Virtual Library

 x 
My Library Virtual Library FAQ

Start Your Search

W.L. Lam



Author of

  • +

    MO15 - Novel Genes and Pathways (ID 89)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Biology
    • Presentations: 2
    • +

      MO15.09 - Amplification of YEATS4, a novel oncogene in NSCLC, inhibits the p53 pathway and increases resistance to cisplatin (ID 1073)

      17:05 - 17:10  |  Author(s): W.L. Lam

      • Abstract
      • Presentation
      • Slides

      Background
      Characterization of lung cancer genomes has revealed a number of genes critical to tumorigenesis (e.g. EGFR, KRAS, EML4-ALK), resulting in significant changes to the treatment of lung cancer and an increase in survival for a subset of patients. These successes have prompted the search for additional driver alterations, leading to the discovery of a number of recurrently mutated or amplified genes and gene fusions with promising clinical utility. Distinguishing the key mechanisms and causal events driving tumorigenesis will lead not only to a better understanding of lung cancer phenotypes and biology, but also to new molecular markers and therapeutic targets. Using an integrative analysis of gene expression and copy number data to identify novel candidate oncogenes, we identified the chromosomal region at 12q13-15, and more specifically, the putative transcription factor YEATS4 (YEATS domain containing 4) as frequently amplified and overexpressed in NSCLC. Amplification of YEATS4 has been reported in dedifferentiated liposarcomas and in the earliest stages of glioma and astrocytoma.

      Methods
      Copy number profiles were generated for 261 NSCLC tumors (169 adenocarcinomas (AC) and 92 squamous cell carcinomas (SqCC)) and expression profiles for a subset of tumors with matched non-malignant tissue. Recurrent DNA amplifications were identified using the GISTIC algorithm. Copy number data were integrated with gene expression data to identify genes frequently amplified and overexpressed (defined as a 2-fold difference in expression between tumor and matched non-malignant tissue). The functional significance of YEATS4 was assessed by lentiviral knockdown in lung cancer cell lines with and without YEATS4 amplification and ectopic expression in human bronchial epithelial cells (HBECs). In vitro and in vivo assays measuring proliferation, anchorage independent growth, senescence, apoptosis, drug sensitivity and tumor growth were used to assess the phenotypic effect of YEATS4 gene expression manipulation.

      Results
      YEATS4 is gained or amplified and concomitantly overexpressed in over 20% of NSCLC tumors, with similar frequencies of amplification in both AC and SqCC. Although frequently co-amplified with MDM2, amplification of YEATS4 was observed to occur in the absence of MDM2 amplification, suggesting it is not merely a passenger event. Overexpression of YEATS4 in HBECs abrogated senescence, whereas knockdown reduced cell proliferation, impaired colony formation and induced cellular senescence in cell lines with YEATS4 amplification. Western blotting revealed increased p21, cleaved PARP and p53 in knockdown lines compared to empty vector controls, implicating YEATS4 as a negative regulator of the p21-p53 pathway. Moreover, YEAST4 expression was found to correlate with cisplatin sensitivity, as overexpression increased resistance and knockdown conferred sensitivity. Consistent with our in vitro findings, tumor size and growth were significantly reduced in mice injected with YEATS4 knockdown cells relative to control mice. Furthermore, survival analysis revealed that patients expressing high levels of YEATS display poorer outcomes.

      Conclusion
      Our findings reveal YEATS4 as a novel candidate oncogene frequently amplified and overexpressed in NSCLC. Gene expression manipulation resulted in distinct phenotypic changes consistent with oncogenic function, and suggesting YEATS4 amplification is a novel mechanism contributing to NSCLC tumorigenesis.

      Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.

      Only Active Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login or select "Add to Cart" and proceed to checkout.

