Journal of Experimental & Clinical Cancer Research
Background Bronchial premalignant lesions (PMLs) are composed primarily of cells resembling basal... more Background Bronchial premalignant lesions (PMLs) are composed primarily of cells resembling basal epithelial cells of the airways, which through poorly understood mechanisms have the potential to progress to lung squamous cell carcinoma (LUSC). Despite ongoing efforts that have mapped gene expression and cell diversity across bronchial PML pathologies, signaling and transcriptional events driving malignancy are poorly understood. Evidence has suggested key roles for the Hippo pathway effectors YAP and TAZ and associated TEAD and TP63 transcription factor families in bronchial basal cell biology and LUSC. In this study we examine the functional association of YAP/TAZ, TEADs and TP63 in bronchial epithelial cells and PMLs. Methods We performed RNA-seq in primary human bronchial epithelial cells following small interfering RNA (siRNA)-mediated depletion of YAP/TAZ, TEADs or TP63, and combined these data with ChIP-seq analysis of these factors. Directly activated or repressed genes were...
Molecular events that drive the development of precancerous lesions in the bronchial epithelium, ... more Molecular events that drive the development of precancerous lesions in the bronchial epithelium, which are precursors of lung squamous cell carcinoma (LUSC), are poorly understood. We demonstrate that disruption of epithelial cellular polarity, via the conditional deletion of the apical determinant Crumbs3 (Crb3), initiates and sustains precancerous airway pathology. The loss of Crb3 in adult luminal airway epithelium promotes the uncontrolled activation of the transcriptional regulators YAP and TAZ, which stimulate intrinsic signals that promote epithelial cell plasticity and paracrine signals that induce basal-like cell growth. We show that aberrant polarity and YAP/TAZ-regulated gene expression associates with human bronchial precancer pathology and disease progression. Analyses of YAP/TAZ-regulated genes further identified the ERBB receptor ligand Neuregulin-1 (NRG1) as a key transcriptional target and therapeutic targeting of ERBB receptors as a means of preventing and treating...
Vaping is suggested to be a risk factor for poor wound healing akin to smoking. However, the mole... more Vaping is suggested to be a risk factor for poor wound healing akin to smoking. However, the molecular and histologic mechanisms underlying this postulation remain unknown. Our study sought to compare molecular and histologic changes in cutaneous flap and non-flap tissue between vaping, smoking and control cohorts. Animal study of 15 male Sprague-Dawley rats was randomized to three cohorts: negative control (n = 5), e-cigarette (n = 5) and cigarette (n = 5) and exposed to their respective treatments with serum cotinine monitoring. After 30 days, random pattern flaps were raised and healed for 2 weeks after which skin punch biopsies of flap and non-flap tissues were collected for quantitative-reverse transcription-polymerase chain reaction of three selected wound healing genes (transforming growth factor β [TGF-β], vascular endothelial growth factor [VEGF], matrix metalloproteinase-1 [MMP-1]); then, immunohistochemistry for CD68 expression, α-smooth muscle actin looking at microvessel density (MVD) and in situ hybridization to localize VEGF production were undertaken. In flap tissue, vaping (mean[SEM]) (0.61[0.07]) and smoking (0.70[0.04]) were associated with decreased fold change of VEGF expression compared with controls (0.91[0.03]) (p < 0.05, p < 0.05, respectively). In non-flap tissue, only vaping was associated with decreased VEGF expression (mean[SEM]) (0.81[0.07]), compared with controls (1.17[0.10]) (p < 0.05) with expression primarily localized to basal keratinocytes and dermal capillaries. Immunohistochemistry showed decreased MVD in smoking (0.27[0.06]) and vaping (0.26[0.04]) flap tissue compared to matched controls (0.65[0.14]) (p < 0.05, p < 0.05, respectively) and decreased areas of fibrosis compared with controls on gross histology. Vaping and smoking were similarly associated with decreased VEGF expression, MVD and fibrotic changes in flap tissue. The results suggest attenuated angiogenesis via decreased VEGF expression as a mechanism for poor wound healing in vaping-exposed rats.
