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1.  Piwi-interacting RNAs in cancer: emerging functions and clinical utility 
Molecular Cancer  2016;15:5.
PIWI-interacting RNAs (piRNAs) are emerging players in cancer genomics. Originally described in the germline, there are over 20,000 piRNA genes in the human genome. In contrast to microRNAs, piRNAs interact with PIWI proteins, another member of the Argonaute family, and function primarily in the nucleus. There, they are involved in the epigenetic silencing of transposable elements in addition to the transcriptional regulation of genes. It has recently been demonstrated that piRNAs are also expressed across a variety of human somatic tissue types in a tissue-specific manner. An increasing number of studies have shown that aberrant piRNA expression is a signature feature across multiple tumour types; however, their specific tumorigenic functions remain unclear. In this article, we discuss the emerging functional roles of piRNAs in a variety of cancers, and highlight their potential clinical utilities.
doi:10.1186/s12943-016-0491-9
PMCID: PMC4714483  PMID: 26768585
PIWI-interacting RNA; piRNA; PIWI; Cancer; Tissue specificity; Transcriptome; Small RNA; Non-coding RNA; Epigenetics
2.  Identification of the epigenetic reader CBX2 as a potential drug target in advanced prostate cancer 
Clinical Epigenetics  2016;8:16.
Background
While localized prostate cancer (PCa) can be effectively cured, metastatic disease inevitably progresses to a lethal state called castration-resistant prostate cancer (CRPC). Emerging evidence suggests that aberrant epigenetic repression by the polycomb group (PcG) complexes fuels PCa progression, providing novel therapeutic opportunities.
Results
In the search for potential epigenetic drivers of CRPC, we analyzed the molecular profile of PcG members in patient-derived xenografts and clinical samples. Overall, our results identify the PcG protein and methyl-lysine reader CBX2 as a potential therapeutic target in advanced PCa. We report that CBX2 was recurrently up-regulated in metastatic CRPC and that elevated CBX2 expression was correlated with poor clinical outcome in PCa cohorts. Furthermore, CBX2 depletion abrogated cell viability and induced caspase 3-mediated apoptosis in metastatic PCa cell lines. Mechanistically explaining this phenotype, microarray analysis in CBX2-depleted cells revealed that CBX2 controls the expression of many key regulators of cell proliferation and metastasis.
Conclusions
Taken together, this study provides the first evidence that CBX2 inhibition induces cancer cell death, positioning CBX2 as an attractive drug target in lethal CRPC.
doi:10.1186/s13148-016-0182-9
PMCID: PMC4751702  PMID: 26877821
Castration-resistant prostate cancer; CBX2; Epigenetics; Metastatic prostate cancer; Polycomb
3.  Multiple Components of the VHL Tumor Suppressor Complex Are Frequently Affected by DNA Copy Number Loss in Pheochromocytoma 
Pheochromocytomas (PCC) are rare tumors that arise in chromaffin tissue of the adrenal gland. PCC are frequently inherited through predisposing mutations in genes such as the von Hippel-Lindau (VHL) tumor suppressor. VHL is part of the VHL elongin BC protein complex that also includes CUL2/5, TCEB1, TCEB2, and RBX1; in normoxic conditions this complex targets hypoxia-inducible factor 1 alpha (HIF1A) for degradation, thus preventing a hypoxic response. VHL inactivation by genetic mechanisms, such as mutation and loss of heterozygosity, inhibits HIF1A degradation, even in the presence of oxygen, and induces a pseudohypoxic response. However, the described <10% VHL mutation rate cannot account for the high frequency of hypoxic response observed. Indeed, little is known about genetic mechanisms disrupting other complex component genes. Here, we show that, in a panel of 171 PCC tumors, 59.6% harbored gene copy number loss (CNL) of at least one complex component. CNL significantly reduced gene expression and was associated with enrichment of gene targets controlled by HIF1. Interestingly, we show that VHL-related renal clear cell carcinoma harbored disruption of VHL alone. Our results indicate that VHL elongin BC protein complex components other than VHL could be important for PCC tumorigenesis and merit further investigation.
