BRAFV600E-induced, tumor intrinsic PD-L1 can regulate chemotherapy-induced apoptosis in human colon cancer cells and in tumor xenografts

Abstract

Programmed death ligand 1 (PD-L1) is an immune checkpoint protein; however, emerging data suggest that tumor cell PD-L1 may regulate immune-independent and intrinsic cellular functions. We demonstrate regulation of PD-L1 by oncogenic BRAFV600E and investigated its ability to influence apoptotic susceptibility in colorectal cancer (CRC) cells. Endogenous or exogenous mutant vs. wild-type BRAF were shown to increase PD-L1 messenger RNA (mRNA) and protein expression that was attenuated by MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase) inhibition or c-JUN and YAP knockdown. Deletion of PD-L1 reduced tumor cell growth in vitro and in vivo. Loss of PD-L1 was also shown to attenuate DNA damage and apoptosis induced by diverse anti-cancer drugs that could be reversed by restoration of wild-type PD-L1, but not mutants with deletion of its extra- or intracellular domain. The effect of PD-L1 on chemosensitivity was confirmed in MC38 murine tumor xenografts generated from PD-L1-knockout vs. parental cells. Deletion of PD-L1 suppressed BH3-only BIM and BIK proteins that could be restored by re-expression of PD-L1; re-introduction of BIM enhanced apoptosis. PD-L1 expression was significantly increased in BRAFV600E human colon cancers, and patients whose tumors had high vs. low PD-L1 had significantly better survival. In summary, BRAFV600E can transcriptionally upregulate PD-L1 expression that was shown to induce BIM and BIK to enhance chemotherapy-induced apoptosis. These data indicate an intrinsic, non-immune function of PD-L1, and suggest the potential for tumor cell PD-L1 as a predictive biomarker.

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References

  1. 1.

    Baeten JM, Palanee-Phillips T, Brown ER, Schwartz K, Soto-Torres LE, Govender V, et al. Use of a vaginal ring containing dapivirine for HIV-1 prevention in women. N Engl J Med. 2016;375:2121–32.

  2. 2.

    Dong H, Zhu G, Tamada K, Chen L. B7-H1 a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat Med. 1999;5:1365–9.

  3. 3.

    Hamanishi J, Mandai M, Matsumura N, Abiko K, Baba T, Konishi I. PD-1/PD-L1 blockade in cancer treatment: perspectives and issues. Int J Clin Oncol. 2016;21:462–73.

  4. 4.

    Chen N, Fang W, Zhan J, Hong S, Tang Y, Kang S, et al. Upregulation of PD-L1 by EGFR activation mediates the immune escape in EGFR-driven NSCLC: implication for optional immune targeted therapy for NSCLC patients with EGFR mutation. J Thorac Oncol. 2015;10:910–23.

  5. 5.

    Sznol M, Chen L. Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer-response. Clin Cancer Res. 2013;19:5542.

  6. 6.

    Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med. 2012;366:2455–65.

  7. 7.

    Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB, et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med. 2002;8:793–800.

  8. 8.

    Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer. 2012;12:252–64.

  9. 9.

    Taube JM, Anders RA, Young GD, Xu H, Sharma R, McMiller TL, et al. Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape. Sci Transl Med. 2012;4:127ra137.

  10. 10.

    Topalian SL, Drake CG, Pardoll DM. Targeting the PD-1/B7-H1(PD-L1) pathway to activate anti-tumor immunity. Curr Opin Immunol. 2012;24:207–12.

  11. 11.

    Azuma T, Yao S, Zhu G, Flies AS, Flies SJ, Chen L. B7-H1 is a ubiquitous antiapoptotic receptor on cancer cells. Blood. 2008;111:3635–43.

  12. 12.

    Clark CA, Gupta HB, Sareddy G, Pandeswara S, Lao S, Yuan B, et al. Tumor-intrinsic PD-L1 signals regulate cell growth, pathogenesis, and autophagy in ovarian cancer and melanoma. Cancer Res. 2016;76:6964–74.

