Steven C Kao1, Yuen Yee Cheng2, Marissa Williams3, Michaela B Kirschner3, Jason Madore4, Trina Lum5, Kadir H Sarun2, Anthony Linton6, Brian McCaughan7, Sonja Klebe8, Nico van Zandwijk3, Richard A Scolyer9, Michael J Boyer10, Wendy A Cooper11, Glen Reid12. 1. Asbestos Diseases Research Institute, Sydney, Australia; Department of Medical Oncology, Chris O'Brien Lifehouse, Sydney, Australia; Sydney Medical School, The University of Sydney, Sydney, Australia. 2. Asbestos Diseases Research Institute, Sydney, Australia. 3. Asbestos Diseases Research Institute, Sydney, Australia; Sydney Medical School, The University of Sydney, Sydney, Australia. 4. Sydney Medical School, The University of Sydney, Sydney, Australia; Melanoma Institute Australia, Sydney, Australia. 5. Department of Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Sydney, Australia. 6. Asbestos Diseases Research Institute, Sydney, Australia; Department of Medical Oncology, Concord Cancer Centre, Sydney, Australia. 7. Sydney Medical School, The University of Sydney, Sydney, Australia; Sydney Cardiothoracic Surgeons, RPAH Medical Centre, Sydney, Australia. 8. Department of Anatomical Pathology, Flinders University and SA Pathology at Flinders Medical Centre, Adelaide Australia. 9. Sydney Medical School, The University of Sydney, Sydney, Australia; Department of Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Sydney, Australia; Department of Medical Oncology, Concord Cancer Centre, Sydney, Australia. 10. Department of Medical Oncology, Chris O'Brien Lifehouse, Sydney, Australia; Sydney Medical School, The University of Sydney, Sydney, Australia. 11. Sydney Medical School, The University of Sydney, Sydney, Australia; Department of Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Sydney, Australia; School of Medicine, Western Sydney University, Sydney, Australia. 12. Asbestos Diseases Research Institute, Sydney, Australia; Sydney Medical School, The University of Sydney, Sydney, Australia. Electronic address: glen.reid@sydney.edu.au.
Abstract
INTRODUCTION: The upregulation of programmed death ligand 1 (PD-L1) is found in many cancers and contributes to evasion of the host's immune defense. In malignant pleural mesothelioma (MPM), PD-L1 expression is associated with the nonepithelioid histological subtype and poor prognosis, but the pathways involved in control of PD-L1 expression in MPM are poorly understood. To address one possible means of PD-L1 regulation we investigated the relationship between dysregulated microRNA levels and PD-L1 expression. METHODS: PD-L1 expression was analyzed by immunohistochemistry in tissue microarrays prepared from samples from patients undergoing an operation (pleurectomy with or without decortication). MicroRNA expression was analyzed by reverse-transcriptase quantitative polymerase chain reaction. Regulation of PD-L1 expression in cell lines was assessed after transfection with microRNA mimics and small interfering RNAs. Interaction between microRNAs and PD-L1 was analyzed by using argonaute-2 immunoprecipitation and a luciferase reporter assay. RESULTS: In a series of 72 patients with MPM, 18 (25%) had positive PD-L1 staining, and this was more common in patients with the nonepithelioid subtype (p = 0.01). PD-L1 expression was associated with poor survival (median overall survival 4.0 versus 9.2 months with positive versus negative PD-L1 expression [p < 0.001]), and in multivariate analyses, PD-L1 expression remained a significant adverse prognostic indicator (hazard ratio = 2.2, 95% confidence interval: 1.2-4.1, p < 0.01). In the same patient series, PD-L1 expression was also associated with downregulation of microRNAs previously shown to have tumor suppressor activity in MPM. The median microRNA expression levels of miR-15b, miR-16, miR-193a-3p, miR-195, and miR-200c were significantly lower in the PD-L1-positive samples. Transfecting MPM cell lines with mimics corresponding to miR-15a and miR-16, both of which are predicted to target PD-L1, led to downregulation of PD-L1 mRNA and protein. In addition, miR-193a-3p, with an alternative G-U-containing target site, also caused PD-L1 downregulation. CONCLUSIONS: Together, these data suggest that tumor suppressor microRNAs contribute to the regulation of PD-L1 expression in MPM.
INTRODUCTION: The upregulation of programmed death ligand 1 (PD-L1) is found in many cancers and contributes to evasion of the host's immune defense. In malignant pleural mesothelioma (MPM), PD-L1 expression is associated with the nonepithelioid histological subtype and poor prognosis, but the pathways involved in control of PD-L1 expression in MPM are poorly understood. To address one possible means of PD-L1 regulation we investigated the relationship between dysregulated microRNA levels and PD-L1 expression. METHODS:PD-L1 expression was analyzed by immunohistochemistry in tissue microarrays prepared from samples from patients undergoing an operation (pleurectomy with or without decortication). MicroRNA expression was analyzed by reverse-transcriptase quantitative polymerase chain reaction. Regulation of PD-L1 expression in cell lines was assessed after transfection with microRNA mimics and small interfering RNAs. Interaction between microRNAs and PD-L1 was analyzed by using argonaute-2 immunoprecipitation and a luciferase reporter assay. RESULTS: In a series of 72 patients with MPM, 18 (25%) had positive PD-L1 staining, and this was more common in patients with the nonepithelioid subtype (p = 0.01). PD-L1 expression was associated with poor survival (median overall survival 4.0 versus 9.2 months with positive versus negative PD-L1 expression [p < 0.001]), and in multivariate analyses, PD-L1 expression remained a significant adverse prognostic indicator (hazard ratio = 2.2, 95% confidence interval: 1.2-4.1, p < 0.01). In the same patient series, PD-L1 expression was also associated with downregulation of microRNAs previously shown to have tumor suppressor activity in MPM. The median microRNA expression levels of miR-15b, miR-16, miR-193a-3p, miR-195, and miR-200c were significantly lower in the PD-L1-positive samples. Transfecting MPM cell lines with mimics corresponding to miR-15a and miR-16, both of which are predicted to target PD-L1, led to downregulation of PD-L1 mRNA and protein. In addition, miR-193a-3p, with an alternative G-U-containing target site, also caused PD-L1 downregulation. CONCLUSIONS: Together, these data suggest that tumor suppressor microRNAs contribute to the regulation of PD-L1 expression in MPM.