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1.  SMAD6 Contributes to Patient Survival in Non–Small Cell Lung Cancer and Its Knockdown Reestablishes TGF-β Homeostasis in Lung Cancer Cells 
Cancer research  2008;68(23):9686-9692.
The malignant transformation in several types of cancer, including lung cancer, results in a loss of growth inhibition by transforming growth factor-β (TGF-β). Here, we show that SMAD6 expression is associated with a reduced survival in lung cancer patients. Short hairpin RNA (shRNA)–mediated knockdown of SMAD6 in lung cancer cell lines resulted in reduced cell viability and increased apoptosis as well as inhibition of cell cycle progression. However, these results were not seen in Beas2B, a normal bronchial epithelial cell line. To better understand the mechanism underlying the association of SMAD6 with poor patient survival, we used a lentivirus construct carrying shRNA for SMAD6 to knock down expression of the targeted gene. Through gene expression analysis, we observed that knockdown of SMAD6 led to the activation of TGF-β signaling through up-regulation of plasminogen activator inhibitor-1 and phosphorylation of SMAD2/3. Furthermore, SMAD6 knockdown activated the c-Jun NH2-terminal kinase pathway and reduced phosphorylation of Rb-1, resulting in increased G0-G1 cell arrest and apoptosis in the lung cancer cell line H1299. These results jointly suggest that SMAD6 plays a critical role in supporting lung cancer cell growth and survival. Targeted inactivation of SMAD6 may provide a novel therapeutic strategy for lung cancers expressing this gene.
PMCID: PMC3617041  PMID: 19047146
2.  Investigating the Genome Diversity of B. cereus and Evolutionary Aspects of B. anthracis Emergence 
Genomics  2011;98(1):26-39.
Here we report the use of a multi-genome DNA microarray to investigate the genome diversity of Bacillus cereus group members and elucidate the events associated with the emergence of B. anthracis the causative agent of anthrax–a lethal zoonotic disease. We initially performed directed genome sequencing of seven diverse B. cereus strains to identify novel sequences encoded in those genomes. The novel genes identified, combined with those publicly available, allowed the design of a “species” DNA microarray. Comparative genomic hybridization analyses of 41 strains indicates that substantial heterogeneity exists with respect to the genes comprising functional role categories. While the acquisition of the plasmid-encoded pathogenicity island (pXO1) and capsule genes (pXO2) represent a crucial landmark dictating the emergence of B. anthracis, the evolution of this species and its close relatives was associated with an overall a shift in the fraction of genes devoted to energy metabolism, cellular processes, transport, as well as virulence.
PMCID: PMC3129444  PMID: 21447378
3.  TLR signaling is required for virulence of an intracellular pathogen 
Cell  2011;144(5):675-688.
Toll-like receptors (TLRs) contribute to host resistance to microbial pathogens and drive the evolution of virulence mechanisms. We have examined the relationship between host resistance and pathogen virulence using mice with a functional allele of the Nramp-1 gene and lacking combinations of TLRs. Mice deficient in both TLR2 and TLR4 were highly susceptible to the intracellular bacterial pathogen Salmonella typhimurium, consistent with reduced innate immune function. However, mice lacking additional TLRs involved in S. typhimurium recognition were less susceptible to infection. In these TLR-deficient cells, bacteria failed to upregulate Salmonella pathogenicity island 2 (SPI-2) genes and did not form a replicative compartment. We demonstrate that TLR signaling enhances the rate of acidification of the Salmonella containing phagosome, and inhibition of this acidification prevents SPI-2 induction. Our results indicate that S. typhimurium requires cues from the innate immune system to regulate virulence genes necessary for intracellular survival, growth, and systemic infection.
PMCID: PMC3063366  PMID: 21376231
4.  Gene Expression Signature of Cigarette Smoking and Its Role in Lung Adenocarcinoma Development and Survival 
PLoS ONE  2008;3(2):e1651.
Tobacco smoking is responsible for over 90% of lung cancer cases, and yet the precise molecular alterations induced by smoking in lung that develop into cancer and impact survival have remained obscure.