Background: SARS-CoV-2 infection and disease severity are influenced by viral entry (VE) gene exp... more Background: SARS-CoV-2 infection and disease severity are influenced by viral entry (VE) gene expression patterns in airway epithelium. The similarities and differences of VE gene expression (ACE2, TMPRSS2, and CTSL) across nasal and bronchial compartments has not been fully characterized using matched samples from large cohorts. Results: Gene expression data from 793 nasal and 1,673 bronchial brushes obtained from individuals participating in lung cancer screening or diagnostic workup revealed that smoking was the only clinical factor significantly and reproducibly associated with VE gene expression. ACE2 and TMPRSS2 expression were higher in smokers in the bronchus but not in the nose. scRNA-seq of nasal brushings indicated that ACE2 co-expressed genes were highly expressed in club and C15orf48+ secretory cells while TMPRSS2 co-expressed genes were highly expressed in keratinizing epithelial cells. In contrast, these ACE2 and TMPRSS2 modules were highly expressed in goblet cells i...
ObjectiveThe immune response to invasive carcinoma has been the focus of published work, but litt... more ObjectiveThe immune response to invasive carcinoma has been the focus of published work, but little is known about the adaptive immune response to bronchial premalignant lesions (PMLs), precursors of lung squamous cell carcinoma. This study was designed to characterize the T cell receptor (TCR) repertoire in PMLs and its association with clinical, pathological, and molecular features.MethodsEndobronchial biopsies (n=295) and brushings (n=137) from high-risk subjects (n=50), undergoing lung cancer screening at approximately 1-year intervals via autofluorescence bronchoscopy and CT, were profiled by RNA-seq. We applied the TCR Repertoire Utilities for Solid Tissue/Tumor (TRUST) tool to the RNA-seq data to identify TCR CDR3 sequences across all samples. In the biopsies, we measured the correlation of TCR diversity with previously derived immune-associated PML transcriptional signatures and outcome. We also quantified the spatial and temporal distribution of shared and clonally expanded...
SummaryThere is an increasing appreciation for the heterogeneity of myeloid lineages in the respi... more SummaryThere is an increasing appreciation for the heterogeneity of myeloid lineages in the respiratory system, but whether distinct populations associate with the conducting airways remains unknown. We use single cell RNA sequencing, flow cytometry and immunofluorescence to characterize myeloid cells of the mouse trachea during homeostasis and epithelial injury/repair. We identify submucosal macrophages that are similar to lung interstitial macrophages and intraepithelial macrophages, and find that repair of the tracheal epithelium is impaired in Ccr2-deficient mice. Following injury there are early increases in neutrophils and submucosal macrophages, including M2-like macrophages. Unexpectedly, intraepithelial macrophages are initially lost but later replaced from CCR2+ monocytes. Mast cells and group 2 innate lymphoid cells are sources of IL13 that polarizes macrophages and directly influences basal cell behaviors. Their proximity to the airway epithelium establishes these myeloi...
102 Background: The immune response to frank malignancy has been the focus of published work, but... more 102 Background: The immune response to frank malignancy has been the focus of published work, but little is known about the adaptive immune response to bronchial premalignant lesions (PMLs), precursors of lung squamous cell carcinoma. This study was designed to characterize the T cell receptor (TCR) repertoire in PMLs and its association with clinical, pathological and molecular features. Methods: Endobronchial biopsies (n=294) and brushings (n=137) from high-risk subjects (n=50), undergoing lung cancer screening at approximately 1-year intervals via auto fluorescence bronchoscopy and CT, were profiled by RNA-Seq as part of the Pre-Cancer Genome Atlas (PCGA). We applied the TCR Repertoire Utilities for Solid Tissue/Tumor (TRUST) tool to identify TCR CDR3 sequences in the RNA-Seq data. We quantified the ratio of private (i.e., found in one patient only) and public (i.e. found in two or more patients) TCRs that are found in curated databases with known antigen specificities. We measur...