    • +

      MO15.10 - ELF3 is a novel oncogene frequently activated by genetic and epigenetic mechanisms in lung adenocarcinoma (ID 1024)

      17:10 - 17:15  |  Author(s): W.L. Lam

      • Abstract
      • Presentation
      • Slides

      Background
      Lung cancer remains the cause of the most cancer-related deaths each year, with a 5 year survival rate of less than 15%. The predominant type of lung cancer is non-small cell lung cancer, and the majority of these cases consist of the adenocarcinoma (AC) histology. Oncogenes such as EGFR and KRAS are well defined drivers of AC, but in approximately 50% of cases the driver alterations are unknown. Furthermore, not all defined drivers are drugable. Additional oncogenes are clearly involved in driving this subtype, and must be elucidated to better understand AC biology and improve treatment. ELF3 is an member of the E-Twenty Six (ETS) transcription factor family, which includes several well known oncogenes such as ETS1. Expression of ELF3 is uniquely epithelial-specific, with high expression in fetal but not adult lung tissue. ELF3 overexpression has been reported in a handful of clinical AC cases and cell lines, however a comprehensive analysis of the extent and impact of this overexpression is lacking. Therefore we conducted a multi-'omic, functional analysis of ELF3, and hypothesize ELF3 represents a novel oncogene in lung AC.

      Methods
      ELF3 was interrogated in a multidimensional integrative manner by assessing copy number (SNP 6.0), methylation (Illumina HM27), and expression (Illumina) data from a panel of 83 AC tumors and matched adjacent non-malignant tissues. ELF3 expression was also assessed in The Cancer Genome Atlas (TCGA) public database. Stable ELF3 mRNA knock-down models were established in AC cell lines with high ELF3 expression, and these models were used to assess the role of ELF3 in cell viability and proliferation via MTT and BrdU incorporation assay, respectively. Knock-down models were also used to assess the impact of ELF3 overexpression on tumor growth in vitro and in vivo by soft agar colony formation assay and flank injections of NOD-SCID mice. Subcellular localization of ELF3 was determined by western blot and confirmed with immunofluorescence. In addition, an ELF3 overexpression model was established in immortalized Human Bronchial Epithelial Cells (HBECs) to assess proliferation and soft agar colony formation in a non-malignant model system.

      Results
      ELF3 was found to be frequently overexpressed in our cohort (72%) and the TCGA cohort (80%). This upregulation correlated significantly with high frequencies of sequence gain (49%) and hypomethylation (71%), often seen within the same tumor. In fact, 82% of tumors with ELF3 overexpression had concurrent gain and/or hypomethylation of the ELF3 locus. Knock-down of ELF3 in cell models led to significantly reduced cell viability and proliferation. Western blot and IF revealed ELF3 to be predominantly located in the nucleus, indicating ELF3 likely behaves through its transcription factor activity. A similar hyperproliferative phenotype was seen in the HBEC ELF3 overexpression models.

      Conclusion
      The high frequency of ELF3 overexpression (>70%) observed in lung AC is accompanied by frequent DNA-level selection events. The affect of ELF3 on cell proliferation suggests that ELF3 is a novel oncogene in lung AC. Further studies are warranted to determine the mechanism by which ELF3 drives hyperproliferation and potentially other oncogenic functions to define novel drugable targets for this disease.

      Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.

      Only Active Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login or select "Add to Cart" and proceed to checkout.

  • +

    O12 - Lung Cancer Biology II (ID 87)

    • Event: WCLC 2013
    • Type: Oral Abstract Session
    • Track: Biology
    • Presentations: 1
    • +

      O12.03 - Pseudogenes as miRNA sponges in non-small cell lung cancer (ID 3455)

