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

Linjie Zhao; Shuang Huang; Shenglin Mei; Zhengnan Yang; Lian Xu; Nianxin Zhou; Qilian Yang; Qiuhong Shen; Wei Wang; Xiaobing Le; Wayne Bond Lau; Bonnie Lau; Xin Wang; Tao Yi; Xia Zhao; Yuquan Wei; Margaret Warner; Jan Åke Gustafsson; Shengtao Zhou

doi:10.1073/pnas.1803291115

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

Linjie Zhao, Shuang Huang, Shenglin Mei, Zhengnan Yang, Lian Xu, Nianxin Zhou, Qilian Yang, Qiuhong Shen, Wei Wang, Xiaobing Le, Wayne Bond Lau, Bonnie Lau, Xin Wang, Tao Yi, Xia Zhao, Yuquan Wei, Margaret Warner, Jan Åke Gustafsson, Shengtao Zhou

Research output: Contribution to journal › Article › peer-review

52 Scopus citations

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.

Original language	English (US)
Pages (from-to)	E3673-E3681
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	115
Issue number	16
DOIs	https://doi.org/10.1073/pnas.1803291115
State	Published - Apr 17 2018

Keywords

Cancer metastasis
ERβ
IL-1β
LY500307
Neutrophil

ASJC Scopus subject areas

General

Access to Document

10.1073/pnas.1803291115

Cite this

Zhao, L., Huang, S., Mei, S., Yang, Z., Xu, L., Zhou, N., Yang, Q., Shen, Q., Wang, W., Le, X., Lau, W. B., Lau, B., Wang, X., Yi, T., Zhao, X., Wei, Y., Warner, M., Gustafsson, J. Å., & Zhou, S. (2018). Pharmacological activation of estrogen receptor beta augments innate immunity to suppress cancer metastasis. Proceedings of the National Academy of Sciences of the United States of America, 115(16), E3673-E3681. https://doi.org/10.1073/pnas.1803291115

Zhao, L, Huang, S, Mei, S, Yang, Z, Xu, L, Zhou, N, Yang, Q, Shen, Q, Wang, W, Le, X, Lau, WB, Lau, B, Wang, X, Yi, T, Zhao, X, Wei, Y, Warner, M, Gustafsson, JÅ & Zhou, S 2018, 'Pharmacological activation of estrogen receptor beta augments innate immunity to suppress cancer metastasis', Proceedings of the National Academy of Sciences of the United States of America, vol. 115, no. 16, pp. E3673-E3681. https://doi.org/10.1073/pnas.1803291115

@article{d5ea31f5cd5f4afabb4ab5bc0cd1bff0,

title = "Pharmacological activation of estrogen receptor beta augments innate immunity to suppress cancer metastasis",

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 = "Cancer metastasis, ERβ, IL-1β, LY500307, Neutrophil",

author = "Linjie Zhao and Shuang Huang and Shenglin Mei and Zhengnan Yang and Lian Xu and Nianxin Zhou and Qilian Yang and Qiuhong Shen and Wei Wang and Xiaobing Le and Lau, {Wayne Bond} and Bonnie Lau and Xin Wang and Tao Yi and Xia Zhao and Yuquan Wei and Margaret Warner and Gustafsson, {Jan {\AA}ke} and Shengtao Zhou",

