependent manner and this interaction is important in regulating fetal globin expression and erythroid development.

HDAC2-miRNA183 Epigenetic Circuit Contributes to the Growth of Philadelphia-Positive Acute Lymphoblastic Leukemia Via BCR-ABL/PTEN/p-AKT and c-MYC Signaling Pathways

Purpose: To characterize the expression and function of leukemia related/specific microRNAs (miRNAs) in Philadelphia-positive Acute Lymphoblastic Leukemia (Ph-positive ALL), and to identify the mechanistic signaling pathways in Ph-positive ALL. Method: Differentially expressed miRNAs in Ph-positive ALL patient samples and cell line were identified using RNA-seq and qPCR. Cell proliferation and apoptosis were detected with CCK-8 and flow cytometry assays, respectively. Bioinformatics software predicted the upstream and downstream regulatory pathway of miRNA183; the dual luciferase reporter gene analysis verified the regulatory effect of miRNA183 on the target gene BCR-ABL; ChIP experiment detected the recruitment of HDACs in the miRNA183 promoter region, and western blot detected the expression of related protein in the miRNA183 related pathway. Results: We identified thirty-one differentially expressed miRNAs in three Ph-positive ALL samples vs. normal karyotype B-ALL samples. Among these alternately expressed miRNAs, we found miRNA183 was significantly downregulated in Ph-positive ALL. qPCR verified the down-expression of miRNA183 in Ph-positive ALLcasesamples and Ph-positive ALL cell line SUP-B15. Forced over-expression of miRNA183 inhibited proliferation of SUP-B15 cells, but had no effect on NALM-6 cells (normal karyotype B-ALL). Furthermore, either forced over-expression of miRNA183 or silence down of BCR-ABL1 promoted SUP-B15 cell apoptosis.TargetScan software prediction found that miRNA183 and BCR-ABL (e1a2) had potential binding sites, dual luciferase reporter gene experiments further proved that miRNA183 could direct bind to BCR-ABL gene; qPCR and western blot further showed that miRNA183 inhibited the expression of P190. Our results showed that BCR-ABL (e1a2) is the target gene of miRNA183. In addition, forced over-expression of miRNA183 and silence down of BCR-ABL1 regulated the downstream proteins including PTEN, PARP, p-AKT and c-MYC with the same change trend. In complementary loss-of-function studies, inhibition of miRNA183 restored the expression of these proteins.Treatment of SUP-B15 cells with decitabine (DAC) and histone deacetylase inhibitor (HDACi), respectively, resulted in decrease of cell viability in a dose-dependent manner. Surprisingly, miRNA183 levels were significantly increased in HDACi exposure but not in DAC exposure. Furthermore, miRNA183 inhibitor reduced HDACi-induced upregulation of miRNA183 expression and attenuated HDACi-mediated apoptosis in SUP-B15 cells. Biology software prediction showed the presence of a transcription start site (TSS) 5 kb upstream of miRNA183. ChIP assay further demonstrated that HDAC2 but not HDAC1 was specifically recruited to the miRNA183 promoter region. Additionally, after HDACi treatment, HDACi-induced H3K27ac was recruited and accumulated at the miRNA183 promoter region. Western blotassay further proved the changes of the related proteins. Conclusion :Our results indicate that miRNA183 is a potential biomarker for Ph-positive ALL and suggest that a novel “HDAC2-miRNA183 Epigenetic Circuit” may be involved in the growth and progression of Ph-positive ALL by PTEN/p-AKT and c-MYC signaling pathways. Taken together, this study may provide novel approaches to the design of targeted drugs for Ph-positive ALL.

Shikonin suppresses small cell lung cancer growth via inducing ATF3-mediated ferroptosis to promote ROS accumulation