      10:50 - 11:00  |  Author(s): W.L. Lam

      • Abstract
      • Presentation
      • Slides

      Background
      Lung cancer is the most common cause of cancer death worldwide, with a five-year survival of less than 15%. This poor therapeutic outcome is largely due to complex molecular backgrounds as well as typically late stage at diagnosis, with most patients presenting with unresectable local tumours or metastatic disease. While mutations of driver genes is a well known mechanism of tumorigenesis, approximately half of all non-small cell lung cancer (NSCLC) tumours harbour no known actionable oncogenic drivers, emphasizing the need to explore alternative mechanisms. New sequencing technologies have allowed investigation of previously unexplored areas of the genome and revealed that several classes of non coding RNAs (ncRNAs), those with no protein product, are involved in tumourigenesis, emphasizing the need for further exploration and study. MicroRNAs (miRNAs) have emerged as major players in lung carcinogenesis, displaying both oncogenic and tumor suppressive functions through translational inhibition of genes containing miRNA target sequences. Pseudogenes are non-coding relatives of protein-coding genes that contain a high degree of sequence similarity with their parent genes, thus sharing many of the same miRNA target sequences. As a result, when overexpressed, a pseudogene can function as a miRNA "decoy" protecting its parent gene from miRNA-mediated translational inhibition. DNA copy number (CN) alterations (gain of oncogenes/loss of tumour suppressors), is a major molecular mechanism driving cancer. Like protein coding genes, CN alterations can influence ncRNA expression levels, and several pseudogenes have been reported to be deregulated at the CN level in other cancer types. We hypothesize that pseudogenes of lung cancer-related genes are deregulated at the CN level in NSCLC.

      Methods
      Global CN profiles for 83 lung adenocarcinomas, and 12 squamous cell carcinomas, as well as paired adjacent non-malignant tissues were generated on the Affymetrix SNP 6.0 array. Frequencies of DNA CN alterations were assessed at candidate pseudogene loci (gain>2.3 copies, loss<1.7 copies). Candidate pseudogenes (1) have a parent gene that has been previously reported to play a role in cancer biology, (2) are expressed in human tissue, and (3) share at least one conserved miRNA binding site with its parent gene.

      Results
      Several pseudogenes for OCT4 (octamer-binding transcription factor 4), an early embryonic transcription factor, were found to be frequently gained (46.9-34.9%), and could protect OCT4 from miRNA-mediated translational inhibition. Additionally, pseudogenes for E2F3 (E2F Transcription Factor 3), a potent cell cycle regulator, as well as those for the well known lung cancer oncogene BRAF, were found to have high frequencies of CN alteration (36.1%, and 19.2%, respectively). These high frequencies of alteration suggest that these pseudogenes play an important role in NSCLC.

      Conclusion
      These results suggest that pseudogenes are clonally selected for at the DNA level, and pseudogene-mediated protection of oncogenic transcripts from miRNA-mediated translational inhibition may represent a novel mechanism of oncogenicity in NSCLC. Analyses of pseudogene expression and corresponding parent gene protein level in cell models will yield insight into how this class of ncRNA affects tumourigenesis, potentially leading to improvements in early detection, diagnosis, and treatment.

      Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.

      Only Active Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login or select "Add to Cart" and proceed to checkout.

  • +

    O18 - Cancer Control and Epidemiology II (ID 133)

    • Event: WCLC 2013
    • Type: Oral Abstract Session
    • Track: Prevention & Epidemiology
    • Presentations: 1
    • +

      O18.01 - Multi-'omic analysis of an arsenic-associated lung squamous cell carcinoma reveals specific DNA level signatures (ID 283)

      10:30 - 10:40  |  Author(s): W.L. Lam

      • Abstract
      • Presentation
      • Slides

      Background
      Chronic low-level exposure to arsenic is an emerging cancer risk factor in many parts of the world, including North America. The lung is one anatomical site prominently affected by the carcinogenic effects of arsenic, evident by the striking incidence of lung cancer in never smokers with chronic exposure. Histologically, arsenic related lung tumors are indistinguishable from those induced by other lung carcinogens, and molecularly, arsenic specific DNA copy-number, methylation and expression changes have been identified. Arsenic mediated carcinogenesis occurs through a combination of molecular mechanisms; however, high resolution, multi-'omic analyses of arsenic related tumors have been difficult due to the lack of fresh frozen samples required to obtain high quality DNA and RNA. In this study, we sought to characterize global changes in DNA sequence and methylation levels and their impacts on gene expression in a lung tumor from a patient with chronic arsenic exposure (As-LUSC).

