PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (28)
 

Clipboard (0)
None

Select a Filter Below

Year of Publication
1.  Progenitor Cell Line (hPheo1) Derived from a Human Pheochromocytoma Tumor 
PLoS ONE  2013;8(6):e65624.
Background
Pheochromocytomas are rare tumors generally arising in the medullary region of the adrenal gland. These tumors release excessive epinephrine and norepinephrine resulting in hypertension and cardiovascular crises for which surgery is the only definitive treatment. Molecular mechanisms that control tumor development and hormone production are poorly understood, and progress has been hampered by the lack of human cellular model systems. To study pheochromocytomas, we developed a stable progenitor pheochromocytoma cell line derived from a primary human tumor.
Methods
After IRB approval and written informed consent, human pheochromocytoma tissue was excised, minced, dispersed enzymatically, and cultured in vitro. Primary pheochromocytoma cells were infected with a lentivirus vector carrying the catalytic subunit of human telomerase reverse transcriptase (hTERT). The hTERT immortalized cells (hPheo1) have been passaged >300 population doublings. The resulting cell line was characterized morphologically, biochemically and for expression of neuroendocrine properties. The expression of marker enzymes and proteins was assessed by immunofluorescence staining and immunoblotting. Telomerase activity was determined by using the telomeric repeat amplification protocol (TRAP) assay.
Results
We have established a human pheochromocytoma precursor cell line that expresses the neuroendocrine marker, chromogranin A, when differentiated in the presence of bone morphogenic protein 4 (BMP4), nerve growth factor (NGF), and dexamethasone. Phenylethanolamine N-methyltransferase (PNMT) expression is also detected with this differentiation regimen. CD-56 (also known as NCAM, neural cell adhesion molecule) is expressed in these cells, but CD31 (also known as PECAM-1, a marker of endothelial cells) is negative.
Conclusions
We have maintained hTERT-immortalized progenitor cells derived from a pheochromocytoma (hPheo1) in culture for over 300 population doublings. This progenitor human cell line is normal diploid except for a deletion in the p16 region and has inducible neuroendocrine biomarkers. These cells should be a valuable reagent for studying mechanisms of tumor development and for testing novel therapeutic approaches.
doi:10.1371/journal.pone.0065624
PMCID: PMC3681983  PMID: 23785438
2.  Integrative Genomics Identified RFC3 as an Amplified Candidate Oncogene in Esophageal Adenocarcinoma 
Purpose
Esophageal adenocarcinoma (EAC) is a lethal malignancy that can develop from the premalignant condition, Barrett’s esophagus (BE). Currently, there are no validated simple methods to predict which patients will progress to EAC. A better understanding of the genetic mechanisms driving EAC tumorigenesis is needed to identify new therapeutic targets and develop biomarkers capable of identifying high-risk patients that would benefit from aggressive neoadjuvant therapy. We employed an integrative genomics approach to identify novel genes involved in EAC biology that may serve as useful clinical markers.
Experimental Design
Whole genome tiling-path array CGH was used to identify significant regions of copy number (CN) alteration in 20 EACs and 10 matching BE tissues. CN and gene expression data were integrated to identify candidate oncogenes within regions of amplification and multiple additional sample cohorts were assessed to validate candidate genes.
Results
We identified RFC3 as a novel, candidate oncogene activated by amplification in ~25% of EAC samples. RFC3 was also amplified in BE from a patient whose EAC harbored amplification, and was differentially expressed between non-malignant and EAC tissues. CN gains were detected in other cancer types and RFC3 knockdown inhibited proliferation and anchorage-independent growth of cancer cells with increased CN, but had little effect on those without. Moreover, high RFC3 expression was associated with poor patient outcome in multiple cancer types.
