The SWI/SNF Complex: A Frequently Mutated Chromatin Remodeling Complex in Cancer.

The switch/sucrose non-fermenting (SWI/SNF) chromatin remodeling complex is a global regulator of gene expression known to maintain nucleosome-depleted regions at active enhancers and promoters. The mammalian SWI/SNF protein subunits are encoded by 29 genes and 11-15 subunits including an ATPase domain of either SMARCA4 (BRG1) or SMARCA2 (BRM) are assembled into a complex. Based on the distinct subunits, SWI/SNF are grouped into 3 major types (subfamilies): the canonical BRG1/BRM-associated factor (BAF/cBAF), polybromo-associated BAF (PBAF), and non-canonical BAF (GBAF/ncBAF). Pan-cancer genome sequencing studies have shown that nearly 25% of all cancers bear mutations in subunits of the SWI/SNF complex, many of which are loss of function (LOF) mutations, suggesting a tumor suppressor role. Inactivation of SWI/SNF complex subunits causes widespread epigenetic dysfunction, including increased dependence on antagonistic components such as polycomb repressor complexes (PRC1/2) and altered enhancer regulation, likely promoting an oncogenic state leading to cancer. Despite the prevalence of mutations, most SWI/SNF-mutant cancers lack targeted therapeutic strategies. Defining the dependencies created by LOF mutations in SWI/SNF subunits will identify better targets for these cancers.

Inhibition of GFAT1 in lung cancer cells destabilizes PD-L1 protein.

Immunotherapy using checkpoint blockers (antibodies) has been a major advance in recent years in the management of various types of solid cancers including lung cancer. One target of checkpoint blockers is programmed death ligand 1 (PD-L1) expressed by cancer cells, which engages programmed death 1 on T cells and Natural Killer (NK) cells resulting in suppression of their activation and cancer-killing function, respectively. Apart from antibodies, other clinically relevant agents that can inhibit PD-L1 are limited. PD-L1 protein stability depends on its glycosylation. Here we show that l-glutamine:d-fructose-6-phosphate amidotransferase 1 (GFAT1), a rate-limiting enzyme of the hexosamine biosynthesis pathway, which produces uridine diphosphate-N-acetyl-ß-glucosamine, a precursor for glycosylation, is required for the stability of PD-L1 protein. Inhibition of GFAT1 activity markedly reduced interferon gamma (IFNγ)-induced PD-L1 levels in various lung cancer cell lines. GFAT1 inhibition suppressed glycosylation of PD-L1 and accelerated its proteasomal degradation. Importantly, inhibition of GFAT1 in IFNγ-treated cancer cells enhanced the activation of T cells and the cancer-killing activity of NK cells. These findings support using GFAT1 inhibitors to manipulate PD-L1 protein level that could augment the efficacy of immunotherapy for lung cancer.

Inhalation delivery dramatically improves the efficacy of topotecan for the treatment of local and distant lung cancer.

Topotecan is potent anti-cancer drug approved for various malignancies but hematopoietic toxicities undermine its wider application and use of its most effective dose. This study aims to improve these limitations through inhalation-delivery. The pharmacokinetics, efficacy, and toxicity of 2-5 times lower inhalation doses of topotecan dry-powder were compared with the standard intravenous (IV) delivery once/twice-a-week. Human-derived EGFR-mutant (H1975), KRAS-mutant (A549), and EGFR/KRAS wild-type (H358) orthotopic and distant lung tumors were evaluated in murine models. Inhalation of 1 mg/kg topotecan significantly improved the half-life and drug exposure (area under the curve, AUC) compared to 5 mg/kg via IV-delivery. AUCs (h*ng/mL) for inhaled/IV topotecan in plasma, lung, liver, and brain were, 831/888, 60,000/1080, 8380/4000, and 297/15, respectively; while the half-life was also greatly increased in these tissues. The average lung tumor burden of H358-derived tumors was reduced from 15.0 g to 8.4 g (44%) in rats treated once-a-week with 2 mg/kg IV and 1.8 g (88%) with 1 mg/kg inhaled topotecan, corroborating previous findings using A549- and H1975-derived orthotopic lung tumors. Importantly, inhaled topotecan showed superior efficacy in suppressing lung tumors at distant sites. The growth of H1975- and H358-derived subcutaneous xenografts were completely arrested and A549-derived tumors were significantly reduced in mice treated twice-a-week with 1 mg/kg inhaled topotecan compared to a minor (H1975 and H358) or no reduction (A549) with twice-a-week 5 mg/kg IV topotecan.

