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1.  p120RasGAP Is a Mediator of Rho Pathway Activation and Tumorigenicity in the DLD1 Colorectal Cancer Cell Line 
PLoS ONE  2014;9(1):e86103.
KRAS is mutated in ∼40% of colorectal cancer (CRC), and there are limited effective treatments for advanced KRAS mutant CRC. Therefore, it is crucial that downstream mediators of oncogenic KRAS continue to be studied. We identified p190RhoGAP as being phosphorylated in the DLD1 CRC cell line, which expresses a heterozygous KRAS G13D allele, and not in DKO4 in which the mutant allele has been deleted by somatic recombination. We found that a ubiquitous binding partner of p190RhoGAP, p120RasGAP (RasGAP), is expressed in much lower levels in DKO4 cells compared to DLD1, and this expression is regulated by KRAS. Rescue of RasGAP expression in DKO4 rescued Rho pathway activation and partially rescued tumorigenicity in DKO4 cells, indicating that the combination of mutant KRAS and RasGAP expression is crucial to these phenotypes. We conclude that RasGAP is an important effector of mutant KRAS in CRC.
doi:10.1371/journal.pone.0086103
PMCID: PMC3897622  PMID: 24465899
2.  Loss of Canonical Smad4 Signaling Promotes KRAS Driven Malignant Transformation of Human Pancreatic Duct Epithelial Cells and Metastasis  
PLoS ONE  2013;8(12):e84366.
Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer death in North America. Activating KRAS mutations and Smad4 loss occur in approximately 90% and 55% of PDAC, respectively. While their roles in the early stages of PDAC development have been confirmed in genetically modified mouse models, their roles in the multistep malignant transformation of human pancreatic duct cells have not been directly demonstrated. Here, we report that Smad4 represents a barrier in KRAS-mediated malignant transformation of the near normal immortalized human pancreatic duct epithelial (HPDE) cell line model. Marked Smad4 downregulation by shRNA in KRASG12V expressing HPDE cells failed to cause tumorigenic transformation. However, KRAS-mediated malignant transformation occurred in a new HPDE-TGF-β resistant (TβR) cell line that completely lacks Smad4 protein expression and is resistant to the mito-inhibitory activity of TGF-β. This transformation resulted in tumor formation and development of metastatic phenotype when the cells were implanted orthotopically into the mouse pancreas. Smad4 restoration re-established TGF-β sensitivity, markedly increased tumor latency by promoting apoptosis, and decreased metastatic potential. These results directly establish the critical combination of the KRAS oncogene and complete Smad4 inactivation in the multi-stage malignant transformation and metastatic progression of normal human HPDE cells.
doi:10.1371/journal.pone.0084366
PMCID: PMC3873993  PMID: 24386371
3.  Lipocalin2 Promotes Invasion, Tumorigenicity and Gemcitabine Resistance in Pancreatic Ductal Adenocarcinoma 
PLoS ONE  2012;7(10):e46677.
Lipocalin 2 (LCN2) is a small secreted protein and its elevated expression has been observed in pancreatic as well as other cancer types. LCN2 has been reported to promote resistance to drug-induced apoptosis, enhance invasion through its physical association with matrix metalloproteinase-9, and promote in vivo tumor growth. LCN2 was found to be commonly expressed in patient PDAC samples and its pattern of immunohistochemical staining intensified with increasing severity in high-grade precursor lesions. Downregulation of LCN2 in two pancreatic ductal adenocarcinoma cell lines (BxPC3 and HPAF-II) with high LCN2 expression significantly reduced attachment, invasion, and tumour growth in vivo, but not proliferation or motility. Downregulation of LCN2 in two pancreatic ductal adenocarcinoma cell lines (BxPC3 and HPAF-II) with high expression significantly reduced attachment, invasion, and tumour growth in vivo. In contrast, LCN2 overexpression in PANC1, with low endogenous expression, significantly increased invasion, attachment, and enhanced tumor growth. Suppression of LCN2 in BxPC3 and HPAF-II cells increased their sensitivity to gemcitabine in vitro, and in vivo when BxPC3 was tested. Furthermore, LCN2 promotes expression of VEGF and HIF1A which contribute to enhanced vascularity. These overall results demonstrate that LCN2 plays an important role in the malignant progression of pancreatic ductal carcinoma and is a potential therapeutic target for this disease.
doi:10.1371/journal.pone.0046677
PMCID: PMC3464270  PMID: 23056397
4.  Prognostic and Predictive Gene Signature for Adjuvant Chemotherapy in Resected Non–Small-Cell Lung Cancer 
Journal of Clinical Oncology  2010;28(29):4417-4424.