Conclusions
RFC3 is a candidate oncogene amplified in EAC. RFC3 DNA amplification is also prevalent in other epithelial cancer types and RFC3 expression could serve as a prognostic marker.
doi:10.1158/1078-0432.CCR-11-1431
PMCID: PMC3523177  PMID: 22328562
RFC3; esophageal adenocarcinoma; Barrett’s esophagus; DNA amplification
3.  Genetic Disruption of KEAP1/CUL3 E3 Ubiquitin Ligase Complex Components is a Key Mechanism of NF-kappaB Pathway Activation in Lung Cancer 
Introduction
IKBKB (IKK-β/IKK-2), which activates NF-κB, is a substrate of the KEAP1-CUL3-RBX1 E3-ubiquitin ligase complex, implicating this complex in regulation of NF-κB signaling. We investigated complex component gene disruption as a novel genetic mechanism of NF-κB activation in non-small cell lung cancer (NSCLC).
Methods
644 tumor- and 90 cell line-genomes were analyzed for gene-dosage status of the individual complex components and IKBKB. Gene expression of these genes, and NF-κB target genes were analyzed in 48 tumors. IKBKB protein levels were assessed in tumors with and without complex or IKBKB genetic disruption. Complex component knockdown was performed to assess effects of the E3-ligase complex on IKBKB and NF-κB levels, and phenotypic importance of IKBKB expression was measured by pharmacological inhibition.
Results
We observed strikingly frequent genetic disruption (42%) and aberrant expression (63%) of the E3-ligase complex and IKBKB in the samples examined. While both adenocarcinomas and squamous cell carcinomas showed complex disruption, the patterns of gene disruption differed. IKBKB levels were elevated with complex disruption, knockdown of complex components increased activated forms of IKBKB and NF-κB proteins, and IKBKB inhibition detriments cell viability, highlighting the biological significance of complex disruption. NF-κB target genes were overexpressed in samples with complex disruption, further demonstrating the effect of complex disruption on NF-κB activity.
Conclusions
Gene dosage alteration is a prominent mechanism that disrupts each component of the KEAP1-CUL3-RBX1 complex and its NF-κB stimulating substrate, IKBKB. Here we show that, multiple component disruption of this complex represents a novel mechanism of NF-κB activation in NSCLC.
doi:10.1097/JTO.0b013e3182289479
PMCID: PMC3164321  PMID: 21795997
KEAP1; CUL3; RBX1; IKBKB; NF-κB signaling; genetic disruption
4.  Integrating the multiple dimensions of genomic and epigenomic landscapes of cancer 
Cancer metastasis reviews  2010;29(1):73-93.
Advances in high-throughput, genome-wide profiling technologies have allowed for an unprecedented view of the cancer genome landscape. Specifically, high-density microarrays and sequencing-based strategies have been widely utilized to identify genetic (such as gene dosage, allelic status, and mutations in gene sequence) and epigenetic (such as DNA methylation, histone modification, and micro-RNA) aberrations in cancer. Although the application of these profiling technologies in unidimensional analyses has been instrumental in cancer gene discovery, genes affected by low-frequency events are often overlooked. The integrative approach of analyzing parallel dimensions has enabled the identification of (a) genes that are often disrupted by multiple mechanisms but at low frequencies by any one mechanism and (b) pathways that are often disrupted at multiple components but at low frequencies at individual components. These benefits of using an integrative approach illustrate the concept that the whole is greater than the sum of its parts. As efforts have now turned toward parallel and integrative multidimensional approaches for studying the cancer genome landscape in hopes of obtaining a more insightful understanding of the key genes and pathways driving cancer cells, this review describes key findings disseminating from such high-throughput, integrative analyses, including contributions to our understanding of causative genetic events in cancer cell biology.
doi:10.1007/s10555-010-9199-2
PMCID: PMC3415277  PMID: 20108112
Integrative analysis; Cancer genome; Sequencing; Microarray
5.  Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers 
Nucleic Acids Research  2012;40(15):e117.
