In lung cancer, enrichment of the lower airway microbiota with oral commensals commonly occurs, and
models support that some of these bacteria can trigger host transcriptomic signatures associated ...with carcinogenesis. Here, we show that this lower airway dysbiotic signature was more prevalent in the stage IIIB-IV tumor-node-metastasis lung cancer group and is associated with poor prognosis, as shown by decreased survival among subjects with early-stage disease (I-IIIA) and worse tumor progression as measured by RECIST scores among subjects with stage IIIB-IV disease. In addition, this lower airway microbiota signature was associated with upregulation of the IL17, PI3K, MAPK, and ERK pathways in airway transcriptome, and we identified
as the most abundant taxon driving this association. In a KP lung cancer model, lower airway dysbiosis with
led to decreased survival, increased tumor burden, IL17 inflammatory phenotype, and activation of checkpoint inhibitor markers. SIGNIFICANCE: Multiple lines of investigation have shown that the gut microbiota affects host immune response to immunotherapy in cancer. Here, we support that the local airway microbiota modulates the host immune tone in lung cancer, affecting tumor progression and prognosis.
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To establish a novel panel of cancer-specific methylated genes for cancer detection and prognostic stratification of early-stage non-small cell lung cancer (NSCLC).
Identification of differentially ...methylated regions (DMR) was performed with bumphunter on "The Cancer Genome Atlas (TCGA)" dataset, and clinical utility was assessed using quantitative methylation-specific PCR assay in multiple sets of primary NSCLC and body fluids that included serum, pleural effusion, and ascites samples.
A methylation panel of 6 genes (
, and
) was selected from TCGA dataset. Promoter methylation of the gene panel was detected in 92.2% (83/90) of the training cohort with a specificity of 72.0% (18/25) and in 93.0% (40/43) of an independent cohort of stage IA primary NSCLC. In serum samples from the later 43 stage IA subjects and population-matched 42 control subjects, the gene panel yielded a sensitivity of 72.1% (31/41) and specificity of 71.4% (30/42). Similar diagnostic accuracy was observed in pleural effusion and ascites samples. A prognostic risk category based on the methylation status of
, and
refined the risk stratification for outcomes as an independent prognostic factor for an early-stage disease. Moreover, the paralog group for HOXA9, predominantly overexpressed in subjects with
methylation, showed poor outcomes.
Promoter methylation of a panel of 6 genes has potential for use as a biomarker for early cancer detection and to predict prognosis at the time of diagnosis.
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In lung cancer, upregulation of the PI3K (phosphoinositide 3-kinase) pathway is an early event that contributes to cell proliferation, survival, and tissue invasion. Upregulation of this pathway was ...recently described as associated with enrichment of the lower airways with bacteria identified as oral commensals.
We hypothesize that host-microbe interactions in the lower airways of subjects with lung cancer affect known cancer pathways.
Airway brushings were collected prospectively from subjects with lung nodules at time of diagnostic bronchoscopy, including 39 subjects with final lung cancer diagnoses and 36 subjects with noncancer diagnoses. In addition, samples from 10 healthy control subjects were included. 16S ribosomal RNA gene amplicon sequencing and paired transcriptome sequencing were performed on all airway samples. In addition, an in vitro model with airway epithelial cells exposed to bacteria/bacterial products was performed.
The composition of the lower airway transcriptome in the patients with cancer was significantly different from the control subjects, which included up-regulation of ERK (extracellular signal-regulated kinase) and PI3K signaling pathways. The lower airways of patients with lung cancer were enriched for oral taxa (Streptococcus and Veillonella), which was associated with up-regulation of the ERK and PI3K signaling pathways. In vitro exposure of airway epithelial cells to Veillonella, Prevotella, and Streptococcus led to upregulation of these same signaling pathways.
The data presented here show that several transcriptomic signatures previously identified as relevant to lung cancer pathogenesis are associated with enrichment of the lower airway microbiota with oral commensals.
Cross-sectional human data suggest that enrichment of oral anaerobic bacteria in the lung is associated with an increased T-helper cell type 17 (Th17) inflammatory phenotype.
In this study, we ...evaluated the microbial and host immune-response dynamics after aspiration with oral commensals using a preclinical mouse model.
Aspiration with a mixture of human oral commensals (MOC;
,
, and
) was modeled in mice followed by variable time of killing. The genetic backgrounds of mice included wild-type, MyD88-knockout, and STAT3C backgrounds.
16S-rRNA gene sequencing characterized changes in microbiota. Flow cytometry, cytokine measurement via Luminex and RNA host-transcriptome sequencing was used to characterize the host immune phenotype. Although MOC aspiration correlated with lower-airway dysbiosis that resolved within 5 days, it induced an extended inflammatory response associated with IL-17-producing T cells lasting at least 14 days. MyD88 expression was required for the IL-17 response to MOC aspiration, but not for T-cell activation or IFN-γ expression. MOC aspiration before a respiratory challenge with
led to a decrease in hosts' susceptibility to this pathogen.
Thus, in otherwise healthy mice, a single aspiration event with oral commensals is rapidly cleared from the lower airways but induces a prolonged Th17 response that secondarily decreases susceptibility to
. Translationally, these data implicate an immunoprotective role of episodic microaspiration of oral microbes in the regulation of the lung immune phenotype and mitigation of host susceptibility to infection with lower-airway pathogens.