Identification of novel epigenetic abnormalities as sputum biomarkers for lung cancer risk among smokers and COPD patients.

OBJECTIVES: Smoking is a common risk factor for chronic obstructive pulmonary disease (COPD) and lung cancer. Although COPD patients have higher risk of lung cancer compared to non-COPD smokers, the molecular links between these diseases are not well-defined. This study aims to identify genes that are downregulated by cigarette smoke and commonly repressed in COPD and lung cancer. MATERIALS AND METHODS: Primary human airway epithelial cells (HAEC) were exposed to cigarette-smoke-extract (CSE) for 10-weeks and significantly suppressed genes were identified by transcriptome array. Epigenetic abnormalities of these genes in lung adenocarcinoma (LUAD) from patients with or without COPD were determined using genome-wide and gene-specific assays and by in vitro treatment of cell lines with trichostatin-A or 5-aza-2-deoxycytidine. RESULTS: The ten most commonly downregulated genes following chronic CSE exposure of HAEC and show promoter hypermethylation in LUAD were selected. Among these, expression of CCNA1, SNCA, and ZNF549 was significantly reduced in lung tissues from COPD compared with non-COPD cases while expression of CCNA1 and SNCA was further downregulated in tumors with COPD. The promoter regions of all three genes were hypermethylated in LUAD but not normal or COPD lungs. The reduced expression and aberrant promoter hypermethylation of these genes in LUAD were independently validated using data from the Cancer Genome Atlas project. Importantly, SNCA and ZNF549 methylation detected in sputum DNA from LUAD (52% and 38%) cases were more prevalent compared to cancer-free smokers (26% and 15%), respectively (p < 0.02). CONCLUSIONS: Our data show that suppression of CCNA1, SNCA, and ZNF549 in lung cancer and COPD occurs with or without promoter hypermethylation, respectively. Detecting methylation of these and previously identified genes in sputum of cancer-free smokers may serve as non-invasive biomarkers for early detection of lung cancer among high risk smokers including COPD patients.

5-Azacytidine inhaled dry powder formulation profoundly improves pharmacokinetics and efficacy for lung cancer therapy through genome reprogramming.

BACKGROUND: Epigenetic therapy through demethylation of 5-methylcytosine has been largely ineffective in treating lung cancer, most likely due to poor tissue distribution with oral or subcutaneous delivery of drugs such as 5-azacytidine (5AZA). An inhalable, stable dry powder formulation of 5AZA was developed. METHODS: Pharmacokinetics of inhaled dry powder and aqueous formulations of 5AZA were compared to an injected formulation. Efficacy studies and effect of therapy on the epigenome were conducted in an orthotopic rat lung cancer model for inhaled formulations. RESULTS: Inhaled dry powder 5AZA showed superior pharmacokinetic properties in lung, liver, brain and blood compared to the injected formulation and for all tissues except lung compared to an inhaled aqueous formulation. Only dry powder 5AZA was detected in brain (~4-h half-life). Inhaled dry powder was superior to inhaled aqueous 5AZA in reducing tumour burden 70-95%. Superiority of inhaled 5AZA dry powder was linked to effectively reprogramming the cancer genome through demethylation and gene expression changes in cancer signalling and immune pathways. CONCLUSIONS: These findings could lead to widespread use of this drug as the first inhaled dry powder therapeutic for treating local and metastatic lung cancer, for adjuvant therapy, and in combination with immunotherapy to improve patient survival.

Epigenetic Regulation of RIP3 Suppresses Necroptosis and Increases Resistance to Chemotherapy in NonSmall Cell Lung Cancer.

