Faculty Bibliography

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.

BACKGROUND:In lung cancer, upregulation of the PI3K 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. METHODS:Airway brushes 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 non-cancer diagnosis. Additionally, samples from 10 healthy control subjects were included. 16S rRNA gene amplicon sequencing and paired transcriptome sequencing (RNAseq) were performed on all airway samples. In addition, an in vitro model with airway epithelial cells exposed to bacteria/bacterial products was performed. RESULTS:The composition of the lower airway transcriptome in the cancer patients was significantly different from the controls, which included upregulation of ERK and PI3K signaling pathways. The lower airways of lung cancer patients were enriched for oral taxa (Streptococcus and Veillonella), which was associated with upregulation 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. CONCLUSIONS:The data presented here shows that several transcriptomic signatures previously identified as relevant to lung cancer pathogenesis are associated with enrichment of the lower airway microbiota with oral commensals.

Background: There is growing appreciation for the role of tumordraining lymph nodes (TDLN) in the dynamic of immuno-editing orchestrated by non-small cell lung cancers (NSCLC). By comparing Tcell subsets and gene expression in TDLN and non-draining lymph nodes (NDLN), we aim to determine whether there is tumor-regional variation in immunophenotype. Method: Patients undergoing endobronchial ultrasound-guided transbronchial needle aspiration for the diagnosis/staging of NSCLC were recruited. Aspirates were obtained from TDLN (N1/N2 nodes with increased fluorodeoxyglucose-F-18 (FDG) avidity and/or enlarged >1cm) and NDLN (non-enlarged/non- FDG-avid N2/N3 nodes) along with peripheral blood. Samples were stained with fluorophore-conjugated antibodies (CD4-FITC, CD8-V450, CD25-PECy7, CD127-APCR700, CD45RO-PECF594) and analyzed by flow cytometry. CD4+CD25- and CD8+ effector T-cells (Teff) were sorted. Gene expression profiling was performed on sorted Teff using the NanostringTM platform to measure differential expression between TDLN and NDLNs. Result: We compared T-cell subpopulations in TDLN and paired NDLN from 16 subjects. There were significantly fewer CD4+ T-cells in TDLN vs NDLN (10.1% vs 28.9%, p=0.0039), with more Tregs (12.1% vs 7.3%, p=0.1563) suggesting a pattern of tumorregional immunosuppression in the TDLN. This was more consistent when tumor histology was adenocarcinoma compared to squamous cell cancer with respect to both depletion of Teff and higher proportion of Tregs (Fig 1). A more immunosuppressive TDLN phenotype was also observed with high tumor PD-L1 expression (>50%), with 36% fewer CD4+ T-cells in TDLN relative to paired NDLN when PD-L1 expression was high relative to just 3.2% fewer CD4+ T-cells with low PD-L1 expression. Gene expression in Teff has preliminarily demonstrated upregulation of genes mediating T-cell exhaustion (CTLA-4, PD-1, TGFb) and downregulation of co-stimulatory/recruitment factors (CD28, ICOS, ICAM2) in TDLN suggesting impaired activation of tumorregional Teff. Conclusion: Our findings suggest that TDLNs in patients with NSCLC display a tolerogenic phenotype, with more marked immunosuppression in the setting of adenocarcinoma and high tumor PD-L1 expression. (Figure Presented)

PURPOSE: 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). EXPERIMENTAL DESIGN: Identification of differentially methylated regions (DMRs) 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. RESULTS: A methylation panel of 6 genes (CDO1, HOXA9, AJAP1, PTGDR, UNCX, and MARCH11) 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 CDO1, HOXA9, PTGDR, and AJAP1 refined the risk stratification for outcomes as an independent prognostic factor for an early stage disease. Moreover, the paralogue group for HOXA9, predominantly overexpressed in subjects with HOXA9 methylation, showed poor outcomes. CONCLUSION: 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.

Identification of biomarkers for early detection of lung cancer (LC) is important, in turn leading to more effective treatment and reduction of mortality. Serological proteome analysis (SERPA) was used to identify proteins around 34 kD as ECH1 and HNRNPA2B1, which had been recognized by serum autoantibody from 25 LC patients. In the validation study, including 90 sera from LC patients and 89 sera from normal individuals, autoantibody to ECH1 achieved an area under the curve (AUC) of 0.799 with sensitivity of 62.2% and specificity of 95.5% in discriminating LC from normal individuals, and showed negative correlation with tumor size (rs = -0.256, p = 0.023). Autoantibody to HNRNPA2B1 performed an AUC of 0.874 with sensitivity of 72.2% and specificity of 95.5%, and showed negative correlation with lymph node metastasis (rs = -0.279, p = 0.012). By using longitudinal preclinical samples, autoantibody to ECH1 showed an AUC of 0.763 with sensitivity of 60.0% and specificity of 89.3% in distinguishing early stage LC from matched normal controls, and elevated autoantibody levels could be detected greater than 2 y before LC diagnosis. ECH1 and HNRNPA2B1 are autoantigens that elicit autoimmune responses in LC and their autoantibody can be the potential biomarkers for the early detection of LC.