Genetic variations in cancer-related significantly mutated genes and lung cancer susceptibility.

BACKGROUND: Cancer initiation and development are driven by key mutations in driver genes. Applying high-throughput sequencing technologies and bioinformatic analyses, The Cancer Genome Atlas (TCGA) project has identified panels of somatic mutations that contributed to the etiology of various cancers. However, there are few studies investigating the germline genetic variations in these significantly mutated genes (SMGs) and lung cancer susceptibility. PATIENTS AND METHODS: We comprehensively evaluated 1655 tagged single nucleotide polymorphisms (SNPs) located in 127 SMGs identified by TCGA, and test their association with lung cancer risk in large-scale case-control study. Functional effect of the validated SNPs, gene mutation frequency and pathways were analyzed. RESULTS: We found 11 SNPs in 8 genes showed consistent association (P < 0.1) and 8 SNPs significantly associated with lung cancer risk (P < 0.05) in both discovery and validation phases. The most significant association was rs10412613 in PPP2R1A, with the minor G allele associated with a decreased risk of lung cancer [odds ratio = 0.91, 95% confidence interval (CI): 0.87-0.96, P = 2.3 × 10-4]. Cumulative analysis of risk score built as a weight sum of the 11 SNPs showed consistently elevated risk with increasing risk score (P for trend = 9.5 × 10-9). In stratified analyses, the association of PPP2R1A:rs10412613 and lung cancer risk appeared stronger among population of younger age at diagnosis and never smokers. The expression quantitative trait loci analysis indicated that rs10412613, rs10804682, rs635469 and rs6742399 genotypes significantly correlated with the expression of PPP2R1A, ATR, SETBP1 and ERBB4, respectively. From TCGA data, expression of the identified genes was significantly different in lung tumors compared with normal tissues, and the genes' highest mutation frequency was found in lung cancers. Integrative pathway analysis indicated the identified genes were mainly involved in AKT/NF-κB regulatory pathway suggesting the underlying biological processes. CONCLUSION: This study revealed novel genetic variants in SMGs associated with lung cancer risk, which might contribute to elucidating the biological network involved in lung cancer development.

LZTFL1 suppresses lung tumorigenesis by maintaining differentiation of lung epithelial cells.

Lung cancer is the leading cause of cancer-related death in the United States, and metastatic behavior is largely responsible for this mortality. Mutations in multiple 'driver' oncogenes and tumor suppressors are known to contribute to the lung tumorigenesis and in some cases represent therapeutic targets. Leucine Zipper Transcription Factor-like 1 (LZTFL1) is located in the chromosome region 3p21.3 where allelic loss and genetic alterations occur early and frequently in lung cancers. Previously, we found that LZTFL1 is downregulated in epithelial tumors, including lung cancer, and functions as a tumor suppressor in gastric cancers. However, the functional role of LZTFL1 in lung oncogenesis is undefined. We show here that downregulation of LZTFL1 expression in non-small cell lung cancer is associated with recurrence and poor survival, whereas re-expression of LZTFL1 in lung tumor cells inhibited extravasation/colonization of circulating tumor cells to the lung and inhibited tumor growth in vivo. Mechanistically, we found that LZTFL1 is expressed in ciliated human bronchial epithelial cells (HBECs) and its expression correlates with HBEC differentiation. LZTFL1 inhibits transforming growth factor ß-activated mitogen-activated protein kinase and hedgehog signaling. Alteration of intracellular levels of LZTFL1 resulted in changes of expression of genes associated with epithelial-to-mesenchymal transition (EMT). We conclude that LZTFL1 inhibits lung tumorigenesis, possibly by maintaining epithelial cell differentiation and/or inhibition of signalings that lead to EMT and suggest that reactivation of LZTFL1 expression in tumor cells may be a novel lung cancer therapeutic approach.

Inflammation-related genetic variants predict toxicity following definitive radiotherapy for lung cancer.