      Methods
      Tumor and non-malignant lung tissues were obtained from a never smoker with lung squamous cell carcinoma (LUSC) who had no family history of lung cancer and 50 years of chronic exposure to high levels of arsenic-contaminated drinking water. Whole genome sequencing was performed and the tumor's mutational signature was compared to those observed in 194 previously characterized NSCLC tumors from the cancer genome atlas (TCGA). DNA methylation was measured using high density methylation arrays and gene expression by RNA sequencing.

      Results
      The As-LUSC exhibited alterations typical of LUSC, such as copy number gains at 3q26 (SOX2 locus) and expression of squamous markers including up-regulation of KRT6B, DSG3, MMP12, KRT5, and down-regulation of PDK4, which are consistent with LUSC histology. However, the As-LUSC harbored a low number of point mutations (only 49 non-synonymous mutations affecting coding DNA sequences) and had a remarkably high fraction of T>G/A>C and low fraction of C>A/G>T transversions, which are features uncharacteristic of LUSCs that suggest arsenic is associated with a distinct mutational spectrum. Furthermore, at the gene level, we identified a G>C mutation in TP53 which is rare in lung tumors (<0.2%) but has been observed in other arsenic-related malignancies. Clustering analysis using ~450,000 methylation probes revealed that the As-LUSC methylation profile was completely distinct from never smoker LUSCs from the TCGA. Of interest, the As-LUSC exhibited lower levels of methylation at CpG islands sores that are not associated with genes, although have been described to exhibit cell type specific methylation patterns.

      Conclusion
      By applying whole genome sequencing, methylation and expression profiling of a LUSC from a never-smoker patient chronically exposed to arsenic, we identified a distinct mutational spectrum and methylation pattern in the As-LUSC. Our results support the concept that arsenic induces lung cancers through mechanisms different from tobacco smoke and other carcinogens. Further study of the mutational profiles of additional arsenic-related cancers is warranted and may yield valuable insight into arsenic associated tumourigenesis, leading to the development of novel diagnostic and therapeutic targets for environmental monitoring and treatment.

      Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.

      Only Active Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login or select "Add to Cart" and proceed to checkout.

  • +

    P1.02 - Poster Session 1 - Novel Cancer Genes and Pathways (ID 144)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
    • +

      P1.02-004 - Differential pathway disruption in lung adenocarcinomas from current and never smokers - A multi-omics data integration analysis (ID 1072)

      09:30 - 09:30  |  Author(s): W.L. Lam

      • Abstract

      Background
      Lung cancers in smokers and never smokers (NS) are distinct clinical diseases. Specific molecular differences identified in these two groups include: EGFR and KRAS mutation, DNA methylation levels at specific loci, and most recently, global mutation spectra. However, much remains to be understood about the biology driving lung tumourigenesis in smokers and NS in order to improve treatment outcome. To date, no multi-dimensional integrative genomics (i.e. multi-omics) analysis designed to specifically compare current (CS) and NS lung tumours has been performed. We hypothesize that a multi-omics analysis which considers each tumour as its own unique perturbed system (as opposed to a grouped approach) will reveal molecular mechanisms of lung adenocarcinoma (AC) biology that are common or different in CS and NS.

      Methods
      Copy number, DNA methylation, and gene expression profiles were generated for lung AC and matched non-malignant lung tissues from 34 CS and 30 NS. PCR was performed to determine EGFR and KRAS mutation status. Copy number, methylation and expression alterations were integrated for 14,000 genes on an individual tumour basis. Disrupted genes were ranked according to the magnitude of alterations they exhibited using a novel algorithm we developed denoted MITRA. Of the genes scored by MITRA, those ranking in the 99th and 1st (top) percentiles for up- and downregulation, respectively, were subjected to Ingenuity Pathway Analysis (IPA). IPA was performed separately on all 64 lung tumours and pathway results for CS and NS were compared.

