Loss of SWI/SNF Chromatin Remodeling Alters NRF2 Signaling in Non-Small Cell Lung Carcinoma

Abstract

The NF-E2-related factor 2 (referred to as NRF2) transcription factor binds antioxidant responsive elements within the promoters of cytoprotective genes to induce their expression. Next-generation sequencing studies in lung cancer have shown a significant number of activating mutations within the NRF2 signaling pathway. Mutations in components of the SWI/SNF chromatin-remodeling complex, a general regulator of transcription using either BRG1 or BRM as the catalytic subunit, also frequently occur in lung cancers. Importantly, low BRG1 expression levels in primary human NSCLC correlated with increased NRF2-target gene expression. Here, we show that loss of SWI/SNF complex function activated a subset of NRF2-mediated transcriptional targets. Using a series of isogenic NSCLC lines with reduced or depleted BRG1 and/or BRM expression, we observed significantly increased expression of the NRF2-target genes HMOX1 and GSTM4. In contrast, expression of the NRF2 target genes NQO1 and GCLM modestly increased following BRM reduction. Chromatin immunoprecipitation showed that BRG1 knockdown led to increased NRF2 binding at its respective ARE sites in the HMOX1 promoter but not in NQO1 and GCLM. Our data demonstrate that loss of BRG1 or BRM in lung cancer results in activation of the NRF2/KEAP1 pathway and HMOX1 expression. Therefore, we provide an additional molecular explanation for why patients harboring BRG1 or BRM mutations show poor prognoses. A better understanding of this mechanism may yield novel insights into the design of targeted treatment modalities. IMPLICATIONS: Our study identifies a novel mechanism for how mutations in the SMARCA4 gene may drive progression of human lung adenocarcinomas.

Figure 1.. Analysis of TCGA Lung Adenocarcinoma Cohort.

(A) Density plot of SMARCA4 expression amongst LUAD tumors in TCGA. Contributions of tumors with mutated vs. unaltered SMARCA4 loci are color coded. blue - no alteration. grey - missense or splice site mutation. orange - nonsene or frame shift deletion. dashed line - highest expression of SMARCA4 in a LUAD tumor with SMARCA4 nonsense mutation (6 LCPM). (B) Expression of NRF2 regulatory and target genes by SMARCA4 status. Displayed target genes include the top 15 (by p-value) differentially expressed genes, comparing LUAD tumors with and without NRF2/KEAP1 mutations. Standard NRF2 target genes HMOX1 and GSTM4 were also added for comparison with experimental data. orange - low SMARCA4 expression as defined by range of expression seen in tumors with non-sense mutations in SMARCA4, below 6 LCPM. blue - normal range of SMARCA4 expression, above 6 LCPM.

Figure 2.. BRG1 deficiency activates transcription of a subset of NRF2 target genes.

BRG1 and/or BRM were knocked down in H358 cell line using a lentiviral system or transfection to deliver shRNA. The A427 and H522 lung cancer cell lines possessing inactivating mutations of BRG1 and epigenetic silencing of BRM were used as controls. (A) The indicated human NSCLC cell line was transfected with either a non-targeting siRNA (siCONTROL) or a siRNA targeting BRG1 (siBRG1). 72h after transfection, the indicated proteins were detected and quantified by western blot analysis from whole cell lysates. Relative protein abundance was quantified by densitometry from linear-range film exposures. (B) mRNA levels were determined by qPCR for each gene normalized to B-ACTIN. *P-value< 0.05, (Student T test) and error bars represent ± SEM. qPCR results are representative of two independent experiments and assayed twice. (C) Whole cell lysates were separated by 4–12% SDS-PAGE and probed with the indicated antibodies. (D) Clonal H358 cells stably expressing BRG1 and/or BRM shRNAs were treated with tBHQ for 16h before protein extraction and western blot analysis. Protein abundance was quantified by densitometry from linear-range film exposures. B-actin-normalized values are presented. (E-J) Transcript abundance for the indicated mRNAs was determined by qPCR. B-ACTIN was used for normalization. Data represent two independent experiments, assayed in technical duplicate. * P-value < 0.05 (Student T test) and error bars represent ± SEM.

Figure 3.. BRG1 knockdown leads to a significant increase in NRF2 binding to the HMOX1 promoter.