Methodology/Principal Findings
We performed gene expression analysis using HG-U133A Affymetrix chips on 135 fresh frozen tissue samples of adenocarcinoma and paired noninvolved lung tissue from current, former and never smokers, with biochemically validated smoking information. ANOVA analysis adjusted for potential confounders, multiple testing procedure, Gene Set Enrichment Analysis, and GO-functional classification were conducted for gene selection. Results were confirmed in independent adenocarcinoma and non-tumor tissues from two studies. We identified a gene expression signature characteristic of smoking that includes cell cycle genes, particularly those involved in the mitotic spindle formation (e.g., NEK2, TTK, PRC1). Expression of these genes strongly differentiated both smokers from non-smokers in lung tumors and early stage tumor tissue from non-tumor tissue (p<0.001 and fold-change >1.5, for each comparison), consistent with an important role for this pathway in lung carcinogenesis induced by smoking. These changes persisted many years after smoking cessation. NEK2 (p<0.001) and TTK (p = 0.002) expression in the noninvolved lung tissue was also associated with a 3-fold increased risk of mortality from lung adenocarcinoma in smokers.
Our work provides insight into the smoking-related mechanisms of lung neoplasia, and shows that the very mitotic genes known to be involved in cancer development are induced by smoking and affect survival. These genes are candidate targets for chemoprevention and treatment of lung cancer in smokers.
PMCID: PMC2249927  PMID: 18297132
5.  Distinguishing Lung Tumours From Normal Lung Based on a Small Set of Genes 
Identifying specific molecular markers and developing sensitive detection methods are two of the fundamental requirements for detection and differential diagnosis of cancer. Toward this goal, we first performed cDNA array analysis using 65 non-small cell lung cancer and non-involved normal lung tissues. We then used several complementary statistical and analytical methods to examine gene expression profiles generated by us and others from four independent sets of normal and neoplastic lung tissues. We report here that several sets of roughly 20 genes were sufficient to provide a robust distinction between normal and neoplastic tissues of the lung. Next we assessed the predictive ability of these gene sets by using Flow-Thru Chips® (FTC) (MetriGenix, Baltimore, MD) containing 20 genes to screen 48 primary lung tumours and normal lung tissues. Gene expression changes detected by FTC distinguished lung cancers from the normal lung tissues by using an RNA amount equivalent to that present in as few as 300 cells. We also used an independent set of 24 genes and showed that their expression profile was equally effective when measured by quantitative polymerase chain reaction (Q-PCR). Our results demonstrate that lung cancers can be identified based on the expression patterns of just 20 genes and that this approach is applicable for cancer diagnosis, prognosis, and monitoring using small amount of tumor or biopsy samples.
PMCID: PMC1859858  PMID: 17161497
cancer detection; class prediction; expression profiling; gene chip; non–small cell lung cancer; Flow-Thru Chip
6.  Inactivation of LLC1 gene in nonsmall cell lung cancer 
Serial analysis of gene expression studies led us to identify a previously unknown gene, c20orf85, that is present in the normal lung epithelium, but absent or downregulated in most primary non-small cell lung cancers and lung cancer cell lines. We named this gene LLC1 for Low in Lung Cancer 1. LLC1 is located on chromosome 20q13.3 and has a 70% GC content in the promoter region. It has 4 exons and encodes a protein containing 137 amino acids. By in situ hybridization, we observed that LLC1 message is localized in normal lung bronchial epithelial cells, but absent in 13 of 14 lung adenocarcinoma and 9 out of 10 lung squamous carcinoma samples. Methylation at CpG sites of the LLC1 promoter was frequently observed in lung cancer cell lines and in a fraction of primary lung cancer tissues. Treatment with 5-aza deoxycytidine resulted in a reduced methylation of the LLC1 promoter concomitant with the increase of LLC1 expression. These results suggest that inactivation of LLC1 by means of promoter methylation is a frequent event in nonsmall cell lung cancer and may play a role in lung tumorigenesis.
PMCID: PMC1907378  PMID: 17304513
nonsmall cell lung cancer; serial analysis of gene expression; promoter methylation

Results 1-6 (6)