DNA built from modular repeats presents a challenge for gene synthesis. We present a solid surface-based sequential ligation approach, which we refer to as iterative capped assembly (ICA), that adds DNA repeat monomers individually to a growing chain while using hairpin ‘capping’ oligonucleotides to block incompletely extended chains, greatly increasing the frequency of full-length final products. Applying ICA to a model problem, construction of custom transcription activator-like effector nucleases (TALENs) for genome engineering, we demonstrate efficient synthesis of TALE DNA-binding domains up to 21 monomers long and their ligation into a nuclease-carrying backbone vector all within 3 h. We used ICA to synthesize 20 TALENs of varying DNA target site length and tested their ability to stimulate gene editing by a donor oligonucleotide in human cells. All the TALENS show activity, with the ones >15 monomers long tending to work best. Since ICA builds full-length constructs from individual monomers rather than large exhaustive libraries of pre-fabricated oligomers, it will be trivial to incorporate future modified TALE monomers with improved or expanded function or to synthesize other types of repeat-modular DNA where the diversity of possible monomers makes exhaustive oligomer libraries impractical.
doi:10.1093/nar/gks624
PMCID: PMC3424587  PMID: 22740649
6.  Divergent Genomic and Epigenomic Landscapes of Lung Cancer Subtypes Underscore the Selection of Different Oncogenic Pathways during Tumor Development 
PLoS ONE  2012;7(5):e37775.
For therapeutic purposes, non-small cell lung cancer (NSCLC) has traditionally been regarded as a single disease. However, recent evidence suggest that the two major subtypes of NSCLC, adenocarcinoma (AC) and squamous cell carcinoma (SqCC) respond differently to both molecular targeted and new generation chemotherapies. Therefore, identifying the molecular differences between these tumor types may impact novel treatment strategy. We performed the first large-scale analysis of 261 primary NSCLC tumors (169 AC and 92 SqCC), integrating genome-wide DNA copy number, methylation and gene expression profiles to identify subtype-specific molecular alterations relevant to new agent design and choice of therapy. Comparison of AC and SqCC genomic and epigenomic landscapes revealed 778 altered genes with corresponding expression changes that are selected during tumor development in a subtype-specific manner. Analysis of >200 additional NSCLCs confirmed that these genes are responsible for driving the differential development and resulting phenotypes of AC and SqCC. Importantly, we identified key oncogenic pathways disrupted in each subtype that likely serve as the basis for their differential tumor biology and clinical outcomes. Downregulation of HNF4α target genes was the most common pathway specific to AC, while SqCC demonstrated disruption of numerous histone modifying enzymes as well as the transcription factor E2F1. In silico screening of candidate therapeutic compounds using subtype-specific pathway components identified HDAC and PI3K inhibitors as potential treatments tailored to lung SqCC. Together, our findings suggest that AC and SqCC develop through distinct pathogenetic pathways that have significant implication in our approach to the clinical management of NSCLC.
doi:10.1371/journal.pone.0037775
PMCID: PMC3357406  PMID: 22629454
7.  Lung Adenocarcinoma of Never Smokers and Smokers Harbor Differential Regions of Genetic Alteration and Exhibit Different Levels of Genomic Instability 
PLoS ONE  2012;7(3):e33003.
Recent evidence suggests that the observed clinical distinctions between lung tumors in smokers and never smokers (NS) extend beyond specific gene mutations, such as EGFR, EML4-ALK, and KRAS, some of which have been translated into targeted therapies. However, the molecular alterations identified thus far cannot explain all of the clinical and biological disparities observed in lung tumors of NS and smokers. To this end, we performed an unbiased genome-wide, comparative study to identify novel genomic aberrations that differ between smokers and NS.
High resolution whole genome DNA copy number profiling of 69 lung adenocarcinomas from smokers (n = 39) and NS (n = 30) revealed both global and regional disparities in the tumor genomes of these two groups. We found that NS lung tumors had a greater proportion of their genomes altered than those of smokers. Moreover, copy number gains on chromosomes 5q, 7p, and 16p occurred more frequently in NS. We validated our findings in two independently generated public datasets. Our findings provide a novel line of evidence distinguishing genetic differences between smoker and NS lung tumors, namely, that the extent of segmental genomic alterations is greater in NS tumors. Collectively, our findings provide evidence that these lung tumors are globally and genetically different, which implies they are likely driven by distinct molecular mechanisms.
doi:10.1371/journal.pone.0033003
PMCID: PMC3296775  PMID: 22412972
8.  TRAF6 is an amplified oncogene bridging the RAS and NF-κB pathways in human lung cancer  
The Journal of Clinical Investigation  2011;121(10):4095-4105.