Small cell lung cancer (SCLC) is a subtype of lung cancer with a very poor overall survival rate due to its extremely high proliferation and metastasis predilection. Shikonin is an active ingredient extracted from the roots of Lithospermum erythrorhizon, and exerts multiple anti-tumor functions in many cancers. In the present study, the role and underlying mechanism of shikonin in SCLC were investigated for the first time. We found that shikonin effectively suppressed cell proliferation, apoptosis, migration, invasion, and colony formation and slightly induced apoptosis in SCLC cells. Further experiment indicated the shikonin could also induced ferroptosis in SCLC cells. Shikonin treatment effectively suppressed the activation of ERK, the expression of ferroptosis inhibitor GPX4, and elevated the level of 4-HNE, a biomarker of ferroptosis. Both total ROS and lipid ROS were increased, while the GSH levels were decreased in SCLC cells after shikonin treatment. More importantly, our data identified that the function of shikonin was dependent on the up-regulation of ATF3 by performing rescue experiments using shRNA to silence the expression of ATF3, especially in the total and lipid ROS accumulaiton. Xenograft model was established using SBC-2 cells, and the results revealed that shikonin also significantly inhibited tumor growth by inducing ferroptosis. Finally, our data further confirmed that shikonin activated ATF3 transcription by impairing the recruitment of HDAC1 mediated by c-myc on the ATF3 promoter, and subsequently elevating of histone acetylation. Our data documented that shikonin suppressed SCLC by inducing ferroptosis in a ATF3-dependent manner. Shikonin upregulated the expression of ATF3 expression via promoting the histone acetylation by inhibiting c-myc-mediated HDAC1 binding on ATF3 promoter.

Histone deacetylase 1 maintains lineage integrity through histone acetylome refinement during early embryogenesis.

Histone acetylation is a pivotal epigenetic modification that controls chromatin structure and regulates gene expression. It plays an essential role in modulating zygotic transcription and cell lineage specification of developing embryos. While the outcomes of many inductive signals have been described to require enzymatic activities of histone acetyltransferases and deacetylases (HDACs), the mechanisms by which HDACs confine the utilization of the zygotic genome remain to be elucidated. Here, we show that histone deacetylase 1 (Hdac1) progressively binds to the zygotic genome from mid blastula and onward. The recruitment of Hdac1 to the genome at blastula is instructed maternally. Cis-regulatory modules (CRMs) bound by Hdac1 possess epigenetic signatures underlying distinct functions. We highlight a dual function model of Hdac1 where Hdac1 not only represses gene expression by sustaining a histone hypoacetylation state on inactive chromatin, but also maintains gene expression through participating in dynamic histone acetylation-deacetylation cycles on active chromatin. As a result, Hdac1 maintains differential histone acetylation states of bound CRMs between different germ layers and reinforces the transcriptional program underlying cell lineage identities, both in time and space. Taken together, our study reveals a comprehensive role for Hdac1 during early vertebrate embryogenesis.

Breast cancer plasticity is restricted by a LATS1-NCOR1 repressive axis

Breast cancer, the most frequent cancer in women, is generally classified into several distinct histological and molecular subtypes. However, single-cell technologies have revealed remarkable cellular and functional heterogeneity across subtypes and even within individual breast tumors. Much of this heterogeneity is attributable to dynamic alterations in the epigenetic landscape of the cancer cells, which promote phenotypic plasticity. Such plasticity, including transition from luminal to basal-like cell identity, can promote disease aggressiveness. We now report that the tumor suppressor LATS1, whose expression is often downregulated in human breast cancer, helps maintain luminal breast cancer cell identity by reducing the chromatin accessibility of genes that are characteristic of a “basal-like” state, preventing their spurious activation. This is achieved via interaction of LATS1 with the NCOR1 nuclear corepressor and recruitment of HDAC1, driving histone H3K27 deacetylation near NCOR1-repressed “basal-like” genes. Consequently, decreased expression of LATS1 elevates the expression of such genes and facilitates slippage towards a more basal-like phenotypic identity. We propose that by enforcing rigorous silencing of repressed genes, the LATS1-NCOR1 axis maintains luminal cell identity and restricts breast cancer progression.

DACH1 inhibits breast cancer cell invasion and metastasis by down-regulating the transcription of matrix metalloproteinase 9

Human Dachshund homolog 1 (DACH1) is usually defined as a tumor suppressor, which plays an influential role in tumor growth and metastasis in a variety of cancer cells. However, the underlying mechanisms in these process are not yet fully clarified. In this study, DACH1 inhibited the invasion and metastasis of breast cancer cells by decreasing MMP9 expression. Mechanistically, DACH1 represses the transcriptional level of MMP9 by interacting with p65 and c-Jun at the NF-κB and AP-1 binding sites in MMP9 promoter respectively, and the association of DACH1 and p65 promote the recruitment of HDAC1 to the NF-κB binding site in MMP9 promoter, resulting in the reduction of the acetylation level and the transcriptional activity of p65. Accordingly, the level of MMP9 was decreased. In conclusion, we found a new mechanism that DACH1 could inhibit the metastasis of breast cancer cells by inhibiting the expression of MMP9.

