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Rho-associated protein kinase-dependent moesin phosphorylation is required for PD-L1 stabilization in breast cancer.

ABSTRACT: Expression of programmed cell death ligand (PD-L1) is associated with poor prognosis in breast cancer. Understanding the regulation of PD-L1 expression in breast cancer could provide a new strategy for breast cancer treatment. Here, we demonstrate that moesin (MSN) phosphorylation by Rho-associated protein kinase (ROCK) stabilizes PD-L1 protein levels. Our results indicate that phosphorylated MSN may compete with the E3 ubiquitin ligase SPOP for binding PD-L1. ROCK inhibition via the Y-27632 inhibitor or MSN silencing decreased PD-L1 expression, resulting in T-cell activation both in vitro and in vivo. Administration of Y-27632 into immunocompetent Balb/c mice bearing breast tumors suppressed tumor progression and enhanced CD4+ and CD8+ T-cell infiltration. RNA-seq analysis of Y-27632-treated mouse tumors revealed that ROCK inhibition upregulated several immune response genes. However, the combination of Y-27632 and an anti-PD-1 antibody did not show additive or synergistic effects due to reduced PD-L1 in the presence of Y-27632. Our study unravels a previously unappreciated mechanism of PD-L1 regulation through the ROCK-MSN pathway. Moreover, we found that ROCK inhibitors could be combined with breast cancer immunotherapy.

SUBMITTER: Meng F

PROVIDER: S-EPMC7607174 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Rho-associated protein kinase-dependent moesin phosphorylation is required for PD-L1 stabilization in breast cancer.

Meng Fanbiao F Su Yang Y Xu Bo B

Molecular oncology 20201003 11

Expression of programmed cell death ligand (PD-L1) is associated with poor prognosis in breast cancer. Understanding the regulation of PD-L1 expression in breast cancer could provide a new strategy for breast cancer treatment. Here, we demonstrate that moesin (MSN) phosphorylation by Rho-associated protein kinase (ROCK) stabilizes PD-L1 protein levels. Our results indicate that phosphorylated MSN may compete with the E3 ubiquitin ligase SPOP for binding PD-L1. ROCK inhibition via the Y-27632 inh ...[more]

PMID: 32941674

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Project description:Ectopic programmed cell death ligand 1 (PD-L1) expression in non-small cell lung cancers (NSCLCs) is related to immune evasion by cancer, and it is a molecular target of immune checkpoint therapies. Although some altered signals in NSCLCs are responsible for ectopic PD-L1 expression, the precise mechanisms remain obscure. Because we found a higher frequency of EGFR/KRAS mutations in NSCLC cell lines with high PD-L1 expression (p < 0.001), we evaluated the relationships between downstream signals and PD-L1 expression, particularly in three KRAS-mutant adenocarcinoma cell lines. The MEK inhibitor U0126 (20 μM) significantly decreased the surface PD-L1 levels by 50-60% compared with dimethyl sulfoxide (p < 0.0001). Phorbol 12-myristate 13-acetate stimulation (100 nM, 15 min) increased (p < 0.05) and two ERK2 siRNAs as well as KRAS siRNAs decreased (p < 0.05) PD-L1 expression. The transcriptional activity of the potential AP-1 site (+4785 to +5056 from the transcription start site) in the PD-L1 gene was demonstrated by luciferase assays, which was inhibited by U0126. The chromatin immunoprecipitation assay demonstrated the binding of cJUN to the AP-1 site. Two STAT3 siRNAs decreased PD-L1 expression by 10-32% in two of the three KRAS-mutant lung adenocarcinoma cell lines (p < 0.05), while the PI3K inhibitor LY294002 (40 μM) did not change the expression level. Supervised cluster analysis and gene set enrichment analysis between the PD-L1-high and -low NSCLCs revealed a correlation between PD-L1 expression and genes/pathways related to cell motility/adhesion. These results indicate that MAPK signaling is the dominant downstream signal responsible for ectopic PD-L1 expression, in which STAT3 is also involved to some extent. Furthermore, MAPK signaling may control the expression of PD-L1 and several genes related to enhanced cell motility. Our findings suggest that MAPK, along with STAT3, is important for determining PD-L1 expression, which could be useful for targeted therapies against lung cancers.

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Rho-associated protein kinase-dependent moesin phosphorylation is required for PD-L1 stabilization in breast cancer.

Publications

Rho-associated protein kinase-dependent moesin phosphorylation is required for PD-L1 stabilization in breast cancer.

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