Jessica Vick

Characterizing the Impact of Smoking and Lung Cancer on the Airway Transcriptome Using RNA-Seq

Cigarette smoke creates a molecular field of injury in epithelial cells that line the respiratory tract. We hypothesized that transcriptome sequencing (RNA-Seq) will enhance our understanding of the field of molecular injury in response to tobacco smoke exposure and lung cancer pathogenesis by identifying gene expression differences not interrogated or accurately measured by microarrays. We sequenced the high-molecular-weight fraction of total RNA (>200 nt) from pooled bronchial airway epithelial cell brushings (n = 3 patients per pool) obtained during bronchoscopy from healthy never smoker (NS) and current smoker (S) volunteers and smokers with (C) and without (NC) lung cancer undergoing lung nodule resection surgery. RNA-Seq libraries were prepared using 2 distinct approaches, one capable of capturing non-polyadenylated RNA (the prototype NuGEN Ovation RNA-Seq protocol) and the other designed to measure only polyadenylated RNA (the standard Illumina mRNA-Seq protocol) followed by sequencing generating approximately 29 million 36 nt reads per pool and approximately 22 million 75 nt paired-end reads per pool, respectively. The NuGEN protocol captured additional transcripts not detected by the Illumina protocol at the expense of reduced coverage of polyadenylated transcripts, while longer read lengths and a paired-end sequencing strategy significantly improved the number of reads that could be aligned to the genome. The aligned reads derived from the two complementary protocols were used to define the compendium of genes expressed in the airway epithelium (n = 20,573 genes). Pathways related to the metabolism of xenobiotics by cytochrome P450, retinol metabolism, and oxidoreductase activity were enriched among genes differentially expressed in smokers, whereas chemokine signaling pathways, cytokine–cytokine receptor interactions, and cell adhesion molecules were enriched among genes differentially expressed in smokers with lung cancer. There was a significant correlation between the RNA-Seq gene expression data and Affymetrix microarray data generated from the same samples (P < 0.001); however, the RNA-Seq data detected additional smoking- and cancer-related transcripts whose expression was were either not interrogated by or was not found to be significantly altered when using microarrays, including smoking-related changes in the inflammatory genes S100A8 and S100A9 and cancer-related changes in MUC5AC and secretoglobin (SCGB3A1). Quantitative real-time PCR confirmed differential expression of select genes and non-coding RNAs within individual samples. These results demonstrate that transcriptome sequencing has the potential to provide new insights into the biology of the airway field of injury associated with smoking and lung cancer. The measurement of both coding and non-coding transcripts by RNA-Seq has the potential to help elucidate mechanisms of response to tobacco smoke and to identify additional biomarkers of lung cancer risk and novel targets for chemoprevention. Cancer Prev Res; 4(6); 803–17. ©2011 AACR.

Molecular Profiling of Premalignant Lesions in Lung Squamous Cell Carcinomas Identifies Mechanisms Involved in Stepwise Carcinogenesis

Lung squamous cell carcinoma (SCC) is thought to arise from premalignant lesions in the airway epithelium; therefore, studying these lesions is critical for understanding lung carcinogenesis. Previous microarray and sequencing studies designed to discover early biomarkers and therapeutic targets for lung SCC had limited success identifying key driver events in lung carcinogenesis, mostly due to the cellular heterogeneity of patient samples examined and the interindividual variability associated with difficult to obtain airway premalignant lesions and appropriate normal control samples within the same patient. We performed RNA sequencing on laser-microdissected representative cell populations along the SCC pathologic continuum of patient-matched normal basal cells, premalignant lesions, and tumor cells. We discovered transcriptomic changes and identified genomic pathways altered with initiation and progression of SCC within individual patients. We used immunofluorescent staining to confirm gene expression changes in premalignant lesions and tumor cells, including increased expression of SLC2A1, CEACAM5, and PTBP3 at the protein level and increased activation of MYC via nuclear translocation. Cytoband enrichment analysis revealed coordinated loss and gain of expression in chromosome 3p and 3q regions, respectively, during carcinogenesis. This is the first gene expression profiling study of airway premalignant lesions with patient-matched SCC tumor samples. Our results provide much needed information about the biology of premalignant lesions and the molecular changes that occur during stepwise carcinogenesis of SCC, and it highlights a novel approach for identifying some of the earliest molecular changes associated with initiation and progression of lung carcinogenesis within individual patients. Cancer Prev Res; 7(5); 487–95. ©2014 AACR.