Lung cancer is the leading cause of cancer mortality due in part to an inability to diagnose and ... more Lung cancer is the leading cause of cancer mortality due in part to an inability to diagnose and intercept the disease at its earliest and potentially most curable stage. In order to improve lung cancer prevention and early detection, a detailed understanding of both the genomic alterations driving carcinogenesis and the mechanisms by which altered cells escape immune surveillance is required. Toward this goal, our group is profiling RNA and DNA from bronchial premalignant lesions (PMLs), precursors of lung squamous cell carcinoma (LUSCC), to identify alterations associated with their development and progression. Results from the analysis of 197 human endobronchial biopsies representing all histologic grades profiled by RNA-Seq, identified a class of high-grade PMLs with high activity of pathways associated with epithelial proliferation and differentiation. Among this class of PMLs, progression-associated pathways included immunoproteasome, antigen processing, immune inhibition, and macrophage activation. Further elucidation of the epithelial and immune changes that characterize progression of PMLs and testing of interception strategies based on these human findings require a preclinical model with similar histology and molecular alterations. The N-nitrosotris-(2-choroethyl)urea (NTCU) mouse model of LUSCC is a promising preclinical model that develops histologically comparable PMLs to those found in humans; however, the molecular alterations associated with these histologic changes have not yet been characterized. In order to establish molecular concordance between NTCU-treated mouse and human PMLs, we isolated RNA from 40 fresh-frozen whole lung sections and laser capture microdissected (LCM) tissue from SWR/J and A/J mice treated with NTCU. We profiled by RNA-Seq tissue that represented a range of histologic grades including normal (n=9), metaplasia/mild dysplasia (n=8), moderate/severe dysplasia (n=19), and carcinoma in situ/SCC (n=4). RNA-Seq data was aligned to mm9 and gene-level counts were summarized using Ensembl annotation. After removing samples of low quality, linear modeling adjusting for sample quality, sample type, and mouse strain was used to identify differentially expressed genes associated with histologic grade. Immune cell abundance was estimated using CIBERSORT and EnrichR was used to identify pathways associated with histologic grade. Concordance between the mouse data and human data was established using GSEA and GSVA. We identified 596 genes differentially expressed with increasing histologic severity across all mouse samples (FDR Citation Format: Sarah Mazzilli, Sherry Zhang, Gang Liu, Marc Lenburg, Avrum Spira, Jennifer Beane. The carcinogen-induced NTCU model: A preclinical mouse model for lung cancer interception [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B18.
Journal of Experimental & Clinical Cancer Research
Background Bronchial premalignant lesions (PMLs) are composed primarily of cells resembling basal... more Background Bronchial premalignant lesions (PMLs) are composed primarily of cells resembling basal epithelial cells of the airways, which through poorly understood mechanisms have the potential to progress to lung squamous cell carcinoma (LUSC). Despite ongoing efforts that have mapped gene expression and cell diversity across bronchial PML pathologies, signaling and transcriptional events driving malignancy are poorly understood. Evidence has suggested key roles for the Hippo pathway effectors YAP and TAZ and associated TEAD and TP63 transcription factor families in bronchial basal cell biology and LUSC. In this study we examine the functional association of YAP/TAZ, TEADs and TP63 in bronchial epithelial cells and PMLs. Methods We performed RNA-seq in primary human bronchial epithelial cells following small interfering RNA (siRNA)-mediated depletion of YAP/TAZ, TEADs or TP63, and combined these data with ChIP-seq analysis of these factors. Directly activated or repressed genes were...
Molecular events that drive the development of precancerous lesions in the bronchial epithelium, ... more Molecular events that drive the development of precancerous lesions in the bronchial epithelium, which are precursors of lung squamous cell carcinoma (LUSC), are poorly understood. We demonstrate that disruption of epithelial cellular polarity, via the conditional deletion of the apical determinant Crumbs3 (Crb3), initiates and sustains precancerous airway pathology. The loss of Crb3 in adult luminal airway epithelium promotes the uncontrolled activation of the transcriptional regulators YAP and TAZ, which stimulate intrinsic signals that promote epithelial cell plasticity and paracrine signals that induce basal-like cell growth. We show that aberrant polarity and YAP/TAZ-regulated gene expression associates with human bronchial precancer pathology and disease progression. Analyses of YAP/TAZ-regulated genes further identified the ERBB receptor ligand Neuregulin-1 (NRG1) as a key transcriptional target and therapeutic targeting of ERBB receptors as a means of preventing and treating...
