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Purpose: Promising results in the treatment of non–small cell lung cancer (NSCLC) have been seen with agents targeting immune checkpoints, such as programmed cell death 1 (PD-1) or programmed death ligand-1 (PD-L1). However, only a select group of patients respond to these interventions. The identification of biomarkers that predict clinical benefit to immune checkpoint blockade is critical to successful clinical translation of these agents. Methods: We conducted an integrated analysis of three independent large datasets, including The Cancer Genome Atlas of lung adenocarcinoma and two datasets from MD Anderson Cancer Center (Houston, TX), Profiling of Resistance Patterns and Oncogenic Signaling Pathways in Evaluation of Cancers of the Thorax (named PROSPECT) and Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (named BATTLE-1). Comprehensive analysis of mRNA gene expression, reverse-phase protein array, IHC, and correlation with clinical data were performed. Results: Epithelial–mesenchymal transition (EMT) is highly associated with an inflammatory tumor microenvironment in lung adenocarcinoma, independent of tumor mutational burden. We found immune activation coexistent with elevation of multiple targetable immune checkpoint molecules, including PD-L1, PD-L2, PD-1, TIM-3, B7-H3, BTLA, and CTLA-4, along with increases in tumor infiltration by CD4+Foxp3+ regulatory T cells in lung adenocarcinomas that displayed an EMT phenotype. Furthermore, we identify B7-H3 as a prognostic marker for NSCLC. Conclusions: The strong association between EMT status and an inflammatory tumor microenvironment with elevation of multiple targetable immune checkpoint molecules warrants further investigation of using EMT as a predictive biomarker for immune checkpoint blockade agents and other immunotherapies in NSCLC and possibly a broad range of other cancers. Clin Cancer Res; 22(14); 3630–42. ©2016 AACR. See related commentary by Datar and Schalper, p. 3422

Background: Tumor cell regulation of the recruitment and activity of diverse inflammatory cells creates an immunosuppressive microenvironment that favors both tumor growth and metastasis. However, the mechanistic basis of how tumor cells trigger intra-tumoral immunosuppression have not been fully defined. Here we show that metastasis-prone lung adenocarcinoma cells suppress the proliferation and activity of intra-tumoral CD8+ T cells through a PD-L1 dependent mechanism and that PD-L1 expression is regulated by microRNA-200 (miR-200), a known mediator of tumor cell EMT. Results: Analysis of patient tumors from The Cancer Genome Atlas (TCGA) (230 adenocarcinomas) and a large MDACC cohort of resected tumors (N>150) demonstrated that tumors with a mesenchymal gene expression signature had repression of miR-200 family expression and higher levels of PD-L1 expression. These findings were confirmed experimentally in human and murine NSCLC cell lines. In vivo experiments with a Kras/p53 mutant mouse model of lung adenocarcinoma showed that decreased miR-200 led to increased expression of PD-L1 levels on tumor cells, causing profound CD8+ T cell suppression. Upon suppression of CD8+ T cell activity tumors acquired a growth advantage and metastatic capability. Confirmation of these results with the LLC model in PD-L1 knockout mice also demonstrated that PD-L1 expression on the tumor cells, rather than other cells in the tumor microenvironment, was critical to defining tumor immunity and metastastic ability. The metastases could be suppressed by genetic and pharmacologic blockade of PD-L1. Conclusion: Our findings demonstrate a novel biological role for miR-200 in cancer cells by simultaneous control of the cell-intrinsic EMT program and the cell-non-autonomous PD-L1-mediated immune evasion. These findings support further investigation into the mechanistic effects of miR-200 on metastasis and the use of specific EMT-related biomarkers to select immunomodulatory therapies. Citation Format: Don L. Gibbons, Limo Chen, Sangeeta Goswami, Young-Ho Ahn, Lauren A. Byers, Lixia Diao, Jing Wang, Angelica Cortez, Jonathon Roybal, James Welsh, Xuejun Zhang, David Dwyer, Xiaohui Yi, Chen Lieping, Ignacio Wistuba, John V. Heymach, Jonathan M. Kurie, F. Xiao-Feng Qin. microRNA-200 regulates tumor cell PD-L1 expression to control lung cancer metastasis. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr A21.