Abstract
Purpose
The present study intended to further elucidate the role of G protein-coupled estrogen receptor 1 (GPER-1) in ovarian cancer by comparing the effects of a GPER-1 knockdown and treatment with its agonist G-1 on cell growth, apoptosis, and the transcriptome of two ovarian cancer cell lines. Furthermore, the role of GPER-1 in ovarian cancer survival was examined.
Methods
GPER-1 expression in OVCAR-3 and OAW-42 ovarian cancer cells was knocked down by RNAi. The effects on cell growth were measured by means of the fluorimetric cell titer blue assay and on the transcriptome by Affymetrix GeneChip analysis. The effect of GPER-1 on patient’s survival was examined using open source mRNA and clinical data of 1657 ovarian cancer patients.
Results
GPER-1 knockdown resulted in a significant growth stimulation of both cell lines, whereas treatment with agonist G-1 decreased growth of both cell lines in a dose-dependent manner. Transcriptome analyses revealed a set of 18 genes being conversely regulated after GPER-1 knockdown and G-1 treatment. Generally, treatment with G-1 led to a transcriptome response associated with growth inhibition. In contrast, knockdown of GPER-1 exerted opposite effects, stimulating pathways activating mitosis, but inhibiting pathways associated with apoptosis or interferon signaling. Further analyses using open-access mRNA and clinical data by bioinformatical online tools revealed a longer OS (HR = 0.86, p = 0.057) and PFS (HR = 0.81, p = 0.0035) of ovarian cancer patients with high GPER-1 mRNA expression.
Conclusions
The results of this study clearly support the hypothesis that GPER-1 acts as a tumor suppressor in ovarian cancer.
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source of the mRNA and clinical data as well as the integrated bioinformatical analysis tool is found at https://kmplot.com/ (Győrffy et al. 2012). Clinical data of the included patients can be found in Supplementary Table 4
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Abbreviations
- RT:
-
Reverse transcription
- qPCR:
-
Quantitative polymerase chain reaction
- siRNA:
-
Small interfering RNA
- TP53:
-
Tumor protein 53
- ER:
-
Estrogen receptor
- PR:
-
Progesterone receptor
- DMEM:
-
Dulbecco’s modified eagle medium
- FBS:
-
Fetal bovine serum
References
Aikhionbare FO, Mehrabi S, Kumaresan K, Zavareh M, Olatinwo M, Odunsi K, Partridge E (2007) Mitochondrial DNA sequence variants in epithelial ovarian tumor subtypes and stages. J Carcinog 6:1. https://doi.org/10.1186/1477-3163-6-1
Ariazi EA, Brailoiu E, Yerrum S, Shupp HA, Slifker MJ, Cunliffe HE, Black MA, Donato AL, Arterburn JB, Oprea TI, Prossnitz ER, Dun NJ, Jordan VC (2010) The G protein-coupled receptor GPR30 inhibits proliferation of estrogen receptor-positive breast cancer cells. Cancer Res 70:1184–1194. https://doi.org/10.1158/0008-5472.CAN-09-3068
Au WC, Moore PA, LaFleur DW, Tombal B, Pitha PM (1998) Characterization of the interferon regulatory factor-7 and its potential role in the transcription activation of interferon A genes. J Biol Chem 273:29210–29217. https://doi.org/10.1074/jbc.273.44.29210