doi:10.1155/2014/546347
PMCID: PMC4178909  PMID: 25298778
4.  EZH2 promotes E2F driven SCLC tumorigenesis through modulation of apoptosis and cell cycle regulation 
Introduction
While EZH2 has been associated with both non small cell and small cell lung cancers, current observations suggest different mechanisms of EZH2 activation and overexpression in these lung cancer types. Globally, small cell lung cancer (SCLC) kills 200,000 people yearly. New clinical approaches for SCLC treatment are required to improve the poor survival rate. Given the therapeutic potential of EZH2 as a target, we sought to delineate the downstream consequences of EZH2 disruption to identify the cellular mechanisms by which EZH2 promotes tumorigenesis in SCLC.
Methods
We generated cells with stable expression of shRNA targeting EZH2 and corresponding controls (pLKO.1) and determined the consequences of EZH2 knockdown on the cell cycle and apoptosis by means of propidium iodide staining and fluorescence activated cell sorting, western blot, qRT-PCR as well as cell viability assessment using MTT assays.
Results
We discovered that EZH2 inhibition 1) increased apoptotic activity by up-regulating the pro-apoptotic factors Puma and Bad, 2) decreased the fraction of cells in S or G2/M phases, and 3) elevated p21 protein levels, implicating EZH2 in cell death and cell cycle control in SCLC.
Conclusions
Our findings present evidence for the role of EZH2 in the regulation of cell cycle and apoptosis, providing a biological mechanism to explain the tumorigenicity of EZH2 in SCLC. Our work points to the great potential of EZH2 as a therapeutic target in SCLC.
doi:10.1097/JTO.0b013e318298762f
PMCID: PMC3713495  PMID: 23857401
SCLC; EZH2; oncogene; RB1; E2F
5.  SOX15 and other SOX family members are important mediators of tumorigenesis in multiple cancer types 
Oncoscience  2014;1(5):326-335.
SOX genes are transcription factors with important roles in embryonic development and carcinogenesis. The SOX family of 20 genes is responsible for regulating lineage and tissue specific gene expression patterns, controlling numerous developmental processes including cell differentiation, sex determination, and organogenesis. As is the case with many genes involved in regulating development, SOX genes are frequently deregulated in cancer. In this perspective we provide a brief overview of how SOX proteins can promote or suppress cancer growth. We also present a pan-cancer analysis of aberrant SOX gene expression and highlight potential molecular mechanisms responsible for their disruption in cancer. Our analyses indicate the prominence of SOX deregulation in different cancer types and reveal potential roles for SOX genes not previously described in cancer. Finally, we summarize our recent identification of SOX15 as a candidate tumor suppressor in pancreatic cancer and propose several research avenues to pursue to further delineate the emerging role of SOX15 in development and carcinogenesis.
PMCID: PMC4278306  PMID: 25594027
SOX; SOX15; oncogene; tumor suppressor; development; cancer
6.  Targeting of chemoprevention to high-risk potentially malignant oral lesions: Challenges and opportunities 
Oral oncology  2014;50(12):1123-1130.
Worldwide, oral cancer is responsible for 170,000 deaths per year. Intervention to prevent this disease is a long sought after goal. Chemoprevention studies have focused on clinicopathological features of potentially malignant lesions (PML) in an effort to prevent their progression to cancer. However, prediction of future behavior for such lesions is difficult and remains a major challenge to such intervention.
Different approaches to this problem have been tested in the past 20 years. Early genetic progression models identified critical regions of allelic imbalance at 3p and 9p, and provided the basis for molecular markers to identify progressing PMLs. Subsequently, technological advances, such as genome-wide high-throughput array platforms, computer imaging, visualization technology and next generation sequencing, have broadened the scope for marker development and have the potential of further improving our ability to identify high-risk lesions in the near future either alone or in combination.
In this article, we examine the milestones in the development of markers for PML progression. We emphasize the critical importance of networks among scientists, health professionals and community to facilitate the validation and application of putative markers into clinical practice. With a growing number of new agents to validate, it is necessary to coordinate the design and implementation of strategies for patient recruitment, integration of marker assessment, and the final translation of such approaches into clinical use.
doi:10.1016/j.oraloncology.2014.08.012
PMCID: PMC4539364  PMID: 25240917
Loss-of-heterozygosity; oral potentially malignant lesions; oral premalignant lesions; progression markers; knowledge translation
7.  Gene expression analysis of microtubule affinity-regulating kinase 2 in non-small cell lung cancer 
Genomics Data  2015;6:145-148.