note = "Funding Information: This work was supported by grants from the National Natural Science Foundation of China (81773119 and 81402396), the National Key Research and Development Program of China (2017YFA0106800), the Sichuan Science-Technology Soft Sciences Project (2016ZR0086), the Yi Yao Foundation (14H0563), the Direct Scientific Research Grants from West China Second University Hospital of Sichuan University (KS021), the Robert A. Welch Foundation (E-0004), the Swedish Cancer Foundation, and the Center for Innovative Medicine. Funding Information: 5. Bianchini G, Balko JM, Mayer IA, Sanders ME, Gianni L (2016) Triple-negative breast cancer: Challenges and opportunities of a heterogeneous disease. Nat Rev Clin Oncol 13:674–690. Materials and Methods Animals and Tissue Preparation. Female BALB/c and C57/BL6 (6-to 8-wk-old) mice were purchased from Vital River. IL1B−/− mice were purchased from The Jackson Laboratory. These mice were housed in a specific-pathogen-free environment with a consistent room temperature and humidity. All animal experiments were approved by the Institutional Animal Care and Use Committee and Ethics Committee of Sichuan University. Briefly, 100 μL of tumor cell suspension containing 5 × 105 4T1 cells or 1 × 105 B16 tumor cells were injected i.v. into the tail veins of BALB/c mice or C57/BL6, respectively. The mice were treated by inserting pellets (vehicle or LY500307) 7 d after inoculation of tumors. Hormonal treatment lasted for 3 d. Body weight was assessed every 2 d. After all animals were euthanized, the lungs were harvested, the lung weight was recorded, and the total number of lung metastases was counted. For histological evaluation of lung micrometastases, sections of lung tissue from each mouse were stained by H&E and examined under a light microscope. For neutrophil depletion studies, Ly6G-depletion antibody (1A8; Bio X Cell), 200 μg diluted in PBS, was administered daily via i.p. injection during the pretreatment phase for 3 d. H&E Staining and Immunohistochemistry (IHC). Immunohistochemistry stain- ing of lung sections was described previously (33). Some of the paraffin tumor sections were stained with H&E. The others were stained with Ly6G, MPO, and cleaved caspase 3 antibodies. Immunoblot Analysis and ELISA. Immunoblot analysis was performed as described previously (33), with minor modifications. Briefly, 4T1 (5 × 105cells per well) or B16 cells (3 × 105 cells per well) were seeded in a 10-mL plate for 12 h and treated with LY500307 (5 μM). After treatment for 48 h, cells were washed twice with ice-cold PBS and lysed in RIPA buffer (Sigma-Aldrich). Antibodies to cleaved caspase 3 and β-actin (Abcam) were used. ELISA was used to measure IL-1β concentration in the cultured medium as described elsewhere (34). Isolation of Mouse Neutrophils and ex Vivo Neutrophil Migration Assay. Mouse neutrophils were prepared from isolated bone marrow as previously described (35). More than 85% of the pelleted cells were neutrophils as determined by flow cytometry. Transwell chamber migration assay was performed to assess the ex vivo neutrophil migratory potential as previously described (36, 37) with minor modifications. Briefly, neutrophils in 200 μL of serum-free medium were added in the top chamber, and then 500 μL of medium with 10% FBS, conditioned media, or LY500307 plus conditioned media was added to the bottom chamber. Different concentrations of niclosamide were added in both chambers. Neutrophils were allowed to migrate for 48 h. Nonmigrated cells in the top chamber were removed. The migrated cells were fixed in 4% paraformaldehyde and stained with 0.5% crystal violet. Migrated cells were counted and photographed under a light microscope. Statistical Analysis. For studies comparing differences between two groups, we used unpaired Student{\textquoteright}s t tests. For studies comparing more than two groups, we used ANOVA with appropriate post hoc testing. Differences were considered significant when P < 0.05. Data are presented as mean ± SEM. High-throughput sequencing data have been deposited in the Gene Expression Omnibus (GEO) database under accession numbers GSE110769 and GSE110770. ACKNOWLEDGMENTS. This work was supported by grants from the National Natural Science Foundation of China (81773119 and 81402396), the National Key Research and Development Program of China (2017YFA0106800), the Sichuan Science-Technology Soft Sciences Project (2016ZR0086), the Yi Yao Foundation (14H0563), the Direct Scientific Research Grants from West China Second University Hospital of Sichuan University (KS021), the Robert A. Welch Foundation (E-0004), the Swedish Cancer Foundation, and the Center for Innovative Medicine. 6. Warner M, Huang B, Gustafsson JA (2017) Estrogen receptor β as a pharmaceutical target. Trends Pharmacol Sci 38:92–99. 7. Bado I, Gugala Z, Fuqua SAW, Zhang XH (2017) Estrogen receptors in breast and bone: From virtue of remodeling to vileness of metastasis. Oncogene 36:4527–4537. 8. de Giorgi V, et al. (2011) Estrogens, estrogen receptors and melanoma. Expert Rev Anticancer Ther 11:739–747. 