Bidwell BN, Slaney CY, Withana NP, Forster S, Cao Y, Loi S, Andrews D, Mikeska T, Mangan NE, Samarajiwa SA, de Weerd NA, Gould J, Argani P, Möller A, Smyth MJ, Anderson RL, Hertzog PJ, Parker BS (2012) Silencing of Irf7 pathways in breast cancer cells promotes bone metastasis through immune escape. Nat Med 18:1224–1231. https://doi.org/10.1038/nm.2830
Casaburi I, Avena P, de Luca A, Sirianni R, Rago V, Chimento A, Trotta F, Campana C, Rainey WE, Pezzi V (2017) GPER-independent inhibition of adrenocortical cancer growth by G-1 involves ROS/Egr-1/BAX pathway. Oncotarget 8:115609–115619. https://doi.org/10.18632/oncotarget.23314
Cawthorn TR, Moreno JC, Dharsee M, Tran-Thanh D, Ackloo S, Zhu PH, Sardana G, Chen J, Kupchak P, Jacks LM, Miller NA, Youngson BJ, Iakovlev V, Guidos CJ, Vallis KA, Evans KR, McCready D, Leong WL, Done SJ (2012) Proteomic analyses reveal high expression of decorin and endoplasmin (HSP90B1) are associated with breast cancer metastasis and decreased survival. PLoS ONE 7:e30992. https://doi.org/10.1371/journal.pone.0030992
Chimento A, Sirianni R, Casaburi I, Zolea F, Rizza P, Avena P, Malivindi R, de Luca A, Campana C, Martire E, Domanico F, Fallo F, Carpinelli G, Cerquetti L, Amendola D, Stigliano A, Pezzi V (2015) GPER agonist G-1 decreases adrenocortical carcinoma (ACC) cell growth in vitro and in vivo. Oncotarget 6:19190–19203. https://doi.org/10.18632/oncotarget.4241
Di Malta C, Siciliano D, Calcagni A, Monfregola J, Punzi S, Pastore N, Eastes AN, Davis O, de Cegli R, Zampelli A, Di Giovannantonio LG, Nusco E, Platt N, Guida A, Ogmundsdottir MH, Lanfrancone L, Perera RM, Zoncu R, Pelicci PG, Settembre C, Ballabio A (2017) Transcriptional activation of RagD GTPase controls mTORC1 and promotes cancer growth. Science 356:1188–1192. https://doi.org/10.1126/science.aag2553
Dong Z, Zhu C, Zhan Q, Jiang W (2018) Cdk phosphorylation licenses Kif4A chromosome localization required for early mitotic progression. J Mol Cell Biol 10:358–370. https://doi.org/10.1093/jmcb/mjy033
Ekholm SV, Reed SI (2000) Regulation of G(1) cyclin-dependent kinases in the mammalian cell cycle. Curr Opin Cell Biol 12:676–684. https://doi.org/10.1016/s0955-0674(00)00151-4
Fabregat A, Jupe S, Matthews L, Sidiropoulos K, Gillespie M, Garapati P, Haw R, Jassal B, Korninger F, May B, Milacic M, Roca CD, Rothfels K, Sevilla C, Shamovsky V, Shorser S, Varusai T, Viteri G, Weiser J, Wu G, Stein L, Hermjakob H, D’Eustachio P (2018) The reactome pathway knowledgebase. Nucleic Acids Res 46:D649–D655. https://doi.org/10.1093/nar/gkx1132
Filardo EJ (2002) Epidermal growth factor receptor (EGFR) transactivation by estrogen via the G-protein-coupled receptor, GPR30: a novel signaling pathway with potential significance for breast cancer. J Steroid Biochem Mol Biol 80:231–238. https://doi.org/10.1016/s0960-0760(01)00190-x
Filardo EJ, Thomas P (2005) GPR30: a seven-transmembrane-spanning estrogen receptor that triggers EGF release. Trends Endocrinol Metab 16:362–367. https://doi.org/10.1016/j.tem.2005.08.005
Filardo EJ, Quinn JA, Bland KI, Frackelton AR (2000) Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF. Mol Endocrinol 14:1649–1660. https://doi.org/10.1210/mend.14.10.0532
Filardo EJ, Quinn JA, Frackelton AR, Bland KI (2002) Estrogen action via the G protein-coupled receptor, GPR30: stimulation of adenylyl cyclase and cAMP-mediated attenuation of the epidermal growth factor receptor-to-MAPK signaling axis. Mol Endocrinol 16:70–84. https://doi.org/10.1210/mend.16.1.0758
