Brahma Expression Research Articles

Chromatin remodeling subunit BRM and valine regulate hematopoietic stem/progenitor cell function and self-renewal via intrinsic and extrinsic effects.

Little is known of hematopoietic stem (HSC) and progenitor (HPC) cell self-renewal. The role of Brahma (BRM), a chromatin remodeler, in HSC function is unknown. Bone marrow (BM) from Brm-/- mice manifested increased numbers of long- and short-term HSCs, GMPs, and increased numbers and cycling of functional HPCs. However, increased Brm-/- BM HSC numbers had decreased secondary and tertiary engraftment, suggesting BRM enhances HSC self-renewal. Valine was elevated in lineage negative Brm-/- BM cells, linking intracellular valine with Brm expression. Valine enhanced HPC colony formation, replating of human cord blood (CB) HPC-derived colonies, mouse BM and human CB HPC survival in vitro, and ex vivo expansion of normal mouse BM HSCs and HPCs. Valine increased oxygen consumption rates of WT cells. BRM through CD98 was linked to regulated import of branched chain amino acids, such as valine, in HPCs. Brm-/- LSK cells exhibited upregulated interferon response/cell cycle gene programs. Effects of BRM depletion are less apparent on isolated HSCs compared to HSCs in the presence of HPCs, suggesting cell extrinsic effects on HSCs. Thus, intracellular valine is regulated by BRM expression in HPCs, and the BRM/valine axis regulates HSC and HPC self-renewal, proliferation, and possibly differentiation fate decisions.

Association of two BRM promoter polymorphisms and smoking status with malignant pleural mesothelioma risk and prognosis.

Brahma (BRM), of the SWI/SNF complex, has two 6 to 7 bp insertion promoter polymorphisms (BRM-741/BRM-1321) that cause epigenetic BRM suppression, and are associated with risk of multiple cancers. BRM polymorphisms were genotyped in malignant pleural mesothelioma (MPM) cases and asbestos-exposed controls. Multivariable logistic regression (risk) and Cox regression (prognosis) were performed, including stratified analyses by smoking status to investigate the effect of polymorphisms on MPM risk and prognosis. Although there was no significant association overall between BRM-741/BRM-1321 and risk in patients with MPM, a differential effect by smoking status was observed (P-interaction < .001), where homozygous variants were protective (aOR of 0.18-0.28) in ever smokers, while never smokers had increased risk when carrying homozygous variants (aOR of 2.7-4.4). While there was no association between BRM polymorphisms and OS in ever-smokers, the aHR of carrying homozygous-variants of BRM-741, BRM-1321 or both were 4.0 to 8.6 in never-smokers when compared to wild-type carriers. Mechanistically, lower mRNA expression of BRM was associated with poorer general cancer prognosis. Electrophoretic mobility shift assays and chromatin immunoprecipitation experiments (ChIP) revealed high BRM insertion variant homology to MEF2 regulatory binding sites. ChIP experimentation confirmed MEF2 binding only occurs in the presence of insertion variants. DNA-affinity purification assays revealed YWHA scaffold proteins as vital to BRM mRNA expression. Never-smokers who carry BRM homozygous variants have an increased chance of developing MPM, which results in worse prognosis. In contrast, in ever-smokers, there may be a protective effect, with no difference in overall survival. Mechanisms for the interaction between BRM and smoking require further study.

Association of BRM promoter polymorphisms and esophageal adenocarcinoma outcome.