Vaping is suggested to be a risk factor for poor wound healing akin to smoking. However, the mole... more Vaping is suggested to be a risk factor for poor wound healing akin to smoking. However, the molecular and histologic mechanisms underlying this postulation remain unknown. Our study sought to compare molecular and histologic changes in cutaneous flap and non-flap tissue between vaping, smoking and control cohorts. Animal study of 15 male Sprague-Dawley rats was randomized to three cohorts: negative control (n = 5), e-cigarette (n = 5) and cigarette (n = 5) and exposed to their respective treatments with serum cotinine monitoring. After 30 days, random pattern flaps were raised and healed for 2 weeks after which skin punch biopsies of flap and non-flap tissues were collected for quantitative-reverse transcription-polymerase chain reaction of three selected wound healing genes (transforming growth factor β [TGF-β], vascular endothelial growth factor [VEGF], matrix metalloproteinase-1 [MMP-1]); then, immunohistochemistry for CD68 expression, α-smooth muscle actin looking at microvessel density (MVD) and in situ hybridization to localize VEGF production were undertaken. In flap tissue, vaping (mean[SEM]) (0.61[0.07]) and smoking (0.70[0.04]) were associated with decreased fold change of VEGF expression compared with controls (0.91[0.03]) (p < 0.05, p < 0.05, respectively). In non-flap tissue, only vaping was associated with decreased VEGF expression (mean[SEM]) (0.81[0.07]), compared with controls (1.17[0.10]) (p < 0.05) with expression primarily localized to basal keratinocytes and dermal capillaries. Immunohistochemistry showed decreased MVD in smoking (0.27[0.06]) and vaping (0.26[0.04]) flap tissue compared to matched controls (0.65[0.14]) (p < 0.05, p < 0.05, respectively) and decreased areas of fibrosis compared with controls on gross histology. Vaping and smoking were similarly associated with decreased VEGF expression, MVD and fibrotic changes in flap tissue. The results suggest attenuated angiogenesis via decreased VEGF expression as a mechanism for poor wound healing in vaping-exposed rats.
Background: SARS-CoV-2 infection and disease severity are influenced by viral entry (VE) gene exp... more Background: SARS-CoV-2 infection and disease severity are influenced by viral entry (VE) gene expression patterns in airway epithelium. The similarities and differences of VE gene expression (ACE2, TMPRSS2, and CTSL) across nasal and bronchial compartments has not been fully characterized using matched samples from large cohorts. Results: Gene expression data from 793 nasal and 1,673 bronchial brushes obtained from individuals participating in lung cancer screening or diagnostic workup revealed that smoking was the only clinical factor significantly and reproducibly associated with VE gene expression. ACE2 and TMPRSS2 expression were higher in smokers in the bronchus but not in the nose. scRNA-seq of nasal brushings indicated that ACE2 co-expressed genes were highly expressed in club and C15orf48+ secretory cells while TMPRSS2 co-expressed genes were highly expressed in keratinizing epithelial cells. In contrast, these ACE2 and TMPRSS2 modules were highly expressed in goblet cells i...
ObjectiveThe immune response to invasive carcinoma has been the focus of published work, but litt... more ObjectiveThe immune response to invasive carcinoma has been the focus of published work, but little is known about the adaptive immune response to bronchial premalignant lesions (PMLs), precursors of lung squamous cell carcinoma. This study was designed to characterize the T cell receptor (TCR) repertoire in PMLs and its association with clinical, pathological, and molecular features.MethodsEndobronchial biopsies (n=295) and brushings (n=137) from high-risk subjects (n=50), undergoing lung cancer screening at approximately 1-year intervals via autofluorescence bronchoscopy and CT, were profiled by RNA-seq. We applied the TCR Repertoire Utilities for Solid Tissue/Tumor (TRUST) tool to the RNA-seq data to identify TCR CDR3 sequences across all samples. In the biopsies, we measured the correlation of TCR diversity with previously derived immune-associated PML transcriptional signatures and outcome. We also quantified the spatial and temporal distribution of shared and clonally expanded...