Lung cancer is the leading cause of cancer death worldwide, and has a five-year survival rate of 18% [1]. MARK2 is a serine/threonine-protein kinase, and is a key component in the phosphorylation of microtubule-associated proteins [2], [3]. A recent study published by Hubaux et al. found that microtubule affinity-regulating kinase 2 (MARK2) showed highly frequent DNA and RNA level disruption in lung cancer cell lines and independent non-small cell lung cancer (NSCLC) cohorts [4]. These alterations result in the acquisition of oncogenic properties in cell lines, such as increased viability and anchorage-independent growth. Furthermore, a microarray-based transcriptome analysis of three short hairpin RNA (shRNA)-mediated MARK2 knockdown lung adenocarcinoma cell lines (GEO#: GSE57966) revealed an association between MARK2 gene expression and cell cycle activation and DNA damage response. Here, we present a detailed description of transcriptome analysis to support the described role of MARK2 in promoting a malignant phenotype.
doi:10.1016/j.gdata.2015.08.011
PMCID: PMC4664690  PMID: 26697357
MARK2; Microarray; Affymetrix; Gene set enrichment analysis; Lung cancer
8.  EYA4 is inactivated biallelically at a high frequency in sporadic lung cancer and is associated with familial lung cancer risk 
Oncogene  2013;33(36):4464-4473.
In an effort to identify novel biallelically inactivated tumor suppressor genes (TSG) in sporadic invasive and pre-invasive non-small cell lung cancer (NSCLC) genomes, we applied a comprehensive integrated multi-‘omics approach to investigate patient matched, paired NSCLC tumor and non-malignant parenchymal tissues. By surveying lung tumor genomes for genes concomitantly inactivated within individual tumors by multiple mechanisms, and by the frequency of disruption in tumors across multiple cohorts, we have identified a putative lung cancer TSG, Eyes Absent 4 (EYA4). EYA4 is frequently and concomitantly deleted, hypermethylated and underexpressed in multiple independent lung tumor data sets, in both major NSCLC subtypes, and in the earliest stages of lung cancer. We find not only that decreased EYA4 expression is associated with poor survival in sporadic lung cancers, but EYA4 SNPs are associated with increased familial cancer risk, consistent with EYA4’s proximity to the previously reported lung cancer susceptibility locus on 6q. Functionally, we find that EYA4 displays TSG-like properties with a role in modulating apoptosis and DNA repair. Cross examination of EYA4 expression across multiple tumor types suggests a cell type-specific tumorigenic role for EYA4, consistent with a tumor suppressor function in cancers of epithelial origin. This work shows a clear role for EYA4 as a putative TSG in NSCLC.
doi:10.1038/onc.2013.396
PMCID: PMC4527534  PMID: 24096489
EYA4; two-hit; hypermethylation; tumor suppressor; TSG; non-small cell lung cancer
9.  Potential application of non-small cell lung cancer-associated autoantibodies to early cancer diagnosis 
To identify a panel of tumor associated autoantibodies which can potentially be used as biomarkers for the early diagnosis of non-small cell lung cancer (NSCLC). Thirty-five unique and in-frame expressed phage proteins were isolated. Based on the gene expression profiling, four proteins were selected for further study. Both receiver operating characteristic curve analysis and leave-one-out method revealed that combined measurements of four antibodies produced have better predictive accuracies than any single marker alone. Leave-one-out validation also showed significant relevance with all stages of NSCLC patients. The panel of autoantibodies has a high potential for detecting early stage NSCLC.
doi:10.1016/j.bbrc.2012.06.050
PMCID: PMC4512951  PMID: 22713465
NSCLC; Tumor-associated autoantibodies; SEREX; Early diagnosis
10.  Acquired Resistance to EGFR Inhibitors Is Associated with a Manifestation of Stem cell–like Properties in Cancer Cells 
Cancer research  2013;73(10):3051-3061.