PurposeBrahma (BRM) is a critical catalytic subunit of the SWI/SNF chromatin remodeling complex; expression of BRM is commonly lost in various cancer types. BRM promoter polymorphisms (BRM-741; BRM-1321) are associated with loss of BRM expression, and with cancer risk/survival. We evaluated these two polymorphisms in the overall survival (OS) of esophageal adenocarcinoma (EAC) patients.ResultsOf 270 patients, 37% were stage IV. Minor allele frequencies were 47−49%; 15% were double-homozygotes. When compared to the wild-type genotype, the homozygous variant of BRM-741 carried an adjusted OS hazard ratio (aHR) of 1.64 (95% CI:1.1−2.4); for BRM-1321, the aHR was 2.09 (95% CI:1.4−3.0). Compared to the double wild-type, carrying homozygous variants of both promoter polymorphisms (double-homozygote) yielded an aHR of 2.21 (95% CI:1.4−3.6). Directions/magnitudes of associations were similar in subsets by age, gender, smoking status, use of platinum agents, and disease stage, and for progression-free survival.Materials and MethodsIn a cohort of EAC patients of all stages (84% male; median age of 64 years), two BRM polymorphisms were genotyped. Cox proportional hazards models, adjusted for known prognostic variables, estimated the association of polymorphisms with OS.ConclusionsBRM polymorphisms were associated with OS in EAC in this study. Validation studies are warranted.

Abstract 2117: Epigenetic mechanisms control switching between metastatic and proliferative subtypes in the SW13 adrenal adenocarcinoma cell line

Abstract The human adrenal carcinoma SW13 cell line can switch between a subtype which expresses the tumor-suppressor gene Brahma (BRM), SW13+, and one that does not, SW13-. Unlike many tumor supressor genes, which are often mutated, loss of BRM expression occurs post-transcriptionally and BRM expression can be restored via histone deacetylase (HDAC) inhibition. However, many of the previously characterized BRM inducing HDAC inhibitors are toxic, broad-spectrum inhibitors which have limited downstream use, and provide little insight into the specificity of BRM epigenetic suppression. In this work we sought to further characterize the oncogenic potential of the two SW13 subtypes, and to investigate the potential epigenetic regulators that contribute to the subtype switching. SW13 subtype morphology and growth rates were assessed by immunofluorescence and standard growth assays. Metastatic potential was characterized by measuring anchorage-independent growth with soft agar assays, collagenase activity with fluorescein-conjugated gelatin, and invasiveness into a 3D extracellular matrix. Differences in gene expression were measured by standard qRT-PCR and pathway-specific qRT-PCR profiling arrays. The capacity of specific HDAC inhibitors for their ability to induce switching between the SW13 subtypes was assessed by immunofluorescence and gene expression analyses. BRM deficient SW13- cells have both a faster doubling time and an increased rate of anchorage-independent growth, while SW13+ cells exhibit higher collagenase activity and invasive capacity. Significant differences in the expression of multiple genes involved in epigenetic chromatin remodeling as well as epigenetic chromatin modification were observed, indicating that these two subtypes also have unique gene expression profiles that could contribute to BRM regulation. Of those tested, inhibition of HDAC1 most efficiently increased the rate of conversion between SW13- and SW13+ cells, and did so in a dose dependent manner. After conversion to SW13+ subtype there is also increased acetylation on histone H3 but not histone H4 when compared to levels in SW13-, suggesting that the role of acetylation on the H3 tail may play an essential role in the subtype switch. These studies provide new insight into the functional consequences of BRM loss in the SW13 cell type and reveal potential targets for the reversal of BRM epigenetic silencing. Citation Format: McKale R. Davis, Elizabeth E. Hull. Epigenetic mechanisms control switching between metastatic and proliferative subtypes in the SW13 adrenal adenocarcinoma cell line. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2117. doi:10.1158/1538-7445.AM2015-2117