Purpose
The JBR.10 trial demonstrated benefit from adjuvant cisplatin/vinorelbine (ACT) in early-stage non–small-cell lung cancer (NSCLC). We hypothesized that expression profiling may identify stage-independent subgroups who might benefit from ACT.
Patients and Methods
Gene expression profiling was conducted on mRNA from 133 frozen JBR.10 tumor samples (62 observation [OBS], 71 ACT). The minimum gene set that was selected for the greatest separation of good and poor prognosis patient subgroups in OBS patients was identified. The prognostic value of this gene signature was tested in four independent published microarray data sets and by quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR).
Results
A 15-gene signature separated OBS patients into high-risk and low-risk subgroups with significantly different survival (hazard ratio [HR], 15.02; 95% CI, 5.12 to 44.04; P < .001; stage I HR, 13.31; P < .001; stage II HR, 13.47; P < .001). The prognostic effect was verified in the same 62 OBS patients where gene expression was assessed by qPCR. Furthermore, it was validated consistently in four separate microarray data sets (total 356 stage IB to II patients without adjuvant treatment) and additional JBR.10 OBS patients by qPCR (n = 19). The signature was also predictive of improved survival after ACT in JBR.10 high-risk patients (HR, 0.33; 95% CI, 0.17 to 0.63; P = .0005), but not in low-risk patients (HR, 3.67; 95% CI, 1.22 to 11.06; P = .0133; interaction P < .001). Significant interaction between risk groups and ACT was verified by qPCR.
Conclusion
This 15-gene expression signature is an independent prognostic marker in early-stage, completely resected NSCLC, and to our knowledge, is the first signature that has demonstrated the potential to select patients with stage IB to II NSCLC most likely to benefit from adjuvant chemotherapy with cisplatin/vinorelbine.
doi:10.1200/JCO.2009.26.4325
PMCID: PMC2988634  PMID: 20823422
5.  A sequence-based approach to identify reference genes for gene expression analysis 
BMC Medical Genomics  2010;3:32.
Background
An important consideration when analyzing both microarray and quantitative PCR expression data is the selection of appropriate genes as endogenous controls or reference genes. This step is especially critical when identifying genes differentially expressed between datasets. Moreover, reference genes suitable in one context (e.g. lung cancer) may not be suitable in another (e.g. breast cancer). Currently, the main approach to identify reference genes involves the mining of expression microarray data for highly expressed and relatively constant transcripts across a sample set. A caveat here is the requirement for transcript normalization prior to analysis, and measurements obtained are relative, not absolute. Alternatively, as sequencing-based technologies provide digital quantitative output, absolute quantification ensues, and reference gene identification becomes more accurate.
Methods
Serial analysis of gene expression (SAGE) profiles of non-malignant and malignant lung samples were compared using a permutation test to identify the most stably expressed genes across all samples. Subsequently, the specificity of the reference genes was evaluated across multiple tissue types, their constancy of expression was assessed using quantitative RT-PCR (qPCR), and their impact on differential expression analysis of microarray data was evaluated.
Results
We show that (i) conventional references genes such as ACTB and GAPDH are highly variable between cancerous and non-cancerous samples, (ii) reference genes identified for lung cancer do not perform well for other cancer types (breast and brain), (iii) reference genes identified through SAGE show low variability using qPCR in a different cohort of samples, and (iv) normalization of a lung cancer gene expression microarray dataset with or without our reference genes, yields different results for differential gene expression and subsequent analyses. Specifically, key established pathways in lung cancer exhibit higher statistical significance using a dataset normalized with our reference genes relative to normalization without using our reference genes.
Conclusions
Our analyses found NDUFA1, RPL19, RAB5C, and RPS18 to occupy the top ranking positions among 15 suitable reference genes optimal for normalization of lung tissue expression data. Significantly, the approach used in this study can be applied to data generated using new generation sequencing platforms for the identification of reference genes optimal within diverse contexts.
doi:10.1186/1755-8794-3-32
PMCID: PMC2928167  PMID: 20682026
6.  Integrative Genomic Analyses Identify BRF2 as a Novel Lineage-Specific Oncogene in Lung Squamous Cell Carcinoma 
PLoS Medicine  2010;7(7):e1000315.
William Lockwood and colleagues show that the focal amplification of a gene, BRF2, on Chromosome 8p12 plays a key role in squamous cell carcinoma of the lung.
Background
Traditionally, non-small cell lung cancer is treated as a single disease entity in terms of systemic therapy. Emerging evidence suggests the major subtypes—adenocarcinoma (AC) and squamous cell carcinoma (SqCC)—respond differently to therapy. Identification of the molecular differences between these tumor types will have a significant impact in designing novel therapies that can improve the treatment outcome.