Acquired resistance to EGF receptor (EGFR) tyrosine kinase inhibitor (TKI) is a critical problem in the treatment of lung cancer. Although several mechanisms have been shown to be responsible for acquired resistance, all mechanisms have not been uncovered. In this study, we investigated the molecular and cellular profiles of the acquired resistant cells to EGFR-TKI in EGFR-mutant lung cancers. Four EGFR-mutant cell lines were exposed to gefitinib by stepwise escalation and high-concentration exposure methods, and resistant sublines to gefitinib were established. The molecular profiles and cellular phenotypes of these resistant sublines were characterized. Although previously reported, alterations including secondary EGFR T790M mutation, MET amplification, and appearance of epithelial-to-mesenchymal transition (EMT) features were observed, these 2 drug-exposure methods revealed different resistance mechanisms. The resistant cells with EMT features exhibited downregulation of miRNA-200c by DNA methylation. Furthermore, the HCC827-derived subline characterized by the high-concentration exposure method exhibited not only EMT features but also stem cell–like properties, including aldehyde dehydrogenase isoform 1 (ALDH1A1) overexpression, increase of side-population, and self-renewal capability. Resistant sublines with stem cell–like properties were resistant to conventional chemotherapeutic agents but equally sensitive to histone deacetylase and proteasome inhibitors, compared with their parental cells. ALDH1A1 was upregulated in clinical samples with acquired resistance to gefitinib. In conclusion, our study indicates that the manner of EGFR-TKI exposure influences the mechanism of acquired resistance and the appearance of stem cell–like property with EGFR-TKI treatment.
doi:10.1158/0008-5472.CAN-12-4136
PMCID: PMC4506773  PMID: 23542356
11.  Loss of the Notch effector RBPJ promotes tumorigenesis 
Kulic et al. show that RBPJ, a transcriptional repressor of Notch, is frequently deleted in human cancers and can function as a tumor suppressor. Loss of RBPJ acts to derepress target gene promoters, allowing Notch-independent activation by alternate transcription factors that promote tumor growth.
Aberrant Notch activity is oncogenic in several malignancies, but it is unclear how expression or function of downstream elements in the Notch pathway affects tumor growth. Transcriptional regulation by Notch is dependent on interaction with the DNA-binding transcriptional repressor, RBPJ, and consequent derepression or activation of associated gene promoters. We show here that RBPJ is frequently depleted in human tumors. Depletion of RBPJ in human cancer cell lines xenografted into immunodeficient mice resulted in activation of canonical Notch target genes, and accelerated tumor growth secondary to reduced cell death. Global analysis of activated regions of the genome, as defined by differential acetylation of histone H4 (H4ac), revealed that the cell death pathway was significantly dysregulated in RBPJ-depleted tumors. Analysis of transcription factor binding data identified several transcriptional activators that bind promoters with differential H4ac in RBPJ-depleted cells. Functional studies demonstrated that NF-κB and MYC were essential for survival of RBPJ-depleted cells. Thus, loss of RBPJ derepresses target gene promoters, allowing Notch-independent activation by alternate transcription factors that promote tumorigenesis.
doi:10.1084/jem.20121192
PMCID: PMC4291530  PMID: 25512468
12.  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
13.  Unique somatic and malignant expression patterns implicate PIWI-interacting RNAs in cancer-type specific biology 
Scientific Reports  2015;5:10423.
Human PIWI-interacting RNAs (piRNAs) are known to be expressed in germline cells, functionally silencing LINEs and SINEs. Their expression patterns in somatic tissues are largely uncharted. We analyzed 6,260 human piRNA transcriptomes derived from non-malignant and tumour tissues from 11 organs. We discovered that only 273 of the 20,831 known piRNAs are expressed in somatic non-malignant tissues. However, expression patterns of these piRNAs were able to distinguish tissue-of-origin. A total of 522 piRNAs are expressed in corresponding tumour tissues, largely distinguishing tumour from non-malignant tissues in a cancer-type specific manner. Most expressed piRNAs mapped to known transcripts, contrary to “piRNA clusters” reported in germline cells. We showed that piRNA expression can delineate clinical features, such as histological subgroups, disease stages, and survival. PiRNAs common to many cancer types might represent a core gene-set that facilitates cancer growth, while piRNAs unique to individual cancer types likely contribute to cancer-specific biology.
