Background/Objectives: The BRG1 loss-of-function (LOF) mutation is found in ~10% of non-small cell lung cancer (NSCLC) cases, but its role in lung tumorigenesis is unclear and so it is investigated in this study. Methods: BRG1-knockout (KO) lines were generated from various non-malignant, pre-malignant, and malignant human lung epithelium-derived cell lines using CRISPR. The effects of BRG1-KO on cell growth, the transcriptome, the methylome, and epigenetic therapy were compared with those of wild-type (BRG1-WT) isogenic controls using standard in vitro and in vivo assays. Results: The BRG1 protein was expressed in all non-/pre-malignant lung epithelial cells but lost in 47% (14/30) of NSCLC cell lines. BRG1-KO and cigarette smoke (CS) exposure individually transformed human bronchial epithelial cell lines (HBECs), as evidenced by anchorage-independent growth. BRG1-KO and CS produced additive to synergistic effects on sensitivity to transformation that differed across HBECs. RNA-seq analysis revealed that BRG1-KO significantly changed the expression of over 8500 genes on average, impacting lung development, function, damage repair, and cancer pathways, including axonal guidance, pulmonary wound healing, and epithelial-to-mesenchymal transition (EMT). BRG1-KO also led to the hypermethylation of >47,000 promoter CpGs within ~9500 genes on average in different HBECs, including silencing of epithelial genes involved in EMT and tumor suppressor genes. BRG1-KO also moderately increased the in vitro and in vivo sensitivity of NSCLC cells to some epigenetic drugs. Conclusions: BRG1-LOF is frequent in NSCLC; can drive the transformation of lung epithelial cells such that they acquire properties of pre-malignant cells, indicating a potential role in lung cancer initiation; and sensitizes lung tumors to epigenetic therapy.

PDF file - 59K, Information for primer sequences used in the nested MSP and the CG sites interrogated for methylation

Fig. S3. Induction of WT p53 protein using temperature regulated expression vector.

PDF file - 66K, Genes methylated in lung cancer but not associated with predicting lung cancer in sputum from the Colorado case-control Study

Fig. S2. Growth of WT p53 cells after TET1 KD.

Abstract The role of transcriptional regulator ten-eleven translocation methylcytosine dioxygenease 1 (TET1) has not been well characterized in lung cancer. Here we show that TET1 is overexpressed in adenocarcinoma and squamous cell carcinomas. TET1 knockdown reduced cell growth in vitro and in vivo and induced transcriptome reprogramming independent of its demethylating activity to affect key cancer signaling pathways. Wild-type p53 bound the TET1 promoter to suppress transcription, while p53 transversion mutations were most strongly associated with high TET1 expression. Knockdown of TET1 in p53-mutant cell lines induced senescence through a program involving generalized genomic instability manifested by DNA single- and double-strand breaks and induction of p21 that was synergistic with cisplatin and doxorubicin. These data identify TET1 as an oncogene in lung cancer whose gain of function via loss of p53 may be exploited through targeted therapy–induced senescence. Significance: These studies identify TET1 as an oncogene in lung cancer whose gain of function following loss of p53 may be exploited by targeted therapy–induced senescence. See related commentary by Kondo, p. 1751

Fig. S6. Growth inhibition and senescence induced in p53 mutated NSCLC cells by TET1 depletion in conjunction with chemotherapy.

Analysis of miR-196b and miR-34c-5p hypermethylation in NSCLC.

Objectives E-cigarettes are the most commonly used nicotine containing products among youth. In vitro studies support the potential for e-cigarettes to cause cellular stress in vivo; however, there have been no studies to address whether exposure to e-liquid aerosols can induce cell transformation, a process strongly associated with pre-malignancy. We examined whether weekly exposure of human bronchial epithelial cell (HBEC) lines to e-cigarette aerosols would induce transformation and concomitant changes in gene expression and promoter hypermethylation. Materials and methods An aerosol delivery system exposed three HBEC lines to unflavored e-liquid with 1.2% nicotine, 3 flavored products with nicotine, or the Kentucky reference cigarette once a week for 12 weeks. Colony formation in soft agar, RNA-sequencing, and the EPIC Beadchip were used to evaluate transformation, genome-wide expression and methylation changes. Results Jamestown e-liquid aerosol induced transformation of HBEC2 and HBEC26, while unflavored and Blue Pucker transformed HBEC26. Cigarette smoke aerosol transformed HBEC4 and HBEC26 at efficiencies up to 3-fold greater than e-liquids. Transformed clones exhibited extensive reprogramming of the transcriptome with common and distinct gene expression changes observed between the cigarette and e-liquids. Transformation by e-liquids induced alterations in canonical pathways implicated in lung cancer that included axonal guidance and NRF2. Gene methylation, while prominent in cigarette-induced transformed clones, also affected hundreds of genes in HBEC2 transformed by Jamestown. Many genes with altered expression or epigenetic-mediated silencing were also affected in lung tumors from smokers. Conclusions These studies show that exposure to e-liquid aerosols can induce a pre-malignant phenotype in lung epithelial cells. While the Food and Drug Administration banned the sale of flavored cartridge-based electric cigarettes, consumers switched to using flavored products through other devices. Our findings clearly support expanding studies to evaluate transformation potency for the major categories of e-liquid flavors to better inform risk from these complex mixtures.

<p>Analysis of miR-196b, miR-34c-5p, miR-497/195 hypermethylation in lung cancer cell lines, PBMC, and NBEC.</p>

Supplementary Tables 1-4 from Promoter Methylation of Genes in and around the Candidate Lung Cancer Susceptibility Locus <i>6q23-25</i>

Supplemental Figure 1. Analysis of miR-196b, miR-34c-5p, miR-497/195 hypermethylation in lung cancer cell lines, PBMC, and NBEC. Supplemental Figure 2. Analysis of miR-196b and miR-34c-5p hypermethylation in NSCLC. Supplemental Table 1. Established In Vitro Model of Pre-Malignant Lung Cancer. Supplemental Table 2. PCR Primer Sequences for COBRA, MSP, and ChIP Assays.

<p>Established In Vitro Model of Pre-Malignant Lung Cancer.</p>

Fig. S5. Effect of TET1 depletion in H1299 cells on histone methyltransferases and lysine methylation.

Supplementary Figure 1 from EMT and Stem Cell–Like Properties Associated with miR-205 and miR-200 Epigenetic Silencing Are Early Manifestations during Carcinogen-Induced Transformation of Human Lung Epithelial Cells

Supplementary Tables 1-4 from EMT and Stem Cell–Like Properties Associated with miR-205 and miR-200 Epigenetic Silencing Are Early Manifestations during Carcinogen-Induced Transformation of Human Lung Epithelial Cells

Supplementary methods, figure legends and tables

Fig. S4. Induction of senescence in A549 cells by simultaneous TET1 depletion and p53 inhibition using alternative TET1 siRNA.

Fig. S1. TET1 knockdown in NSCLC cell lines.

Supplementary Figure S1; Supplementary Tables S1-S3.