Somatic mutations and copy number alterations (as a result of deletion or amplification of large portions of a chromosome) are major drivers of human lung cancers. Detailed analysis of lung cancer–associated chromosomal amplifications could identify novel oncogenes. By performing an integrative cytogenetic and gene expression analysis of non–small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) cell lines and tumors, we report here the identification of a frequently recurring amplification at chromosome 11 band p13. Within this region, only TNF receptor–associated factor 6 (TRAF6) exhibited concomitant mRNA overexpression and gene amplification in lung cancers. Inhibition of TRAF6 in human lung cancer cell lines suppressed NF-κB activation, anchorage-independent growth, and tumor formation. In these lung cancer cell lines, RAS required TRAF6 for its oncogenic capabilities. Furthermore, TRAF6 overexpression in NIH3T3 cells resulted in NF-κB activation, anchorage-independent growth, and tumor formation. Our findings show that TRAF6 is an oncogene that is important for RAS-mediated oncogenesis and provide a mechanistic explanation for the previously apparent importance of constitutive NF-κB activation in RAS-driven lung cancers.
doi:10.1172/JCI58818
PMCID: PMC3195480  PMID: 21911935
9.  MicroRNA Gene Dosage Alterations and Drug Response in Lung Cancer 
Chemotherapy resistance is a key contributor to the dismal prognoses for lung cancer patients. While the majority of studies have focused on sequence mutations and expression changes in protein-coding genes, recent reports have suggested that microRNA (miRNA) expression changes also play an influential role in chemotherapy response. However, the role of genetic alterations at miRNA loci in the context of chemotherapy response has yet to be investigated. In this study, we demonstrate the application of an integrative, multidimensional approach in order to identify miRNAs that are associated with chemotherapeutic resistance and sensitivity utilizing publicly available drug response, miRNA loci copy number, miRNA expression, and mRNA expression data from independent resources. By instigating a logical stepwise strategy, we have identified specific miRNAs that are associated with resistance to several chemotherapeutic agents and provide a proof of principle demonstration of how these various databases may be exploited to derive relevant pharmacogenomic results.
doi:10.1155/2011/474632
PMCID: PMC3085440  PMID: 21541180
10.  Deciphering Squamous Cell Carcinoma Using Multidimensional Genomic Approaches 
Journal of Skin Cancer  2010;2011:541405.
Squamous cell carcinomas (SqCCs) arise in a wide range of tissues including skin, lung, and oral mucosa. Although all SqCCs are epithelial in origin and share common nomenclature, these cancers differ greatly with respect to incidence, prognosis, and treatment. Current knowledge of genetic similarities and differences between SqCCs is insufficient to describe the biology of these cancers, which arise from diverse tissue origins. In this paper we provide a general overview of whole genome approaches for gene and pathway discovery and highlight the advancement of integrative genomics as a state-of-the-art technology in the study of SqCC genetics.
doi:10.1155/2011/541405
PMCID: PMC3017908  PMID: 21234096
11.  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
12.  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
13.  FACADE: a fast and sensitive algorithm for the segmentation and calling of high resolution array CGH data 
Nucleic Acids Research  2010;38(15):e157.
The availability of high resolution array comparative genomic hybridization (CGH) platforms has led to increasing complexities in data analysis. Specifically, defining contiguous regions of alterations or segmentation can be computationally intensive and popular algorithms can take hours to days for the processing of arrays comprised of hundreds of thousands to millions of elements. Additionally, tumors tend to demonstrate subtle copy number alterations due to heterogeneity, ploidy and hybridization effects. Thus, there is a need for fast, sensitive array CGH segmentation and alteration calling algorithms. Here, we describe Fast Algorithm for Calling After Detection of Edges (FACADE), a highly sensitive and easy to use algorithm designed to rapidly segment and call high resolution array data.
doi:10.1093/nar/gkq548
PMCID: PMC2926625  PMID: 20551132
14.  An integrative multi-dimensional genetic and epigenetic strategy to identify aberrant genes and pathways in cancer 
BMC Systems Biology  2010;4:67.
