Christina Anderlind

Airway PI3K pathway activation is an early and reversible event in lung cancer development.

A cancer-associated signaling pathway is reversibly activated in the normal airways of smokers before they develop lung cancer, presenting an opportunity for preventive therapy. An Ounce of Prevention for Lung Cancer Lung cancer takes a terrific toll on humankind. Despite our understanding of the contribution of tobacco smoke, this knowledge has not been able to reverse the global increase in lung cancer incidence. New approaches are needed. Is there a way to tell whether a smoker will develop cancer and, even more important, can we see when this process starts so we can stop it? Work from Gustafson and colleagues has defined a biochemical harbinger of cancer in seemingly normal respiratory tissue that can be reversed before cancer begins. Numerous cellular signaling pathways are deregulated in cancers, such as the Ras, p53, and phosphatidylinositol 3-kinase (PI3K) pathways. A molecular understanding of lung cancer may help to develop effective drugs for deterrence. To see whether they could find a predictor of impending cancer, the authors examined normal respiratory tract tissue from smokers with lung cancer or other abnormalities. By looking for previously determined gene expression signatures for various signaling pathways, they found that one of these pathways—PI3K—was clearly activated above normal values. Moreover, the PI3K pathway was already turned on in smokers with abnormal dysplastic lesions, precursors to lung cancer. Lung cancer cells themselves showed even higher expression of the genes in the PI3K pathway. Concluding that elevated PI3K pathway activity precedes the development of lung cancer, the authors assessed gene expression in tissue from patients with dysplasias who had been successfully treated with myo-inositol, an inhibitor of PI3K, finding effective down-regulation of the PI3K pathway. Treatment of cancers with surgery, radiation, and chemotherapy—or, in some cases, targeted molecular therapies—may be the standard of care at present. But prevention should surely be the ultimate goal. The new tool reported in this article—measurement of PI3K pathway activation—and the demonstration that this is an early and reversible step in lung tumorigenesis are hopeful signs. Although only a subset of smokers develop lung cancer, we cannot determine which smokers are at highest risk for cancer development, nor do we know the signaling pathways altered early in the process of tumorigenesis in these individuals. On the basis of the concept that cigarette smoke creates a molecular field of injury throughout the respiratory tract, this study explores oncogenic pathway deregulation in cytologically normal proximal airway epithelial cells of smokers at risk for lung cancer. We observed a significant increase in a genomic signature of phosphatidylinositol 3-kinase (PI3K) pathway activation in the cytologically normal bronchial airway of smokers with lung cancer and smokers with dysplastic lesions, suggesting that PI3K is activated in the proximal airway before tumorigenesis. Further, PI3K activity is decreased in the airway of high-risk smokers who had significant regression of dysplasia after treatment with the chemopreventive agent myo-inositol, and myo-inositol inhibits the PI3K pathway in vitro. These results suggest that deregulation of the PI3K pathway in the bronchial airway epithelium of smokers is an early, measurable, and reversible event in the development of lung cancer and that genomic profiling of these relatively accessible airway cells may enable personalized approaches to chemoprevention and therapy. Our work further suggests that additional lung cancer chemoprevention trials either targeting the PI3K pathway or measuring airway PI3K activation as an intermediate endpoint are warranted.

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.

Airways hyperresponsiveness and the effects of lung inflation

Lung inflation has a beneficial effect on the airways of healthy subjects. It acts as a bronchoprotector, that is to prevent bronchoconstriction, and as a bronchodilator, in that it reverses bronchial obstruction. The bronchoprotective effect of deep inspiration is more potent than the bronchodilatory one, and the two phenomena appear to advocte different mechanisms. Asthmatics and rhinitics with airways hyperresponsiveness show an impairment in bronchoprotection induced by deep breaths, whereas the bronchodilatory effect, although reduced, is still effective. The lack of the bronchoprotective effect of deep inspiration may contribute to the development of airways hyperresponsiveness. The mechanisms through which lung inflation exerts its beneficial role in healthy subjects, and the factors impairing such an effect in those with airways hyperresponsiveness, are currently under investigation.

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.