ChIP assays were carried out using cross-linked chromatin from H358, H358 control, H358 BRG1 knockdown (H358 Brg1.2) and H358 BRG1 and BRM knockdown (H358Brg1i.2/Brmi) cells treated with 100% ethanol (vehicle control) or tBHQ (75μM) for 6h. Nucleoprotein complexes were immunoprecipitated with antibodies against BRG1, NRF2, RNAP II, or a rabbit IgG control and the amount of precipitated DNA was determined by qPCR with oligonucleotide primers complimentary to the ARE, TSS and +500bp sequences of HMOX1 promoter region and the NRF2 ARE binding sites (BS) of the NQO1 and GCLM promoter regions. (A) BRG1 binding is reduced to the 4 sites within the HMOX1 promoter region after knockdown in the H358 Brg1.2 and H358Brg1i.2/Brmi cell lines (B) tBHQ treatment (75μM) for 6h does not affect BRG1 association with 4 sites within the HMOX1 promoter region (C) Differential NRF2 binding to its regulatory regions in the HMOX1 promoter among the H358, H358 control, H358 BRG1 knockdown and H358 BRG1/BRM knockdown cell lines. (D) In response to tBHQ treatment, NRF2 binding was increased ~10 fold to its regulatory regions in the HMOX1 promoter in the H358, H358 control, H358 BRG1 knockdown and H358 BRG1/BRM knockdown cell lines. (E) The recruitment of RNA polymerase II to EN2, TSS, and +500bp was increased in H358 cells after BRG1 loss and/or tBHQ treatment. (F) and (G) Nucleoprotein complexes were immunoprecipitated with antibodies against NRF2, RNAP II, BRG1 or a rabbit IgG control and the amount of precipitated DNA was determined by qPCR with oligonucleotide primers complimentary to the ARE sequences of NQO1 (F) and GCLM (G). Data are representative of one experiment (NRF2-ChIP) and two independent experiments (RNAPII-ChIP, BRG1-ChIP and IgG-ChIP) and assayed twice. *P-value < 0.05, (Student T test) and error bars represent ± SEM.

Figure 4.. Characterization of H358 BRG1 and BRM knockout cell lines.

CRISPR/Cas9 engineering was used to delete BRG1 or BRM from H358 cells. (A) Western blots showing the effects on CRISPR knockouts of BRG1 and BRM on NRF2, KEAP1 and downstream targets. (B) Volcano plot results of RNA-seq differential gene expression (H358 BRG1_KO/Parent) for protein coding genes using DESeq2 (n = 19,879 genes total). Significantly upregulated genes (padj < 0.05 and Log2FoldChange > 1) are colored in red (n = 2,082 genes). Significantly downregulated genes (padj < 0.05 & Log2FoldChange < −1) are colored in blue (n = 2010 genes). Non-significant genes are colored in grey (n = 15,787 genes). SMARCA4/BRG1 and NRF2 target genes are identified. (C) Volcano plot results of RNA-seq differential gene expression (H358 BRM_KO/Parent) for protein coding genes using DESeq2 (n = 19,879 genes total). Significantly upregulated genes (padj < 0.05 and Log2FoldChange > 1) are colored in red (n = 1,785 genes). Significantly downregulated genes (padj < 0.05 & Log2FoldChange < −1) are colored in blue (n = 1,531 genes). Non-significant genes are colored in grey (n = 16,653 genes). SMARCA2/BRM and NRF2 target genes are identified. (D) Venn diagram showing the number of genes that change relative to the H358 parental cell line exclusive to the H358 BRG1 KO cell line (n = 2792 genes), exclusive to the H358 BRM KO cell line (n = 3,518 genes) and the number of genes that change in both cell lines (n = 3, 487 genes). (E) Enrichment plot for NRF2 HALLMARK terms in RNA-seq data (LCPM normalized TPMs) using GSEA (MSigDB GO gene set - C5 all v6.0). (F) mRNA expression of NRF2 target genes derived from the H358 parent, H358 BRG1 KO and the H358 BRM KO cell lines. Poly A capture RNAseq was performed in biological triplicate. Raw counts as quantified by Salmon were normalized by the trimmed means method and Log2 transformed. Indicated q-values were estimated using DESeq2, comparing parental H358 to the indicated knock out cell lines.

Figure 5.. Schematic mechanism for KEAP1-NRF2 activation caused by SWI/SNF chromatin remodeling loss.

BRG1 loss induced KEAP1-NRF2 activation is shown through ROS production and altered nucleosome occupancy.