      Results
      We identified 361 genes that ranked in the top percentiles for up- or downregulation in at least 20% of the lung ACs we assessed. Identification of recurrent RASSF1A downregulation, and EGFR upregulation predominantly in NS demonstrates the ability of our ranking algorithm to prioritize genes known to be involved in lung tumour biology using multi-dimensional genomics data. To determine cellular pathways and functions likely deregulated as a consequence of gene disruption, we performed IPA on each tumour and determined the frequency of individual pathway disruption across tumours. This analysis revealed 88 annotated pathways with a minimum disruption frequency of 15% in either or both CS and NS. Commonly affected pathways involved: adhesion and extravasation implicating tumour invasion and migration; various catabolic and anabolic processes implicating cell metabolism; and several specific signaling pathways including atherosclerosis and Wnt/β-catenin signaling implicating inflammation and cell proliferation. Comparison of the pathways identified in CS and NS revealed 13 differentially disrupted pathways (Fisher's Exact test p < 0.05 and disruption frequency difference > 15%). Eleven pathways were preferentially disrupted in CS and affected metabolic, immune response, and inflammatory pathways. Anandamine degradation and ephrin receptor signaling were preferential to NS.

      Conclusion
      Our novel, multi-omics tumour system based approach revealed genes prominently disrupted in CS and NS lung AC which were associated with several cellular pathways commonly or differentially disrupted in these two groups. Pathways affected by genes disrupted at both the DNA and RNA level may contribute to the distinct clinical characteristics associated with CS and NS lung cancer and may serve as targets for intervention.

  • +

    P2.02 - Poster Session 2 - Novel Cancer Genes and Pathways (ID 148)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
    • +

      P2.02-020 - MicroRNA deregulation associated with node positive lung adenocarcinoma (ID 3426)

      14:53 - 15:10  |  Author(s): W.L. Lam

      • Abstract

      Background
      Lung cancer is the leading cause of cancer-related deaths worldwide, with poor survival largely attributed to late stage of disease at diagnosis and frequent metastasis. Typically, tumor cells colonize the regional lymph nodes before spreading to distant sites, significantly dampening prognosis. The presence and number of affected nodes can be detected by routine imaging procedures, but occasionally, additional affected nodes are not detected until time of surgery, which impacts staging. The identification of biomarkers that stratify node positive (NP) and node negative (NN) patients could improve detection of aggressive disease, while a deeper understanding of the underlying biology of NP lung cancer could lead to the design of novel therapeutic interventions. MicroRNAs (miRNAs) are major regulators of gene expression that control a wide range of cellular processes, and have been shown to be excellent biomarker candidates due to their stability in biofluids. The role of miRNA deregulation in NP lung cancer is poorly understood; therefore, we sought to compare miRNA expression levels of lung adenocarcinoma (AC) cases with and without nodal involvement in order to identify miRNAs associated with tumor aggressiveness. We hypothesize that a subset of miRNAs are specifically deregulated in NP AC, representing potential biomarkers, and that identification of miRNA-mRNA interaction networks will shed light on the biological mechanisms of tumor spread that may be therapeutically exploited.

      Methods
      A panel of 45 NN and 28 NP primary AC tumors were collected along with paired adjacent non-malignant tissues. All samples underwent expression profiling of miRNA (Illumina GAIIx small RNA sequencing) and mRNA (Illumina microarray). NN and NP groups were analyzed separately as follows: matched tumor and normal miRNA normalized read count comparisons were performed (Wilcoxon Signed-Rank test, Bejamini-Hochberg corrected p<0.05), and miRNAs that were significantly deregulated in tumors were further investigated. miRNA fold changes (FC) were calculated on a case by case basis, and FC values were then compared between groups (Mann Whitney U Test p<0.05). miRNAs that (i) displayed a minimum 2-fold frequency of disruption >50% of NP cases, (ii) had a median FC>2 in the NP group, (iii) had a Signed-Rank corrected p<0.05, and (iv) had a MWU p<0.05 were considered to be NP-specific. Target prediction analysis of these miRNAs was then performed, and mRNA expression data was utilized to ensure predicted target expression was anti-correlated with miRNA expression. Candidate targets were input into Ingenuity Pathway Analysis to determine pathways implicated in NP AC.