Definitive radiotherapy improves locoregional control and survival in inoperable non-small cell lung cancer patients. However, radiation-induced toxicities (pneumonitis/esophagitis) are common dose-limiting inflammatory conditions. We therefore conducted a pathway-based analysis to identify inflammation-related single-nucleotide polymorphisms associated with radiation-induced pneumonitis or esophagitis. A total of 11,930 single-nucleotide polymorphisms were genotyped in 201 stage I-III non-small cell lung cancer patients treated with definitive radiotherapy. Validation was performed in an additional 220 non-small cell lung cancer cases. After validation, 19 single-nucleotide polymorphisms remained significant. A polygenic risk score was generated to summarize the effect from validated single-nucleotide polymorphisms. Significant improvements in discriminative ability were observed when the polygenic risk score was added into the clinical/epidemiological variable-based model. We then used 277 lymphoblastoid cell lines to assess radiation sensitivity and expression quantitative trait loci (eQTL) relationships of the identified single-nucleotide polymorphisms. Three genes (PRKCE, DDX58, and TNFSF7) were associated with radiation sensitivity. We concluded that inflammation-related genetic variants could contribute to the development of radiation-induced toxicities.

miR-93-directed downregulation of DAB2 defines a novel oncogenic pathway in lung cancer.

The disabled homolog 2 (DAB2) gene was recently identified as a tumor suppressor gene with its expression downregulated in multiple cancer types. The role of DAB2 in lung tumorigenesis, however, is not fully characterized, and the mechanisms of DAB2 dysregulation in lung cancer are not defined. Here we show that low DAB2 levels in lung tumor specimens are significantly correlated with poor patient survival, and that DAB2 overexpression significantly inhibits cell growth in cultured lung cancer cells, indicating its potent tumor suppressor function. We next identify that microRNA miR-93 functions as a potent repressor of DAB2 expression by directly targeting the 3'UTR of the DAB2 mRNA. Using in vitro and in vivo approaches, we demonstrate that miR-93 overexpression has an important role in promoting lung cancer cell growth, and that its oncogenic function is primarily mediated by downregulating DAB2 expression. Our clinical investigations further indicate that high tumor levels of miR-93 are correlated with poor survival of lung cancer patients. The correlations of both low DAB2 and high miR-93 expression levels with poor patient survival strongly support the critical role of the miR-93/DAB2 pathway in determining lung cancer progression.

Ras regulates kinesin 13 family members to control cell migration pathways in transformed human bronchial epithelial cells.

We show that expression of the microtubule depolymerizing kinesin KIF2C is induced by transformation of immortalized human bronchial epithelial cells (HBEC) by expression of K-Ras(G12V) and knockdown of p53. Further investigation demonstrates that this is due to the K-Ras/ERK1/2 MAPK pathway, as loss of p53 had little effect on KIF2C expression. In addition to KIF2C, we also found that the related kinesin KIF2A is modestly upregulated in this model system; both proteins are expressed more highly in many lung cancer cell lines compared to normal tissue. As a consequence of their depolymerizing activity, these kinesins increase dynamic instability of microtubules. Depletion of either of these kinesins impairs the ability of cells transformed with mutant K-Ras to migrate and invade matrigel. However, depletion of these kinesins does not reverse the epithelial to mesenchymal transition (EMT) caused by mutant K-Ras. Our studies indicate that increased expression of microtubule destabilizing factors can occur during oncogenesis to support enhanced migration and invasion of tumor cells.

NeuroD1 regulation of migration accompanies the differential sensitivity of neuroendocrine carcinomas to TrkB inhibition.

The developmental transcription factor NeuroD1 is anomalously expressed in a subset of aggressive neuroendocrine tumors. Previously, we demonstrated that TrkB and neural cell adhesion molecule (NCAM) are downstream targets of NeuroD1 that contribute to the actions of neurogenic differentiation 1 (NeuroD1) in neuroendocrine lung. We found that several malignant melanoma and prostate cell lines express NeuroD1 and TrkB. Inhibition of TrkB activity decreased invasion in several neuroendocrine pigmented melanoma but not in prostate cell lines. We also found that loss of the tumor suppressor p53 increased NeuroD1 expression in normal human bronchial epithelial cells and cancer cells with neuroendocrine features. Although we found that a major mechanism of action of NeuroD1 is by the regulation of TrkB, effective targeting of TrkB to inhibit invasion may depend on the cell of origin. These findings suggest that NeuroD1 is a lineage-dependent oncogene acting through its downstream target, TrkB, across multiple cancer types, which may provide new insights into the pathogenesis of neuroendocrine cancers.

