Eya3 promotes breast tumor–associated immune suppression via threonine phosphatase–mediated PD-L1 upregulation
Rebecca L. Vartuli, … , Rui Zhao, Heide L. Ford
Rebecca L. Vartuli, … , Rui Zhao, Heide L. Ford
Published May 14, 2018
Citation Information: J Clin Invest. 2018;128(6):2535-2550. https://doi.org/10.1172/JCI96784.
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Research Article Immunology Oncology

Eya3 promotes breast tumor–associated immune suppression via threonine phosphatase–mediated PD-L1 upregulation

Abstract

Eya proteins are critical developmental regulators that are highly expressed in embryogenesis but downregulated after development. Amplification and/or re-expression of Eyas occurs in many tumor types. In breast cancer, Eyas regulate tumor progression by acting as transcriptional cofactors and tyrosine phosphatases. Intriguingly, Eyas harbor a separate threonine (Thr) phosphatase activity, which was previously implicated in innate immunity. Here we describe what we believe to be a novel role for Eya3 in mediating triple-negative breast cancer–associated immune suppression. Eya3 loss decreases tumor growth in immune-competent mice and is associated with increased numbers of infiltrated CD8+ T cells, which, when depleted, reverse the effects of Eya3 knockdown. Mechanistically, Eya3 utilizes its Thr phosphatase activity to dephosphorylate Myc at pT58, resulting in a stabilized form. We show that Myc is required for Eya3-mediated increases in PD-L1, and that rescue of PD-L1 in Eya3-knockdown cells restores tumor progression. Finally, we demonstrate that Eya3 significantly correlates with PD-L1 in human breast tumors, and that tumors expressing high levels of Eya3 have a decreased CD8+ T cell signature. Our data uncover a role for Eya3 in mediating tumor-associated immune suppression, and suggest that its inhibition may enhance checkpoint therapies.

Authors

Rebecca L. Vartuli, Hengbo Zhou, Lingdi Zhang, Rani K. Powers, Jared Klarquist, Pratyaydipta Rudra, Melanie Y. Vincent, Debashis Ghosh, James C. Costello, Ross M. Kedl, Jill E. Slansky, Rui Zhao, Heide L. Ford

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Figure 7

Eya3 regulates CD8+ T cell exhaustion and promotes apoptosis of mammary carcinoma cells.

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Eya3 regulates CD8+ T cell exhaustion and promotes apoptosis of mammary ...
(A–I) Number of immune cells per gram of 66cl4-SCR and Eya3 KD3 tumors. Tumors were isolated 3 weeks after cancer cell injection (SCR, n = 5; KD3, n = 5), digested, and treated with brefeldin A, ionomycin, and PMA for 4 hours, and analyzed by flow cytometry. Representative experiment (n = 2). Data represent mean ± SEM. Significance was measured using a 2-tailed Student’s t test. (A) Tim3+CD8+ T cells were defined as GhostRed780–B220–MHCII–CD3+CD8+CD4–Tim3+ (Ghost Red 780, Tonbo Biosciences). (B) Blimp1+CD8+ T cells were defined as GhostRed780–B220–MHCII–CD3+CD8+CD4–Blimp1+. (C) T-bethiCD8+ T cells were defined as GhostRed780–B220–MHCII–CD3+CD8+CD4–T-bethi. (D) EomeshiCD8+ T cells were defined as GhostRed780–B220–MHCII–CD3+CD8+CD4–Eomeshi. (E) PD-1+CD8+ T cells were defined as GhostRed780–B220–MHCII–CD45+CD3+CD8+CD4–PD-1+. (F) IL-2+CD8+ T cells were defined as GhostRed780–B220–MHCII–CD3+CD8+CD4–IL-2+. (G) IFN-γ+CD8+ T cells were defined as GhostRed780–B220–MHCII–CD45+CD3+CD8+CD4–IFN-γ+. (H) CD44+CD8+ T cells were defined as GhostRed780–B220–MHCII–CD3+CD8+CD4–CD44+. (I) TNF-α+CD8+ T cells were defined as GhostRed780–B220–MHCII–CD3+CD8+CD4–TNF-α+. Number of apoptotic cells per gram of 66cl4-SCR and Eya3 KD3 tumors. (J and K) Tumors were isolated 5 weeks after cancer cell injection (SCR, n = 5; SCR2, n = 5; KD2, n = 5; KD3, n = 5) and treated as outlined above. Data represent mean ± SEM. Significance was measured using ANOVA with sum contrasts in R. (J) Early-apoptotic carcinoma cells were defined as B220–MHCII–CD45–annexinV+PI–. (K) Late-apoptotic carcinoma cells were defined as B220–MHCII–CD45–annexinV+PI+.