      Results
      Fourteen miRNAs were NP-specific according to our criteria. These miRNAs included those previously associated with biology and metastasis of epithelial cancers, as well as miRNAs novel to lung AC. Target analysis implicated several pathways and functions previously associated with a metastatic phenotype such as the TGFβ pathway.

      Conclusion
      These results indicate that miRNA expression differs between NP and NN lung AC, further demonstrating the involvement of miRNAs in the regulation of the metastatic process. The role of these miRNAs as prognostic markers and serum biomarkers, as well as the mechanism of action of these miRNAs in AC biology must be further explored.

  • +

    P3.02 - Poster Session 3 - Novel Cancer Genes and Pathways (ID 149)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
    • +

      P3.02-006 - Downregulation of the candidate tumor suppressor gene SIRPA induces senescence mediated by Rb and p27 and is associated with mutation of EGFR (ID 1074)

      09:30 - 09:30  |  Author(s): W.L. Lam

      • Abstract

      Background
      The epidermal growth factor receptor (EGFR) signaling pathway is involved in numerous biological processes including proliferation and apoptosis, migration/invasion, and angiogenesis, and has emerged as one of the most important and frequently deregulated pathways in NSCLC. The discovery of oncogenic, activating mutations in the tyrosine kinase domain of EGFR and DNA amplification of EGFR have led to the development of multiple targeted therapeutics against this pathway. While effective at prolonging survival, these targeted therapies are only applicable to a subset of patients (~15-20%) that harbour these alterations and resistance to treatment ultimately develops. As multiple genomic and epigenomic mechanisms can disrupt genes, a comprehensive understanding of the genetic alterations affecting genes within this pathway is required. An integrative, multi-dimensional genomics approach can detect genes disrupted by multiple mechanisms which may otherwise be overlooked if only a single genomic dimension were assessed, improving the ability to identify causal genetic events and decipher downstream consequences.

      Methods
      A multi-dimensional integrative analysis of copy number, DNA methylation and gene expression profiles on 77 adenocarcinomas and matched non-malignant tissue, was performed to investigate the complement of genetic alterations affecting the EGFR pathway. Novel candidate genes were validated in external datasets and immunohistochemical analysis of a tissue microarray was used to verify disruption at the protein level and to correlate expression with clinical features. The tumor suppressive effects of SIRPA were assessed by stable knockdown and in vitro assays on a panel of lung cancer cell lines. The effect of SIRPA downregulation on TKI sensitivity was assessed by dose response assays.

      Results
      Of the 35 genes examined, 11 were aberrantly expressed in over 50% of tumors, with 6 (RRAS, SIRPA, PIK3R1, TGFA, ERBB2 and EGFR) ranking in the 95th percentile of altered genes. Of these genes, all but SIRPA are known to be frequently disrupted in NSCLC and play a role in tumorigenesis. SIRPA is a transmembrane protein that negatively regulates receptor tyrosine kinsase activity and is frequently downregulated at both the mRNA and protein level in NSCLC tumors and cell lines. Underexpression of SIRPA is associated with EGFR mutations and is more prominent in adenocarcinoma than squamous cell carcinoma. Downregulation of SIRPA enhanced colony formation and wound healing but impaired viability and suppressed proliferation. Interestingly, SIRPA knockdown induced a senescent phenotype through the accumulation of p27 and Rb in its unphosphorylated state thereby blocking progression of the cell cycle. These results suggest senescence induced by SIRPA downregulation is a tumor suppressive mechanism that must be overcome to develop tumors.

      Conclusion
      Our integrative analysis of the EGFR pathway revealed SIRPA as one of the most frequently deregulated genes within the pathway. SIRPA functions as a tumor suppressor gene, controlling a number of biological functions through the inhibition of singaling pathways downstream of EGFR. To our knowledge, this is the first study to report a role for SIRPA in NSCLC tumorigenesis.