Methods and Findings
We used an integrative genomics approach, combing high-resolution comparative genomic hybridization and gene expression microarray profiles, to compare AC and SqCC tumors in order to uncover alterations at the DNA level, with corresponding gene transcription changes, which are selected for during development of lung cancer subtypes. Through the analysis of multiple independent cohorts of clinical tumor samples (>330), normal lung tissues and bronchial epithelial cells obtained by bronchial brushing in smokers without lung cancer, we identified the overexpression of BRF2, a gene on Chromosome 8p12, which is specific for development of SqCC of lung. Genetic activation of BRF2, which encodes a RNA polymerase III (Pol III) transcription initiation factor, was found to be associated with increased expression of small nuclear RNAs (snRNAs) that are involved in processes essential for cell growth, such as RNA splicing. Ectopic expression of BRF2 in human bronchial epithelial cells induced a transformed phenotype and demonstrates downstream oncogenic effects, whereas RNA interference (RNAi)-mediated knockdown suppressed growth and colony formation of SqCC cells overexpressing BRF2, but not AC cells. Frequent activation of BRF2 in >35% preinvasive bronchial carcinoma in situ, as well as in dysplastic lesions, provides evidence that BRF2 expression is an early event in cancer development of this cell lineage.
Conclusions
This is the first study, to our knowledge, to show that the focal amplification of a gene in Chromosome 8p12, plays a key role in squamous cell lineage specificity of the disease. Our data suggest that genetic activation of BRF2 represents a unique mechanism of SqCC lung tumorigenesis through the increase of Pol III-mediated transcription. It can serve as a marker for lung SqCC and may provide a novel target for therapy.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Lung cancer is the commonest cause of cancer-related death. Every year, 1.3 million people die from this disease, which is mainly caused by smoking. Most cases of lung cancer are “non-small cell lung cancers” (NSCLCs). Like all cancers, NSCLC starts when cells begin to divide uncontrollably and to move round the body (metastasize) because of changes (mutations) in their genes. These mutations are often in “oncogenes,” genes that, when activated, encourage cell division. Oncogenes can be activated by mutations that alter the properties of the proteins they encode or by mutations that increase the amount of protein made from them, such as gene amplification (an increase in the number of copies of a gene). If NSCLC is diagnosed before it has spread from the lungs (stage I disease), it can be surgically removed and many patients with stage I NSCLC survive for more than 5 years after their diagnosis. Unfortunately, in more than half of patients, NSCLC has metastasized before it is diagnosed. This stage IV NSCLC can be treated with chemotherapy (toxic chemicals that kill fast-growing cancer cells) but only 2% of patients with stage IV lung cancer are alive 5 years after diagnosis.
Why Was This Study Done?
Traditionally, NSCLC has been regarded as a single disease in terms of treatment. However, emerging evidence suggests that the two major subtypes of NSCLC—adenocarcinoma and squamous cell carcinoma (SqCC)—respond differently to chemotherapy. Adenocarcinoma and SqCC start in different types of lung cell and experts think that for each cell type in the body, specific combinations of mutations interact with the cell type's own unique characteristics to provide the growth and survival advantage needed for cancer development. If this is true, then identifying the molecular differences between adenocarcinoma and SqCC could provide targets for more effective therapies for these major subtypes of NSCLC. Amplification of a chromosome region called 8p12 is very common in NSCLC, which suggests that an oncogene that drives lung cancer development is present in this chromosome region. In this study, the researchers investigate this possibility by looking for an amplified gene in the 8p12 chromosome region that makes increased amounts of protein in lung SqCC but not in lung adenocarcinoma.
What Did the Researchers Do and Find?
The researchers used a technique called comparative genomic hybridization to show that focal regions of Chromosome 8p are amplified in about 40% of lung SqCCs, but that DNA loss in this region is the most common alteration in lung adenocarcinomas. Ten genes in the 8p12 chromosome region were expressed at higher levels in the SqCC samples that they examined than in adenocarcinoma samples, they report, and overexpression of five of these genes correlated with amplification of the 8p12 region in the SqCC samples. Only one of the genes—BRF2—was more highly expressed in squamous carcinoma cells than in normal bronchial epithelial cells (the cell type that lines the tubes that take air into the lungs and from which SqCC develops). Artificially induced expression of BRF2 in bronchial epithelial cells made these normal cells behave like tumor cells, whereas reduction of BRF2 expression in squamous carcinoma cells made them behave more like normal bronchial epithelial cells. Finally, BRF2 was frequently activated in two early stages of squamous cell carcinoma—bronchial carcinoma in situ and dysplastic lesions.
What Do These Findings Mean?