INTRODUCTION: The efficacy of chemotherapeutic agents in killing cancer cells is mainly attributed to the induction of apoptosis. However, the tremendous efforts on enhancing apoptosis-related mechanisms have only moderately improved lung cancer chemotherapy, suggesting that other cell death mechanisms such as necroptosis could be involved. In this study, we investigated the role of the necroptosis pathway in the responsiveness of nonsmall cell lung cancer (NSCLC) to chemotherapy. METHODS: In vitro cell culture and in vivo xenograft tumor therapy models and clinical sample studies are combined in studying the role of necroptosis in chemotherapy and mechanism of necroptosis suppression involving RIP3 expression regulation. RESULTS: While chemotherapeutic drugs were able to induce necroptotic cell death, this pathway was suppressed in lung cancer cells at least partly through downregulation of RIP3 expression. Ectopic RIP3 expression significantly sensitized lung cancer cells to the cytotoxicity of anticancer drugs such as cisplatin, etoposide, vincristine, and adriamycin. In addition, RIP3 suppression was associated with RIP3 promoter methylation, and demethylation partly restored RIP3 expression and increased chemotherapeutic-induced necroptotic cell death. In a xenograft tumor therapy model, ectopic RIP3 expression significantly sensitized anticancer activity of cisplatin in vivo. Furthermore, lower RIP3 expression was associated with worse chemotherapy response in NSCLC patients. CONCLUSION: Our results indicate that the necroptosis pathway is suppressed in lung cancer through RIP3 promoter methylation, and reactivating this pathway should be exploited for improving lung cancer chemotherapy.

Vasorin/ATIA Promotes Cigarette Smoke-Induced Transformation of Human Bronchial Epithelial Cells by Suppressing Autophagy-Mediated Apoptosis.

BACKGROUND: Escaping cell death pathways is an important event during carcinogenesis. We previously identified anti-TNFα-induced apoptosis (ATIA, also known as vasorin) as an antiapoptotic factor that suppresses reactive oxygen species (ROS) production. However, the role of vasorin in lung carcinogenesis has not been investigated. METHODS: Vasorin expression was examined in human lung cancer tissues with immunohistochemistry and database analysis. Genetic and pharmacological approaches were used to manipulate protein expression and autophagy activity in human bronchial epithelial cells (HBECs). ROS generation was measured with fluorescent indicator, apoptosis with release of lactate dehydrogenase, and cell transformation was assessed with colony formation in soft agar. RESULTS: Vasorin expression was increased in human lung cancer tissues and cell lines, which was inversely associated with lung cancer patient survival. Cigarette smoke extract (CSE) and benzo[a]pyrene diol epoxide (BPDE)-induced vasorin expression in HBECs. Vasorin knockdown in HBECs significantly suppressed CSE-induced transformation in association with enhanced ROS accumulation and autophagy. Scavenging ROS attenuated autophagy and cytotoxicity in vasorin knockdown cells, suggesting that vasorin potentiates transformation by impeding ROS-mediated CSE cytotoxicity and improving survival of the premalignant cells. Suppression of autophagy effectively inhibited CSE-induced apoptosis, suggesting that autophagy was pro-apoptotic in CSE-treated cells. Importantly, blocking autophagy strongly potentiated CSE-induced transformation. CONCLUSION: These results suggest that vasorin is a potential lung cancer-promoting factor that facilitates cigarette smoke-induced bronchial epithelial cell transformation by suppressing autophagy-mediated apoptosis, which could be exploited for lung cancer prevention.

Inhibition of the hexosamine biosynthesis pathway potentiates cisplatin cytotoxicity by decreasing BiP expression in non-small-cell lung cancer cells.

Platinum anticancer agents are essential components in chemotherapeutic regimens for non-small-cell lung cancer (NSCLC) patients ineligible for targeted therapy. However, platinum-based regimens have reached a plateau of therapeutic efficacy; therefore, it is critical to implement novel approaches for improvement. The hexosamine biosynthesis pathway (HBP), which produces amino-sugar N-acetyl-glucosamine for protein glycosylation, is important for protein function and cell survival. Here we show a beneficial effect by the combination of cisplatin with HBP inhibition. Expression of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme of HBP, was increased in NSCLC cell lines and tissues. Pharmacological inhibition of GFAT activity or knockdown of GFATimpaired cell proliferation and exerted synergistic or additive cytotoxicity to the cells treated with cisplatin. Mechanistically, GFAT positively regulated the expression of binding immunoglobulin protein (BiP; also known as glucose-regulated protein 78, GRP78), an endoplasmic reticulum chaperone involved in unfolded protein response (UPR). Suppressing GFAT activity resulted in downregulation of BiP that activated inositol-requiring enzyme 1α, a sensor protein of UPR, and exacerbated cisplatin-induced cell apoptosis. These data identify GFAT-mediated HBP as a target for improving platinum-based chemotherapy for NSCLC.

