MET-mediated regulation of immune checkpoint molecules and suppression of immune cell function
MET의 면역제어분자 발현조절을 통한 면역세포 기능 억제기전

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의과대학 협동과정 종양생물학전공
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서울대학교 대학원
학위논문 (석사)-- 서울대학교 대학원 : 의과대학 협동과정 종양생물학전공, 2018. 8. 전윤경.
Purpose: MET is a receptor tyrosine kinase playing an important role for the development and progression of variable tumors through diverse mechanisms. However, the role of MET in the modulation of anti-tumor immune response remains unclear. Cancer immunotherapy targeting immune checkpoint pathways (co-inhibitory pathways) and co-stimulatory pathways has shown clinical and preclinical benefits. Thus, this study was intended to investigate whether MET expressed on tumor cells is involved in the regulation of immune checkpoint pathways and immune cell function.

Methods: Lung adenocarcinoma cell lines including H596 (harboring MET exon 14 skipping mutation), H1993 (harboring MET gene amplification), H23 and H522 (MET wild-type), and a gastric carcinoma cell line, Hs746T (harboring MET exon 14 skipping mutation and gene amplification) were used. Cells were treated with MET inhibitor (PHA665752 or crizotinib) or recombinant HGF (a MET ligand), or transfected with MET siRNA and subject to oligonucleotide microarray, qRT-PCR, Western blotting and flow cytometry. Co-culture experiment using human peripheral blood mononuclear cells (PBMC) and Hs746T cells was performed to evaluate if PD-L1 expressed on tumor cells affect the immune cell function. Cancer Cell Line Encyclopedia (CCLE) and the Cancer Genome Atlas (TCGA) data were analyzed and immunohistochemistry was also performed using human cancer tissues.

Result: Microarray analysis for Hs746T cells with MET inhibition or knockdown showed that co-inhibitory molecules were down-regulated and co-stimulatory molecules and HLA class I molecules were up-regulated by MET suppression. Among the co-inhibitory molecules, PD-L1 was one of the most significantly down-regulated genes by MET suppression. CCLE analysis revealed that a significant positive correlation between MET and PD-L1, rather than PD-L2, throughout the cancer cell lines. Immunohistochemistry using lung cancer tissues also showed a significant positive correlation between MET and PD-L1 expression in tumor cells. Stimulation of H596, H23 and H522 cells with HGF increased PD-L1 expression at the mRNA and protein levels. Treatment of H1993 and Hs746T cells with MET inhibitor or MET knockdown using siRNA resulted in down-regulation of PD-L1 at the mRNA, total protein, and surface expression levels. Co-culture of PBMC with Hs746T cells suppressed IFN production from PBMC, which was reversed by PD-L1 blocking antibody or crizotinib treatment. Finally, TCGA analysis showed that MET and PD-L1 expression are significantly positively correlates with each other in variable tumor tissues, including non-small cell lung cancer (NSCLC, rho=0.219 [p<0.001]
in adenocarcinoma, r=0.401 [p<0.001]
in squamous cell carcinoma, r=0.144 [p=0.001]) and stomach cancer (rho=0.137 [p=0.005]). Moreover, TCGA analysis of NSCLC exhibited that MET expression is inversely correlated with expression level of CD8 effector function-related genes, including PDCD1, granzyme A, granzyme B, perforin, IFN, CXCL9 and CXCL10, in CD274-high group rather than CD274-low group.

Conclusion This study demonstrates that MET signaling increases PD-L1 transcription and protein overexpression in tumor cells, which might contribute to immune escape of tumor via PD-1/PD-L1 pathway.

Keywords: MET, immune evasion, immune checkpoint, PD-1/PD-L1 pathway, PD-L1, cancer immunotherapy, lung cancer

Student number: 2016-22021
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College of Medicine/School of Medicine (의과대학/대학원)Program in Cancer Biology (협동과정-종양생물학전공)Theses (Master's Degree_협동과정-종양생물학전공)
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