doi:10.1038/srep10423
PMCID: PMC4444957  PMID: 26013764
14.  DNA Methylation Is Globally Disrupted and Associated with Expression Changes in Chronic Obstructive Pulmonary Disease Small Airways 
DNA methylation is an epigenetic modification that is highly disrupted in response to cigarette smoke and involved in a wide spectrum of malignant and nonmalignant diseases, but surprisingly not previously assessed in small airways of patients with chronic obstructive pulmonary disease (COPD). Small airways are the primary sites of airflow obstruction in COPD. We sought to determine whether DNA methylation patterns are disrupted in small airway epithelia of patients with COPD, and evaluate whether changes in gene expression are associated with these disruptions. Genome-wide methylation and gene expression analysis were performed on small airway epithelial DNA and RNA obtained from the same patient during bronchoscopy, using Illumina’s Infinium HM27 and Affymetrix’s Genechip Human Gene 1.0 ST arrays. To control for known effects of cigarette smoking on DNA methylation, methylation and gene expression profiles were compared between former smokers with and without COPD matched for age, pack-years, and years of smoking cessation. Our results indicate that aberrant DNA methylation is (1) a genome-wide phenomenon in small airways of patients with COPD, and (2) associated with altered expression of genes and pathways important to COPD, such as the NF-E2–related factor 2 oxidative response pathway. DNA methylation is likely an important mechanism contributing to modulation of genes important to COPD pathology. Because these methylation events may underlie disease-specific gene expression changes, their characterization is a critical first step toward the development of epigenetic markers and an opportunity for developing novel epigenetic therapeutic interventions for COPD.
doi:10.1165/rcmb.2013-0304OC
PMCID: PMC4068945  PMID: 24298892
chronic obstructive pulmonary disease; small airways; epigenetic regulation; DNA methylation; integrative omics
15.  A search for novel cancer/testis antigens in lung cancer identifies VCX/Y genes expanding the repertoire of potential immunotherapeutic targets 
Cancer research  2014;74(17):4694-4705.
Cancer/testis (CT) antigens are potential immunotherapeutic targets in cancer. However, the expression of particular antigens is limited to a subset of tumors of a given type. Thus, there is a need to identify antigens with complementary expression patterns for effective therapeutic intervention. In this study, we searched for genes that were distinctly expressed at a higher level in lung tumor tissue and the testes compared to other non-tumor tissues and identified members of the VCX/Y gene family as novel CT antigens. VCX3A, a member of the VCX/Y gene family, was expressed at the protein level in approximately 20% of lung adenocarcinomas and 35% of squamous cell carcinomas, but not expressed in normal lung tissues. Among CT antigens with concordant mRNA and protein expression levels, four CT antigens, XAGE1, VCX, IL13RA2, and SYCE1, were expressed, alone or in combination, in about 80% of lung adenocarcinoma tumors. The CT antigen VCX/Y gene family broadens the spectrum of CT antigens expressed in lung adenocarcinomas for clinical applications.
doi:10.1158/0008-5472.CAN-13-3725
PMCID: PMC4398029  PMID: 24970476
Cancer/testis antigen; VCX/Y; lung cancer; immunotherapy
16.  Polycomb-mediated silencing in neuroendocrine prostate cancer 
Clinical Epigenetics  2015;7(1):40.
Background
Neuroendocrine prostate cancer (NEPC) is a highly aggressive subtype of prostate cancer (PCa) for which the median survival remains less than a year. Current treatments are only palliative in nature, and the lack of suitable pre-clinical models has hampered previous efforts to develop novel therapeutic strategies. Addressing this need, we have recently established the first in vivo model of complete neuroendocrine transdifferentiation using patient-derived xenografts. Few genetic differences were observed between parental PCa and relapsed NEPC, suggesting that NEPC likely results from alterations that are epigenetic in nature. Thus, we sought to identify targetable epigenetic regulators whose expression was elevated in NEPC using genome-wide profiling of patient-derived xenografts and clinical samples.