Abstract 5186: Reduced expression of BRM in hepatocellular carcinoma

Abstract Background: Hepatocellular carcinoma (HCC) is the fifth most common malignancy in men and the eighth most common in women worldwide. The eukaryotic genome is organized into highly ordered chromatin structures in the nucleus. Chromatin creates a barrier to nuclear processes, such as transcription, by obstructing the access of the transcriptional machinery and gene-specific regulators to recognition sequences within target promoters. The SWI/SNF protein complex, an ATPase-dependent chromatin remodeling enzyme, mobilizes nucleosomes and functions as a master regulator of gene expression and chromatin dynamics. SWI/SNF is a large multiprotein complex that contains either BRG1 or brahma (BRM) as the catalytic ATPase. Each SWI/SNF complex thus incorporates one of two possible ATPases, BRG1 or BRM. Aims: To examine alterations of BRG1 and BRM in HCC. Methods: We investigated DNA copy number aberrations in human HCC cell lines using a high-density oligonucleotide microarray. We determined DNA copy numbers and expression levels of BRG1 and BRM genes in primary HCC tumors, and conducted further searches for mutations in BRG1 and BRM genes. Results: Homozygous deletion of the BRG1 gene was found in HCC cell line SNU398. Copy number losses of BRG1 and BRM genes were observed in 14 (26%) and 7 (13%) of 54 primary HCC tumors respectively. We found four somatic missense mutations in the BRG1 gene in two of 36 primary HCC tumors, but no mutations in BRM gene. Expression of BRM mRNA, but not BRG1 mRNA, was significantly reduced in primary HCC tumors, compared to non-tumor tissue counterparts. Immunohistochemical analyses of non-tumour liver tissues showed that BRM protein was expressed in hepatocytes and bile-duct epithelial cells, whereas BRG1 protein was expressed in bile-duct epithelial cells, but not in hepatocytes. BRM protein expression was lost in nine (22.5%) of 40 HCC tumours. Loss of BRM protein expression was significantly associated with poor overall survival. Conclusion: Reduced expression of BRM may contribute to the carcinogenesis of HCC. Although deletions and mutations in BRG1 gene were identified, the role of BRG1 in HCC tumourigenesis remains unclear. Citation Format: Kohichiroh Yasui, Yasuyuki Gen, Akira Tomie, Tomoko Kitaichi, Yoshito Itoh. Reduced expression of BRM in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5186. doi:10.1158/1538-7445.AM2014-5186

Modulation of BRAHMA expression by the mitogen-activated protein kinase/extracellular signal regulated kinase pathway is associated with changes in melanoma proliferation

Brahma (BRM) and Brahma-related gene 1(BRG1) are catalytic subunits of SWItch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complexes. BRM is epigenetically silenced in a wide-range of tumors. Mutations in the v-raf murine sarcoma viral oncogene homolog B1 (BRAF) gene occur frequently in melanoma and lead to constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK1/2) pathway. We tested the hypothesis that BRM expression is modulated by oncogenic BRAF and phosphorylation of ERK1/2 in melanocytes and melanoma cells. Expression of oncogenic BRAF in melanocytes and melanoma cells that are wild-type for BRAF decreased BRM expression and increased BRG1 expression. Inhibition of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) or selective inhibition of BRAF in melanoma cells that harbor oncogenic BRAF increased BRM expression and decreased BRG1 expression. Increased BRM expression was associated with increased histone acetylation on the BRM promoter. Over-expression of BRM in melanoma cells that harbor oncogenic BRAF promoted changes in cell cycle progression and apoptosis consistent with a tumor suppressive role. Upon inhibition of BRAF(V600E) with PLX4032, BRM promoted survival. PLX4032 induced changes in BRM function were correlated with increased acetylation of the BRM protein. This study provides insights into the epigenetic consequences of inhibiting oncogenic BRAF in melanoma through modulation of SWI/SNF subunit expression and function.

Flavonoids from each of the six structural groups reactivate BRM, a possible cofactor for the anticancer effects of flavonoids.

Flavonoids have been extensively studied and are well documented to have anticancer effects, but it is not entirely known how they impact cellular mechanisms to elicit these effects. In the course of this study, we found that a variety of different flavonoids readily restored Brahma (BRM) in BRM-deficient cancer cell lines. Flavonoids from each of the six different structural groups were effective at inducing BRM expression as well as inhibiting growth in these BRM-deficient cancer cells. By blocking the induction of BRM with shRNA, we found that flavonoid-induced growth inhibition was BRM dependent. We also found that flavonoids can restore BRM functionality by reversing BRM acetylation. In addition, we observed that an array of natural flavonoid-containing products both induced BRM expression as well as deacetylated the BRM protein. We also tested two of the BRM-inducing flavonoids (Rutin and Diosmin) at both a low and a high dose on the development of tumors in an established murine lung cancer model. We found that these flavonoids effectively blocked development of adenomas in the lungs of wild-type mice but not in that of BRMnull mice. These data demonstrate that BRM expression and function are regulated by flavonoids and that functional BRM appears to be a prerequisite for the anticancer effects of flavonoids both in vitro and in vivo.