MicroRNA 4423 is a primate-specific regulator of airway epithelial cell differentiation and lung carcinogenesis

Significance MicroRNAs are small noncoding RNAs that negatively regulate gene expression and have been implicated in a variety of cellular processes. Using small RNA sequencing, we identified microRNA 4423 (miR-4423) as a primate-specific microRNA whose expression is largely restricted to airway epithelium and which functions as a regulator of airway epithelium differentiation and a repressor of lung carcinogenesis. Understanding miR-4423’s role in airway development may provide insights into primate-specific aspects of airway biology and the evolution of primate-specific tumor suppressors. Moreover, this study opens the possibility that microRNAs might be useful for the early detection of lung cancer in the proximal airway and that miR-4423 mimetics might also be used as therapeutic agents to specifically target lung cancer. Smoking is a significant risk factor for lung cancer, the leading cause of cancer-related deaths worldwide. Although microRNAs are regulators of many airway gene-expression changes induced by smoking, their role in modulating changes associated with lung cancer in these cells remains unknown. Here, we use next-generation sequencing of small RNAs in the airway to identify microRNA 4423 (miR-4423) as a primate-specific microRNA associated with lung cancer and expressed primarily in mucociliary epithelium. The endogenous expression of miR-4423 increases as bronchial epithelial cells undergo differentiation into mucociliary epithelium in vitro, and its overexpression during this process causes an increase in the number of ciliated cells. Furthermore, expression of miR-4423 is reduced in most lung tumors and in cytologically normal epithelium of the mainstem bronchus of smokers with lung cancer. In addition, ectopic expression of miR-4423 in a subset of lung cancer cell lines reduces their anchorage-independent growth and significantly decreases the size of the tumors formed in a mouse xenograft model. Consistent with these phenotypes, overexpression of miR-4423 induces a differentiated-like pattern of airway epithelium gene expression and reverses the expression of many genes that are altered in lung cancer. Together, our results indicate that miR-4423 is a regulator of airway epithelium differentiation and that the abrogation of its function contributes to lung carcinogenesis.

Translating the transcriptome into tools for the early detection and prevention of lung cancer

Despite advances in the management of lung cancer, this disease remains a significant global health burden with survival rates that have not significantly improved in decades. The mortality reduction achieved by low-dose helical CT (LDCT) screening of select high-risk patients is challenged by the high false positive rate of this screening modality and the potential for morbidity associated with follow-up diagnostic evaluation in patients with high risk for iatrogenic complications. The diagnostic dilemma of the indeterminate nodule incidentally identified on diagnostic or screening CT has created a need for reliable biomarkers capable of distinguishing benign from malignant disease. Furthermore, there is an urgent need to develop molecular biomarkers to supplement clinical risk models in order to identify patients at highest risk for having an early stage lung cancer that may derive the greatest benefit from LDCT screening, as well as identifying patients at high-risk for developing lung cancer that may be candidates for emerging chemopreventive strategies. Evolving bioinformatic techniques and the application of these algorithms to analyse the transcriptomic changes associated with lung cancer promise translational discoveries that can bridge these large clinical gaps. The identification of lung cancer associated transcriptomic alterations in readily accessible tissue sampling sites offers the potential to develop early diagnostic and risk stratification strategies applicable to large populations. This review summarises the challenges associated with the early detection, screening and chemoprevention of lung cancer with an emphasis on how genomic information encapsulated by the transcriptome can facilitate future innovations in these clinical settings.

Genomic approaches to accelerate cancer interception

Although major advances have been reported in the last decade in the treatment of late-stage cancer with targeted and immune-based therapies, there is a crucial unmet need to develop new approaches to improve the prevention and early detection of cancer. Advances in genomics and computational biology offer unprecedented opportunities to understand the earliest molecular events associated with carcinogenesis, enabling novel strategies to intercept the development of invasive cancers. This Series paper will highlight emerging big data genomic approaches with the potential to accelerate advances in cancer prevention, screening, and early detection across various tumour types, and the challenges inherent in the development of these tools for clinical use. Through coordinated multicentre consortia, these genomic approaches are likely to transform the landscape of cancer interception in the coming years.