Oncogenic KRAS-induced epiregulin overexpression contributes to aggressive phenotype and is a promising therapeutic target in non-small-cell lung cancer.

KRAS mutations are one of the most common driver mutations in non-small-cell lung cancer (NSCLC) and finding druggable target molecules to inhibit oncogenic KRAS signaling is a significant challenge in NSCLC therapy. We recently identified epiregulin (EREG) as one of several putative transcriptional targets of oncogenic KRAS signaling in both KRAS-mutant NSCLC cells and immortalized bronchial epithelial cells expressing ectopic mutant KRAS. In the current study, we found that EREG is overexpressed in NSCLCs harboring KRAS, BRAF or EGFR mutations compared with NSCLCs with wild-type KRAS/BRAF/EGFR. Small interfering RNAs (siRNAs) targeting mutant KRAS, but not an siRNA targeting wild-type KRAS, significantly reduced EREG expression in KRAS-mutant and EREG-overexpressing NSCLC cell lines. In these cell lines, EREG expression was downregulated by MEK and ERK inhibitors. Importantly, EREG expression significantly correlated with KRAS expression or KRAS copy number in KRAS-mutant NSCLC cell lines. Further expression analysis using 89 NSCLC specimens showed that EREG was predominantly expressed in NSCLCs with pleural involvement, lymphatic permeation or vascular invasion and in KRAS-mutant adenocarcinomas. In addition, multivariate analysis revealed that EREG expression is an independent prognostic marker and EREG overexpression in combination with KRAS mutations was associated with an unfavorable prognosis for lung adenocarcinoma patients. In KRAS-mutant and EREG overexpressing NSCLC cells, siRNA-mediated EREG silencing inhibited anchorage-dependent and -independent growth and induced apoptosis. Our findings suggest that oncogenic KRAS-induced EREG overexpression contributes to an aggressive phenotype and could be a promising therapeutic target in oncogenic KRAS-driven NSCLC.

Smac mimetics increase cancer cell response to chemotherapeutics in a TNF-α-dependent manner.

Second mitochondria-derived activator of caspase (Smac) is a mitochondrial protein released into the cytosol during apoptosis. Smac mimetics have recently been touted as a novel therapeutic to induce apoptosis in cancer cells. The ability of Smac mimetics to induce apoptosis in vitro has been shown to be dependent upon both XIAP neutralization and cancer cell autocrine tumor necrosis factor-α (TNF-α) production. In this study we provide new evidence for the utility of Smac mimetics in combination with conventional chemotherapy agents to exacerbate caspase activation and induce cancer cell death. Furthermore, we find that the combination effect is because of a multifaceted mechanism involving both inhibition of cell proliferation by the chemotherapy agents and an enhanced autocrine TNF-α feedback loop by the Smac mimetic/chemotherapy agent combination. Surprisingly, although genotoxic agents typically induce apoptosis through the mitochondrial intrinsic pathway, we show that this synergism is mediated through a TNF-α/RIP1-dependent pathway, leading to activation of the extrinsic apoptotic pathway. Finally, we report that autocrine TNF-α contributes to Smac mimetic-induced tumor regression as a single agent or in combination with chemotherapeutics in xenograft mouse models. Collectively, we provide mechanistic and applicable data to support translational studies in the use of a Smac mimetic/chemotherapy antineoplasm modality.

Clinicopathological and therapeutic significance of CXCL12 expression in lung cancer.

