Abstract
SummaryThe immunosuppressive transmembrane protein PD-L1 was shown to traffic via the multivesicular body (MVB) and to be released on exosomes. A high-content siRNA screen identified the endosomal sorting complexes required for transport (ESCRT)-associated protein ALIX as a regulator of both EGFR activity and PD-L1 surface presentation in basal-like breast cancer (BLBC) cells. ALIX depletion results in prolonged and enhanced stimulation-induced EGFR activity as well as defective PD-L1 trafficking through the MVB, reduced exosomal secretion, and its redistribution to the cell surface. Increased surface PD-L1 expression confers an EGFR-dependent immunosuppressive phenotype on ALIX-depleted cells. An inverse association between ALIX and PD-L1 expression was observed in human breast cancer tissues, while an immunocompetent mouse model of breast cancer revealed that ALIX-deficient tumors are larger and show an increased immunosuppressive environment. Our data suggest that ALIX modulates immunosuppression through regulation of PD-L1 and EGFR and may, therefore, present a diagnostic and therapeutic target for BLBC.
Highlights
Epidermal growth factor receptor (EGFR) is either amplified or mutated in a variety of cancers and contributes significantly to tumorigenesis (Chong and Janne, 2013; Ciardiello and Tortora, 2008)
We found that cells lacking the endosomal sorting complexes required for transport (ESCRT) component ALIX, a critical mediator of exosome biogenesis (Bissig and Gruenberg, 2014; Carlton, 2010), displayed enhanced EGFR activation, suggesting unexpected parallels between mechanisms of exosome biogenesis and regulation of EGFR activity
We found that programmed death-ligand 1 (PD-L1) is secreted on exosomes in an ALIX-dependent manner, and impaired exosomal release conferred an enhanced immunosuppressive phenotype on tumor cells that was dependent upon EGFR kinase activity
Summary
Epidermal growth factor receptor (EGFR) is either amplified or mutated in a variety of cancers and contributes significantly to tumorigenesis (Chong and Janne, 2013; Ciardiello and Tortora, 2008). The analysis confirmed that the association between EGF treatment and lifetime was unaffected by the level of biosensor expression (Figure 1E). This stringent method of analysis was applied to all lifetime data acquired from the high-content small interfering RNA (siRNA) screen. Identification of Regulators of EGFR Using a FLIM-Based High-Content Screen We determined the effects of the targeted knockdown of 533 candidate proteins on EGFR signaling in situ to identify regulators of EGFR among a network of candidate proteins extracted from a bioinformatics-led analysis of proteininteraction databases (Figure S1). Twenty hits were identified that abrogated the biosensor response to ligand (Figure 2A, gray nodes; Figure 2B, cf difference in the slopes of linear regression lines for EGF versus non-EGF treatment cell populations within the example ‘‘non-hit’’ and ‘‘hit’’ siRNA experimental groups; Table S1). Details of the 20 hits identified in the screen are provided in Table S1, and their known interactions with members of the EGFR subnetwork are illustrated in Figure S1 (pink nodes)
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