Rab37-mediated exocytosis of IL-6 skews M2 polarization and activates STAT3 signaling in macrophages to promote lung cancer progression

• Main Authors: You-EnYang, 楊侑恩
• Other Authors: Yi-Ching Wang
• Format: Others
• Language: en_US
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/3at7kz

碩士 === 國立成功大學 === 藥理學研究所 === 107 === Background: Recent studies have revealed that bidirectional interactions between tumor cells and immune cells in the tumor microenvironment contribute to tumor progression. Our preliminary data showed that mice systemically lacking Rab37 (Rab37 KO) suppressed tumor growth of subcutaneously injected with Lewis lung carcinoma (LLC) cells. Interestingly, Rab37 KO mice displayed low infiltration of pro-tumor M2 macrophages in LLC allografts, suggesting that Rab37 possesses pro-tumor functions in tumor microenvironment. However, the role of Rab37 small GTPase-mediated exocytosis in stromal macrophages remains unknown. Purpose: This study aims to investigate whether Rab37-mediated exocytosis skews macrophages toward M2 polarization and explore the mechanism involved. Results: Our quantitative RT-PCR (qRT-PCR) and flow cytometry data showed that bone marrow derived macrophages (BMDMs) from Rab37 KO mice suppressed M2 macrophages polarization. Next, we performed the cytokine/chemokine arrays to reveal Rab37-mediated cytokines using conditioned media of BMDMs derived from wild-type (WT) or Rab37 KO mice. Interestingly, among the differential cytokines/chemokines, the secreted level of interleukin-6 (IL-6) correlated with Rab37 expression in BMDMs. Vesicle isolation revealed that IL-6 enriched in Rab37-specific vesicles in RAW264.7 macrophage cell line. ELISA and immunofluorescence (IF) images confirmed that Rab37 mediated IL-6 secretion in RAW264.7 cell in a GTPase nucleotide-dependent manner, i.e., IL6 secretion was upregulated in RAW264.7 cells expressing Rab37WT and GTP-bound Rab37Q89L. In addition, our cDNA microarray data showed that BMDMs from Rab37 KO mice displayed increased IFN-I signaling pathways compared to those from WT BMDMs after LLC conditional medium treatment. Gene Set Enrichment Analysis (GSEA) and pathway analysis demonstrated that IFNα/β genes and STAT1 downstream interferon stimulated genes (ISGs) were upregulated in Rab37 KO BMDMs. qRT-PCR assays confirmed the increased expression of IFNβ and ISGs genes, including IFIT3, IRF7 and Mx1 in BMDMs from Rab37 KO mice. In contrast, expression of these genes was decreased in Rab37 overexpressing RAW264.7 cells, which led to increased expression of M2 marker genes Arg1 and Ym1. Indeed, ELISA data showed that the level of IFN-β was decreased in Rab37WT and Rab37Q89L RAW264.7 cells. Among type I IFN upstream transcription factors, we validated that STAT3 inhibited STAT1/IRF3 nuclear translocalization and suppressed type I IFN-associated genes expression through Rab37 mediated IL-6 secretion by immunoblotting and IF image. Conclusions: Our results uncover a novel role of Rab37-mediated IL-6 secretion in regulating type I IFN signaling to skew macrophage polarization toward M2. Therapeutic strategies targeting Rab37/IL-6 axis in stromal macrophages in tumor microenvironment is a testable approach in the future.