Common cancer-driver mutations and their association with abnormally methylated genes in lung adenocarcinoma from never-smokers.

OBJECTIVES: Lung adenocarcinoma in never-smokers accounts for 15-20% of all lung cancer. Although targetable mutations are more prevalent in these tumors, the biological and clinical importance of coexisting and/or mutually exclusive abnormalities is just emerging. This study evaluates the relationships between common genetic and epigenetic aberrations in these tumors. MATERIALS AND METHODS: Next-generation sequencing was employed to screen 20 commonly mutated cancer-driver genes in 112 lung adenocarcinomas from never-smokers. The relationship of these mutations with cancer-related methylation of 59 genes, and geographical/ethnic differences in the prevalence for mutations compared to multiple East Asian never-smoker lung adenocarcinoma cohorts was studied. RESULTS: The most common driver mutation detected in 40% (45/112) of the tumors was EGFR, followed by TP53 (18%), SETD2 (11%), and SMARCA4 (11%). Over 72% (81/112) of the cases have mutation of at least one driver gene. While 30% (34/112) of the tumors have co-mutations of two or more genes, 42% (47/112) have only one driver gene mutation. Differences in the prevalence for some of these mutations were seen between adenocarcinomas in East Asian versus US (mainly Caucasian) never-smokers including a significantly lower rate of EGFR mutation among the US patients. Interestingly, aberrant methylation of multiple cancer-related genes was significantly associated with EGFR wildtype tumors. Among 15 differentially methylated genes by EGFR mutation, 14 were more commonly methylated in EGFR wildtype compared to mutant tumors. These findings were independently validated using publicly available data. CONCLUSION: Most lung adenocarcinomas from never-smokers harbor targetable mutation/co-mutations. In the absence of EGFR mutation that drives 40% of these tumors, EGFR wildtype tumors appear to develop by acquiring aberrant promoter methylation that silences tumor-suppressor genes.

Inhalation delivery of topotecan is superior to intravenous exposure for suppressing lung cancer in a preclinical model.

Intravenous (IV) topotecan is approved for the treatment of various malignancies including lung cancer but its clinical use is greatly undermined by severe hematopoietic toxicity. We hypothesized that inhalation delivery of topotecan would increase local exposure and efficacy against lung cancer while reducing systemic exposure and toxicity. These hypotheses were tested in a preclinical setting using a novel inhalable formulation of topotecan against the standard IV dose. Respirable dry-powder of topotecan was manufactured through spray-drying technology and the pharmacokinetics of 0.14 and 0.79 mg/kg inhalation doses were compared with 0.7 mg/kg IV dose. The efficacy of four weekly treatments with 1 mg/kg inhaled vs. 2 mg/kg IV topotecan were compared to untreated control using an established orthotopic lung cancer model for a fast (H1975) and moderately growing (A549) human lung tumors in the nude rat. Inhalation delivery increased topotecan exposure of lung tissue by approximately 30-fold, lung and plasma half-life by 5- and 4-folds, respectively, and reduced the maximum plasma concentration by 2-fold than the comparable IV dose. Inhaled topotecan improved the survival of rats with the fast-growing lung tumors from 7 to 80% and reduced the tumor burden of the moderately-growing lung tumors over 5- and 10-folds, respectively, than the 2-times higher IV topotecan and untreated control (p < .00001). These results indicate that inhalation delivery increases topotecan exposure of lung tissue and improves its efficacy against lung cancer while also lowering the effective dose and maximum systemic concentration that is responsible for its dose-limiting toxicity.

ANK1 Methylation regulates expression of MicroRNA-486-5p and discriminates lung tumors by histology and smoking status.