  13. 13.

    Kleffel S, Posch C, Barthel SR, Mueller H, Schlapbach C, Guenova E, et al. Melanoma cell-intrinsic PD-1 receptor functions promote tumor growth. Cell. 2015;162:1242–56.

  14. 14.

    Orzechowski ADH. B7-H1 confers tumor chemoresistance by regulating MAPK/ERK activation. Cancer Res. 2014;74:abstract 5026.

  15. 15.

    Roberts PJ, Der CJ. Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene. 2007;26:3291–310.

  16. 16.

    Phipps AI, Ahnen DJ, Cheng I, Newcomb PA, Win AK, Burnett T. PIK3CA somatic mutation status in relation to patient and tumor factors in racial/ethnic minorities with colorectal cancer. Cancer Epidemiol Biomark Prev. 2015;24:1046–51.

  17. 17.

    Zaanan A, Okamoto K, Kawakami H, Khazaie K, Huang S, Sinicrope FA. The mutant KRAS gene up-regulates BCL-XL protein via STAT3 to confer apoptosis resistance that is reversed by BIM protein induction and BCL-XL antagonism. J Biol Chem. 2015;290:23838–49.

  18. 18.

    Kopetz S, Desai J, Chan E, Hecht JR, O'Dwyer PJ, Maru D, et al. Phase II pilot study of vemurafenib in patients with metastatic BRAF-mutated colorectal cancer. J Clin Oncol. 2015;33:4032–8.

  19. 19.

    Missiaglia E, Jacobs B, D'Ario G, Di Narzo AF, Soneson C, Budinska E, et al. Distal and proximal colon cancers differ in terms of molecular, pathological, and clinical features. Ann Oncol. 2014;25:1995–2001.

  20. 20.

    Cancer Genome Atlas N. Comprehensive molecular characterization of human colon and rectal cancer. Nature. 2012;487:330–7.

  21. 21.

    Weisenberger DJ, Siegmund KD, Campan M, Young J, Long TI, Faasse MA, et al. CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nat Genet. 2006;38:787–93.

  22. 22.

    Le DT, Hubbard-Lucey VM, Morse MA, Heery CR, Dwyer A, Marsilje TH, et al. A blueprint to advance colorectal cancer immunotherapies. Cancer Immunol Res. 2017;5:942–9.

  23. 23.

    Taieb J, Le Malicot K, Shi Q, Penault-Llorca F, Bouche O, Tabernero J, et al. Prognostic value of BRAF and KRAS mutations in MSI and MSS stage III colon cancer. J Natl Cancer Inst. 2017;109:djw272.

  24. 24.

    Shaul YD, Seger R. The MEK/ERK cascade: from signaling specificity to diverse functions. Biochim Biophys Acta. 2007;1773:1213–26.

  25. 25.

    Yang SM, Park YK, Kim JI, Lee YH, Lee TY, Jang BC. LY3009120, a pan-Raf kinase inhibitor, inhibits adipogenesis of 3T3-L1 cells by controlling the expression and phosphorylation of C/EBP-alpha, PPAR-gamma, STAT3, FAS, ACC, perilipin A, and AMPK. Int J Mol Med. 2018;42:3477–84.

  26. 26.

    King AJ, Arnone MR, Bleam MR, Moss KG, Yang J, Fedorowicz KE, et al. Dabrafenib; preclinical characterization, increased efficacy when combined with trametinib, while BRAF/MEK tool combination reduced skin lesions. PLoS ONE. 2013;8:e67583.

  27. 27.

    Hersey P, Gallagher S. A focus on PD-L1 in human melanoma. Clin Cancer Res. 2013;19:514–6.

  28. 28.

    Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646–74.

  29. 29.

    Bhalla S, Evens AM, Dai B, Prachand S, Gordon LI, Gartenhaus RB. The novel anti-MEK small molecule AZD6244 induces BIM-dependent and AKT-independent apoptosis in diffuse large B-cell lymphoma. Blood. 2011;118:1052–61.

