BRM Promoter Polymorphisms and Survival of Advanced Non–Small Cell Lung Cancer Patients in the Princess Margaret Cohort and CCTG BR.24 Trial

Abstract

Introduction BRM, a key catalytic subunit of the SWI/SNF chromatin remodeling complex, is a putative tumor susceptibility gene that is silenced in 15% of non-small cell lung cancer (NSCLC). Two novel BRM promoter polymorphisms (BRM-741, BRM-1321) are associated with reversible epigenetic silencing of BRM protein expression. Methods Advanced NSCLC patients from the Princess Margaret (PM) cohort study and from the CCTG BR.24 clinical trial were genotyped for BRM promoter polymorphisms. Associations of BRM variants with survival were assessed using log-rank tests, the method of Kaplan and Meier, and Cox proportional hazards models. Promoter swap, luciferase assays, and chromatin immunoprecipitation (ChIP) experiments evaluated polymorphism function. In silico analysis of publicly available gene expression datasets with outcome were performed. Results Carrying the homozygous variants of both polymorphisms (“double homozygotes”, DH) when compared with those carrying the double wild-type, was associated with worse overall survival, with an adjusted hazard ratios (aHR) of 2.74 (95%CI: 1.9–4.0). This was confirmed in the BR.24 trial (aHR 8.97, 95%CI: 3.3–18.5). Lower BRM gene expression (by RNA-Seq or microarray) was associated with worse outcome (p<0.04). ChIP and promoter swap experiments confirmed binding of MEF2D and HDAC9 only to homozygotes of each polymorphism, associated with reduced promoter activity in the DH. Conclusion Epigenetic regulatory molecules bind to two BRM promoter sequence variants but not to their wild-type sequences. These variants are associated with adverse overall and progression free survival. Decreased BRM gene expression, seen with these variants, is also associated with worse overall survival.

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

“…In assessing our patient sample, we did not find an association between BRM polymorphisms and risk of developing MPM, consistent with liver, pancreatic, and colorectal cancer but in contrast to upper aerodigestive (head and neck and lung cancer), suggesting heterogeneity in mechanism and cancer‐site specificity . This is the first study, however, to identify an effect modification by smoking.…”

“…Recently, several mechanisms by which BRM polymorphisms are linked to various cancer risks and prognosis have been proposed . Homozygous 6 to 7 bp insertion variants at BRM‐741 and BRM‐1321 produce the MEF2D binding site . MEF2D is known to recruit HDACs and cause epigenetic suppression of BRM expression, which is a critical ATPase subunit of the SWI/SNF chromatin remodeling complex.…”

“…However, two exploratory analysis, one restricted to Caucasian males (which allowed for improved matching between cases and controls) and one restricted to occupationally‐exposed individuals both yielded ORs similar to the entire sample, including the differential effect by smoking status. Differential effect by disease sites raises concerns about technical differences in genotyping; however, we utilized an identical genotyping technique by the same technician, in similarly processed samples, as with our other studies . In our study, we did not explore BAP1, a widely studied susceptibility and prognostic marker for MPM .…”

“…However, in liver and pancreatic tumors, BRM polymorphisms were not associated with risk, suggesting the role of BRM may differ between tumor types . In efforts to explain the molecular basis of BRM polymorphisms on malignancies, in vitro studies revealed increases in MEF2D binding and five‐fold decreases in luciferase reporter gene activity in cancer cell lines in the presence of the promoter insertion variant . MEF2 proteins typically form transcription binding complexes that regulate gene function by dampening gene expression.…”

Two BRM promoter polymorphisms predict poor survival in patients with hepatocellular carcinoma

“…In many BRM ‐deficient cell lines, BRM expression can be restored by HDAC inhibitors, flavonoids, or certain non‐steroidal anti‐inflammatory drugs . Investigations into the reversibility of BRM suppression led to the identification of two BRM promoter polymorphisms, BRM‐741 (a 7‐base pair [bp] insertion‐deletion [indel]) and BRM‐1321 (a 6‐bp indel) which were located 741 and 1321 bp upstream from the transcription start site, respectively . BRM silencing is correlated with the presence of one or both promoter polymorphisms.…”