The intragenic tumor-suppressor microRNA miR-486-5p is often down-regulated in non-small cell lung cancer (NSCLC) but the mechanism is unclear. This study investigated epigenetic co-regulation of miR-486-5p and its host gene ANK1. MiR-486-5p expression in lung tumors and cell lines was significantly reduced compared to normal lung (p < 0.001) and is strongly correlated with ANK1 expression. In vitro, siRNA-mediated ANK1 knockdown in NSCLC cells also reduced miR-486-5p while the DNA methylation inhibitor 5-aza-2'-deoxycytidine induced expression of both. ANK1 promoter CpG island was unmethylated in normal lung but methylated in 45% (118/262) lung tumors and 55% (17/31) NSCLC cell lines. After adjustment for tumor histology and smoking, methylation was significantly more prevalent in adenocarcinoma (101/200, 51%) compared to squamous cell carcinoma (17/62, 27%), p < 0.001; HR = 3.513 (CI: 1.818-6.788); and in smokers (73/128, 57%) than never-smokers (28/72, 39%), p = 0.014; HR = 2.086 (CI: 1.157-3.759). These results were independently validated using quantitative methylation data for 809 NSCLC cases from The Cancer Genome Atlas project. Together, our data indicate that aberrant ANK1 methylation is highly prevalent in lung cancer, discriminate tumors by histology and patients' smoking history, and contributes to miR-486-5p repression.

Muc1 knockout potentiates murine lung carcinogenesis involving an epiregulin-mediated EGFR activation feedback loop.

Mucin 1 (MUC1) is a tumor antigen that is aberrantly overexpressed in various cancers, including lung cancer. Our previous in vitro studies showed that MUC1 facilitates carcinogen-induced EGFR activation and transformation in human lung bronchial epithelial cells (HBECs), which along with other reports suggests an oncogenic property for MUC1 in lung cancer. However, direct evidence for the role of MUC1 in lung carcinogenesis is lacking. In this study, we used the 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced A/J mouse lung tumor model to investigate the effect of whole-body Muc1 knockout (KO) on carcinogen-induced lung carcinogenesis. Surprisingly, lung tumor multiplicity was significantly increased in Muc1 KO compared to wild-type (WT) mice. The EGFR/AKT pathway was unexpectedly activated, and expression of the EGFR ligand epiregulin (EREG) was increased in the lung tissues of the Muc1 KO compared to the WT mice. EREG stimulated proliferation and protected against cigarette smoke extract (CSE)-induced cytotoxicity in in vitro cultured human bronchial epithelial cells. Additionally, we determined that MUC1 was expressed in human fibroblast cell lines where it suppressed CSE-induced EREG production. Further, suppression of MUC1 cellular activity with GO-201 enhanced EREG production in lung cancer cells, which in turn protected cancer cells from GO-201-induced cell death. Moreover, an inverse association between MUC1 and EREG was detected in human lung cancer, and EREG expression was inversely associated with patient survival. Together, these results support a promiscuous role of MUC1 in lung cancer development that may be related to cell-type specific functions of MUC1 in the tumor microenvironment, and MUC1 deficiency in fibroblasts and malignant cells results in increased EREG production that activates the EGFR pathway for lung carcinogenesis.

Epigenetic Repression of CCDC37 and MAP1B Links Chronic Obstructive Pulmonary Disease to Lung Cancer.

