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MET Inhibitors Promote Liver Tumor Evasion of the Immune Response by Stabilizing PDL1.

ABSTRACT: BACKGROUND & AIMS:Inhibitors of MET have not produced satisfactory outcomes in trials of patients with liver cancer. We investigated the mechanisms of liver tumor resistance to MET inhibitors in mice. METHODS:We tested the effects of MET inhibitors tivantinib and capmatinib in the mouse hepatocellular carcinoma (HCC) cell line HCA-1 and in immune-competent and immunodeficient mice with subcutaneous tumors grown from this cell line. Tumors were collected from mice and tumor cells were analyzed by time-of-flight mass cytometry. We used short hairpin RNAs to weaken expression of MET in Hep3B, SK-HEP-1, HA59T, and HA22T liver cancer cell lines and analyzed cells by immunoblot, immunofluorescence, and immunoprecipitation assays. Mass spectrometry was used to assess interactions between MET and glycogen synthase kinase 3? (GSK3B), and GSK3B phosphorylation, in liver cancer cell lines. C57/BL6 mice with orthotopic tumors grown from Hep1-6 cells were given combinations of capmatinib or tivantinib and antibodies against programmed cell death 1 (PDCD1; also called PD1); tumors were collected and analyzed by immunofluorescence. We analyzed 268 HCCsamples in a tissue microarray by immunohistochemistry. RESULTS:Exposure of liver cancer cell lines to MET inhibitors increased their expression of PD ligand 1 (PDL1) and inactivated cocultured T cells. MET phosphorylated and activated GSK3B at tyrosine 56, which decreased the expression of PDL1 by liver cancer cells. In orthotopic tumors grown in immune-competent mice, MET inhibitors decreased the antitumor activity of T cells. However, addition of anti-PD1 decreased orthotopic tumor growth and prolonged survival of mice compared with anti-PD1 or MET inhibitors alone. Tissue microarray analysis of HCC samples showed an inverse correlation between levels of MET and PDL1 and a positive correlation between levels of MET and phosphorylated GSK3B. CONCLUSIONS:In studies of liver cancer cell lines and mice with orthotopic tumors, MET mediated phosphorylation and activated GSK3B, leading to decreased expression of PDL1. Combined with a MET inhibitor, anti-PD1 and anti-PDL1 produced additive effect to slow growth of HCCs in mice.

SUBMITTER: Li H

PROVIDER: S-EPMC6904924 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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MET Inhibitors Promote Liver Tumor Evasion of the Immune Response by Stabilizing PDL1.

Li Hui H Li Chia-Wei CW Li Xiaoqiang X Ding Qingqing Q Guo Lei L Liu Shuang S Liu Chunxiao C Lai Chien-Chen CC Hsu Jung-Mao JM Dong Qiongzhu Q Xia Weiya W Hsu Jennifer L JL Yamaguchi Hirohito H Du Yi Y Lai Yun-Ju YJ Sun Xian X Koller Paul B PB Ye Qinghai Q Hung Mien-Chie MC

Gastroenterology 20190131 6

<h4>Background & aims</h4>Inhibitors of MET have not produced satisfactory outcomes in trials of patients with liver cancer. We investigated the mechanisms of liver tumor resistance to MET inhibitors in mice.<h4>Methods</h4>We tested the effects of MET inhibitors tivantinib and capmatinib in the mouse hepatocellular carcinoma (HCC) cell line HCA-1 and in immune-competent and immunodeficient mice with subcutaneous tumors grown from this cell line. Tumors were collected from mice and tumor cells w ...[more]