Background
Genomics has substantially changed our approach to cancer research. Gene expression profiling, for example, has been utilized to delineate subtypes of cancer, and facilitated derivation of predictive and prognostic signatures. The emergence of technologies for the high resolution and genome-wide description of genetic and epigenetic features has enabled the identification of a multitude of causal DNA events in tumors. This has afforded the potential for large scale integration of genome and transcriptome data generated from a variety of technology platforms to acquire a better understanding of cancer.
Results
Here we show how multi-dimensional genomics data analysis would enable the deciphering of mechanisms that disrupt regulatory/signaling cascades and downstream effects. Since not all gene expression changes observed in a tumor are causal to cancer development, we demonstrate an approach based on multiple concerted disruption (MCD) analysis of genes that facilitates the rational deduction of aberrant genes and pathways, which otherwise would be overlooked in single genomic dimension investigations.
Conclusions
Notably, this is the first comprehensive study of breast cancer cells by parallel integrative genome wide analyses of DNA copy number, LOH, and DNA methylation status to interpret changes in gene expression pattern. Our findings demonstrate the power of a multi-dimensional approach to elucidate events which would escape conventional single dimensional analysis and as such, reduce the cohort sample size for cancer gene discovery.
doi:10.1186/1752-0509-4-67
PMCID: PMC2880289  PMID: 20478067
15.  Disruption of the non-canonical WNT pathway in lung squamous cell carcinoma 
Clinical medicine. Oncology  2008;2008(2):169-179.
Disruptions of beta-catenin and the canonical Wnt pathway are well documented in cancer. However, little is known of the non-canonical branch of the Wnt pathway. In this study, we investigate the transcript level patterns of genes in the Wnt pathway in squamous cell lung cancer using reverse-transcriptase (RT)-PCR. It was found that over half of the samples examined exhibited dysregulated gene expression of multiple components of the non-canonical branch of the WNT pathway. In the cases where beta catenin (CTNNB1) was not over-expressed, we identified strong relationships of expression between wingless-type MMTV integration site family member 5A (WNT5A)/ frizzled homolog 2 (FZD2), frizzled homolog 3 (FZD3) / dishevelled 2 (DVL2), and low density lipoprotein receptor-related protein 5 (LRP5)/ secreted frizzled-related protein 4 (SFRP4). This is one of the first studies to demonstrate expression of genes in the non-canonical pathway in normal lung tissue and its disruption in lung squamous cell carcinoma. These findings suggest that the non-canonical pathway may have a more prominent role in lung cancer than previously reported.
PMCID: PMC2855195  PMID: 20401333
WNT pathway; lung cancer; gene expression; NSCLC; non-canonical; squamous cell carcinoma
16.  Transcriptome Profiles of Carcinoma-in-Situ and Invasive Non-Small Cell Lung Cancer as Revealed by SAGE 
PLoS ONE  2010;5(2):e9162.
Background
Non-small cell lung cancer (NSCLC) presents as a progressive disease spanning precancerous, preinvasive, locally invasive, and metastatic lesions. Identification of biological pathways reflective of these progressive stages, and aberrantly expressed genes associated with these pathways, would conceivably enhance therapeutic approaches to this devastating disease.
Methodology/Principal Findings
Through the construction and analysis of SAGE libraries, we have determined transcriptome profiles for preinvasive carcinoma-in-situ (CIS) and invasive squamous cell carcinoma (SCC) of the lung, and compared these with expression profiles generated from both bronchial epithelium, and precancerous metaplastic and dysplastic lesions using Ingenuity Pathway Analysis. Expression of genes associated with epidermal development, and loss of expression of genes associated with mucociliary biology, are predominant features of CIS, largely shared with precancerous lesions. Additionally, expression of genes associated with xenobiotic metabolism/detoxification is a notable feature of CIS, and is largely maintained in invasive cancer. Genes related to tissue fibrosis and acute phase immune response are characteristic of the invasive SCC phenotype. Moreover, the data presented here suggests that tissue remodeling/fibrosis is initiated at the early stages of CIS. Additionally, this study indicates that alteration in copy-number status represents a plausible mechanism for differential gene expression in CIS and invasive SCC.