9. Marzagalli M, et al. (2016) Estrogen receptor beta in melanoma: From molecular in-sights to potential clinical utility. Front Endocrinol (Lausanne) 7:140. 10. Brabletz T, Kalluri R, Nieto MA, Weinberg RA (2018) EMT in cancer. Nat Rev Cancer 18:128–134. 11. Katt ME, Wong AD, Searson PC (2018) Dissemination from a solid tumor: Examining the multiple parallel pathways. Trends Cancer 4:20–37. 12. Braeuer RR, et al. (2014) Why is melanoma so metastatic? Pigment Cell Melanoma Res 27:19–36. 13. Press DJ, Miller ME, Liederbach E, Yao K, Huo D (2017) De novo metastasis in breast cancer: Occurrence and overall survival stratified by molecular subtype. Clin Exp Metastasis, 10.1007/s10585-017-9871-9. 14. Bajikar SS, et al. (2017) Tumor-suppressor inactivation of GDF11 occurs by precursor sequestration in triple-negative breast cancer. Dev Cell 43:418–435.e413. 15. Ossio R, Rold{\'a}n-Mar{\'i}n R, Mart{\'i}nez-Said H, Adams DJ, Robles-Espinoza CD (2017) Melanoma: A global perspective. Nat Rev Cancer 17:393–394. 16. Mann S, et al. (2001) Estrogen receptor beta expression in invasive breast cancer. Hum Pathol 32:113–118. 17. Ma R, et al. (2017) Estrogen receptor beta as a therapeutic target in breast cancer stem cells. J Natl Cancer Inst 109:1–14. 18. Renoir JM, Marsaud V, Lazennec G (2013) Estrogen receptor signaling as a target for novel breast cancer therapeutics. Biochem Pharmacol 85:449–465. 19. Honma N, et al. (2008) Clinical importance of estrogen receptor-beta evaluation in breast cancer patients treated with adjuvant tamoxifen therapy. J Clin Oncol 26:3727–3734. 20. Hinsche O, Girgert R, Emons G, Gr{\"u}ndker C (2015) Estrogen receptor β selective ag-onists reduce invasiveness of triple-negative breast cancer cells. Int J Oncol 46: 878–884. 21. Ruddy SC, et al. (2014) Preferential estrogen receptor β ligands reduce Bcl-2 expres-sion in hormone-resistant breast cancer cells to increase autophagy. Mol Cancer Ther 13:1882–1893. 22. Cotrim CZ, et al. (2013) Estrogen receptor beta growth-inhibitory effects are re-pressed through activation of MAPK and PI3K signalling in mammary epithelial and breast cancer cells. Oncogene 32:2390–2402. 23. Marzagalli M, Casati L, Moretti RM, Montagnani Marelli M, Limonta P (2015) Estrogen receptor beta agonists differentially affect the growth of human melanoma cell lines. PLoS One 10:e0134396. BIOCHEMISTRY 24. Papayannopoulos V (2018) Neutrophil extracellular traps in immunity and disease. Nat Rev Immunol 18:134–147. 25. Sch{\"u}rmann N, et al. (2017) Myeloperoxidase targets oxidative host attacks to Sal-monella and prevents collateral tissue damage. Nat Microbiol 2:16268. 26. Harbort CJ, et al. (2015) Neutrophil oxidative burst activates ATM to regulate cytokine production and apoptosis. Blood 126:2842–2851. 27. Coffelt SB, Wellenstein MD, de Visser KE (2016) Neutrophils in cancer: Neutral no more. Nat Rev Cancer 16:431–446. 28. Mouchemore KA, Anderson RL, Hamilton JA (2018) Neutrophils, G-CSF and their contribution to breast cancer metastasis. FEBS J 285:665–679. 29. Dissemond J, et al. (2003) Activated neutrophils exert antitumor activity against human melanoma cells: Reactive oxygen species-induced mechanisms and their modulation by granulocyte-macrophage-colony-stimulating factor. J Invest Dermatol 121:936–938. 30. Granot Z, et al. (2011) Tumor entrained neutrophils inhibit seeding in the premeta-static lung. Cancer Cell 20:300–314. 31. Andzinski L, et al. (2016) Type I IFNs induce anti-tumor polarization of tumor asso-ciated neutrophils in mice and human. Int J Cancer 138:1982–1993. 32. Finisguerra V, et al. (2015) MET is required for the recruitment of anti-tumoural neutrophils. Nature 522:349–353. 33. Zhou S, et al. (2012) Proteomics identification of annexin A2 as a key mediator in the metastasis and proangiogenesis of endometrial cells in human adenomyosis. Mol Cell Proteomics 11:M112.017988. 34. Bai Y, et al. (2009) VEGF-targeted short hairpin RNA inhibits intraperitoneal ovarian cancer growth in nude mice. Oncology 77:385–394. 35. Partida-Sanchez S, et al. (2007) Chemotaxis of mouse bone marrow neutrophils and dendritic cells is controlled by adp-ribose, the major product generated by the CD38 enzyme reaction. J Immunol 179:7827–7839. 36. Zhao L, et al. (2017) Long noncoding RNA LINC00092 acts in cancer-associated fi-broblasts to drive glycolysis and progression of ovarian cancer. Cancer Res 77: 1369–1382. 37. Zhao L, et al. (2017) An integrated analysis identifies STAT4 as a key regulator of ovarian cancer metastasis. Oncogene 36:3384–3396. PNAS PLUS Funding Information: ACKNOWLEDGMENTS. This work was supported by grants from the National Natural Science Foundation of China (81773119 and 81402396), the National Key Research and Development Program of China (2017YFA0106800), the Sichuan Science-Technology Soft Sciences Project (2016ZR0086), the Yi Yao Foundation (14H0563), the Direct Scientific Research Grants from West China Second University Hospital of Sichuan University (KS021), the Robert A. Welch Foundation (E-0004), the Swedish Cancer Foundation, and the Center for Innovative Medicine. Publisher Copyright: {\textcopyright} 2018 National Academy of Sciences. All Rights Reserved.",

