Eya3 promotes breast tumor–associated immune suppression via threonine phosphatase–mediated PD-L1 upregulation
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
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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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 8

Eya3 Thr phosphatase activity regulates PD-L1 expression in 66cl4 mammary carcinoma cells.

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Eya3 Thr phosphatase activity regulates PD-L1 expression in 66cl4 mammar...
(A) RT-qPCR analysis on cDNA derived from RNA isolated from 66cl4-SCR and Eya3-KD cells. PD-L1 was normalized to GAPDH levels. Data represent mean ± SEM, and significance was measured using ANOVA with sum contrasts in R for biological triplicates for 3 combined experiments. (B) Calculated percentage of PD-L1+ cancer cells present per gram of 66cl4-SCR and Eya3-KD tumors. Tumors were isolated (SCR, n = 5; SCR2, n = 5; KD2, n = 4; KD3, n = 5) and analyzed by flow cytometry. Data represent mean ± SEM, and significance was measured using ANOVA with sum contrasts in R. PD-L1 cancer cells were defined as GhostRed780–luciferase+CD45–PD-L1+. (C) FLAG-Eya3 WT and H79A Thr phosphatase–dead protein was isolated from HEK293FT cells using FLAG tag pull-down. Thr phosphatase activity was measured using an equal amount of Eya3 WT and H79A mutant protein in a malachite green phosphorylation assay in duplicate. Control indicates reaction with buffer and phospho-substrate without Eya3 protein added. Data represent mean ± SD. Significance was measured using ANOVA. Representative experiment (n = 4). (D) 66cl4-Eya3-KD cells were stably rescued with empty vector (EV), Eya3 WT, Eya3 H79A, Eya3 D262N, or Eya3 A520R. Western blot analysis performed on membranes containing whole cell lysates from these cells, as well as the 66cl4-SCR +EV control cells, and antibodies against Eya3 and GAPDH were used to probe the membranes. Representative image of experiments performed at least 3 times. (E) RT-qPCR analysis for PD-L1 using cDNA derived from cell lines shown in D. PD-L1 normalized to GAPDH. Data represent mean ± SEM. Significance was measured using ANOVA. Representative experiment (n = 3). **P < 0.01, ***P < 0.001.