Shared Gene Expression Alterations in Nasal and Bronchial Epithelium for Lung Cancer Detection

Background: We previously derived and validated a bronchial epithelial gene expression biomarker to detect lung cancer in current and former smokers. Given that bronchial and nasal epithelial gene expression are similarly altered by cigarette smoke exposure, we sought to determine if cancer-associated gene expression might also be detectable in the more readily accessible nasal epithelium. Methods: Nasal epithelial brushings were prospectively collected from current and former smokers undergoing diagnostic evaluation for pulmonary lesions suspicious for lung cancer in the AEGIS-1 (n = 375) and AEGIS-2 (n = 130) clinical trials and gene expression profiled using microarrays. All statistical tests were two-sided. Results: We identified 535 genes that were differentially expressed in the nasal epithelium of AEGIS-1 patients diagnosed with lung cancer vs those with benign disease after one year of follow-up (P < .001). Using bronchial gene expression data from the AEGIS-1 patients, we found statistically significant concordant cancer-associated gene expression alterations between the two airway sites (P < .001). Differentially expressed genes in the nose were enriched for genes associated with the regulation of apoptosis and immune system signaling. A nasal lung cancer classifier derived in the AEGIS-1 cohort that combined clinical factors (age, smoking status, time since quit, mass size) and nasal gene expression (30 genes) had statistically significantly higher area under the curve (0.81; 95% confidence interval [CI] = 0.74 to 0.89, P = .01) and sensitivity (0.91; 95% CI = 0.81 to 0.97, P = .03) than a clinical-factor only model in independent samples from the AEGIS-2 cohort. Conclusions: These results support that the airway epithelial field of lung cancer–associated injury in ever smokers extends to the nose and demonstrates the potential of using nasal gene expression as a noninvasive biomarker for lung cancer detection.

Abstract A1-66: Leveraging Gene Expression in the Bronchial Airway to Develop a Nasal Biomarker for Early Detection of Lung Cancer

Rationale: Lung cancer results in five times more deaths per year than car accidents in the United States. Approximately 57% of lung cancers diagnosed this year will be diagnosed at a late stage and these patients will exhibit a 5-year survival rate of only 4%. Annual screening of high-risk current and former smokers by chest-CT can reduce cancer mortality, however this procedure has a 95% false positive rate. It is therefore critical to develop methods to rapidly and accurately determine which patients with nodules on chest CT have lung cancer and potentially spare those with benign disease an unnecessary invasive procedure. We have previously demonstrated that specific gene expression alterations in cytologically normal bronchial epithelial cells from patients with lung cancer can be leveraged to form a clinically informative lung cancer biomarker in the population of patients undergoing bronchoscopy for suspect lung cancer. We hypothesized that there might be similar expression differences in nasal epithelium and that these could form the basis of a less invasive test that could be applied more broadly to individuals with screen detected nodules on chest CT. Methods: Bronchial (n=676) and nasal (n=280) epithelial brushings were collected from current and former smokers undergoing bronchoscopy for clinical suspicion of lung cancer within the AEGIS clinical trial. 271 subjects had matched bronchial and nasal samples. RNA was extracted and hybridized to Affymetrix Human Gene ST 1.0 Arrays. To establish a connection between bronchial and nasal epithelial gene expression signal for cancer, we first applied the bronchial gene expression-based diagnostic test, BronchoGen, directly to our nasal cohort. Gene Set Enrichment Analysis was then used to determine the concordance of cancer signal between the bronchial and nasal epithelium. To develop the nasal gene expression biomarker for lung cancer detection, we examined the correlation of each gene between the bronchial and nasal epithelium as well as the significance of each gene9s association with cancer in each tissue. Genes passing our selection criteria were passed to a biomarker discovery pipeline in which we examined the performance of different biomarker algorithm configurations (e.g. feature-selection algorithms, classification algorithms, and other biomarker parameters) using cross-validation. Results: Direct application of BronchoGen to our nasal cohort resulted in an AUC of 0.64 on a set of NE samples (n=110) with a matched bronchial sample in the training set used to develop the test. On an independent set of nasal samples (n=109), BronchoGen achieved an AUC of 0.67. Gene Set Enrichment Analysis revealed high levels of concordance between cancer-associated nasal and bronchial gene expression. Using a cross-validation approach, we found that nasal biomarkers built from sets of genes showing significant correlation (p Conclusions: Given the larger sample size, more isolated location in the airway, and higher RIN scores that characterize the bronchial cohort, we sought to leverage bronchial airway epithelial gene-expression to inform which genes in the nasal epithelium should be indicative of the presence of cancer. We have shown that gene expression in the nasal epithelium reflects the presence of lung cancer and can serve as a diagnostic biomarker. We have further demonstrated concordance between bronchial and nasal airway gene expression differences associated with lung cancer. These results suggest the potential to develop a robust nasal gene expression biomarker for lung cancer diagnosis that leverages cancer-associated gene expression differences occurring at other airway sites. Citation Format: Joseph F. Perez-Rogers, Joseph Gerrein, Christina Anderlind, Rebecca L. Kusko, Joshua D. Campbell, Teresa W. Wang, Kate Porta, Duncan Whitney, Avrum Spira, Marc Lenburg. Leveraging Gene Expression in the Bronchial Airway to Develop a Nasal Biomarker for Early Detection of Lung Cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-66.