Results
Our data indicate that multiple members of the polycomb group (PcG) family of transcriptional repressors were selectively upregulated in NEPC. Notably, CBX2 and EZH2 were consistently the most highly overexpressed epigenetic regulators across multiple datasets from clinical and xenograft tumor tissues. Given the striking upregulation of PcG genes and other transcriptional repressors, we derived a 185-gene list termed ‘neuroendocrine-associated repression signature’ (NEARS) by overlapping transcripts downregulated across multiple in vivo NEPC models. In line with the striking upregulation of PcG family members, NEARS was preferentially enriched with PcG target genes, suggesting a driving role for PcG silencing in NEPC. Importantly, NEARS was significantly associated with high-grade tumors, metastatic progression, and poor outcome in multiple clinical datasets, consistent with extensive literature linking PcG genes and aggressive disease progression.
Conclusions
We have explored the epigenetic landscape of NEPC and provided evidence of increased PcG-mediated silencing associated with aberrant transcriptional regulation of key differentiation genes. Our results position CBX2 and EZH2 as potential therapeutic targets in NEPC, providing opportunities to explore novel strategies aimed at reversing epigenetic alterations driving this lethal disease.
Electronic supplementary material
The online version of this article (doi:10.1186/s13148-015-0074-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s13148-015-0074-4
PMCID: PMC4391120  PMID: 25859291
Neuroendocrine prostate cancer; Patient-derived xenografts; Epigenetics; Polycomb; CBX2; EZH2; Therapeutics; Prognosis
17.  YEATS4 is a novel oncogene amplified in non-small cell lung cancer that regulates the p53 pathway 
Cancer research  2013;73(24):7301-7312.
Genetic analyses of lung cancer have helped found new treatments in this disease. We conducted an integrative analysis of gene expression and copy number in 261 non-small cell lung cancers (NSCLC) relative to matched normal tissues to define novel candidate oncogenes, identifying 12q13-15 and more specifically the YEATS4 gene as amplified and overexpressed in ~20% of the NSCLC cases examined. Overexpression of YEATS4 abrogated senescence in human bronchial epithelial cells (HBECs). Conversely, RNAi-mediated attenuation of YEATS4 in human lung cancer cells reduced their proliferation and tumor growth, impairing colony formation and inducing cellular senescence. These effects were associated with increased levels of p21WAF1 and p53 and cleavage of PARP, implicating YEATS4 as a negative regulator of the p21-p53 pathway. We also found that YEATS4 expression affected cellular responses to cisplastin, with increased levels associated with resistance and decreased levels with sensitivity. Taken together, our findings reveal YEATS4 as a candidate oncogene amplified in NSCLC, and a novel mechanism contributing to NSCLC pathogenesis.
doi:10.1158/0008-5472.CAN-13-1897
PMCID: PMC3959870  PMID: 24170126
YEATS4; NSCLC; oncogene; p53; integrative analysis
18.  CDKN2A/p16 inactivation mechanisms and their relationship to smoke exposure and molecular features in non-small cell lung cancer 
Introduction
CDKN2A(p16) inactivation is common in lung cancer and occurs via homozygous deletions (HD), methylation of promoter region, or point mutations. While p16 promoter methylation has been linked to KRAS mutation and smoking, the associations between p16 inactivation mechanisms and other common genetic mutations and smoking status are still controversial or unknown.
Methods
We determined all three p16 inactivation mechanisms using multiple methodologies for genomic status, methylation, RNA and protein expression, and correlated them with EGFR, KRAS, STK11 mutations and smoking status in 40 cell lines and 45 tumor samples of primary NSCLC. We also performed meta-analyses to investigate the impact of smoke exposure on p16 inactivation.
Results
p16 inactivation was the major mechanism of RB pathway perturbation in NSCLC, with HD being the most frequent method, followed by methylation and the rarer point mutations. Inactivating mechanisms were tightly correlated with loss of mRNA and protein expression. p16 inactivation occurred at comparable frequencies regardless of mutational status of EGFR, KRAS and STK11, however, the major inactivation mechanism of p16 varied. p16 methylation was linked to KRAS mutation but was mutually exclusive with EGFR mutation. Cell lines and tumor samples demonstrated similar results. Our meta-analyses confirmed a modest positive association between p16 promoter methylation and smoking.