Conclusions/Significance
This study is the first report of large-scale expression profiling of CIS of the lung. Unbiased expression profiling of these preinvasive and invasive lesions provides a platform for further investigations into the molecular genetic events relevant to early stages of squamous NSCLC development. Additionally, up-regulated genes detected at extreme differences between CIS and invasive cancer may have potential to serve as biomarkers for early detection.
doi:10.1371/journal.pone.0009162
PMCID: PMC2820080  PMID: 20161782
17.  Public Databases and Software for the Pathway Analysis of Cancer Genomes 
Cancer informatics  2007;3:379-397.
The study of pathway disruption is key to understanding cancer biology. Advances in high throughput technologies have led to the rapid accumulation of genomic data. The explosion in available data has generated opportunities for investigation of concerted changes that disrupt biological functions, this in turns created a need for computational tools for pathway analysis. In this review, we discuss approaches to the analysis of genomic data and describe the publicly available resources for studying biological pathways.
PMCID: PMC2410087  PMID: 19455256
18.  Oncogene Mutations, Copy Number Gains and Mutant Allele Specific Imbalance (MASI) Frequently Occur Together in Tumor Cells 
PLoS ONE  2009;4(10):e7464.
Background
Activating mutations in one allele of an oncogene (heterozygous mutations) are widely believed to be sufficient for tumorigenesis. However, mutant allele specific imbalance (MASI) has been observed in tumors and cell lines harboring mutations of oncogenes.
Methodology/Principal Findings
We determined 1) mutational status, 2) copy number gains (CNGs) and 3) relative ratio between mutant and wild type alleles of KRAS, BRAF, PIK3CA and EGFR genes by direct sequencing and quantitative PCR assay in over 400 human tumors, cell lines, and xenografts of lung, colorectal, and pancreatic cancers. Examination of a public database indicated that homozygous mutations of five oncogenes were frequent (20%) in 833 cell lines of 12 tumor types. Our data indicated two major forms of MASI: 1) MASI with CNG, either complete or partial; and 2) MASI without CNG (uniparental disomy; UPD), due to complete loss of wild type allele. MASI was a frequent event in mutant EGFR (75%) and was due mainly to CNGs, while MASI, also frequent in mutant KRAS (58%), was mainly due to UPD. Mutant: wild type allelic ratios at the genomic level were precisely maintained after transcription. KRAS mutations or CNGs were significantly associated with increased ras GTPase activity, as measured by ELISA, and the two molecular changes were synergistic. Of 237 lung adenocarcinoma tumors, the small number with both KRAS mutation and CNG were associated with shortened survival.
Conclusions
MASI is frequently present in mutant EGFR and KRAS tumor cells, and is associated with increased mutant allele transcription and gene activity. The frequent finding of mutations, CNGs and MASI occurring together in tumor cells indicates that these three genetic alterations, acting together, may have a greater role in the development or maintenance of the malignant phenotype than any individual alteration.
doi:10.1371/journal.pone.0007464
PMCID: PMC2757721  PMID: 19826477
19.  Genomic imbalances in precancerous tissues signal oral cancer risk 
Molecular Cancer  2009;8:50.
Oral cancer develops through a series of histopathological stages: through mild (low grade), moderate, and severe (high grade) dysplasia to carcinoma in situ and then invasive disease. Early detection of those oral premalignant lesions (OPLs) that will develop into invasive tumors is necessary to improve the poor prognosis of oral cancer. Because no tools exist for delineating progression risk in low grade oral lesions, we cannot determine which of these cases require aggressive intervention. We undertook whole genome analysis by tiling-path array comparative genomic hybridization for a rare panel of early and late stage OPLs (n = 62), all of which had extensive longitudinal follow up (>10 years). Genome profiles for oral squamous cell carcinomas (n = 24) were generated for comparison. Parallel analysis of genome alterations and clinical parameters was performed to identify features associated with disease progression. Genome alterations in low grade dysplasias progressing to invasive disease more closely resembled those observed for later stage disease than they did those observed for non-progressing low grade dysplasias. This was despite the histopathological similarity between progressing and non-progressing cases. Strikingly, unbiased computational analysis of genomic alteration data correctly classified nearly all progressing low grade dysplasia cases. Our data demonstrate that high resolution genomic analysis can be used to evaluate progression risk in low grade OPLs, a marked improvement over present histopathological approaches which cannot delineate progression risk. Taken together, our data suggest that whole genome technologies could be used in management strategies for patients presenting with precancerous oral lesions.