year = "2018",

month = apr,

day = "17",

doi = "10.1073/pnas.1803291115",

language = "English (US)",

volume = "115",

pages = "E3673--E3681",

journal = "Proceedings of the National Academy of Sciences of the United States of America",

issn = "0027-8424",

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}

TY - JOUR

T1 - Pharmacological activation of estrogen receptor beta augments innate immunity to suppress cancer metastasis

AU - Zhao, Linjie

AU - Huang, Shuang

AU - Mei, Shenglin

AU - Yang, Zhengnan

AU - Xu, Lian

AU - Zhou, Nianxin

AU - Yang, Qilian

AU - Shen, Qiuhong

AU - Wang, Wei

AU - Le, Xiaobing

AU - Lau, Wayne Bond

AU - Lau, Bonnie

AU - Wang, Xin

AU - Yi, Tao

AU - Zhao, Xia

AU - Wei, Yuquan

AU - Warner, Margaret

AU - Gustafsson, Jan Åke

AU - Zhou, Shengtao

N1 - Funding Information: This work was supported by grants from the National Natural Science Foundation of China (81773119 and 81402396), the National Key Research and Development Program of China (2017YFA0106800), the Sichuan Science-Technology Soft Sciences Project (2016ZR0086), the Yi Yao Foundation (14H0563), the Direct Scientific Research Grants from West China Second University Hospital of Sichuan University (KS021), the Robert A. Welch Foundation (E-0004), the Swedish Cancer Foundation, and the Center for Innovative Medicine. Funding Information: 5. Bianchini G, Balko JM, Mayer IA, Sanders ME, Gianni L (2016) Triple-negative breast cancer: Challenges and opportunities of a heterogeneous disease. Nat Rev Clin Oncol 13:674–690. Materials and Methods Animals and Tissue Preparation. Female BALB/c and C57/BL6 (6-to 8-wk-old) mice were purchased from Vital River. IL1B−/− mice were purchased from The Jackson Laboratory. These mice were housed in a specific-pathogen-free environment with a consistent room temperature and humidity. All animal experiments were approved by the Institutional Animal Care and Use Committee and Ethics Committee of Sichuan University. Briefly, 100 μL of tumor cell suspension containing 5 × 105 4T1 cells or 1 × 105 B16 tumor cells were injected i.v. into the tail veins of BALB/c mice or C57/BL6, respectively. The mice were treated by inserting pellets (vehicle or LY500307) 7 d after inoculation of tumors. Hormonal treatment lasted for 3 d. Body weight was assessed every 2 d. After all animals were euthanized, the lungs were harvested, the lung weight was recorded, and the total number of lung metastases was counted. For histological evaluation of lung micrometastases, sections of lung tissue from each mouse were stained by H&E and examined under a light microscope. For neutrophil depletion studies, Ly6G-depletion antibody (1A8; Bio X Cell), 200 μg diluted in PBS, was administered daily via i.p. injection during the pretreatment phase for 3 d. H&E Staining and Immunohistochemistry (IHC). Immunohistochemistry stain- ing of lung sections was described previously (33). Some of the paraffin tumor sections were stained with H&E. The others were stained with Ly6G, MPO, and cleaved caspase 3 antibodies. Immunoblot Analysis and ELISA. Immunoblot analysis was performed as described previously (33), with minor modifications. Briefly, 4T1 (5 × 105cells per well) or B16 cells (3 × 105 cells per well) were seeded in a 10-mL plate for 12 h and treated with LY500307 (5 μM). After treatment for 48 h, cells were washed twice with ice-cold PBS and lysed in RIPA buffer (Sigma-Aldrich). Antibodies to cleaved caspase 3 and β-actin (Abcam) were used. ELISA was used to measure IL-1β concentration in the cultured medium as described elsewhere (34). Isolation of Mouse Neutrophils and ex Vivo Neutrophil Migration Assay. Mouse neutrophils were prepared from isolated bone marrow as previously described (35). More than 85% of the pelleted cells were neutrophils as determined by flow cytometry. Transwell chamber migration assay was performed to assess the ex vivo neutrophil