INTRODUCTION: Lung cancer and chronic obstructive pulmonary disease (COPD) share environmental risk factors. COPD also increases the risk of lung cancer; however, the molecular mechanisms are unclear. METHODS: An epigenome-wide association study of lung tumors and cancer-free lung tissue (CFLT) pairs from non-small-cell lung cancer cases with (n = 18) or without (n = 17) COPD was conducted using the HumanMethylation450 beadchip (HM450K). COPD-associated methylation of top-ranked genes was confirmed in a larger sample set, independently validated, and their potential as sputum-based biomarkers was investigated. RESULTS: Methylation of CCDC37 and MAP1B was more prevalent in lung tumors from COPD than non-COPD cases [54 of 71 (76%) versus 20 of 46 (43%), p = 0.0013] and [48 of 71 (68%) versus 17 of 46 (37%), p = 0.0035], respectively, after adjustment for age, sex, smoking status, and tumor histology. HM450K probes across CCDC37 and MAP1B promoters showed higher methylation in tumors than CFLT with the highest methylation seen in tumors from COPD cases (p < 0.05). These results were independently validated using The Cancer Genome Atlas data. CCDC37 methylation was more prevalent in sputum from COPD than non-COPD smokers (p < 0.005) from two cohorts. CCDC37 and MAP1B expression was dramatically repressed in tumors and CFLT from COPD than non-COPD cases, p less than 0.02. CONCLUSIONS: The reduced expression of CCDC37 and MAP1B associated with COPD likely predisposes these genes to methylation that in turn, may contribute to lung cancer.

GATA2 is epigenetically repressed in human and mouse lung tumors and is not requisite for survival of KRAS mutant lung cancer.

INTRODUCTION: GATA2 was recently described as a critical survival factor and therapeutic target for KRAS mutant non-small-cell lung cancer (NSCLC). However, whether this role is affected by epigenetic repression of GATA2 in lung cancer is unclear. METHODS: GATA2 expression and promoter CpG island methylation were evaluated using human and mouse NSCLC cell lines and tumor-normal pairs. In vitro assays were used to study GATA2 repression on cell survival and during tobacco carcinogen-induced transformation. RESULTS: GATA2 expression in KRAS wild-type (n = 15) and mutant (n = 10) NSCLC cell lines and primary lung tumors (n = 24) was significantly lower, 1.3- to 33.6-fold (p = 2.2 × 10(9)), compared with corresponding normal lung. GATA2 promoter was unmethylated in normal lung (0 of 10) but frequently methylated in lung tumors (96%, 159 of 165) and NSCLC cell lines (97%, 30 of 31). This highly prevalent aberrant methylation was independently validated using The Cancer Genome Atlas data for 369 NSCLC tumor-normal pairs. In vitro studies using an established carcinogen-induced premalignancy model revealed that GATA2 expression was initially repressed by chromatin remodeling followed by cytosine methylation during transformation. Similarly, expression of GATA2 in NNK-induced mouse lung tumors (n = 6) and cell lines (n = 5) was fivefold and 100-fold lower, respectively, than normal mouse lung. Finally, siRNA-mediated knockdown of GATA2 in KRAS mutant (human [n = 4] and murine [n = 5]) and wild-type (human [n = 4]) NSCLC cell lines showed that further reduction of expression (up to 95%) does not induce cell death. CONCLUSION: GATA2 is epigenetically repressed in human and mouse lung tumors and its further inhibition is not a valid therapeutic strategy for KRAS mutant lung cancer.

Functional identification of cancer-specific methylation of CDO1, HOXA9, and TAC1 for the diagnosis of lung cancer.

PURPOSE: Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality in the world. Novel diagnostic biomarkers may augment both existing NSCLC screening methods as well as molecular diagnostic tests of surgical specimens to more accurately stratify and stage candidates for adjuvant chemotherapy. Hypermethylation of CpG islands is a common and important alteration in the transition from normal tissue to cancer. EXPERIMENTAL DESIGN: Following previously validated methods for the discovery of cancer-specific hypermethylation changes, we treated eight NSCLC cell lines with the hypomethylating agent deoxyazacitidine or trichostatin A. We validated the findings using a large publicly available database and two independent cohorts of primary samples. RESULTS: We identified >300 candidate genes. Using The Cancer Genome Atlas (TCGA) and extensive filtering to refine our candidate genes for the greatest ability to distinguish tumor from normal, we define a three-gene panel, CDO1, HOXA9, and TAC1, which we subsequently validate in two independent cohorts of primary NSCLC samples. This three-gene panel is 100% specific, showing no methylation in 75 TCGA normal and seven primary normal samples and is 83% to 99% sensitive for NSCLC depending on the cohort. CONCLUSION: This degree of sensitivity and specificity may be of high value to diagnose the earliest stages of NSCLC. Addition of this three-gene panel to other previously validated methylation biomarkers holds great promise in both early diagnosis and molecular staging of NSCLC.