doi:10.1186/1476-4598-8-50
PMCID: PMC2726119  PMID: 19627613
20.  SIGMA2: A system for the integrative genomic multi-dimensional analysis of cancer genomes, epigenomes, and transcriptomes 
BMC Bioinformatics  2008;9:422.
Background
High throughput microarray technologies have afforded the investigation of genomes, epigenomes, and transcriptomes at unprecedented resolution. However, software packages to handle, analyze, and visualize data from these multiple 'omics disciplines have not been adequately developed.
Results
Here, we present SIGMA2, a system for the integrative genomic multi-dimensional analysis of cancer genomes, epigenomes, and transcriptomes. Multi-dimensional datasets can be simultaneously visualized and analyzed with respect to each dimension, allowing combinatorial integration of the different assays belonging to the different 'omics.
Conclusion
The identification of genes altered at multiple levels such as copy number, loss of heterozygosity (LOH), DNA methylation and the detection of consequential changes in gene expression can be concertedly performed, establishing SIGMA2 as a novel tool to facilitate the high throughput systems biology analysis of cancer.
doi:10.1186/1471-2105-9-422
PMCID: PMC2571113  PMID: 18840289
21.  Disruption of the Non-Canonical WNT Pathway in Lung Squamous Cell Carcinoma 
Clinical Medicine. Oncology  2008;2:169-179.
Disruptions of beta-catenin and the canonical Wnt pathway are well documented in cancer. However, little is known of the non-canonical branch of the Wnt pathway. In this study, we investigate the transcript level patterns of genes in the Wnt pathway in squamous cell lung cancer using reverse-transcriptase (RT)-PCR. It was found that over half of the samples examined exhibited dysregulated gene expression of multiple components of the non-canonical branch of the WNT pathway. In the cases where beta catenin (CTNNB1) was not over-expressed, we identified strong relationships of expression between wingless-type MMTV integration site family member 5A (WNT5A)/frizzled homolog 2 (FZD2), frizzled homolog 3 (FZD3)/dishevelled 2 (DVL2), and low density lipoprotein receptor-related protein 5 (LRP5)/secreted frizzled-related protein 4 (SFRP4). This is one of the first studies to demonstrate expression of genes in the non-canonical pathway in normal lung tissue and its disruption in lung squamous cell carcinoma. These findings suggest that the non-canonical pathway may have a more prominent role in lung cancer than previously reported.
PMCID: PMC2855195  PMID: 20401333
WNT pathway; lung cancer; gene expression; NSCLC; non-canonical; squamous cell carcinoma
22.  Up regulation in gene expression of chromatin remodelling factors in cervical intraepithelial neoplasia 
BMC Genomics  2008;9:64.
Background
The highest rates of cervical cancer are found in developing countries. Frontline monitoring has reduced these rates in developed countries and present day screening programs primarily identify precancerous lesions termed cervical intraepithelial neoplasias (CIN). CIN lesions described as mild dysplasia (CIN I) are likely to spontaneously regress while CIN III lesions (severe dysplasia) are likely to progress if untreated. Thoughtful consideration of gene expression changes paralleling the progressive pre invasive neoplastic development will yield insight into the key casual events involved in cervical cancer development.