migratory potential as previously described (36, 37) with minor modifications. Briefly, neutrophils in 200 μL of serum-free medium were added in the top chamber, and then 500 μL of medium with 10% FBS, conditioned media, or LY500307 plus conditioned media was added to the bottom chamber. Different concentrations of niclosamide were added in both chambers. Neutrophils were allowed to migrate for 48 h. Nonmigrated cells in the top chamber were removed. The migrated cells were fixed in 4% paraformaldehyde and stained with 0.5% crystal violet. Migrated cells were counted and photographed under a light microscope. Statistical Analysis. For studies comparing differences between two groups, we used unpaired Student’s t tests. For studies comparing more than two groups, we used ANOVA with appropriate post hoc testing. Differences were considered significant when P < 0.05. Data are presented as mean ± SEM. High-throughput sequencing data have been deposited in the Gene Expression Omnibus (GEO) database under accession numbers GSE110769 and GSE110770. ACKNOWLEDGMENTS. This work was supported by grants from the National Natural Science Foundation of China (81773119 and 81402396), the National Key Research and Development Program of China (2017YFA0106800), the Sichuan Science-Technology Soft Sciences Project (2016ZR0086), the Yi Yao Foundation (14H0563), the Direct Scientific Research Grants from West China Second University Hospital of Sichuan University (KS021), the Robert A. Welch Foundation (E-0004), the Swedish Cancer Foundation, and the Center for Innovative Medicine. 6. Warner M, Huang B, Gustafsson JA (2017) Estrogen receptor β as a pharmaceutical target. Trends Pharmacol Sci 38:92–99. 7. Bado I, Gugala Z, Fuqua SAW, Zhang XH (2017) Estrogen receptors in breast and bone: From virtue of remodeling to vileness of metastasis. Oncogene 36:4527–4537. 8. de Giorgi V, et al. (2011) Estrogens, estrogen receptors and melanoma. Expert Rev Anticancer Ther 11:739–747. 9. Marzagalli M, et al. (2016) Estrogen receptor beta in melanoma: From molecular in-sights to potential clinical utility. Front Endocrinol (Lausanne) 7:140. 10. Brabletz T, Kalluri R, Nieto MA, Weinberg RA (2018) EMT in cancer. Nat Rev Cancer 18:128–134. 11. Katt ME, Wong AD, Searson PC (2018) Dissemination from a solid tumor: Examining the multiple parallel pathways. Trends Cancer 4:20–37. 12. Braeuer RR, et al. (2014) Why is melanoma so metastatic? Pigment Cell Melanoma Res 27:19–36. 13. Press DJ, Miller ME, Liederbach E, Yao K, Huo D (2017) De novo metastasis in breast cancer: Occurrence and overall survival stratified by molecular subtype. Clin Exp Metastasis, 10.1007/s10585-017-9871-9. 14. Bajikar SS, et al. (2017) Tumor-suppressor inactivation of GDF11 occurs by precursor sequestration in triple-negative breast cancer. Dev Cell 43:418–435.e413. 15. Ossio R, Roldán-Marín R, Martínez-Said H, Adams DJ, Robles-Espinoza CD (2017) Melanoma: A global perspective. Nat Rev Cancer 17:393–394. 16. Mann S, et al. (2001) Estrogen receptor beta expression in invasive breast cancer. Hum Pathol 32:113–118. 17. Ma R, et al. (2017) Estrogen receptor beta as a therapeutic target in breast cancer stem cells. J Natl Cancer Inst 109:1–14. 18. Renoir JM, Marsaud V, Lazennec G (2013) Estrogen receptor signaling as a target for novel breast cancer therapeutics. Biochem Pharmacol 85:449–465. 19. Honma N, et al. (2008) Clinical importance of estrogen receptor-beta evaluation in breast cancer patients treated with adjuvant tamoxifen therapy. J Clin Oncol 26:3727–3734. 20. Hinsche O, Girgert R, Emons G, Gründker C (2015) Estrogen receptor β selective ag-onists reduce invasiveness of triple-negative breast cancer cells. Int J Oncol 46: 878–884. 21. Ruddy SC, et al. (2014) Preferential estrogen receptor β ligands reduce Bcl-2 expres-sion in hormone-resistant breast cancer cells to increase autophagy. Mol Cancer Ther 13:1882–1893. 