SummaryThere is an increasing appreciation for the heterogeneity of myeloid lineages in the respi... more SummaryThere is an increasing appreciation for the heterogeneity of myeloid lineages in the respiratory system, but whether distinct populations associate with the conducting airways remains unknown. We use single cell RNA sequencing, flow cytometry and immunofluorescence to characterize myeloid cells of the mouse trachea during homeostasis and epithelial injury/repair. We identify submucosal macrophages that are similar to lung interstitial macrophages and intraepithelial macrophages, and find that repair of the tracheal epithelium is impaired in Ccr2-deficient mice. Following injury there are early increases in neutrophils and submucosal macrophages, including M2-like macrophages. Unexpectedly, intraepithelial macrophages are initially lost but later replaced from CCR2+ monocytes. Mast cells and group 2 innate lymphoid cells are sources of IL13 that polarizes macrophages and directly influences basal cell behaviors. Their proximity to the airway epithelium establishes these myeloi...
102 Background: The immune response to frank malignancy has been the focus of published work, but... more 102 Background: The immune response to frank malignancy has been the focus of published work, but little is known about the adaptive immune response to bronchial premalignant lesions (PMLs), precursors of lung squamous cell carcinoma. This study was designed to characterize the T cell receptor (TCR) repertoire in PMLs and its association with clinical, pathological and molecular features. Methods: Endobronchial biopsies (n=294) and brushings (n=137) from high-risk subjects (n=50), undergoing lung cancer screening at approximately 1-year intervals via auto fluorescence bronchoscopy and CT, were profiled by RNA-Seq as part of the Pre-Cancer Genome Atlas (PCGA). We applied the TCR Repertoire Utilities for Solid Tissue/Tumor (TRUST) tool to identify TCR CDR3 sequences in the RNA-Seq data. We quantified the ratio of private (i.e., found in one patient only) and public (i.e. found in two or more patients) TCRs that are found in curated databases with known antigen specificities. We measur...
Lung cancer is the leading cause of cancer mortality due in part to an inability to diagnose and ... more Lung cancer is the leading cause of cancer mortality due in part to an inability to diagnose and intercept the disease at its earliest and potentially most curable stage. In order to improve lung cancer prevention and early detection, a detailed understanding of both the genomic alterations driving carcinogenesis and the mechanisms by which altered cells escape immune surveillance is required. Toward this goal, our group is profiling RNA and DNA from bronchial premalignant lesions (PMLs), precursors of lung squamous cell carcinoma (LUSCC), to identify alterations associated with their development and progression. Results from the analysis of 197 human endobronchial biopsies representing all histologic grades profiled by RNA-Seq, identified a class of high-grade PMLs with high activity of pathways associated with epithelial proliferation and differentiation. Among this class of PMLs, progression-associated pathways included immunoproteasome, antigen processing, immune inhibition, and macrophage activation. Further elucidation of the epithelial and immune changes that characterize progression of PMLs and testing of interception strategies based on these human findings require a preclinical model with similar histology and molecular alterations. The N-nitrosotris-(2-choroethyl)urea (NTCU) mouse model of LUSCC is a promising preclinical model that develops histologically comparable PMLs to those found in humans; however, the molecular alterations associated with these histologic changes have not yet been characterized. In order to establish molecular concordance between NTCU-treated mouse and human PMLs, we isolated RNA from 40 fresh-frozen whole lung sections and laser capture microdissected (LCM) tissue from SWR/J and A/J mice treated with NTCU. We profiled by RNA-Seq tissue that represented a range of histologic grades including normal (n=9), metaplasia/mild dysplasia (n=8), moderate/severe dysplasia (n=19), and carcinoma in situ/SCC (n=4). RNA-Seq data was aligned to mm9 and gene-level counts were summarized using Ensembl annotation. After removing samples of low quality, linear modeling adjusting for sample quality, sample type, and mouse strain was used to identify differentially expressed genes associated with histologic grade. Immune cell abundance was estimated using CIBERSORT and EnrichR was used to identify pathways associated with histologic grade. Concordance between the mouse data and human data was established using GSEA and GSVA. We identified 596 genes differentially expressed with increasing histologic severity across all mouse samples (FDR Citation Format: Sarah Mazzilli, Sherry Zhang, Gang Liu, Marc Lenburg, Avrum Spira, Jennifer Beane. The carcinogen-induced NTCU model: A preclinical mouse model for lung cancer interception [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B18.
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