Abstract 2878: Development of the pre-cancer genome atlas (PCGA) for squamous cell lung carcinoma

Squamous cell cancer (SCC) of the lung is a leading cause of cancer mortality in the US, due to late stage diagnosis and lack of effective treatments. Lung SCC arises in the epithelial layer of the bronchial airways and is often preceded by the development of premalignant lesions (PMLs). The molecular events involved in the progression of PMLs to lung SCC are not clearly understood and not all PMLs go on to form carcinoma. By molecularly characterizing PMLs and non-lesion areas in the airway of individuals with PMLs we hypothesize that we will be able to identify early events in the process of lung carcinogenesis that lead to SCC. We used next-generation sequencing to profile bronchial brushings and biopsies obtained from high-risk smokers undergoing lung cancer screening by auto-fluorescence bronchoscopy and CT at the Roswell Park Cancer Institute in Buffalo, NY. For each subject (n = 26), we sampled the PML(s) and the mainstem bronchus repeatedly over time (394 +/- 170 days) with serial bronchoscopies (5 +/- 3 biopsies/subject) as the PML progressed towards or regressed away from frank malignancy. mRNA-Seq (n = 192) and miRNA-Seq (n = 183) were performed on the endobronchial biopsies and brushings and exome-Seq was performed on blood DNA from these subjects. RNA-seq data was aligned to the hg19 and gene/transcript levels were summarized using RSEM/Ensembl 74 or Bedtools/ mirBase 18. Single nucleotide variants were quantified using a modified PRADA pipeline and GATK. We identified gene and miRNA expression changes as well as pathways that are associated with biopsy histological grade as well as progressive/stable disease. HE 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2878. doi:10.1158/1538-7445.AM2015-2878

Molecular Subtyping reveals Immune Alterations associated with Progression of Bronchial Premalignant Lesions

Bronchial premalignant lesions (PMLs) are precursors of lung squamous cell carcinoma, but have variable outcome, and we lack tools to identify and treat PMLs at highest risk for progression to invasive cancer. Profiling endobronchial biopsies of PMLs obtained from high-risk smokers by RNA-Seq identified four PML subtypes with differences in epithelial and immune processes. One molecular subtype (Proliferative) is enriched with dysplastic lesions and exhibits up-regulation of metabolic and cell cycle pathways and down-regulation of ciliary processes. RNA-Seq profiles from normal-appearing uninvolved large airway brushings could identify subjects with Proliferative lesions with high specificity. Expression of interferon signaling and antigen processing/presentation pathways are decreased in progressive/persistent Proliferative lesions and immunofluorescence indicates a depletion of innate and adaptive immune cells in these lesions. Molecular biomarkers measured in PMLs or the uninvolved airway can enhance histopathological grading and suggests that immunoprevention strategies may be effective in intercepting the progression of PMLs to lung cancer.

Abstract A05: Bronchial premalignant lesions have distinct molecular subtypes associated with future histologic progression

Squamous cell carcinoma (SCC) of the lung is a leading cause of cancer mortality in the U.S. due to late-stage diagnosis and lack of effective treatments. Lung SCC arises in the epithelial layer of the bronchial airways and is often preceded by the development of premalignant lesions (PMLs). The molecular alterations involved in the progression of PMLs to lung SCC are not clearly understood as not all PMLs progress to carcinoma. We hypothesize that molecular characterization of PMLs and nonlesion areas will allow us to identify alterations associated with histology and lesion progression. We used mRNA sequencing to profile biopsies obtained from high-risk smokers undergoing lung cancer screening by auto-fluorescence bronchoscopy and CT at the Roswell Park Cancer Institute in Buffalo, NY. For each subject (n=49), a brushing of the airway field (normal fluorescing area) and endobronchial biopsies were collected over time in repeat locations with serial bronchoscopies. The discovery cohort, included 29 subjects, 197 biopsies, and 91 brushes, while the validation cohort included 20 subjects, 111 biopsies and 49 brushes. The mRNA-Seq data were aligned to hg19 using STAR, and gene/transcript levels were summarized using RSEM. Immune, stromal, and epithelial cell content were inferred using xCell. Biopsy molecular subtypes were discovered using consensus clustering in the discovery cohort and used to train a nearest centroid subtype predictor to assign subtypes in the validation cohort and the brushes. We identified four distinct molecular subtypes in the discovery cohort bronchial biopsies using genes (n=3936) co-expressed across the the discovery cohort brushes and biopsies and two additional RNA-seq lung SCC-related datasets. One of the four molecular subtypes is enriched (p We have identified four molecular subclasses of premalignant lung SCC lesions that may associate with prognosis. Molecular classification of PMLs may lead to biomarkers of future disease progression that could be used to stratify patients into prevention trials and to monitor efficacy of the treatment. Additionally, the results suggest that personalized lung cancer chemoprevention that targets specific cancer-related pathways or the immune system may have potential therapeutic benefits. Citation Format: Jennifer E. Beane, Sarah Mazzilli, Ania Tassinari, Joshua Campbell, Christopher Moy, Michael Schaffer, Catalina Perdomo, David Jenkins, Mary Beth Pine, Gang Liu, Sherry Zhang, Hangqio Lin, Jessica Vick, Evan Johnson, Suso Platero, Christopher Stevenson, Marc Lenburg, Mary Reid, Samjot Dhillon, Avrum Spira. Bronchial premalignant lesions have distinct molecular subtypes associated with future histologic progression [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A05.