Interactions between CXCL12 and its receptors CXCR4 or CXCR7 are involved in tumor growth and metastasis in various types of human cancer. However, CXCL12 expression and its role in lung cancer are not fully elucidated. Here we examined the expression of CXCL12 in 54 lung cancer cell lines consisting of 23 small cell lung cancers (SCLCs) and 31 non-small cell lung cancers (NSCLCs). CXCL12 was overexpressed in lung cancer cell lines compared to non-malignant human bronchial epithelial cell lines (N = 6). CXCL12 expression was positively but weakly correlated with the expression of CXCR4 or CXCR7. We also examined CXCL12 expression in 89 NSCLC specimens and found that CXCL12 expression was significantly higher in tumor specimens from female patients, non-smokers and adenocarcinoma patients. Small interfering RNAs targeting CXCL12 inhibited cellular proliferation, colony formation and migration of CXCL12-overexpressing lung cancer cells; however, this inhibition did not occur in lung cancer cells that lacked CXCL12. Furthermore, the anti-CXCL12 neutralizing antibody mediated inhibitory effects in three lung cancer cell lines that overexpressed CXCL12, but not in two CXCL12 non-expressing lung cancer cell lines nor two non-malignant bronchial epithelial cell lines. The present study demonstrates that: CXCL12 is concomitantly overexpressed with CXCR4 or CXCR7 in lung cancers; CXCL12 is highly expressed in NSCLCs from females, non-smokers and adenocarcinoma patients; and disruption of CXCL12 inhibits the growth and migration of lung cancer cells. Our findings indicate that CXCL12 is required for tumor growth and provide a rationale for the anti-CXCL12 treatment strategy in lung cancer.

Epidermal growth factor receptor regulates MET levels and invasiveness through hypoxia-inducible factor-1alpha in non-small cell lung cancer cells.

Recent studies have established that amplification of the MET proto-oncogene can cause resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) cell lines with EGFR-activating mutations. The role of non-amplified MET in EGFR-dependent signaling before TKI resistance, however, is not well understood. Using NSCLC cell lines and transgenic models, we demonstrate here that EGFR activation by either mutation or ligand binding increases MET gene expression and protein levels. Our analysis of 202 NSCLC patient specimens was consistent with these observations: levels of MET were significantly higher in NSCLC with EGFR mutations than in NSCLC with wild-type EGFR. EGFR regulation of MET levels in cell lines occurred through the hypoxia-inducible factor (HIF)-1alpha pathway in a hypoxia-independent manner. This regulation was lost, however, after MET gene amplification or overexpression of a constitutively active form of HIF-1alpha. EGFR- and hypoxia-induced invasiveness of NSCLC cells, but not cell survival, were found to be MET dependent. These findings establish that, absent MET amplification, EGFR signaling can regulate MET levels through HIF-1alpha and that MET is a key downstream mediator of EGFR-induced invasiveness in EGFR-dependent NSCLC cells.

Genomic and functional analysis identifies CRKL as an oncogene amplified in lung cancer.

DNA amplifications, leading to the overexpression of oncogenes, are a cardinal feature of lung cancer and directly contribute to its pathogenesis. To uncover such novel alterations, we performed an array-based comparative genomic hybridization survey of 128 non-small-cell lung cancer cell lines and tumors. Prominent among our findings, we identified recurrent high-level amplification at cytoband 22q11.21 in 3% of lung cancer specimens, with another 11% of specimens exhibiting low-level gain spanning that locus. The 22q11.21 amplicon core contained eight named genes, only four of which were overexpressed (by transcript profiling) when amplified. Among these, CRKL encodes an adapter protein functioning in signal transduction, best known as a substrate of the BCR-ABL kinase in chronic myelogenous leukemia. RNA-interference-mediated knockdown of CRKL in lung cancer cell lines with (but not without) amplification led to significantly decreased cell proliferation, cell-cycle progression, cell survival, and cell motility and invasion. In addition, overexpression of CRKL in immortalized human bronchial epithelial cells led to enhanced growth factor-independent cell growth. Our findings indicate that amplification and resultant overexpression of CRKL contribute to diverse oncogenic phenotypes in lung cancer, with implications for targeted therapy, and highlight a role of adapter proteins as primary genetic drivers of tumorigenesis.