LSD1 Facilitates Pro-Inflammatory Polarization of Macrophages by Repressing Catalase.

The increased level of hydrogen peroxide accompanies some modes of macrophage specification and is linked to ROS-based antimicrobial activity of these phagocytes. In this study, we show that activation of toll-like receptors with bacterial components such as LPS is accompanied by the decline in transcription of hydrogen peroxide decomposing enzyme-catalase, suppression of which facilitates the polarization of human macrophages towards the pro-inflammatory phenotype. The chromatin remodeling at the CAT promoter involves LSD1 and HDAC1, but activity of the first enzyme defines abundance of the two proteins on chromatin, histone acetylation status and the CAT transcription. LSD1 inhibition prior to macrophage activation with LPS prevents CAT repression by enhancing the LSD1 and interfering with the HDAC1 recruitment to the gene promoter. The maintenance of catalase level with LSD1 inhibitors during M1 polarization considerably limits LPS-triggered expression of some pro-inflammatory cytokines and markers such as IL1β, TNFα, COX2, CD14, TLR2, and IFNAR, but the effect of LSD1 inhibitors is lost upon catalase deficiency. Summarizing, activity of LSD1 allows for the CAT repression in LPS stimulated macrophages, which negatively controls expression of some key pro-inflammatory markers. LSD1 inhibitors can be considered as possible immunosuppressive drugs capable of limiting macrophage M1 specialization.

Transcriptional Repression of RASSF2 Impairs Hippo Kinase-Dependent p53 Activation and Tumor Suppression in t(8;21) AML

The t(8;21) chromosomal translocation is a recurring cytogenetic abnormality in acute myeloid leukemia (AML), found in ~10% of AML patients. This translocation results in the stable fusion of the RUNX1 (AML1) and RUNX1T1 genes, and formation of the oncofusion protein RUNX1-ETO (RE). One function of RE in promoting leukemia development is the recruitment of aberrant transcription factor complexes to regulatory regions of RUNX1 target genes known to be critical for myeloid differentiation, such as CEBPA, SPI1, NFE2, and CSF1R . Despite this knowledge, additional RE target genes remain poorly characterized, and the complete molecular mechanism through which RE leads to leukemic transformation remains to be elucidated.Previously, we used a mouse model of t(8;21) AML to compare gene expression in RE-expressing leukemia cells to the normal mouse hematopoietic progenitor (lin-cKit+) compartment (Lo et al, 2012). Among the most significantly downregulated genes was Ras-association domain family member 2 (Rassf2). RASSF genes are frequently epigenetically silenced in human tumors, making them highly relevant to study in the context of cancer development. Using public gene expression data, RT-qPCR, and western blotting, we found that RASSF2 expression is specifically downregulated in t(8;21) AML cells compared to non-t(8;21) AML cells and normal CD34+ controls. Furthermore, RE knockdown in a t(8;21) AML cell line led to marked upregulation of RASSF2 mRNA. Based on ChiP-seq data analysis this repression appears to be mediated by direct RE binding and HDAC recruitment to the promoter region of both alternative RASSF2 transcriptional start sites in the genomic locus.To investigate the significance of this repression we used retroviral vectors to re-express RASSF2 in t(8;21) AML cell lines and RE-transduced primary murine hematopoietic cells in vivo and in vitro . Re-expression of RASSF2 significantly improved survival and reduced the total leukemic burden in a primary mouse model of RE-driven AML in vivo . RASSF2 inhibited leukemic transformation and the self-renewal ability of RE-transduced mouse bone marrow cells in a serial replating/colony formation assay, without a noticeable effect on normal hematopoietic progenitors. This inhibition correlated with increased apoptotic cells in the context of RE and RASSF2 co-expression.Given that RASSF2 is a non-enzymatic scaffolding protein, we hypothesized that it mediates tumor suppressive functions via its Ras-association or SARAH effector domains. We saw no detectable effect of RASSF2 expression on oncogenic Ras signaling in t(8;21) AML cells. This led us to investigate the SARAH domain-mediated interaction of RASSF2 with the pro-apoptotic Hippo kinases, MST1 and MST2. RASSF2 expression in t(8;21) AML cells led to an increased protein amount of both MST1 and MST2 in a SARAH domain-dependent manner. Importantly, use of either a RASSF2 SARAH domain deletion mutant, or Mst1(-/-)Mst2(-/-) mouse bone marrow cells, completely abrogated the ability of RASSF2 to inhibit RE leukemia cell self-renewal in the serial replating/colony formation assay. We found this inhibition to be independent of canonical Hippo pathway signaling, and therefore utilized a proximity-dependent biotin identification approach (BioID, Roux et al, 2012) to map the protein interactome of RASSF2-MST1/2 in a t(8;21) AML cell line. Interestingly, this revealed an interaction between RASSF2 and the protein deacetylase SIRT1. This highlighted a role for RASSF2 in promoting p53 acetylation in response to cellular stress, which is dependent on MST1/2-mediated negative regulation of SIRT1. Overall, our study revealed a critical tumor suppressive role of RASSF2-MST1/2 signaling via the SIRT1-p53 axis in t(8;21) AML. DisclosuresNo relevant conflicts of interest to declare.