Conclusions
Our results confirm that all of the inactivation mechanisms are truly associated with loss of gene product and identify specific associations between p16 inactivation mechanisms and other genetic changes and smoking status.
doi:10.1097/JTO.0b013e3182a46c0c
PMCID: PMC3951422  PMID: 24077454
p16; CDKN2A; inactivation; homozygous deletion; methylation; lung cancer; adenocarcinoma; meta-analysis
20.  Smoking status impacts microRNA mediated prognosis and lung adenocarcinoma biology 
BMC Cancer  2014;14:778.
Background
Cigarette smoke is associated with the majority of lung cancers: however, 25% of lung cancer patients are non-smokers, and half of all newly diagnosed lung cancer patients are former smokers. Lung tumors exhibit distinct epidemiological, clinical, pathological, and molecular features depending on smoking status, suggesting divergent mechanisms underlie tumorigenesis in smokers and non-smokers. MicroRNAs (miRNAs) are integral contributors to tumorigenesis and mediate biological responses to smoking. Based on the hypothesis that smoking-specific miRNA differences in lung adenocarcinomas reflect distinct tumorigenic processes selected by different smoking and non-smoking environments, we investigated the contribution of miRNA disruption to lung tumor biology and patient outcome in the context of smoking status.
Methods
We applied a whole transcriptome sequencing based approach to interrogate miRNA levels in 94 patient-matched lung adenocarcinoma and non-malignant lung parenchymal tissue pairs from current, former and never smokers.
Results
We discovered novel and distinct smoking status-specific patterns of miRNA and miRNA-mediated gene networks, and identified miRNAs that were prognostically significant in a smoking dependent manner.
Conclusions
We conclude that miRNAs disrupted in a smoking status-dependent manner affect distinct cellular pathways and differentially influence lung cancer patient prognosis in current, former and never smokers. Our findings may represent promising biologically relevant markers for lung cancer prognosis or therapeutic intervention.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-778) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2407-14-778
PMCID: PMC4216369  PMID: 25342220
Lung adenocarcinoma; miRNA; Current smoker; Former smoker; Never smoker; Reversible; Survival; Smoking specific
22.  MicroRNAs As Biomarkers For Clinical Features Of Lung Cancer 
Metabolomics : open access  2012;2(3):1000108-.
Each year about 1.4 million people die from lung cancer worldwide. Despite efforts in prevention, diagnosis and treatment, survival rate remains poor for this disease. This unfortunate situation is largely due to the fact that a high proportion of cases are diagnosed at advanced stages, highlighting the great need for identifying new biomarkers in order to improve early diagnosis and treatment. Recent studies on microRNAs have not only shed light on their involvement in tumor development and progression, but also suggested their potential utility as biomarkers for subtype diagnostics, staging and prediction of treatment response. This review article summarizes the impact of microRNAs on lung cancer biology, and highlights their role in the detection and classification of lung cancer as well as direct targets for drug development.
doi:10.4172/2153-0769.1000108
PMCID: PMC4159950  PMID: 25221729
23.  Unique Pattern of Component Gene Disruption in the NRF2 Inhibitor KEAP1/CUL3/RBX1 E3-Ubiquitin Ligase Complex in Serous Ovarian Cancer 
BioMed Research International  2014;2014:159459.
The NFE2-related factor 2 (NRF2) pathway is critical to initiate responses to oxidative stress; however, constitutive activation occurs in different cancer types, including serous ovarian carcinomas (OVCA). The KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex is a regulator of NRF2 levels. Hence, we investigated the DNA-level mechanisms affecting these genes in OVCA. DNA copy-number loss (CNL), promoter hypermethylation, mRNA expression, and sequence mutation for KEAP1, CUL3, and RBX1 were assessed in a cohort of 568 OVCA from The Cancer Genome Atlas. Almost 90% of cases exhibited loss-of-function alterations in any components of the NRF2 inhibitory complex. CNL is the most prominent mechanism of component disruption, with RBX1 being the most frequently disrupted component. These alterations were associated with reduced mRNA expression of complex components, and NRF2 target gene expression was positively enriched in 90% of samples harboring altered complex components. Disruption occurs through a unique DNA-level alteration pattern in OVCA. We conclude that a remarkably high frequency of DNA and mRNA alterations affects components of the KEAP1/CUL3/RBX1 complex, through a unique pattern of genetic mechanisms. Together, these results suggest a key role for the KEAP1/CUL3/RBX1 complex and NRF2 pathway deregulation in OVCA.