DNA amplification is a ubiquitous mechanism of oncogene activation in lung and other cancers.

Chromosomal translocation is the best-characterized genetic mechanism for oncogene activation. However, there are documented examples of activation by alternate mechanisms, for example gene dosage increase, though its prevalence is unclear. Here, we answered the fundamental question of the contribution of DNA amplification as a molecular mechanism driving oncogenesis. Comparing 104 cancer lines representing diverse tissue origins identified genes residing in amplification 'hotspots' and discovered an unexpected frequency of genes activated by this mechanism. The 3431 amplicons identified represent approximately 10 per hematological and approximately 36 per epithelial cancer genome. Many recurrently amplified oncogenes were previously known to be activated only by disease-specific translocations. The 135 hotspots identified contain 538 unique genes and are enriched for proliferation, apoptosis and linage-dependency genes, reflecting functions advantageous to tumor growth. Integrating gene dosage with expression data validated the downstream impact of the novel amplification events in both cell lines and clinical samples. For example, multiple downstream components of the EGFR-family-signaling pathway, including CDK5, AKT1 and SHC1, are overexpressed as a direct result of gene amplification in lung cancer. Our findings suggest that amplification is far more common a mechanism of oncogene activation than previously believed and that specific regions of the genome are hotspots of amplification.

Genomic profiling identifies TITF1 as a lineage-specific oncogene amplified in lung cancer.

Lung cancer is a leading cause of cancer death, where the amplification of oncogenes contributes to tumorigenesis. Genomic profiling of 128 lung cancer cell lines and tumors revealed frequent focal DNA amplification at cytoband 14q13.3, a locus not amplified in other tumor types. The smallest region of recurrent amplification spanned the homeobox transcription factor TITF1 (thyroid transcription factor 1; also called NKX2-1), previously linked to normal lung development and function. When amplified, TITF1 exhibited increased expression at both the RNA and protein levels. Small interfering RNA (siRNA)-mediated knockdown of TITF1 in lung cancer cell lines with amplification led to reduced cell proliferation, manifested by both decreased cell-cycle progression and increased apoptosis. Our findings indicate that TITF1 amplification and overexpression contribute to lung cancer cell proliferation rates and survival and implicate TITF1 as a lineage-specific oncogene in lung cancer.

Prevalence and specificity of LKB1 genetic alterations in lung cancers.

Germline LKB1 mutations cause Peutz-Jeghers syndrome, a hereditary disorder that predisposes to gastrointestinal hamartomatous polyposis and several types of malignant tumors. Somatic LKB1 alterations are rare in sporadic cancers, however, a few reports showed the presence of somatic alterations in a considerable fraction of lung cancers. To determine the prevalence and the specificity of LKB1 alterations in lung cancers, we examined a large number of lung cancer cell lines and lung adenocarcinoma (AdC) specimens for the alterations. LKB1 genetic alterations were frequently detected in the cell lines (21/70, 30%), especially in non-small cell lung cancers (NSCLCs) (20/51, 39%), and were significantly more frequent in cell lines with KRAS mutations. Point mutations were detected only in AdCs and large cell carcinomas, whereas homozygous deletions were detected in all histological types of lung cancer. Among lung AdC specimens, LKB1 mutations were found in seven (8%) of 91 male smokers but in none of 64 females and/or nonsmokers, and were significantly more frequent in poorly differentiated tumors. The difference in the frequency of LKB1 alterations between cell lines and tumor specimens was likely to be owing to masking of deletions by the contamination of noncancerous cells in the tumor specimens. These results indicate that somatic LKB1 genetic alterations preferentially occur in a subset of poorly differentiated lung AdCs that appear to correlate with smoking males.

c-Jun N-terminal kinase is activated in non-small-cell lung cancer and promotes neoplastic transformation in human bronchial epithelial cells.