Calcitriol and Retinoic acid antagonize each other to suppress the production of IL-9 by Th9 cells

Distinct T helper cells, including Th9 cells help maintain homeostasis in the immune system. Vitamins play pivotal role in the immune system through many mechanisms, including regulating the differentiation of T helper cells. Calcitriol (1,25-dihydroxyvitamin D3) and retinoic acid possess hormone-like properties and are the bioactive metabolites of vitamin D and A, respectively, that signal through heterodimers containing the common retinoid X receptor. In contrast to individual treatment with the vitamins that significantly attenuates IL-9 production from Th9 cells, Th9 cells treated with both vitamins demonstrated IL-9 production similar to untreated Th9 cells. This is associated with reciprocal expression of PU.1 and Foxp3. While the recruitment of PU.1 was significantly impaired to the Il9 gene in the presence of calcitriol or retinoic acid in Th9 cells, addition of both vitamins together increased the recruitment of PU.1 to the Il9 gene. Calcitriol and retinoic acid together impaired the recruitment of HDAC1 to the Il9 gene without impacting Gcn5 recruitment. Importantly, retinoic acid negated the effect of calcitriol and impaired the binding of VDR on the Il9 gene by dampened VDR-RXR formation. Collectively, our data show that calcitriol and retinoic acid antagonize each other to regulate the differentiation of Th9 cells.

BRMS1L inhibits bone metastasis of breast cancer cells through epigenetic silence of CXCR4.

e13002 Background: We previously demonstrated that BRMS1L (breast cancer metastasis suppressor 1 like) suppresses breast cancer metastasis through HDAC1 recruitment and histone H3K9 deacetylation at the promoter of FZD10, a receptor for Wnt signaling. It is still unclear whether BRMS1L regulates organ-specific metastases, such as bone metastasis, the most prevalent metastatic site of breast cancer. Methods: Examination of the expression of BRMS1L in primary tumors, bone metastatic and other metastatic tissues from breast cancer patients was implemented using qRT-PCR and immunohistochemistry staining. To investigate the mechanism by which BRMS1L drives breast cancer bone metastasis, we tested the mRNA expression by qRT-PCR of a set of potential bone related genes (BRGs) based on PubMed database in MDA-MB-231 cells over expressing BRMS1L and MCF-7 cells knocking-down BRMS1L, and detected the expression of CXCR4 in these established cells by western blot. Transwell assays were performed to assess the migration abilities of breast cancer cells towards osteoblasts. ChIP (Chromatin Immuno-Precipitation) were employed to test the interaction between BRMS1L and CXCR4. Results: At both mRNA and protein levels, the expression of BRMS1L was significantly lower in bone metastatic sites than that in primary cancer tissues and other metastatic sites of breast cancer patients. CXCR4 was screened out in a set of BRGs and negatively correlated with the expression of BRMS1L in breast cancer cell lines. BRMS1L inhibited the migration of breast cancer cells towards osteoblasts through CXCL12/CXCR4 axis. In the presence of TSA treatment, breast cancer cell lines showed an increased expression of CXCR4 in a TSA concentration-dependent manner. In addition, ChIP assays verified that BRMS1L directly bound to the promoter region of CXCR4 and inhibited its transcription through promoter histone deacetylation. Conclusions: BRMS1L mediates the migration abilities of breast cancer cells to bone microenvironment via targeting CXCR4 and contributes to bone metastasis of breast cancer cells. Thus, BRMS1L may be a potential biomarker for predicting bone metastasis in breast cancer.