Fujiwara S, Terai Y, Kawaguchi H, Takai M, Yoo S, Tanaka Y, Tanaka T, Tsunetoh S, Sasaki H, Kanemura M, Tanabe A, Yamashita Y, Ohmichi M (2012) GPR30 regulates the EGFR-Akt cascade and predicts lower survival in patients with ovarian cancer. J Ovarian Res 5:35. https://doi.org/10.1186/1757-2215-5-35
Gui Y, Shi Z, Wang Z, Li J-J, Xu C, Tian R, Song X, Walsh MP, Li D, Gao J, Zheng X-L (2015) The GPER agonist G-1 induces mitotic arrest and apoptosis in human vascular smooth muscle cells independent of GPER. J Cell Physiol 230:885–895. https://doi.org/10.1002/jcp.24817
Győrffy B, Lánczky A, Szállási Z (2012) Implementing an online tool for genome-wide validation of survival-associated biomarkers in ovarian-cancer using microarray data from 1287 patients. Endocr Relat Cancer 19:197–208. https://doi.org/10.1530/ERC-11-0329
Heublein S, Mayr D, Vrekoussis T, Friese K, Hofmann SS, Jeschke U, Lenhard M (2013) The G-protein coupled estrogen receptor (GPER/GPR30) is a gonadotropin receptor dependent positive prognosticator in ovarian carcinoma patients. PLoS ONE 8:e71791. https://doi.org/10.1371/journal.pone.0071791
Holmberg Olausson K, Elsir T, Moazemi Goudarzi K, Nistér M, Lindström MS (2015) NPM1 histone chaperone is upregulated in glioblastoma to promote cell survival and maintain nucleolar shape. Sci Rep 5:166. https://doi.org/10.1038/srep16495
Ignatov T, Modl S, Thulig M, Weißenborn C, Treeck O, Ortmann O, Zenclussen AC, Costa S, Kalinski T, Ignatov A (2013a) GPER-1 acts as a tumor suppressor in ovarian cancer. J Ovarian Res 6:51. https://doi.org/10.1186/1757-2215-6-51
Ignatov T, Weißenborn C, Poehlmann A, Lemke A, Semczuk A, Roessner A, Costa SD, Kalinski T, Ignatov A (2013b) GPER-1 expression decreases during breast cancer tumorigenesis. Cancer Invest 31:309–315. https://doi.org/10.3109/07357907.2013.789901
Jeon S-Y, Hwang K-A, Choi K-C (2016) Effect of steroid hormones, estrogen and progesterone, on epithelial mesenchymal transition in ovarian cancer development. J Steroid Biochem Mol Biol 158:1–8. https://doi.org/10.1016/j.jsbmb.2016.02.005
Jiang X, Ye X, Ma J, Li W, Wu R, Jun L (2015) G protein-coupled estrogen receptor 1 (GPER 1) mediates estrogen-induced, proliferation of leiomyoma cells. Gynecol Endocrinol 31:894–898. https://doi.org/10.3109/09513590.2015.1092022
Khachaturov V, Xiao G-Q, Kinoshita Y, Unger PD, Burstein DE (2014) Histone H1.5, a novel prostatic cancer marker: an immunohistochemical study. Hum Pathol 45:2115–2119. https://doi.org/10.1016/j.humpath.2014.06.015
Kolkova Z, Casslén V, Henic E, Ahmadi S, Ehinger A, Jirström K, Casslén B (2012) The G protein-coupled estrogen receptor 1 (GPER/GPR30) does not predict survival in patients with ovarian cancer. J Ovarian Res 5:9. https://doi.org/10.1186/1757-2215-5-9
Kurt AH, Çelik A, Kelleci BM (2015) Oxidative/antioxidative enzyme-mediated antiproliferative and proapoptotic effects of the GPER1 agonist G-1 on lung cancer cells. Oncol Lett 10:3177–3182. https://doi.org/10.3892/ol.2015.3711
Levy DE, Darnell JE (2002) Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol 3:651–662. https://doi.org/10.1038/nrm909