  30. 30.

    Merino D, Giam M, Hughes PD, Siggs OM, Heger K, O'Reilly LA, et al. The role of BH3-only protein Bim extends beyond inhibiting Bcl-2-like prosurvival proteins. J Cell Biol. 2009;186:355–62.

  31. 31.

    Kim MH, Kim CG, Kim SK, Shin SJ, Choe EA, Park SH, et al. YAP-induced PD-L1 expression drives immune evasion in BRAFi-resistant melanoma. Cancer Immunol Res. 2018;6:255–66.

  32. 32.

    Lee BS, Park DI, Lee DH, Lee JE, Yeo MK, Park YH, et al. Hippo effector YAP directly regulates the expression of PD-L1 transcripts in EGFR-TKI-resistant lung adenocarcinoma. Biochem Biophys Res Commun. 2017;491:493–9.

  33. 33.

    Monje P, Hernandez-Losa J, Lyons RJ, Castellone MD, Gutkind JS. Regulation of the transcriptional activity of c-Fos by ERK. A novel role for the prolyl isomerase PIN1. J Biol Chem. 2005;280:35081–4.

  34. 34.

    Liu J, Hamrouni A, Wolowiec D, Coiteux V, Kuliczkowski K, Hetuin D, et al. Plasma cells from multiple myeloma patients express B7-H1 (PD-L1) and increase expression after stimulation with IFN-{gamma} and TLR ligands via a MyD88-, TRAF6-, and MEK-dependent pathway. Blood. 2007;110:296–304.

  35. 35.

    Loi S, Dushyanthen S, Beavis PA, Salgado R, Denkert C, Savas P, et al. RAS/MAPK activation is associated with reduced tumor-infiltrating lymphocytes in triple-negative breast cancer: therapeutic cooperation between MEK and PD-1/PD-L1 immune checkpoint inhibitors. Clin Cancer Res. 2016;22:1499–509.

  36. 36.

    Audrito V, Serra S, Stingi A, Orso F, Gaudino F, Bologna C, et al. PD-L1 up-regulation in melanoma increases disease aggressiveness and is mediated through miR-17-5p. Oncotarget. 2017;8:15894–911.

  37. 37.

    Atefi M, Avramis E, Lassen A, Wong DJ, Robert L, Foulad D, et al. Effects of MAPK and PI3K pathways on PD-L1 expression in melanoma. Clin Cancer Res. 2014;20:3446–57.

  38. 38.

    Prahallad A, Sun C, Huang S, Di Nicolantonio F, Salazar R, Zecchin D, et al. Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR. Nature. 2012;483:100–3.

  39. 39.

    Corcoran RB, Andre T, Atreya CE, Schellens JHM, Yoshino T, Bendell JC, et al. Combined BRAF, EGFR, and MEK inhibition in patients with BRAF(V600E)-mutant colorectal cancer. Cancer Discov. 2018;8:428–43.

  40. 40.

    Ghebeh H, Lehe C, Barhoush E, Al-Romaih K, Tulbah A, Al-Alwan M, et al. Doxorubicin downregulates cell surface B7-H1 expression and upregulates its nuclear expression in breast cancer cells: role of B7-H1 as an anti-apoptotic molecule. Breast Cancer Res. 2010;12:R48.

  41. 41.

    Liu J, Quan L, Zhang C, Liu A, Tong D, Wang J. Over-activated PD-1/PD-L1 axis facilitates the chemoresistance of diffuse large B-cell lymphoma cells to the CHOP regimen. Oncol Lett. 2018;15:3321–8.

  42. 42.

    Yan F, Pang J, Peng Y, Molina JR, Yang P, Liu S. Elevated cellular PD1/PD-L1 expression confers acquired resistance to cisplatin in small cell lung cancer cells. PLoS ONE. 2016;11:e0162925.

  43. 43.

