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. 2018 Apr 17;115(16):E3673-E3681.
doi: 10.1073/pnas.1803291115. Epub 2018 Mar 28.

Pharmacological activation of estrogen receptor beta augments innate immunity to suppress cancer metastasis

Linjie Zhao  1 , Shuang Huang  1 , Shenglin Mei  2 , Zhengnan Yang  1 , Lian Xu  3 , Nianxin Zhou  1 , Qilian Yang  1 , Qiuhong Shen  1 , Wei Wang  4 , Xiaobing Le  1 , Wayne Bond Lau  5 , Bonnie Lau  6 , Xin Wang  4 , Tao Yi  1 , Xia Zhao  1 , Yuquan Wei  1 , Margaret Warner  7   8 , Jan-Åke Gustafsson  9   8 , Shengtao Zhou  10
Affiliations

Pharmacological activation of estrogen receptor beta augments innate immunity to suppress cancer metastasis

Linjie Zhao et al. Proc Natl Acad Sci U S A. .

Abstract

Metastases constitute the greatest causes of deaths from cancer. However, no effective therapeutic options currently exist for cancer patients with metastasis. Estrogen receptor β (ERβ), as a member of the nuclear receptor superfamily, shows potent tumor-suppressive activities in many cancers. To investigate whether modulation of ERβ could serve as a therapeutic strategy for cancer metastasis, we examined whether the selective ERβ agonist LY500307 could suppress lung metastasis of triple-negative breast cancer (TNBC) and melanoma. Mechanistically, while we observed that LY500307 potently induced cell death of cancer cells metastasized to lung in vivo, it does not mediate apoptosis of cancer cells in vitro, indicating that the cell death-inducing effects of LY500307 might be mediated by the tumor microenvironment. Pathological examination combined with flow cytometry assays indicated that LY500307 treatment induced significant infiltration of neutrophils in the metastatic niche. Functional experiments demonstrated that LY500307-treated cancer cells show chemotactic effects for neutrophils and that in vivo neutrophil depletion by Ly6G antibody administration could reverse the effects of LY500307-mediated metastasis suppression. RNA sequencing analysis showed that LY500307 could induce up-regulation of IL-1β in TNBC and melanoma cells, which further triggered antitumor neutrophil chemotaxis. However, the therapeutic effects of LY500307 treatment for suppression of lung metastasis was attenuated in IL1B-/- murine models, due to failure to induce antitumor neutrophil infiltration in the metastatic niche. Collectively, our study demonstrated that pharmacological activation of ERβ could augment innate immunity to suppress cancer metastatic colonization to lung, thus providing alternative therapeutic options for cancer patients with metastasis.