Liang Y-K, Lin H-Y, Chen C-F, Zeng D (2017) Prognostic values of distinct CBX family members in breast cancer. Oncotarget 8:92375–92387. https://doi.org/10.18632/oncotarget.21325
Liu H, Yan Y, Wen H, Jiang X, Cao X, Zhang G, Liu G (2014) A novel estrogen receptor GPER mediates proliferation induced by 17β-estradiol and selective GPER agonist G-1 in estrogen receptor α (ERα)-negative ovarian cancer cells. Cell Biol Int 38:631–638. https://doi.org/10.1002/cbin.10243
Luo H, Yang G, Yu T, Luo S, Wu C, Sun Y, Liu M, Tu G (2014) GPER-mediated proliferation and estradiol production in breast cancer-associated fibroblasts. Endocr Relat Cancer 21:355–369. https://doi.org/10.1530/ERC-13-0237
Martin SG, Lebot MN, Sukkarn B, Ball G, Green AR, Rakha EA, Ellis IO, Storr SJ (2018) Low expression of G protein-coupled oestrogen receptor 1 (GPER) is associated with adverse survival of breast cancer patients. Oncotarget 9:25946–25956. https://doi.org/10.18632/oncotarget.25408
Mazumdar M, Sung M-H, Misteli T (2011) Chromatin maintenance by a molecular motor protein. Nucleus 2:591–600. https://doi.org/10.4161/nucl.2.6.18044
Mi H, Muruganujan A, Thomas PD (2012) PANTHER in 2013: modeling the evolution of gene function, and other gene attributes, in the context of phylogenetic trees. Nucleic Acids Res 41:D377–D386. https://doi.org/10.1093/nar/gks1118
Mori T, Ito F, Matsushima H, Takaoka O, Tanaka Y, Koshiba A, Kusuki I, Kitawaki J (2015) G protein-coupled estrogen receptor 1 agonist G-1 induces cell cycle arrest in the mitotic phase, leading to apoptosis in endometriosis. Fertil Steril 103:1228–35.e1. https://doi.org/10.1016/j.fertnstert.2015.01.026
Nakamura S, Kahyo T, Tao H, Shibata K, Kurabe N, Yamada H, Shinmura K, Ohnishi K, Sugimura H (2015) Novel roles for LIX1L in promoting cancer cell proliferation through ROS1-mediated LIX1L phosphorylation. Sci Rep 5:e28011. https://doi.org/10.1038/srep13474
Ning G, Huang Y-L, Zhen L-M, Xu W-X, Jiao Q, Yang F-J, Wu L-N, Zheng Y-Y, Song J, Wang Y-S, Xie C, Peng L (2018) Transcriptional expressions of chromobox 1/2/3/6/8 as independent indicators for survivals in hepatocellular carcinoma patients. Aging 10:3450–3473. https://doi.org/10.18632/aging.101658
Nozawa R-S, Nagao K, Masuda H-T, Iwasaki O, Hirota T, Nozaki N, Kimura H, Obuse C (2010) Human POGZ modulates dissociation of HP1alpha from mitotic chromosome arms through Aurora B activation. Nat Cell Biol 12:719–727. https://doi.org/10.1038/ncb2075
Ohshima K, Hatakeyama K, Nagashima T, Watanabe Y, Kanto K, Doi Y, Ide T, Shimoda Y, Tanabe T, Ohnami S, Ohnami S, Serizawa M, Maruyama K, Akiyama Y, Urakami K, Kusuhara M, Mochizuki T, Yamaguchi K (2017) Integrated analysis of gene expression and copy number identified potential cancer driver genes with amplification-dependent overexpression in 1,454 solid tumors. Sci Rep 7:1546. https://doi.org/10.1038/s41598-017-00219-3
Parker BS, Rautela J, Hertzog PJ (2016) Antitumour actions of interferons: implications for cancer therapy. Nat Rev Cancer 16:131–144. https://doi.org/10.1038/nrc.2016.14
Polakis P (2000) Wnt signaling and cancer. Genes Dev 14:1837–1851
Prossnitz ER, Arterburn JB, Sklar LA (2007) GPR30: a G protein-coupled receptor for estrogen. Mol Cell Endocrinol 265–266:138–142. https://doi.org/10.1016/j.mce.2006.12.010
