Lung cancer development is driven by the expression of mutant oncogenes, with EGFR and KRAS being the most frequent mutations in lung adenocarcinoma. However, additional factors may influence lung tumor development and progression, including the balance of antitumor immune effector cells and pro-tumorigenic immune suppressor cells within the lung and lung tumor microenvironment. Tumor cells can evade immune attack by producing cytokines that recruit immune modulatory cells, such as regulatory T cells (Tregs), that promote a localized immune suppressive environment We hypothesized that oncogene signaling regulates the production of cytokines by tumor cells at the earliest stages of transformation that can recruit immune suppressive cells and promote lung tumour development. We used CIBERSORT-based analysis of gene expression data to quantify 22 different immune cell types from over 300 human lung adenocarcinomas and 100 matched normal lung tissues. We found that Tregs were significantly enriched in early-stage lung adenocarcinoma tumors compared to matched normal tissue from the same patient, and validated these findings with immunohistochemistry staining of lung sections. To identify cytokines that could recruit Tregs early in lung tumorigenesis, we used normal cells expressing doxycycline-inducible wild-type EGFR, mutant EGFRL858R or mutant KRASG12V. Secreted cytokines were quantified using a multiplex LUMINEX assay with subsequent validation by ELISA. Induction of EGFRL858R and KRASG12V expression in normal cells rapidly increased the production of CCL5 (RANTES), as did expression of wild-type EGFR in the presence of exogenous EGF. To elucidate the mechanism of oncogene-driven CCL5 secretion, we treated lung cancer cells harboring EGFR or KRAS mutations with a MEK inhibitor (trametinib) to disrupt oncogenic signaling downstream of EGFR and KRAS. In KRAS mutant lung cancer cells, treatment with trametinib decreased CCL5 production and inhibited both ERK and AKT signaling. To determine if oncogene-driven cytokines could induce migration of Tregs ex vivo, we used a trans-well assay with conditioned media from cells expressing doxycycline inducible EGFRL858R or KRASG12V. Conditioned media from EGFRL858R and KRASG12V-expressing cells induced Treg migration, which was mitigated by the addition of an anti-CCL5 antibody. These data indicate that oncogenic EGFR and KRAS signaling regulates expression of CCL5 in lung tumor cells, and that CCL5-mediated Treg recruitment to lung tumors may occur in early stages of lung tumor development. Therefore, targeted inhibition of CCL5, Tregs, and/or oncogenic EGFR and KRAS signaling may represent therapeutic strategies to block recruitment and function of immunosuppressive Tregs during lung tumor development.
Citation Format: Elizabeth Franks, Elizabeth C. Halvorsen, Etienne Melese, Unni Arun, Jenna L. Collier, Bryant T. Harbourne, Min Hee Oh, Lam Vivian, Gerry Krystal, John C. English, Wan L. Lam, Stephen Lam, Ninan Abraham, Kevin L. Bennewith, William W. Lockwood. Oncogenic drivers of lung cancer induce production of CCL5 and recruitment of regulatory T-cells [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A02.