Loss of BRM expression is a frequently observed event in poorly differentiated clear cell renal cell carcinoma

The aim of this study was to examine the status of Brahma (BRM), a key SWI/SNF complex subunit, in clear cell renal cell carcinomas (RCCs), and to analyse the histopathology, immunophenotype, molecular features and prognosis of the BRM-negative cases. We identified 19 cases of grade 4 tumours lacking BRM expression among 625 clear cell RCCs. All 19 cases exhibited features of poor differentiation: 13 showed pure poorly differentiated morphology, while six were composite tumours with an admixed typical low-grade component. Besides negative BRM expression, the immunophenotype of these cases was similar to clear cell RCC. VHL gene mutations were identified in nine of the 19 patients (47%). Chromosome 3p deletion was detected in 11 of 13 poorly differentiated RCCs and both areas of five of five composite tumours. All poorly differentiated tumour areas showed polysomy of chromosome 3. No losses or gains of chromosome 3 were observed in low-grade tumour areas of five of five composite RCCs. We have shown that loss of BRM expression is a common feature among poorly differentiated tumours in clear cell RCCs. We hypothesize that loss of BRM expression is involved in tumor de-differentiation in clear cell RCCs and may play an important role during tumour progression.

Oncogenic targeting of BRM drives malignancy through C/EBPβ-dependent induction of α5 integrin.

Integrin expression and activity are altered in tumors, and aberrant integrin signaling promotes malignancy. However, how integrins become altered in tumors remains poorly understood. We discovered that oncogenic activation of MEK signaling induces cell growth and survival, and promotes the malignant phenotype of mammary epithelial cells (MECs) by increasing α5 integrin expression. We determined that MEK activates c-Myc to reduce the transcription of the SWI/SNF chromatin remodeling enzyme Brahma (BRM). Our studies revealed that reduced BRM expression and/or activity drives the malignant behavior of MECs by epigenetically promoting C/EBPβ expression to directly induce α5 integrin transcription. Consistently, we could show that restoring BRM levels normalized the malignant behavior of transformed MECs in culture and in vivo by preventing C/EBPβ-dependent α5 integrin transcription. Our findings identify a novel mechanism whereby oncogenic signaling promotes malignant transformation by regulating transcription of a key chromatin remodeling molecule that regulates integrin-dependent stromal-epithelial interactions.

Promoter polymorphisms of the SWI/SNF chromatin remodeling complex molecule, BRM, and esophageal adenocarcinoma outcome.

4077 Background: Better understanding of the biology of esophageal cancer may help improve its treatment. The SWI/SNF chromatin remodeling complex is an important regulator of gene expression that has been linked to cancer development and outcome. Expression of Brahma (BRM), a critical catalytic subunit of SWI/SNF, is lost in a variety of solid tumors. Two novel BRM promoter polymorphisms (BRM -741 and BRM -1321) have been correlated with BRM loss and elevated cancer risk in upper aerodigestive cancers (Wang et al, Carcinogenesis, 2012) and more recently in lung cancer outcome (Cuffe et al, ESMO, 2012) by our research teams. Objectives: We evaluated BRM polymorphisms and their role in the survival of esophageal cancer patients. Methods: 223 histologically-confirmed esophageal adenocarcinoma patients of all stages were evaluated. The two BRM polymorphisms utilized Taqman genotyping. Cox proportional hazards models adjusted for clinical prognostic variables and determined the association of polymorphisms with overall survival (OS) and progression free survival (PFS). Adjusted hazard ratio (aHR) and 95% confidence intervals (CI) were calculated. Results: Among our patients, 85% were male; the mean age was 63 years. 37% had stage IV advanced tumors. The median PFS was 1.03 years, while median OS was 1.82 years. After adjustment for known prognostic clinical variables, carrying homozygous variants of both BRM polymorphisms (double homozygotes) was associated with a worse outcome: aHR 1.84 (1.06-2.34, p=0.03) for OS and aHR 1.93 (1.10-2.48, p=0.02) for PFS. The direction and magnitude of associations were similar in subsets of patients by age, gender, smoking status, use of platinum agents, and disease stage. Non-significant trends in the same direction but of aHR magnitudes 1.34-1.54 were seen in the patients who carried one homozygous variant or who were double heterozygotes. Conclusions: We report the initial association of BRM polymorphisms with survival in esophageal cancer. We plan to explore additional relationships and validate these findings in other datasets.