Z-Ligustilide Selectively Targets AML by Restoring Nuclear Receptors Nur77 and NOR-1-mediated Apoptosis and Differentiation

BackgroundAcute myeloid leukemia (AML) is a devastating hematologic malignancy with a high mortality. The nuclear receptors Nur77 and NOR-1 are commonly downregulated in human AML blasts and have emerged as key therapeutic targets for AML. MethodsThis study aimed to identify Z-ligustilide (Z-LIG), the main phthalide of Rhizoma Chuanxiong, as a potential agent that can selectively target AML. The anti-AML activity of Z-LIG was evaluated in vitro and in vivo, and the effect and underlying mechanisms of Z-LIG on the restoration of Nur77 and NOR-1 was determined. Moreover, the role of Nur77 and NOR-1 in the regulation of Z-LIG-induced apoptosis and differentiation of AML cells was explored. ResultsZ-LIG preferentially inhibited the viability of human AML cells, as well as suppressed the proliferation and colony formation ability. Notably, a concentration-dependent dual effect of Z-LIG was observed in AML cells: inducing apoptosis at relatively high concentrations (25 μM to 100 μM) and promoting differentiation at relatively low concentrations (10 μM and 25 μM). Importantly, Z-LIG restored Nur77 and NOR-1 expression in AML cells by increasing Ace-H3 (lys9/14) enrichment in their promoters. Meanwhile, Z-LIG enhanced the recruitment of p300 and reduced the recruitment of HDAC1, HDAC4/5/7, and MTA1 in the Nur77 promoter and enhanced the recruitment of p-CREB and reduced HDAC1 and HDAC3 in the NOR-1 promoter. Furthermore, Z-LIG-induced apoptosis was shown to be correlated with the mitochondria localization of Nur77/NOR-1 and subsequent Bcl-2 conformational change, converting Bcl-2 from a cyto-protective phenotype into a cyto-destructive phenotype. Z-LIG-promoted differentiation was found to be related to Nur77/NOR-1-mediated myeloid differentiation-associated transcription factors Jun B, c-Jun, and C/EBPβ. Finally, silencing of Nur77 and NOR-1 attenuated anti-AML activity of Z-LIG in NOD/SCID mice. ConclusionsOur study suggests that Z-LIG may serve as a novel bifunctional agent for AML by restoring Nur77/NOR-1-mediated apoptosis and differentiation

SMAR1 repression by pluripotency factors and consequent chemoresistance in breast cancer stem-like cells is reversed by aspirin.

The high abundance of drug efflux pumps in cancer stem cells (CSCs) contributes to chemotherapy resistance. The transcriptional regulator SMAR1 suppresses CSC expansion in colorectal cancer, and increased abundance of SMAR1 is associated with better prognosis. Here, we found in breast tumors that the expression of SMAR1 was decreased in CSCs through the cooperative interaction of the pluripotency factors Oct4 and Sox2 with the histone deacetylase HDAC1. Overexpressing SMAR1 sensitized CSCs to chemotherapy through SMAR1-dependent recruitment of HDAC2 to the promoter of the gene encoding the drug efflux pump ABCG2. Treating cultured CSCs or 4T1 tumor-bearing mice with the nonsteroidal anti-inflammatory drug aspirin restored SMAR1 expression and ABCG2 repression and enhanced tumor sensitivity to doxorubicin. Our findings reveal transcriptional mechanisms regulating SMAR1 that also regulate cancer stemness and chemoresistance and suggest that, by restoring SMAR1 expression, aspirin might enhance chemotherapeutic efficacy in patients with stem-like tumors.

