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 4

Eya3 regulates CD8+ T cells in Met1 mammary carcinoma tumors.

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Eya3 regulates CD8+ T cells in Met1 mammary carcinoma tumors. (A) Repres...
(A) Representative bioluminescence images of FVB mice bearing Met1-SCR or Eya3-KD tumors at week 5 after injection. One Eya3 KD was used, as Eya3 KD2 lost Eya3 suppression. (B) Tumor volume of Met1-SCR and Eya3-KD tumors in FVB mice as measured by luciferase signal of primary tumor over time. Points represent the tumor size at a given time after injection ± SEM. A mixed effects model was used to measure significance. n = 10 mice per cell line. (C) Representative pictures, original magnification ×40, of IHC for Ki67 staining performed on 66cl4-SCR or Eya3-KD tumors. Five sections per tumor were stained and 5 fields of view photographed for each tumor. (D) Quantification of Ki67 IHC performed on Met1-SCR or Eya3-KD tumors. Data represent mean ± SEM. Significance was determined using a 2-tailed Student’s t test on quantification from 5 tumor sections. Five fields of view were scored for each section. (E) Representative pictures, original magnification ×20, of IHC for CD8+ performed on Met1-SCR or Eya3-KD tumors. Five sections per tumor were stained and 5 fields of view photographed for each tumor. (F) Quantification of CD8+ IHC performed on Met1-SCR or Eya3-KD tumors. Data represent mean ± SEM. Significance was measured using a 2-tailed Student’s t test for 5 tumor sections with 5 fields of view scored for each section. (G and H) Calculated number of CD8+ and CD4+ T cells present per gram of Met1-SCR and Eya3-KD tumors. Tumors were isolated (SCR, n = 7; KD3, n = 8) and analyzed using flow cytometry. CD8+ T cells defined as CD45+CD3+CD8+CD4–. CD4+ T cells defined as CD45+CD3+CD8–CD4+. Data represent mean ± SEM. Significance was measured using a 2-tailed Student’s t test. *P < 0.05, ***P < 0.001. C–H are from a repeated experiment and are not from the mice pictured in A and B.