Abstract A13: Pharmacologic targeting of IL-6 suppresses tumor progression in a non-small cell lung cancer mouse model with K-ras mutation through re-educating the tumor microenvironment.
Authors: Mauricio S. Caetano, Amber M. Cumpian, Lei Gong, Cesar E. Ochoa, Seon H. Chang, Huiyuan Zhang, Stephanie Watowich, Cinthya Sternberg, Carlos G. Ferreira, Seyed J. Moghaddam
Journal: Clinical Cancer Research (2014)

Abstract Activating mutations of K-ras are one of the most common molecular alterations associated with lung cancer development. Several attempts to develop therapies directly targeting K-ras have failed thus far, clearly state the need for new strategies to bring clinical benefits to patients displaying such molecular profile. Using a conditional K-ras mutated lung cancer mouse model (CC-LR) we previously showed that K-ras mutated lung tumors have intrinsic inflammatory characteristics and we also demonstrated that chronic obstructive pulmonary disease (COPD)-like airway inflammation, an independent risk factor for lung cancer, promotes lung tumor in this model. CC-LR mice shows high protein level of interleukin 6 (IL-6) and increased STAT3 (downstream signal to IL6) gene expression, and we have further shown that genetic ablation of IL-6 in CC-LR mice results in significant tumor reduction. High levels of IL-6 and up-regulation of STAT3 and its target genes have been observed in the lungs of smokers with COPD, and in 50% of human lung adenocarcinomas. These indicate an essential role for IL-6 in lung cancer promotion, and introduce IL-6 as a potential druggable target for lung cancer patients who harbor K-ras mutations. This prompted us to block IL-6 pathway using a monoclonal anti-IL-6 immunotherapy in our K-ras mutated lung cancer mouse model. Six week old CC-LR mice were injected intraperitoneally (I.P.) with 20 mg/kg dose of an anti-IL-6 monoclonal IgG1 antibody, twice a week, for a period of eight weeks. Flowcytometry analysis of total lung inflammatory cell population in non-treated CC-LR mice showed 2-fold increase in the population of tumor associated macrophage (TAM) (CD45+/CD11b+/GR1+/F4/80+/MCSFR+) during tumor progression from age 6 weeks to 14 weeks. qPCR analysis of total lung RNA from non-treated CC-LR mice showed that there is an increase in the level of Arginase-1 (3 fold) with no change in the level of inducible Nitric Oxide Synthetize (NOS2). This suggests that epithelial signal initiated by K-ras mutation may dictate the fate of lung microenvironment by creating an inflammatory pro-tumor type 2 response. Then we found that anti-IL-6 treatment suppresses tumor progression by ~78% (4.6-fold) in CC-LR mice which is associated with a significant reduction in the quantity of inflammatory cells in bronchoalveolar lavage fluid (BALF). It also decreased the expression of proliferation marker: Ki-67, and pro angiogenic factors: VEGF, MMP9 and CD31 and phospho-STAT3 in lung tissue. ELISA analysis of BALF from CC-LR mice also showed that anti-IL-6 treatment reduces TGFβ and KC protein levels with no change in level of IL-17. qPCR and flowcytometry analysis of total lung homogenate of CC-LR mice further showed that IL-6 blockade induces a significant decrease in Arginase1 expression (3-fold) and TAM population (5-fold). Taking together here we showed that K-ras mutated lung tumor drives an immunosuppressive pro-tumor microenvironment, with an M2 TAM polarization and blocking the suppressive actions of IL-6 shifts this to an anti-tumor lung microenvironment. Therefore we propose pharmacological targeting of IL-6 as a potential therapeutic strategy alone or in combination with conventional cytotoxic therapy for lung cancer patients with K-ras mutation. Funded by: American Cancer Society, RSG-11-115-01-CNE, and American Lung Association/LUNGevity Foundation, LCD-114696-N Citation Format: Mauricio S. Caetano, Amber M. Cumpian, Lei Gong, Cesar E. Ochoa, Seon H. Chang, Huiyuan Zhang, Stephanie Watowich, Cinthya Sternberg, Carlos G. Ferreira, Seyed J. Moghaddam. Pharmacologic targeting of IL-6 suppresses tumor progression in a non-small cell lung cancer mouse model with K-ras mutation through re-educating the tumor microenvironment. [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 A13.