Genome-wide unmasking of epigenetically silenced genes in lung adenocarcinoma from smokers and never smokers.

Lung cancer in never smokers (NS) shows striking demographic, clinicopathological and molecular distinctions from the disease in smokers (S). Studies on selected genetic and epigenetic alterations in lung cancer identified that the frequency and profile of some abnormalities significantly differ by smoking status. This study compared the transcriptome of lung adenocarcinoma cell lines derived from S (n = 3) and NS (n = 3) each treated with vehicle (control), histone deacetylation inhibitor (trichostatin A) or DNA methylation inhibitor (5-aza-2'-deoxycytidine). Among 122 genes reexpressed following 5-aza-2'-deoxycytidine but not trichostatin A treatment in two or more cell lines (including 32 genes in S-only and 12 NS-only), methylation was validated for 80% (98/122 genes). After methylation analysis of 20 normal tissue samples and 14 additional non-small cell lung cancer cell lines (total 20), 39 genes frequently methylated in normal (>20%, 4/20) and 21 genes rarely methylated in non-small cell lung cancer (≤10%, 2/20) were excluded. The prevalence for methylation of the remaining 38 genes in lung adenocarcinomas from S (n = 97) and NS (n = 75) ranged from 8-89% and significantly differs between S and NS for CPEB1, CST6, EMILIN2, LAYN and MARVELD3 (P < 0.05). Furthermore, methylation of EMILIN2, ROBO3 and IGDCC4 was more prevalent in advanced (Stage II-IV, n = 61) than early (Stage I, n = 110) tumors. Knockdown of MARVELD3, one of the novel epigenetically silenced genes, by small interfering RNA significantly reduced anchorage-independent growth of lung cancer cells (P < 0.001). Collectively, this study has identified multiple, novel, epigenetically silenced genes in lung cancer and provides invaluable resources for the development of diagnostic and prognostic biomarkers.

Phase II study of topotecan and bevacizumab in advanced, refractory non--small-cell lung cancer.

BACKGROUND: This clinical trial evaluated whether topotecan in combination with bevacizumab improved progression-free survival (PFS) in patients with advanced, refractory non--small-cell lung cancer in a second-line setting. PATIENT AND METHODS: Patients aged 18 years old and older received topotecan (4.0 mg/m(2)) on days 1, 8, and 15, and bevacizumab (10 mg/kg) on days 1 and 15 as intravenous infusions on a 28-day treatment cycle. Available tumor specimens were analyzed for ISG15 gene expression as a biomarker of response to topotecan. RESULTS: Forty-two patients were enrolled in the study, with a median age of 62.5 years and a median of 3 (range, 1-7) prior treatment regimens. Almost half (n = 18, 42.9%) of the patients received prior bevacizumab therapy. PFS was 5.1 months (95% CI, 3.7-7.8 months), and overall survival was 11.5 months (95% CI, 6.8-15.5 months). Response rates were as follows: 14.3% partial response, 54.8% stable disease, and 28.6% progressive disease. Hematologic toxicities included grade 3 thrombocytopenia (n = 7, 16.7%), neutropenia (n = 4, 9.5%), and anemia (n = 2, 4.8%). One toxic death occurred due to pulmonary hemorrhage, and one patient experienced a grade 4 pulmonary embolism. Grade 3 nonhematologic adverse events were uncommon (< 8%). There was a trend for improved median PFS, 3.5 months vs. 1.8 months (P = .26), in patients with high ISG15 expression. CONCLUSION: Bevacizumab in combination with topotecan as a salvage therapy for metastatic non--small-cell lung cancer is well tolerated and is worthy of further investigation.

Differential epigenetic regulation of TOX subfamily high mobility group box genes in lung and breast cancers.