doi:10.1155/2014/159459
PMCID: PMC4121105  PMID: 25114896
24.  The Hedgehog processing pathway is required for NSCLC growth and survival 
Oncogene  2012;32(18):10.1038/onc.2012.243.
Considerable interest has been generated from the results of recent clinical trials using SMOOTHENED (SMO) antagonists to inhibit the growth of HEDGEHOG (HH) signaling dependent tumors. This interest is tempered by the discovery of SMO mutations mediating resistance, underscoring the rationale for developing therapeutic strategies that interrupt HH signaling at levels distinct from those inhibiting SMO function. Here, we demonstrate that HH dependent non-small cell lung carcinoma (NSCLC) growth is sensitive to blockade of the HH pathway upstream of SMO, at the level of HH ligand processing. Individually, the use of different lentivirally delivered shRNA constructs targeting two functionally distinct HH-processing proteins, SKINNY HEDGEHOG (SKN) or DISPATCHED-1 (DISP-1), in NSCLC cell lines produced similar decreases in cell proliferation and increased cell death. Further, providing either an exogenous source of processed HH or a SMO agonist reverses these effects. The attenuation of HH processing, by knocking down either of these gene products, also abrogated tumor growth in mouse xenografts. Finally, we extended these findings to primary clinical specimens, showing that SKN is frequently over-expressed in NSCLC and that higher DISP-1 expression is associated with an unfavorable clinical outcome. Our results show a critical role for HH processing in HH-dependent tumors, identifies two potential druggable targets in the HH pathway, and suggest that similar therapeutic strategies could be explored to treat patients harboring HH ligand dependent cancers.
doi:10.1038/onc.2012.243
PMCID: PMC3821972  PMID: 22733134
DISPATCHED; Lung Cancer; Hedgehog; HEDGEHOG ACYLTRANSFERASE; SKINNY HEDGEHOG
25.  Comprehensive Analysis of HPV16 Integration in OSCC Reveals No Significant Impact of Physical Status on Viral Oncogene and Virally Disrupted Human Gene Expression 
PLoS ONE  2014;9(2):e88718.
Infection with high-risk human papillomavirus (HPV) type 16 is an independent risk factor for the development of oropharyngeal squamous cell carcinomas (OSCC). However, it is unclear whether viral integration is an essential hallmark in the carcinogenic process of OSCC and whether HPV integration correlates with the level of viral gene transcription and influences the expression of disrupted host genes. We analyzed 75 patients with OSCC. HPV16-positivity was proven by p16INK4A immunohistochemistry, PCR and FISH. Viral integration was examined using DIPS- as well as APOT-PCR. Viral E2, E6 and E7 gene expression levels were quantified by quantitative reverse transcriptase (RT-q)PCR. Expression levels of 7 human genes disrupted by the virus were extracted from mRNA expression profiling data of 32 OSCCs. Viral copy numbers were assessed by qPCR in 73 tumors. We identified 37 HPV16-human fusion products indicating viral integration in 29 (39%) OSCC. In the remaining tumors (61%) only episome-derived PCR products were detected. When comparing OSCC with or without an integration-derived fusion product, we did not find significant differences in the mean RNA expression of viral genes E2, E6 and E7 or the viral copy numbers per cell, nor did the RNA expression of the HPV-disrupted genes differ from either group of OSCC. In conclusion, our data do not support the hypothesis that integration affects the levels of viral and/or HPV-disrupted human gene transcripts. Thus constitutive, rather than a high level, of expression of oncogene transcripts appears to be required in HPV-related OSCC.
doi:10.1371/journal.pone.0088718
PMCID: PMC3933331  PMID: 24586376

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