PMID: 30711629

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Project description:Background: The up-regulation of PD-L1 is recognized as an adaption of cancer cells to evade immune surveillance and attack. However, the intrinsic mechanisms of the induction of PD-L1 by interferon-γ (IFN-γ) in tumor microenvironment remain incompletely characterized. Ubiquitin ligase E3 component N-recognition protein 5 (UBR5) has a critical role in tumorigenesis of triple negative breast cancer (TNBC) by triggering specific immune responses to the tumor. Dual targeting of UBR5 and PD-L1 exhibited superior therapeutic benefits in a preclinical TNBC model in short term. Methods: The regulation of UBR5 to PD-L1 upon IFN-γ stimulation was evaluated through in UBR5 deficiency, reconstitution or overexpression cell line models by quantitative PCR, immunohistochemistry and RNA-seq. The effects of PD-L1 regulation by UBR5 and double blockade of both genes were evaluated in mouse TNBC model. Luciferase reporter assay, chromatin immunoprecipitation-qPCR and bioinformatics analysis were performed to explore the transcription factors involved in the regulation of UBR5 to PD-L1. Results: E3 ubiquitin ligase UBR5 plays a key role in IFN-γ-induced PDL1 transcription in TNBC in an E3 ubiquitination activity-independent manner. RNA-seq-based transcriptomic analyses reveal that UBR5 globally affects the genes in the IFN-γ-induced signaling pathway. Through its poly adenylate binding (PABC) domain, UBR5 enhances the transactivation of PDL1 by upregulating protein kinase RNA-activated (PKR), and PKR's downstream factors including signal transducers and activators of transcription 1 (STAT1) and interferon regulatory factor 1 (IRF1). Restoration of PD-L1 expression in UBR5-deficient tumor cells recoups their malignancy in vivo, whereas CRISPR/Cas9-mediated simultaneous abrogation of UBR5 and PD-L1 expression yields synergistic therapeutic benefits than either blockade alone, with a strong impact on the tumor microenvironment. Conclusions: This study identifies a novel regulator of PDL1 transcription, elucidates the underlying molecular mechanisms and provides a strong rationale for combination cancer immunotherapies targeting UBR5 and PD-L1.

Project description:BackgroundLATS1/2 are frequently mutated and down-regulated in endometrial cancer (EC), but the contributions of LATS1/2 in EC progression remains unclear. Impaired antigen presentation due to mutations or downregulation of the major histocompatibility complex class I (MHC-I) has been implicated in tumor immune evasion. Herein, we elucidate the oncogenic role that dysregulation of LATS1/2 in EC leads to immune evasion through the down-regulation of MHC-I.MethodsThe mutation and expression as well as the clinical significance of LATS1/2 in EC was assessed in the TCGA cohort and our sample cohort. CRISPR-Cas9 was used to construct knockout cell lines of LATS1/2 in EC. Differentially expressed genes were analyzed by RNA-seq. The interaction between LATS1/2 and STAT1 was verified using co-immunoprecipitation and GST pull-down assays. Mass spectrometry, in vitro kinase assays, ChIP-qPCR, flow cytometry, immunohistochemistry, immunofluorescence and confocal microscopy were performed to investigate the regulation of LATS1/2 on MHC-I through interaction with and phosphorylate STAT1. The killing effect of activated PBMCs on EC cells were used to monitor anti-tumor activity.ResultsHere, we demonstrate that LATS1/2 are frequently mutated and down-regulated in EC. Moreover, LATS1/2 loss was found to be associated with a significant down-regulation of MHC-I, independently of the Hippo-YAP pathway. Instead, LATS1/2 were found to directly interact with and phosphorylate STAT1 at Ser727, a crucial transcription factor for MHC-I upregulation in response to interferon-gamma (IFN-γ) signaling, to promote STAT1 accumulating and moving into the nucleus to enhance the transcriptional activation of IRF1/NLRC5 on MHC-I. Additionally, the loss of LATS1/2 was observed to confer increased resistance of EC cells to immune cell-mediated killing and this resistance could be reversed by over-expression of MHC-I.ConclusionOur findings indicate that dysregulation of LATS1/2 in EC leads to immune evasion through the down-regulation of MHC-I, leading to the suppression of infiltrating activated CD8 + T cells and highlight the importance of LATS1/2 in IFN-γ signaling-mediated tumor immune response, suggesting that LATS1/2 is a promising target for immune checkpoint blockade therapy in EC.

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MET Inhibitors Promote Liver Tumor Evasion of the Immune Response by Stabilizing PDL1.

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MET Inhibitors Promote Liver Tumor Evasion of the Immune Response by Stabilizing PDL1.

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