It remains unclear whether PAX6 acts as a crucial transcription factor for lung cancer stem cell (CSC) traits. We demonstrate that PAX6 acts as an oncogene responsible for induction of cancer ...stemness properties in lung adenocarcinoma (LUAD). Mechanistically, PAX6 promotes GLI transcription, resulting in SOX2 upregulation directly by the binding of GLI to the proximal promoter region of the SOX2 gene. The overexpressed SOX2 enhances the expression of key pluripotent factors (OCT4 and NANOG) and suppresses differentiation lineage factors (HOPX and NKX2-1), driving cancer cells toward a stem-like state. In contrast, in the differentiated non-CSCs, PAX6 is transcriptionally silenced by its promoter methylation. In human lung cancer tissues, the positive linear correlations of PAX6 expression with GLI and SOX2 expression and its negative correlations with HOPX and NKX2-1 expression were observed. Therapeutically, the blockade of the PAX6-GLI-SOX2 signaling axis elicits a long-lasting therapeutic efficacy by limiting CSC expansion following chemotherapy. Furthermore, a methylation panel including the PAX6 gene yielded a sensitivity of 79.1% and specificity of 83.3% for cancer detection using serum DNA from stage IA LUAD. Our findings provide a rationale for targeting the PAX6-GLI-SOX2 signaling axis with chemotherapy as an effective therapeutic strategy and support the clinical utility of PAX6 gene promoter methylation as a biomarker for early lung cancer detection.
Approximately 30% of early-stage lung adenocarcinoma patients present with disease progression after successful surgical resection. Despite efforts of mapping the genetic landscape, there has been ...limited success in discovering predictive biomarkers of disease outcomes. Here we performed a systematic multi-omic assessment of 143 tumors and matched tumor-adjacent, histologically-normal lung tissue with long-term patient follow-up. Through histologic, mutational, and transcriptomic profiling of tumor and adjacent-normal tissue, we identified an inflammatory gene signature in tumor-adjacent tissue as the strongest clinical predictor of disease progression. Single-cell transcriptomic analysis demonstrated the progression-associated inflammatory signature was expressed in both immune and non-immune cells, and cell type-specific profiling in monocytes further improved outcome predictions. Additional analyses of tumor-adjacent transcriptomic data from The Cancer Genome Atlas validated the association of the inflammatory signature with worse outcomes across cancers. Collectively, our study suggests that molecular profiling of tumor-adjacent tissue can identify patients at high risk for disease progression.
Microaspiration is a common phenomenon in healthy subjects, but its frequency is increased in chronic inflammatory airway diseases, and its role in inflammatory and immune phenotypes is unclear. We ...have previously demonstrated that acellular bronchoalveolar lavage samples from half of the healthy people examined are enriched with oral taxa (here called pneumotypeSPT) and this finding is associated with increased numbers of lymphocytes and neutrophils in bronchoalveolar lavage. Here, we have characterized the inflammatory phenotype using a multi-omic approach. By evaluating both upper airway and acellular bronchoalveolar lavage samples from 49 subjects from three cohorts without known pulmonary disease, we observed that pneumotypeSPT was associated with a distinct metabolic profile, enhanced expression of inflammatory cytokines, a pro-inflammatory phenotype characterized by elevated Th-17 lymphocytes and, conversely, a blunted alveolar macrophage TLR4 response. The cellular immune responses observed in the lower airways of humans with pneumotypeSPT indicate a role for the aspiration-derived microbiota in regulating the basal inflammatory status at the pulmonary mucosal surface.
Cancer recurrence after tumor resection in early-stage non-small cell lung cancer (NSCLC) is common, yet difficult to predict. The lung microbiota and systemic immunity may be important modulators of ...risk for lung cancer recurrence, yet biomarkers from the lung microbiome and peripheral immune environment are understudied. Such markers may hold promise for prediction as well as improved etiologic understanding of lung cancer recurrence.
In tumor and distant normal lung samples from 46 stage II NSCLC patients with curative resection (39 tumor samples, 41 normal lung samples), we conducted 16S rRNA gene sequencing. We also measured peripheral blood immune gene expression with nanoString®. We examined associations of lung microbiota and peripheral gene expression with recurrence-free survival (RFS) and disease-free survival (DFS) using 500 × 10-fold cross-validated elastic-net penalized Cox regression, and examined predictive accuracy using time-dependent receiver operating characteristic (ROC) curves.
Over a median of 4.8 years of follow-up (range 0.2-12.2 years), 43% of patients experienced a recurrence, and 50% died. In normal lung tissue, a higher abundance of classes Bacteroidia and Clostridia, and orders Bacteroidales and Clostridiales, were associated with worse RFS, while a higher abundance of classes Alphaproteobacteria and Betaproteobacteria, and orders Burkholderiales and Neisseriales, were associated with better RFS. In tumor tissue, a higher abundance of orders Actinomycetales and Pseudomonadales were associated with worse DFS. Among these taxa, normal lung Clostridiales and Bacteroidales were also related to worse survival in a previous small pilot study and an additional independent validation cohort. In peripheral blood, higher expression of genes TAP1, TAPBP, CSF2RB, and IFITM2 were associated with better DFS. Analysis of ROC curves revealed that lung microbiome and peripheral gene expression biomarkers provided significant additional recurrence risk discrimination over standard demographic and clinical covariates, with microbiome biomarkers contributing more to short-term (1-year) prediction and gene biomarkers contributing to longer-term (2-5-year) prediction.
We identified compelling biomarkers in under-explored data types, the lung microbiome, and peripheral blood gene expression, which may improve risk prediction of recurrence in early-stage NSCLC patients. These findings will require validation in a larger cohort.
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