Aberrant cytosine methylation affects regulation of hundreds of genes during cancer development. In this study, a novel aberrantly hypermethylated CpG island in cancer was discovered within the TOX2 promoter. TOX2 was unmethylated in normal cells but 28% lung (n = 190) and 23% breast (n = 80) tumors were methylated. Expression of two novel TOX2 transcripts identified was significantly reduced in primary lung tumors than distant normal lung (p<0.05). These transcripts were silenced in methylated lung and breast cancer cells and 5-Aza-2-deoxycytidine treatment re-expressed both. Extension of these assays to TOX, TOX3, and TOX4 genes that share similar genomic structure and protein homology with TOX2 revealed distinct methylation profiles by smoking status, histology, and cancer type. TOX was almost exclusively methylated in breast (43%) than lung (5%) cancer, whereas TOX3 was frequently methylated in lung (58%) than breast (30%) tumors. TOX4 was unmethylated in all samples and showed the highest expression in normal lung. Compared to TOX4, expression of TOX, TOX2 and TOX3 in normal lung was 25, 44, and 88% lower, respectively, supporting the premise that reduced promoter activity confers increased susceptibility to methylation during lung carcinogenesis. Genome-wide assays revealed that siRNA-mediated TOX2 knockdown modulated multiple pathways while TOX3 inactivation targeted neuronal development and function. Although these knockdowns did not result in further phenotypic changes of lung cancer cells in vitro, the impact on tissue remodeling, inflammatory response, and cell differentiation pathways suggest a potential role for TOX2 in modulating tumor microenvironment.

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.

Epithelial-to-mesenchymal transition (EMT) is strongly associated with cancer progression, but its potential role during premalignant development has not been studied. Here, we show that a 4-week exposure of immortalized human bronchial epithelial cells (HBEC) to tobacco carcinogens can induce a persistent, irreversible, and multifaceted dedifferentiation program marked by EMT and the emergence of stem cell-like properties. EMT induction was epigenetically driven, initially by chromatin remodeling through H3K27me3 enrichment and later by ensuing DNA methylation to sustain silencing of tumor-suppressive microRNAs (miRNA), miR-200b, miR-200c, and miR-205, which were implicated in the dedifferentiation program in HBECs and also in primary lung tumors. Carcinogen-treated HBECs acquired stem cell-like features characterized by their ability to form spheroids with branching tubules and enrichment of the CD44(high)/CD24(low), CD133, and ALDH1 stem cell-like markers. miRNA overexpression studies indicated that regulation of the EMT, stem-like, and transformed phenotypes in HBECs were distinct events. Our findings extend present concepts of how EMT participates in cancer pathophysiology by showing that EMT induction can participate in cancer initiation to promote the clonal expansion of premalignant lung epithelial cells.

The A/G allele of rs16906252 predicts for MGMT methylation and is selectively silenced in premalignant lesions from smokers and in lung adenocarcinomas.

PURPOSE: To address the association between sequence variants within the MGMT (O(6)-methylguanine-DNA methyltransferase) promoter-enhancer region and methylation of MGMT in premalignant lesions from smokers and lung adenocarcinomas, their biological effects on gene regulation, and targeting MGMT for therapy. EXPERIMENTAL DESIGN: Single nucleotide polymorphisms (SNP) identified through sequencing a 1.9 kb fragment 5' of MGMT were examined in relation to MGMT methylation in 169 lung adenocarcinomas and 1,731 sputum samples from smokers. The effect of promoter haplotypes on MGMT expression was tested using a luciferase reporter assay and cDNA expression analysis along with allele-specific sequencing for methylation. The response of MGMT methylated lung cancer cell lines to the alkylating agent temozolomide (TMZ) was assessed. RESULTS: The A allele of rs16906252 and the haplotype containing this SNP were strongly associated with increased risk for MGMT methylation in adenocarcinomas (ORs ≥ 94). This association was observed to a lesser extent in sputum samples in both smoker cohorts. The A allele was selectively methylated in primary lung tumors and cell lines heterozygous for rs16906252. With the most common haplotype as the reference, a 20 to 41% reduction in promoter activity was seen for the haplotype carrying the A allele that correlated with lower MGMT expression. The sensitivity of lung cancer cell lines to TMZ was strongly correlated with levels of MGMT methylation and expression. CONCLUSIONS: These studies provide strong evidence that the A allele of a MGMT promoter-enhancer SNP is a key determinant for MGMT methylation in lung carcinogenesis. Moreover, TMZ treatment may benefit a subset of lung cancer patients methylated for MGMT.