The Airway Transcriptome as a Biomarker for Early Lung Cancer Detection

Lung cancer remains the leading cause of cancer-related death due to its advanced stage at diagnosis. Early detection of lung cancer can be improved by better defining who should be screened radiographically and determining which imaging-detected pulmonary nodules are malignant. Gene expression biomarkers measured in normal-appearing airway epithelium provide an opportunity to use lung cancer–associated molecular changes in this tissue for early detection of lung cancer. Molecular changes in the airway may result from an etiologic field of injury and/or field cancerization. The etiologic field of injury reflects the aberrant physiologic response to carcinogen exposure that creates a susceptible microenvironment for cancer initiation. In contrast, field cancerization reflects effects of “first-hit” mutations in a clone of cells from which the tumor ultimately arises or the effects of the tumor on the surrounding tissue. These fields might have value both for assessing lung cancer risk and diagnosis. Cancer-associated gene expression changes in the bronchial airway have recently been used to develop and validate a 23-gene classifier that improves the diagnostic yield of bronchoscopy for lung cancer among intermediate-risk patients. Recent studies have demonstrated that these lung cancer–related gene expression changes extend to nasal epithelial cells that can be sampled noninvasively. While the bronchial gene expression biomarker is being adopted clinically, further work is necessary to explore the potential clinical utility of gene expression profiling in the nasal epithelium for lung cancer diagnosis, lung cancer risk assessment, and precision medicine for lung cancer treatment and chemoprevention. Clin Cancer Res; 24(13); 2984–92. ©2018 AACR.

MicroRNA 4423 is a primate-specific regulator of airway epithelial cell differentiation and lung carcinogenesis - eScholarship

Significance MicroRNAs are small noncoding RNAs that negatively regulate gene expression and have been implicated in a variety of cellular processes. Using small RNA sequencing, we identified microRNA 4423 (miR-4423) as a primate-specific microRNA whose expression is largely restricted to airway epithelium and which functions as a regulator of airway epithelium differentiation and a repressor of lung carcinogenesis. Understanding miR-4423’s role in airway development may provide insights into primate-specific aspects of airway biology and the evolution of primate-specific tumor suppressors. Moreover, this study opens the possibility that microRNAs might be useful for the early detection of lung cancer in the proximal airway and that miR-4423 mimetics might also be used as therapeutic agents to specifically target lung cancer. Smoking is a significant risk factor for lung cancer, the leading cause of cancer-related deaths worldwide. Although microRNAs are regulators of many airway gene-expression changes induced by smoking, their role in modulating changes associated with lung cancer in these cells remains unknown. Here, we use next-generation sequencing of small RNAs in the airway to identify microRNA 4423 (miR-4423) as a primate-specific microRNA associated with lung cancer and expressed primarily in mucociliary epithelium. The endogenous expression of miR-4423 increases as bronchial epithelial cells undergo differentiation into mucociliary epithelium in vitro, and its overexpression during this process causes an increase in the number of ciliated cells. Furthermore, expression of miR-4423 is reduced in most lung tumors and in cytologically normal epithelium of the mainstem bronchus of smokers with lung cancer. In addition, ectopic expression of miR-4423 in a subset of lung cancer cell lines reduces their anchorage-independent growth and significantly decreases the size of the tumors formed in a mouse xenograft model. Consistent with these phenotypes, overexpression of miR-4423 induces a differentiated-like pattern of airway epithelium gene expression and reverses the expression of many genes that are altered in lung cancer. Together, our results indicate that miR-4423 is a regulator of airway epithelium differentiation and that the abrogation of its function contributes to lung carcinogenesis.