    Zhang XH, Tee LY, Wang XG, Huang QS, Yang SH. Off-target effects in CRISPR/Cas9-mediated genome engineering. Mol Ther Nucleic Acids. 2015;4:e264.

  44. 44.

    Adams JM, Cory S. The BCL-2 arbiters of apoptosis and their growing role as cancer targets. Cell Death Differ. 2018;25:27–36.

  45. 45.

    Luo H, Yang Y, Duan J, Wu P, Jiang Q, Xu C. PTEN-regulated AKT/FoxO3a/Bim signaling contributes to reactive oxygen species-mediated apoptosis in selenite-treated colorectal cancer cells. Cell Death Dis. 2013;4:e481.

  46. 46.

    Bae SU, Jeong WK, Baek SK, Kim NK, Hwang I. Prognostic impact of programmed cell death ligand 1 expression on long-term oncologic outcomes in colorectal cancer. Oncol Lett. 2018;16:5214–22.

  47. 47.

    Droeser RA, Hirt C, Viehl CT, Frey DM, Nebiker C, Huber X, et al. Clinical impact of programmed cell death ligand 1 expression in colorectal cancer. Eur J Cancer. 2013;49:2233–42.

  48. 48.

    Li Y, Liang L, Dai W, Cai G, Xu Y, Li X, et al. Prognostic impact of programed cell death-1 (PD-1) and PD-ligand 1 (PD-L1) expression in cancer cells and tumor infiltrating lymphocytes in colorectal cancer. Mol Cancer. 2016;15:55.

  49. 49.

    Song M, Chen D, Lu B, Wang C, Zhang J, Huang L, et al. PTEN loss increases PD-L1 protein expression and affects the correlation between PD-L1 expression and clinical parameters in colorectal cancer. PLoS ONE. 2013;8:e65821.

  50. 50.

    Tang H, Liang Y, Anders RA, Taube JM, Qiu X, Mulgaonkar A, et al. PD-L1 on host cells is essential for PD-L1 blockade-mediated tumor regression. J Clin Invest. 2018;128:580–8.

  51. 51.

    Tu X, Qin B, Zhang Y, Zhang C, Kahila M, Nowsheen S, et al. PD-L1 (B7-H1) competes with the RNA exosome to regulate the DNA damage response and can be targeted to sensitize to radiation or chemotherapy. Molecular Cell. 2019;74:1215–26.e4.

  52. 52.

    Kawakami H, Huang S, Pal K, Dutta SK, Mukhopadhyay D, Sinicrope FA. Mutant BRAF upregulates MCL-1 to confer apoptosis resistance that is reversed by MCL-1 antagonism and cobimetinib in colorectal cancer. Mol Cancer Ther. 2016;15:3015–27.

  53. 53.

    Zhang P, Kawakami H, Liu W, Zeng X, Strebhardt K, Tao K, et al. Targeting CDK1 and MEK/ERK overcomes apoptotic resistance in BRAF-mutant human colorectal cancer. Mol Cancer Res. 2018;16:378–89.

  54. 54.

    Guzman C, Bagga M, Kaur A, Westermarck J, Abankwa D. Colony rea: an ImageJ plugin to automatically quantify colony formation in clonogenic assays. PLoS ONE. 2014;9:e92444.

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Acknowledgements

This study was supported, in part, by NCI R01 (CA210509-01A1) to F.A.S. D.F. was supported by the Scientific Research Training Program for Young Talents of Tianjin Medical University General Hospital, PRC; current address is Department of General Surgery, Tianjin Medical University General Hospital, Tianjin, China. L.S. is supported by the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, China. The authors’ express their gratitude to Mr. Matthew A. Bockol for downloading TCGA data.

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Correspondence to Frank A. Sinicrope.

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Feng, D., Qin, B., Pal, K. et al. BRAFV600E-induced, tumor intrinsic PD-L1 can regulate chemotherapy-induced apoptosis in human colon cancer cells and in tumor xenografts. Oncogene 38, 6752–6766 (2019) doi:10.1038/s41388-019-0919-y

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