Identifying targets for the restoration and reactivation of BRM

Brahma (BRM) is a novel anticancer gene, which is frequently inactivated in a variety of tumor types. Unlike many anticancer genes, BRM is not mutated, but rather epigenetically silenced. In addition, histone deacetylase complex (HDAC) inhibitors are known to reverse BRM silencing, but they also inactivate it via acetylation of its C-terminus. High-throughput screening has uncovered many compounds that are effective at pharmacologically restoring BRM and thereby inhibit cancer cell growth. As we do not know which specific proteins, if any, regulate BRM, we sought to identify the proteins, which underlie the epigenetic suppression of BRM. By selectively knocking down each HDAC, we found that HDAC3 and HDAC9 regulate BRM expression, whereas HDAC2 controls its acetylation. Similarly, we ectopically overexpressed 21 different histone acetyltransferases and found that KAT6A, KAT6B and KAT7 induce BRM expression, whereas KAT2B and KAT8 induce its acetylation. We also investigated the role of two transcription factors (TFs) linked to either BRM (GATA3) or HDAC9 (MEF2D) expression. Knockdown of either GATA3 and/or MEF2D downregulated HDAC9 and induced BRM. As targets for molecular biotherapy are typically uniquely, or simply differentially expressed in cancer cells, we also determined if any of these proteins are dysregulated. However, by sequencing, no mutations were found in any of these BRM-regulating HDACs, HATs or TFs. We selectively knocked down GATA3, MEF2D, HDAC3 and HDAC9, and found that each gene-specific knockdown induced growth inhibition. We observed that both GATA3 and HDAC9 were greatly overexpressed only in BRM-negative cell lines indicating that HDAC9 may be a good target for therapy. We also found that the mitogen-activated protein (MAP) kinase pathway regulates both BRM acetylation and BRM silencing as MAP kinase pathway inhibitors both induced BRM as well as caused BRM deacetylation. Together, these data identify a cadre of key proteins, which underlie the epigenetic regulation of BRM.

Association of two BRM promoter polymorphisms with head and neck squamous cell carcinoma risk

The SWI/SNF chromatin remodeling complex is an important regulator of gene expression that has been linked to cancer development. Expression of Brahma (BRM), a critical catalytic subunit of SWI/SNF, is lost in a variety of solid tumors. Two novel BRM promoter polymorphisms (BRM-741 and BRM-1321) have been correlated with BRM loss and elevated cancer risk. The aim(s) of this study were to examine BRM expression in head and neck squamous cell carcinoma (HNSCC) and to correlate BRM polymorphisms with HNSCC risk. BRM expression studies were performed on eight HNSCC cell lines and 76 surgically resected tumor samples. A case-control study was conducted on 668 HNSCC patients (oral cavity, oropharynx, larynx and hypopharynx) and 700 healthy matched controls. BRM expression was lost in 25% of cell lines and 16% of tumors. The homozygous genotype of each polymorphism was significantly associated with increased HNSCC risk [BRM-741: adjusted odds ratio (aOR) 1.75, 95% CI 1.2-2.3, P < 0.001; BRM-1321: aOR 1.65, 95% CI 1.2-2.2, P < 0.001]. Individuals that were homozygous for both BRM polymorphisms had a more than 2-fold increase in the risk of HNSCC (aOR 2.23, 95% CI 1.5-3.4, P < 0.001). A particularly elevated risk was seen within the oropharynx, human papillomavirus-positive subgroup for carriers of both homozygous variants (aOR 3.09, 95% CI 1.5-6.8, P = 0.004). BRM promoter polymorphisms appear to act as susceptibility markers of HNSCC with potential utility in screening, prevention and treatment.