Results
In this study, we have identified gene expression changes across 16 cervical cases (CIN I, CIN II, CIN III and normal cervical epithelium) using the unbiased long serial analysis of gene expression (L-SAGE) method. The 16 L-SAGE libraries were sequenced to the level of 2,481,387 tags, creating the largest SAGE data collection for cervical tissue worldwide. We have identified 222 genes differentially expressed between normal cervical tissue and CIN III. Many of these genes influence biological functions characteristic of cancer, such as cell death, cell growth/proliferation and cellular movement. Evaluation of these genes through network interactions identified multiple candidates that influence regulation of cellular transcription through chromatin remodelling (SMARCC1, NCOR1, MRFAP1 and MORF4L2). Further, these expression events are focused at the critical junction in disease development of moderate dysplasia (CIN II) indicating a role for chromatin remodelling as part of cervical cancer development.
Conclusion
We have created a valuable publically available resource for the study of gene expression in precancerous cervical lesions. Our results indicate deregulation of the chromatin remodelling complex components and its influencing factors occur in the development of CIN lesions. The increase in SWI/SNF stabilizing molecule SMARCC1 and other novel genes has not been previously illustrated as events in the early stages of dysplasia development and thus not only provides novel candidate markers for screening but a biological function for targeting treatment.
doi:10.1186/1471-2164-9-64
PMCID: PMC2277413  PMID: 18248679
23.  Public Databases and Software for the Pathway Analysis of Cancer Genomes 
Cancer Informatics  2007;3:379-397.
The study of pathway disruption is key to understanding cancer biology. Advances in high throughput technologies have led to the rapid accumulation of genomic data. The explosion in available data has generated opportunities for investigation of concerted changes that disrupt biological functions, this in turns created a need for computational tools for pathway analysis. In this review, we discuss approaches to the analysis of genomic data and describe the publicly available resources for studying biological pathways.
PMCID: PMC2410087  PMID: 19455256
24.  Computational Methods for the Analysis of Array Comparative Genomic Hybridization 
Cancer informatics  2006;2:48-58.
Array comparative genomic hybridization (array CGH) is a technique for assaying the copy number status of cancer genomes. The widespread use of this technology has lead to a rapid accumulation of high throughput data, which in turn has prompted the development of computational strategies for the analysis of array CGH data. Here we explain the principles behind array image processing, data visualization and genomic profile analysis, review currently available software packages, and raise considerations for future software development.
PMCID: PMC2067254  PMID: 17992253
Array CGH; microarray; cancer genome; software; bioinformatics; alteration detection
25.  Effect of active smoking on the human bronchial epithelium transcriptome 
BMC Genomics  2007;8:297.
Background
Lung cancer is the most common cause of cancer-related deaths. Tobacco smoke exposure is the strongest aetiological factor associated with lung cancer. In this study, using serial analysis of gene expression (SAGE), we comprehensively examined the effect of active smoking by comparing the transcriptomes of clinical specimens obtained from current, former and never smokers, and identified genes showing both reversible and irreversible expression changes upon smoking cessation.
Results
Twenty-four SAGE profiles of the bronchial epithelium of eight current, twelve former and four never smokers were generated and analyzed. In total, 3,111,471 SAGE tags representing over 110 thousand potentially unique transcripts were generated, comprising the largest human SAGE study to date. We identified 1,733 constitutively expressed genes in current, former and never smoker transcriptomes. We have also identified both reversible and irreversible gene expression changes upon cessation of smoking; reversible changes were frequently associated with either xenobiotic metabolism, nucleotide metabolism or mucus secretion. Increased expression of TFF3, CABYR, and ENTPD8 were found to be reversible upon smoking cessation. Expression of GSK3B, which regulates COX2 expression, was irreversibly decreased. MUC5AC expression was only partially reversed. Validation of select genes was performed using quantitative RT-PCR on a secondary cohort of nine current smokers, seven former smokers and six never smokers.
Conclusion
Expression levels of some of the genes related to tobacco smoking return to levels similar to never smokers upon cessation of smoking, while expression of others appears to be permanently altered despite prolonged smoking cessation. These irreversible changes may account for the persistent lung cancer risk despite smoking cessation.
doi:10.1186/1471-2164-8-297
PMCID: PMC2001199  PMID: 17727719

Results 1-25 (28)