Keywords: ERβ; IL-1β; LY500307; cancer metastasis; neutrophil.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Selective ERβ agonist LY500307 suppresses lung metastasis of cancer and prolongs survival in murine models. (A) Representative photos showing the lung metastatic nodules of the 4T1 murine model. (B) The number of lung metastatic nodules in the two groups (n = 10 in each group) of the 4T1 murine model. (C) The overall survival of mice in the two groups (n = 10 in each group) of the 4T1 model. (D) The lung weight in each group of the 4T1 murine model at indicated time points (n = 10 in each group). (E) The body weight of mice in each group of the 4T1 model at indicated time points (n = 10 in each group). (F) Representative photos showing the lung metastatic nodules of the B16 murine model. (G) The number of lung metastatic nodules in the two groups (n = 12 in each group) of the B16 murine model. (H) The overall survival of mice in the two groups (n = 12 in each group) of the B16 model. (I) The lung weight in each group of the B16 murine model at indicated time points (n = 12 in each group). (J) The body weight of mice in each group of the B16 model at indicated time points (n = 12 in each group). Data are shown as mean ± SEM. *P < 0.05; ***P < 0.001.
Fig. 2.
Fig. 2.
Selective ERβ agonist LY500307 induces apoptosis of lung metastatic cancer cells in vivo, but not in vitro. (A) Western blotting analysis of caspase 3 in 4T1 cells treated with control or LY500307 in vitro. (B) Immunostaining of cleaved caspase 3 in the lung metastatic niche in the LY500307-treated 4T1 murine model. (C) Flow cytometry analysis of propidium iodide (PI)/Annexin V staining in 4T1 cells treated with different concentrations of LY500307 in vitro. (D) Western blotting analysis of caspase 3 in B16 cells treated with control or LY500307 in vitro. (E) Immunostaining of cleaved caspase 3 in the lung metastatic niche in the LY500307-treated B16 murine model. (F) Flow cytometry analysis of PI/Annexin V staining in B16 cells treated with different concentrations of LY500307 in vitro.
Fig. 3.
Fig. 3.
Selective ERβ agonist LY500307 induces infiltration of neutrophils in the lung metastatic niche. (A) Flow cytometry analysis of dissociated 4T1 tumors treated with control or LY500307 at indicated time points (staining with CD11b and Ly6G antibodies). (B) H&E, Ly6G, and MPO staining of the lung metastatic niche in the LY500307-treated 4T1 murine model. (C) Flow cytometry analysis of dissociated B16 tumors treated with control or LY500307 at indicated time points (staining with CD11b and Ly6G antibodies). (D) H&E, Ly6G, and MPO staining of the lung metastatic niche in the LY500307-treated B16 murine model.
Fig. 4.
Fig. 4.
Neutrophil depletion hampers the therapeutic efficacy of LY500307 in the suppression of cancer metastasis to the lung. (A) Representative photos showing the lung metastatic nodules of the 4T1 murine model groups treated with control, LY500307, and a combination of LY500307 and 1A8 (Ly6G antibody) (n = 10 in each group). (B–D) Lung weight (B), number of lung metastatic nodules (C), and Ly6G, MPO, and cleaved caspase 3 staining of the lung metastatic niche (D) in each treatment group (n = 10 in each group) of the 4T1 murine model. (E) Representative photos showing the lung metastatic nodules of the B16 murine model groups treated with control, LY500307, and a combination of LY500307 and 1A8 (Ly6G antibody) (n = 10 in each group). (F) The lung weight in each treatment group (n = 10 in each group) of the B16 murine model. (G) The number of lung metastatic nodules in each treatment group (n = 10 in each group) of the B16 murine model. (H) Ly6G, MPO, and cleaved caspase 3 staining of the lung metastatic niche in each treatment group (n = 10 in each group) of the B16 murine model. Data are shown as mean ± SEM. *P < 0.05; **P < 0.01.
Fig. 5.
Fig. 5.
ERβ activation induces IL-1β secretion in tumor cells. (A) Heatmap showing the differentially expressed mRNAs in 4T1 and B16 cells treated with control and LY500307. (B) Venn diagram showing the genes commonly up-regulated in LY500307-treated 4T1 and B16 cells compared with control-treated 4T1 and B16 cells. (C and D) Volcano plots showing the differentially expressed mRNAs in LY500307-treated compared with control-treated 4T1 cells (C) and B16 cells (D). (E and F) qPCR assays to measure the mRNA levels of IL-1β in control-treated and LY500307-treated 4T1 cells (E) and B16 cells (F). (G and H) The relative concentrations of IL-1β in the conditioned media of control-treated and LY500307-treated 4T1 cells (G) and B16 cells (H). Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 6.
Fig. 6.
IL1B knockout reduces infiltration of neutrophils to the lung metastatic niche and reduces the therapeutic effects of LY500307. (A) Representative photos showing the lung metastatic nodules (arrows) in IL1B WT, LY500307-treated IL1B WT, and LY500307-treated IL1B−/− mice (n = 10 in each group) of the B16 model. (B–D) Number of lung metastatic nodules (B), lung weight (C), and body weight (D) of IL1B WT, LY500307-treated IL1B WT, and LY500307-treated IL1B−/− mice in each treatment group (n = 10 in each group) of the B16 model. (E) Flow cytometry analysis of dissociated B16 tumors in IL1B WT, LY500307-treated IL1B WT, and LY500307-treated IL1B−/− mice (n = 10 in each group) (staining with CD11b and Ly6G antibodies). (F) Schematic model for LY500307 effects on neutrophil-mediated activation of antitumor innate immunity within the tumor microenvironment, resulting in invasive cancer clearance. The blue cells with red nuclei are tumor cells. The yellow cells with green nuclei are tumor cells undergoing cell death. The white cells with lobed nuclei are neutrophils. The yellow squared particles are IL-1β. Data are shown as mean ± SEM. *P < 0.05.
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References

    1. Steeg PS. Targeting metastasis. Nat Rev Cancer. 2016;16:201–218. - PMC - PubMed
    1. Nilsson S, Koehler KF, Gustafsson JA. Development of subtype-selective oestrogen receptor-based therapeutics. Nat Rev Drug Discov. 2011;10:778–792. - PubMed
    1. Thomas C, Gustafsson JA. The different roles of ER subtypes in cancer biology and therapy. Nat Rev Cancer. 2011;11:597–608. - PubMed
    1. Jeselsohn R, Buchwalter G, De Angelis C, Brown M, Schiff R. ESR1 mutations—A mechanism for acquired endocrine resistance in breast cancer. Nat Rev Clin Oncol. 2015;12:573–583. - PMC - PubMed
    1. Bianchini G, Balko JM, Mayer IA, Sanders ME, Gianni L. Triple-negative breast cancer: Challenges and opportunities of a heterogeneous disease. Nat Rev Clin Oncol. 2016;13:674–690. - PMC - PubMed

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