Together, these findings show that the focal amplification of chromosome region 8p12 plays a role in the development of lung SqCC but not in the development of lung adenocarcinoma, the other major subtype of NSCLC. These findings identify BRF2 (which encodes a RNA polymerase III transcription initiation factor, a protein that is required for the synthesis of RNA molecules that help to control cell growth) as a lung SqCC-specific oncogene and uncover a unique mechanism for lung SqCC development. Most importantly, these findings suggest that genetic activation of BRF2 could be used as a marker for lung SqCC, which might facilitate the early detection of this type of NSCLC and that BRF2 might provide a new target for therapy.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000315.
The US National Cancer Institute provides detailed information for patients and professionals about all aspects of lung cancer, including information on non-small cell carcinoma (in English and Spanish)
Cancer Research UK also provides information about lung cancer and information on how cancer starts
MedlinePlus has links to other resources about lung cancer (in English and Spanish)
doi:10.1371/journal.pmed.1000315
PMCID: PMC2910599  PMID: 20668658
7.  SOX2 Is an Amplified Lineage Survival Oncogene in Lung and Esophageal Squamous Cell Carcinomas 
Nature genetics  2009;41(11):1238-1242.
Lineage survival oncogenes are activated by somatic DNA alterations in cancers arising from the cell lineages in which these genes play a role in normal development.1,2 Here we show that a peak of genomic amplification on chromosome 3q26.33, found in squamous cell carcinomas (SCCs) of the lung and esophagus, contains the transcription factor gene SOX2—which is mutated in hereditary human esophageal malformations3 and necessary for normal esophageal squamous development4, promotes differentiation and proliferation of basal tracheal cells5 and co-operates in induction of pluripotent stem cells.6,7,8 SOX2 expression is required for proliferation and anchorage-independent growth of lung and esophageal cell lines, as shown by RNA interference experiments. Furthermore, ectopic expression of SOX2 cooperated with FOXE1 or FGFR2 to transform immortalized tracheobronchial epithelial cells. SOX2-driven tumors show expression of markers of both squamous differentiation and pluripotency. These observations identify SOX2 as a novel lineage survival oncogene in lung and esophageal SCC.
doi:10.1038/ng.465
PMCID: PMC2783775  PMID: 19801978
8.  Co-overexpression of Met and Hepatocyte Growth Factor Promotes Systemic Metastasis in NCI-H460 Non-Small Cell Lung Carcinoma Cells12 
Neoplasia (New York, N.Y.)  2009;11(12):1292-1300.
Complete resection of early-stage non-small cell lung cancer (NSCLC) is potentially curative, yet approximately 50% of patients are at risk for developing metastatic recurrence. Met, the receptor for hepatocyte growth factor (HGF) is a receptor tyrosine kinase with demonstrated roles in regulating cellular proliferation, motility, morphogenesis, and apoptosis. Met receptor and its ligand, HGF, are commonly overexpressed in NSCLC, and their overexpression has been associated with poor prognosis, which could potentially involve a paracrine and/or autocrine activation loop. However, there is as yet no direct evidence that HGF-Met signaling directly promotes metastasis in NSCLC cells. Using retroviral transduction, we overexpressed the human c-met and hgf complementary DNA, alone or in combination in the NCI-H460 human large cell carcinoma cell line. The HGF/Met co-overexpressing (H460-HGF/Met) cells demonstrated enhanced tumorigenicity in xenograft SCID mice. When these cells are implanted orthotopically into the lungs of nude rats, only the H460-HGF/Met cells showed higher spontaneous metastases to distant organs including bone, brain, and kidney. These results provide evidence that autocrine overactivation of the Met- HGF loop enhances systemic metastases in NSCLC. Targeted interference of this loop may potentially be an effective adjuvant therapy to improve survival of early-stage NSCLC patients.
PMCID: PMC2794510  PMID: 20019837
9.  Gene Expression-Based Survival Prediction in Lung Adenocarcinoma: A Multi-Site, Blinded Validation Study 
Nature medicine  2008;14(8):822-827.
Although prognostic gene expression signatures for survival in early stage lung cancer have been proposed, for clinical application it is critical to establish their performance across different subject populations and in different laboratories. Here we report a large, training-testing, multi-site blinded validation study to characterize the performance of several prognostic models based on gene expression for 442 lung adenocarcinomas. The hypotheses proposed examined whether microarray measurements of gene expression either alone or combined with basic clinical covariates (stage, age, sex) can be used to predict overall survival in lung cancer subjects. Several models examined produced risk scores that substantially correlated with actual subject outcome. Most methods performed better with clinical data, supporting the combined use of clinical and molecular information when building prognostic models for early stage lung cancer. This study also provides the largest available set of microarray data with extensive pathological and clinical annotation for lung adenocarcinomas.
doi:10.1038/nm.1790
PMCID: PMC2667337  PMID: 18641660

Results 1-9 (9)