IKAROS and CK2 regulate expression of BCL-XL and chemosensitivity in high-risk B-cell acute lymphoblastic leukemia

AbstractHigh-risk B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive disease, often characterized by resistance to chemotherapy. A frequent feature of high-risk B-ALL is loss of function of the IKAROS (encoded by the IKZF1 gene) tumor suppressor. Here, we report that IKAROS regulates expression of the BCL2L1 gene (encodes the BCL-XL protein) in human B-ALL. Gain-of-function and loss-of-function experiments demonstrate that IKAROS binds to the BCL2L1 promoter, recruits histone deacetylase HDAC1, and represses BCL2L1 expression via chromatin remodeling. In leukemia, IKAROS' function is impaired by oncogenic casein kinase II (CK2), which is overexpressed in B-ALL. Phosphorylation by CK2 reduces IKAROS binding and recruitment of HDAC1 to the BCL2L1 promoter. This results in a loss of IKAROS-mediated repression of BCL2L1 and increased expression of BCL-XL. Increased expression of BCL-XL and/or CK2, as well as reduced IKAROS expression, are associated with resistance to doxorubicin treatment. Molecular and pharmacological inhibition of CK2 with a specific inhibitor CX-4945, increases binding of IKAROS to the BCL2L1 promoter and enhances IKAROS-mediated repression of BCL2L1 in B-ALL. Treatment with CX-4945 increases sensitivity to doxorubicin in B-ALL, and reverses resistance to doxorubicin in multidrug-resistant B-ALL. Combination treatment with CX-4945 and doxorubicin show synergistic therapeutic effects in vitro and in preclinical models of high-risk B-ALL. Results reveal a novel signaling network that regulates chemoresistance in leukemia. These data lay the groundwork for clinical testing of a rationally designed, targeted therapy that combines the CK2 inhibitor, CX-4945, with doxorubicin for the treatment of hematopoietic malignancies.

Che-1/AATF binds to RNA polymerase I machinery and sustains ribosomal RNA gene transcription.

Originally identified as an RNA polymerase II interactor, Che-1/AATF (Che-1) has now been recognized as a multifunctional protein involved in cell-cycle regulation and cancer progression, as well as apoptosis inhibition and response to stress. This protein displays a peculiar nucleolar localization and it has recently been implicated in pre-rRNA processing and ribosome biogenesis. Here, we report the identification of a novel function of Che-1 in the regulation of ribosomal RNA (rRNA) synthesis, in both cancer and normal cells. We demonstrate that Che-1 interacts with RNA polymerase I and nucleolar upstream binding factor (UBF) and promotes RNA polymerase I-dependent transcription. Furthermore, this protein binds to the rRNA gene (rDNA) promoter and modulates its epigenetic state by contrasting the recruitment of HDAC1. Che-1 downregulation affects RNA polymerase I and UBF recruitment on rDNA and leads to reducing rDNA promoter activity and 47S pre-rRNA production. Interestingly, Che-1 depletion induces abnormal nucleolar morphology associated with re-distribution of nucleolar proteins. Finally, we show that upon DNA damage Che-1 re-localizes from rDNA to TP53 gene promoter to induce cell-cycle arrest. This previously uncharacterized function of Che-1 confirms the important role of this protein in the regulation of ribosome biogenesis, cellular proliferation and response to stress.

Adenosine Receptor A1-A2a Heteromers Regulate EAAT2 Expression and Glutamate Uptake via YY1-Induced Repression of PPARγ Transcription.

Adenosine receptors A1 (A1AR) and A2a (A2aAR) play an important role in regulating glutamate uptake to avoid glutamate accumulation that causes excitotoxicity in the brain; however, the precise mechanism of the effects of A1AR and A2aAR is unclear. Herein, we report that expression of the A1AR protein in the astrocyte membrane and the level of intracellular glutamate were decreased, while expression of the A2aR protein was elevated in cells exposed to oxygen-glucose deprivation (OGD) conditions. Coimmunoprecipitation (Co-IP) experiments showed that A1AR interacts with A2aAR under OGD conditions. The activation of A1AR and inactivation of A2aAR by 2-chloro-N6-cyclopentyladenosine (CCPA) and SCH58251, respectively, partly reversed OGD-mediated glutamate uptake dysfunction, elevated EAAT2, and PPARγ protein levels, and suppressed the expression of Ying Yang 1 (YY1). Both the silencing of YY1 and the activation of PPARγ upregulated EAAT2 expression. Moreover, YY1 silencing elevated the PPARγ level under both normal and OGD conditions. Histone deacetylase (HDAC)1 was found to interact with YY1, and HDAC1 silencing improved PPARγ promoter activity. Taken together, our findings suggest that A1AR-A2aAR heteromers regulate EAAT2 expression and glutamate uptake through the YY1-mediated recruitment of HDAC1 to the PPARγ promoter region.