Abstract 1574: Leveraging bronchial airway gene expression to develop a nasal biomarker for lung cancer detection

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Rationale: Using nasal gene expression to predict the presence of lung cancer would offer a less invasive alternative to diagnostic approaches we have pioneered using bronchial airway epithelial (BE) gene expression. We have previously demonstrated that cytologically normal BE and nasal epithelial (NE) cells harbor gene expression differences that reflect tobacco-related lung disease and that these changes in the BE form the basis of a clinically informative lung cancer biomarker. Given the concordance of BE and NE gene-expression, we hypothesized that gene signatures associated with the presence of lung cancer extend from the airway to the nose and that lung cancer associated BE gene-expression could be leveraged to develop more accurate nasal lung cancer biomarkers. Methods: BE (n = 676) and NE (n = 280) brushings were collected from current and former smokers undergoing bronchoscopy for clinical suspicion of lung cancer. We leveraged two methods to determine the concordance between BE and NE gene-expression signal for cancer. First we applied the bronchial gene expression-based diagnostic test directly to our nasal cohort. Second, we used Gene Set Enrichment Analysis (GSEA) to quantify the relationship between the BE and NE. To develop the nasal gene expression biomarker, we examined the correlation of each gene between the BE and NE. Genes passing our selection criteria were passed to a biomarker discovery pipeline in which we examined the performance of different biomarker algorithm configurations using cross-validation. Results: Direct application of the bronchial airway gene-expression classifier to an independent set of nasal samples (n = 109) resulted in an AUC of 0.67. GSEA revealed high concordance (p<0.001) between cancer-associated nasal and bronchial gene expression profiles from the same patients. Using a cross-validation approach, we found that nasal biomarkers built from sets of genes showing significant correlation (p<0.05) between the BE and NE as well as significance for cancer in both tissues (p<0.05) perform better, on average, than biomarkers built from genes significant for cancer (p<0.05) in the NE alone. Conclusions. We have demonstrated concordance between BE and NE gene expression differences associated with lung cancer. We have further shown that gene expression in the NE reflects the presence of lung cancer and can serve as a diagnostic biomarker. These results demonstrate the feasibility of leveraging cancer-associated gene expression changes throughout the airway to develop a minimally invasive and robust nasal gene expression biomarker for lung cancer diagnosis. Citation Format: Joseph Perez-Rogers, Joseph Gerrein, Christina Anderlind, Xiaohui Xiao, Hanqiao Liu, Rebecca Kusko, Joshua Campbell, Teresa Wang, Yuriy Alekseyev, Gang Liu, Kate Porta, Duncan Whitney, Avrum Spira, Marc Lenburg. Leveraging bronchial airway gene expression to develop a nasal biomarker for lung cancer detection. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1574. doi:10.1158/1538-7445.AM2015-1574

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.

From the Cover: 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.

Abstract 3173: Identification of miR-4423 as a primate-specific microRNA highly expressed in airway epithelium and associated with lung cancer

Smoking is a significant risk factor for lung cancer, the leading cause of cancer-related deaths worldwide. Our group has previously shown that epithelial gene expression is altered throughout the airway of smokers and that some of these changes are regulated by microRNAs. Moreover, we have previously identified gene expression differences in cytologically normal bronchial airway epithelial cells between smokers with and without lung cancer that can serve as an early diagnostic biomarker for lung cancer. Here, we use next-generation sequencing of small RNAs to identify novel microRNAs expressed in airway epithelium and associated with lung cancer. We identify miR-4423 as a primate-specific microRNA highly expressed in the airway epithelium. In vitro, the expression of miR-4423 increases as Normal Human Bronchial Epithelial cells are differentiated into mucociliary epithelium at an Air Liquid Interface, while its mRNA targets decrease in expression. Furthermore, the expression of miR-4423 is reduced in lung tumors and in the cytologically normal bronchial airway epithelium of smokers with lung cancer. In gain-of-function experiments, ectopic expression of miR-4423 in lung cancer cell lines resulted in reduced colony formation in soft agar. Taken together, these data support the power of next-generation sequencing in identifying novel cell type- specific transcripts and provides evidence that this newly characterized microRNA may play a role in promoting the differentiation and/or maintenance of airway epithelium, and can reduce anchorage-independent lung cancer cell growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3173. doi:1538-7445.AM2012-3173