Alterations of the SWI/SNF chromatin remodelling subunit‐BRG1 and BRM in hepatocellular carcinoma

The SWI/SNF chromatin remodelling complex, which contains either brahma-related gene-1 (BRG1) or brahma (BRM) as the catalytic ATPase, functions as a master regulator of gene expression. To examine alterations of BRG1 and BRM in hepatocellular carcinoma (HCC). We investigated DNA copy number aberrations in human HCC cell lines using a high-density oligonucleotide microarray. We determined DNA copy numbers and expression levels of BRG1 and BRM genes in primary HCC tumours, and conducted further searches for mutations in BRG1 and BRM genes. Homozygous deletion of the BRG1 gene was found in HCC cell line SNU398. Copy number losses of BRG1 and BRM genes were observed in 14 (26%) and 7 (13%) of 54 primary HCC tumours respectively. We found four somatic missense mutations in the BRG1 gene in two of 36 primary HCC tumours, but no mutations in BRM gene. Expression of BRM mRNA, but not BRG1 mRNA, was significantly reduced in primary HCC tumours, compared to non-tumour tissue counterparts. Immunohistochemical analyses of non-tumour liver tissues showed that BRM protein was expressed in hepatocytes and bile-duct epithelial cells, whereas BRG1 protein was expressed in bile-duct epithelial cells, but not in hepatocytes. BRM protein expression was lost in nine (22.5%) of 40 HCC tumours. Loss of BRM protein expression was significantly associated with poor overall survival. Reduced expression of BRM may contribute to the carcinogenesis of HCC. Although deletions and mutations in BRG1 gene were identified, the role of BRG1 in HCC tumourigenesis remains unclear.

Methylation patterns of Brahma during spermatogenesis and oogenesis: potential implications

To compare methylation profiles and expression levels of Brahma at different stages of spermatogenesis, and to identify the methylation pattern during oogenesis, we analyzed gene expression and methylation patterns in murine germ cells at various developmental stages. The methylation levels of CpG islands within Brahma increased during spermatogenesis and decreased during oogenesis. This change in methylation pattern correlates with the change in expression of Brahma during spermatogenesis. As the degree of methylation increases, the expression decreases. The change in methylation is opposite during oogenesis, which suggests opposite expression levels.

The reversible epigenetic silencing of BRM: implications for clinical targeted therapy

The SWI/SNF chromatin-remodeling complex serves as a master switch that directs and limits the execution of specific cellular programs, such as differentiation and growth control. SWI/SNF function requires one of two paralogous ATPase subunits, Brahma (BRM) or BRM-related gene 1 (BRG1), which we previously found are lost together in cancer cell lines and primary lung cancers. Although BRG1 has been found to be mutated in cancer cell lines, the mechanisms underlying BRM silencing are not known. To address this question, we sequenced BRM in 10 BRM/BRG1-deficient cancer cell lines and found that BRM was devoid of abrogating mutations. Moreover, histone deacetylase (HDAC) inhibitors restored BRM expression in each of these BRG1/BRM-deficient cancer cell lines, indicating that epigenetic silencing is a major mechanism underlying the loss of BRM expression. Despite their ability to restore BRM expression, these HDAC inhibitors also blocked BRM function when present. However, after their removal, we observed that BRM expression remained elevated for several days, and during this period, BRM activity was detected. We also found that the suppression of BRM occurs in a broad range of human tumor types and that loss of one or both BRM alleles potentiated tumor development in mice. Thus, BRG1 and BRM are silenced by different mechanisms, and it may be possible to clinically target and reexpress BRM in a number of tumor types, potentially impacting tumor development.