c-Jun N-terminal kinase (JNK) has been reported to either potentiate or inhibit oncogenesis, depending upon the cellular context, but its role in lung neoplasia is unclear. Here we sought to define the role of JNK in lung neoplasia by examining evidence of JNK phosphorylation in non-small-cell lung cancer (NSCLC) biopsy samples and by using genetic and pharmacologic approaches to modulate JNK expression and activity in cultured cells. Immunohistochemical staining for JNK phosphorylation was detected in 114 (45%) of 252 NSCLC biopsy samples and was predominantly nuclear, providing evidence of JNK activation in a subset of NSCLC cases. Introduction of a doxycycline-inducible, constitutively active, mutant mitogen-activated protein kinase kinase 4 (MKK4) into the human bronchial epithelial cell lines BEAS-2B and HB56B increased the cells' proliferation, migration, invasion and clonogenicity. Depletion of JNK in MKK4 mutant-transformed BEAS-2B cells by introduction of JNK1/2 short hairpin RNA reversed the transformed phenotype, indicating that JNK activation is oncogenic and MKK4 confers neoplastic properties in these cells. The proliferation of NSCLC cell lines HCC827 and H2009, in which JNK and its substrate c-Jun are constitutively phosphorylated, was inhibited by SP600125, a JNK kinase inhibitor. We conclude that JNK is activated in a subset of NSCLC biopsy samples and promotes oncogenesis in the bronchial epithelium, suggesting that strategies to inhibit the JNK pathway should be considered for the prevention and treatment of NSCLC.

The DRD2 TaqI-B polymorphism and its relationship to smoking abstinence and withdrawal symptoms.

The dopamine receptor D2 (DRD2) gene has polymorphisms that have been linked to regulation of the dopamine system and to an increased prevalence of smoking. The present study examined the relationship of the DRD2 TaqI-A and -B polymorphisms with short-term clinical outcome (abstinence and withdrawal symptoms), collected from daily (14 pre-quit and 42 post-quit) diary data among smokers (n=116) treated with the nicotine patch plus either venlafaxine or placebo. The results showed that B1/B1 or B1/B2 smokers were slightly less likely to be abstinent on a given day than those homozygous for the TaqI-B2 allele. Significant DRD2 TaqI-B x time interactions were found for several of the withdrawal scales, indicating that those smokers with the B1/B1 or B1/B2 genotypes tended to report more symptoms over time compared to those with the B2/B2 genotype. No interactions or main effects were found for the DRD2 TaqI-A polymorphism. The findings demonstrate that smokers homozygous for the TaqI-B2 allele experience progressive improvement in self-reported withdrawal symptoms while smokers with the TaqI-B1 allele showing little change.

Differential disruption of cell cycle pathways in small cell and non-small cell lung cancer.

Lung cancer is the leading cause of cancer-related mortality in the world, with small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) comprising the two major cell types. Although these cell types can be distinguished readily at the histological level, knowledge of their underlying molecular differences is very limited. In this study, we compared 14 SCLC cell lines against 27 NSCLC cell lines using an integrated array comparative genomic hybridisation and gene expression profiling approach to identify subtype-specific disruptions. Using stringent criteria, we have identified 159 of the genes that are responsible for the different biology of these cell types. Sorting of these genes by their biological functions revealed the differential disruption of key components involved in cell cycle pathways. Our novel comparative combined genome and transcriptome analysis not only identified differentially altered genes, but also revealed that certain shared pathways are preferentially disrupted at different steps in these cell types. Small cell lung cancer exhibited increased expression of MRP5, activation of Wnt pathway inhibitors, and upregulation of p38 MAPK activating genes, while NSCLC showed downregulation of CDKN2A, and upregulation of MAPK9 and EGFR. This information suggests that cell cycle upregulation in SCLC and NSCLC occurs through drastically different mechanisms, highlighting the need for differential molecular target selection in the treatment of these cancers.

ASPP1, a common activator of TP53, is inactivated by aberrant methylation of its promoter in acute lymphoblastic leukemia.

