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 10

PD-L1 upregulation is required for Eya3-enhanced mammary carcinoma growth.

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PD-L1 upregulation is required for Eya3-enhanced mammary carcinoma growt...
66cl4-SCR and Eya3 KD3 cell lines were stably rescued with empty vector (EV) or PD-L1. (A) RT-qPCR analysis on cDNA derived from RNA isolated from 66cl4-SCR+EV, Eya3 KD3+EV, and Eya3 KD3+PD-L1 rescue cells. PD-L1 normalized to GAPDH. Data represent mean ± SEM. Significance was measured using ANOVA for biological triplicates from 3 combined experiments. (B) Percentage of PD-L1+ cancer cells present per gram of 66cl4-SCR+EV, Eya3 KD3+EV, and Eya3 KD3+PD-L1 rescue tumors. Tumors were isolated (n = 10 mice per group) and analyzed using flow cytometry. Data represent mean ± SEM. Significance was measured using ANOVA. PD-L1+ cancer cells were defined as GhostRed780–luciferase+CD45–PD-L1+. (C) Tumor volume of 66cl4-SCR+EV, Eya3 KD3+EV, and Eya3 KD3+PD-L1 rescue tumors in BALB/c mice as measured using calipers. Each point represents the mean tumor size of mice at that time point after injection ± SEM, and a mixed effects model was used to measure significance. n = 10 mice per group. (D) Representative bioluminescence images of BALB/c mice bearing 66cl4-SCR, Eya3 KD3+EV, and Eya3 KD3+PD-L1 tumors at week 4 after injection. (E–G) Tumors were isolated (n = 10 mice per group) and analyzed by flow cytometry. Data represent mean ± SEM. Significance was measured using ANOVA. (E) Calculated percentage of BrdU+ cancer cells present per gram of 66cl4-SCR and Eya3 KD3+PD-L1 rescue tumors. BrdU+ cancer cells were defined as GhostRed780–luciferase+CD45–BrdU+. (F) Percentage of live CD8+ T cells per gram of 66cl4-SCR, Eya3 KD3+EV, and Eya3 KD3+PD-L1 rescue tumors. Live CD8+ T cells were defined as GhostRed780–luciferase–CD45+CD3+CD8+. (G) Percentage of dead CD8+ T cells present per gram of 66cl4-SCR, Eya3 KD3+EV, and Eya3 KD3+PD-L1 rescue tumors. Dead CD8+ T cells were defined as GhostRed780+luciferase–CD45+CD3+CD8+. *P < 0.05, **P < 0.01, ***P < 0.001.