22. Cotrim CZ, et al. (2013) Estrogen receptor beta growth-inhibitory effects are re-pressed through activation of MAPK and PI3K signalling in mammary epithelial and breast cancer cells. Oncogene 32:2390–2402. 23. Marzagalli M, Casati L, Moretti RM, Montagnani Marelli M, Limonta P (2015) Estrogen receptor beta agonists differentially affect the growth of human melanoma cell lines. PLoS One 10:e0134396. BIOCHEMISTRY 24. Papayannopoulos V (2018) Neutrophil extracellular traps in immunity and disease. Nat Rev Immunol 18:134–147. 25. Schürmann N, et al. (2017) Myeloperoxidase targets oxidative host attacks to Sal-monella and prevents collateral tissue damage. Nat Microbiol 2:16268. 26. Harbort CJ, et al. (2015) Neutrophil oxidative burst activates ATM to regulate cytokine production and apoptosis. Blood 126:2842–2851. 27. Coffelt SB, Wellenstein MD, de Visser KE (2016) Neutrophils in cancer: Neutral no more. Nat Rev Cancer 16:431–446. 28. Mouchemore KA, Anderson RL, Hamilton JA (2018) Neutrophils, G-CSF and their contribution to breast cancer metastasis. FEBS J 285:665–679. 29. Dissemond J, et al. (2003) Activated neutrophils exert antitumor activity against human melanoma cells: Reactive oxygen species-induced mechanisms and their modulation by granulocyte-macrophage-colony-stimulating factor. J Invest Dermatol 121:936–938. 30. Granot Z, et al. (2011) Tumor entrained neutrophils inhibit seeding in the premeta-static lung. Cancer Cell 20:300–314. 31. Andzinski L, et al. (2016) Type I IFNs induce anti-tumor polarization of tumor asso-ciated neutrophils in mice and human. Int J Cancer 138:1982–1993. 32. Finisguerra V, et al. (2015) MET is required for the recruitment of anti-tumoural neutrophils. Nature 522:349–353. 33. Zhou S, et al. (2012) Proteomics identification of annexin A2 as a key mediator in the metastasis and proangiogenesis of endometrial cells in human adenomyosis. Mol Cell Proteomics 11:M112.017988. 34. Bai Y, et al. (2009) VEGF-targeted short hairpin RNA inhibits intraperitoneal ovarian cancer growth in nude mice. Oncology 77:385–394. 35. Partida-Sanchez S, et al. (2007) Chemotaxis of mouse bone marrow neutrophils and dendritic cells is controlled by adp-ribose, the major product generated by the CD38 enzyme reaction. J Immunol 179:7827–7839. 36. Zhao L, et al. (2017) Long noncoding RNA LINC00092 acts in cancer-associated fi-broblasts to drive glycolysis and progression of ovarian cancer. Cancer Res 77: 1369–1382. 37. Zhao L, et al. (2017) An integrated analysis identifies STAT4 as a key regulator of ovarian cancer metastasis. Oncogene 36:3384–3396. PNAS PLUS Funding Information: ACKNOWLEDGMENTS. This work was supported by grants from the National Natural Science Foundation of China (81773119 and 81402396), the National Key Research and Development Program of China (2017YFA0106800), the Sichuan Science-Technology Soft Sciences Project (2016ZR0086), the Yi Yao Foundation (14H0563), the Direct Scientific Research Grants from West China Second University Hospital of Sichuan University (KS021), the Robert A. Welch Foundation (E-0004), the Swedish Cancer Foundation, and the Center for Innovative Medicine. Publisher Copyright: © 2018 National Academy of Sciences. All Rights Reserved.

PY - 2018/4/17

Y1 - 2018/4/17

N2 - 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.

AB - 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.

KW - Cancer metastasis

KW - ERβ

KW - IL-1β

KW - LY500307

KW - Neutrophil

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UR - http://www.scopus.com/inward/citedby.url?scp=85045525110&partnerID=8YFLogxK

U2 - 10.1073/pnas.1803291115

DO - 10.1073/pnas.1803291115

M3 - Article

C2 - 29592953

AN - SCOPUS:85045525110

VL - 115

SP - E3673-E3681

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 16

ER -

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

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