We have analyzed the regulation and expression of ASPP members, genes implicated in the regulation of the apoptotic function of the TP53 tumor-suppressor gene, in acute lymphoblastic leukemia (ALL). Expression of ASPP1 was significantly reduced in ALL and was dependent on hypermethylation of the ASPP1 gene promoter. Abnormal ASPP1 expression was associated with normal function of the tumor-suppressor gene TP53 in ALL. The analyses of 180 patients with ALL at diagnosis showed that the ASPP1 promoter was hypermethylated in 25% of cases with decreased mRNA expression. Methylation was significantly higher in adult ALL vs childhood ALL (32 vs 17%, P = 0.03) and T-ALL vs B-ALL (50 vs 9%, P = 0.001). Relapse rate (62 vs 44%, P = 0.05) and mortality (59 vs 43%, P = 0.05) were significantly higher in patients with methylated ASPP1. DFS and OS were 32.8 and 33.7% for patients with unmethylated ASPP1 and 6.1 and 9.9% for methylated patients (P < 0.001 y P < 0.02, respectively). On the multivariate analysis, methylation of the ASPP1 gene promoter was an independent poor prognosis factor in ALL patients. Our results demonstrate that decreased expression of ASPP1 in patients with ALL is due to an abnormal methylation of its promoter and is associated with a poor prognosis.

Combined microarray analysis of small cell lung cancer reveals altered apoptotic balance and distinct expression signatures of MYC family gene amplification.

DNA amplifications and deletions frequently contribute to the development and progression of lung cancer. To identify such novel alterations in small cell lung cancer (SCLC), we performed comparative genomic hybridization on a set of 24 SCLC cell lines, using cDNA microarrays representing approximately 22,000 human genes (providing an average mapping resolution of <70 kb). We identified localized DNA amplifications corresponding to oncogenes known to be amplified in SCLC, including MYC (8q24), MYCN (2p24) and MYCL1 (1p34). Additional highly localized DNA amplifications suggested candidate oncogenes not previously identified as amplified in SCLC, including the antiapoptotic genes TNFRSF4 (1p36), DAD1 (14q11), BCL2L1 (20q11) and BCL2L2 (14q11). Likewise, newly discovered PCR-validated homozygous deletions suggested candidate tumor-suppressor genes, including the proapoptotic genes MAPK10 (4q21) and TNFRSF6 (10q23). To characterize the effect of DNA amplification on gene expression patterns, we performed expression profiling using the same microarray platform. Among our findings, we identified sets of genes whose expression correlated with MYC, MYCN or MYCL1 amplification, with surprisingly little overlap among gene sets. While both MYC and MYCN amplification were associated with increased and decreased expression of known MYC upregulated and downregulated targets, respectively, MYCL1 amplification was associated only with the latter. Our findings support a role of altered apoptotic balance in the pathogenesis of SCLC, and suggest that MYC family genes might affect oncogenesis through distinct sets of targets, in particular implicating the importance of transcriptional repression.

DNA methylation-associated inactivation of TGFbeta-related genes DRM/Gremlin, RUNX3, and HPP1 in human cancers.

The transforming growth factor beta (TGFbeta)-signalling pathway is deregulated in many cancers. We examined the role of gene silencing via aberrant methylation of DRM/Gremlin and HPP1, which inhibit TGFbeta signalling, and RUNX3, which facilitates TGFbeta-signalling, of all genes that are thought to be tumour suppressors, are aberrantly expressed, and are thus thought to have important role in human cancers. We examined DRM/Gremlin mRNA expression in 44 cell lines and the promoter methylation status of DRM/Gremlin, HPP1, and RUNX3 in 44 cell lines and 511 primary tumours. The loss of DRM/Gremlin mRNA expression in human cancer cell lines is associated with DNA methylation, and treatment with the methylation inhibitor-reactivated mRNA expression (n=13). Methylation percentages of the three genes ranged from 0-83% in adult tumours and 0-50% in paediatric tumours. Methylation of DRM/Gremlin was more frequent in lung tumours in smokers, and methylation of all three genes was inversely correlated with prognosis in patients with bladder or prostate cancer. Our results provide strong evidence that these TGFbeta-related genes are frequently deregulated through aberrant methylation in many human malignancies.