Prossnitz ER, Arterburn JB, Smith HO, Oprea TI, Sklar LA, Hathaway HJ (2008) Estrogen signaling through the transmembrane G protein-coupled receptor GPR30. Annu Rev Physiol 70:165–190. https://doi.org/10.1146/annurev.physiol.70.113006.100518
Rudelius M, Rauert-Wunderlich H, Hartmann E, Hoster E, Dreyling M, Klapper W, Ott G, Rosenwald A (2015) The G protein-coupled estrogen receptor 1 (GPER-1) contributes to the proliferation and survival of mantle cell lymphoma cells. Haematologica 100:e458–e461. https://doi.org/10.3324/haematol.2015.127399
Sathya S, Sudhagar S, Lakshmi BS (2015) Estrogen suppresses breast cancer proliferation through GPER/p38 MAPK axis during hypoxia. Mol Cell Endocrinol 417:200–210. https://doi.org/10.1016/j.mce.2015.09.032
Scaling AL, Prossnitz ER, Hathaway HJ (2014) GPER mediates estrogen-induced signaling and proliferation in human breast epithelial cells and normal and malignant breast. Horm Cancer 5:146–160. https://doi.org/10.1007/s12672-014-0174-1
Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3:1101–1108. https://doi.org/10.1038/nprot.2008.73
Shou J, Soriano R, Hayward SW, Cunha GR, Williams PM, Gao W-Q (2002) Expression profiling of a human cell line model of prostatic cancer reveals a direct involvement of interferon signaling in prostate tumor progression. Proc Natl Acad Sci 99:2830–2835. https://doi.org/10.1073/pnas.052705299
Siegel RL, Miller KD, Jemal A (2015) Cancer statistics, 2015. Cancer J Clin 65:5–29. https://doi.org/10.3322/caac.21254
Skrzypczak M, Schüler S, Lattrich C, Ignatov A, Ortmann O, Treeck O (2013) G protein-coupled estrogen receptor (GPER) expression in endometrial adenocarcinoma and effect of agonist G-1 on growth of endometrial adenocarcinoma cell lines. Steroids 78:1087–1091. https://doi.org/10.1016/j.steroids.2013.07.007
Smith HO, Arias-Pulido H, Kuo DY, Howard T, Qualls CR, Lee S-J, Verschraegen CF, Hathaway HJ, Joste NE, Prossnitz ER (2009) GPR30 predicts poor survival for ovarian cancer. Gynecol Oncol 114:465–471. https://doi.org/10.1016/j.ygyno.2009.05.015
Wang C, Lv X, Jiang C, Davis JS (2012) The putative G-protein coupled estrogen receptor agonist G-1 suppresses proliferation of ovarian and breast cancer cells in a GPER-independent manner. Am J Transl Res 4:390–402
Weißenborn C, Ignatov T, Poehlmann A, Wege AK, Costa SD, Zenclussen AC, Ignatov A (2014a) GPER functions as a tumor suppressor in MCF-7 and SK-BR-3 breast cancer cells. J Cancer Res Clin Oncol 140:663–671. https://doi.org/10.1007/s00432-014-1598-2
Weißenborn C, Ignatov T, Ochel H-J, Costa SD, Zenclussen AC, Ignatova Z, Ignatov A (2014b) GPER functions as a tumor suppressor in triple-negative breast cancer cells. J Cancer Res Clin Oncol 140:713–723. https://doi.org/10.1007/s00432-014-1620-8
Xu Y, Chen Z, Zhang G, Xi Y, Sun R, Wang X, Wang W, Chai F, Li X (2016) HSP90B1 overexpression predicts poor prognosis in NSCLC patients. Tumor Biol 37:14321–14328. https://doi.org/10.1007/s13277-016-5304-7
Yan Y, Jiang X, Zhao Y, Wen H, Liu G (2015) Role of GPER on proliferation, migration and invasion in ligand-independent manner in human ovarian cancer cell line SKOV3. Cell Biochem Funct 33:552–559. https://doi.org/10.1002/cbf.3154
Yang Z, Zhuang L, Szatmary P, Wen L, Sun H, Lu Y, Xu Q, Chen X (2015) Upregulation of heat shock proteins (HSPA12A, HSP90B1, HSPA4, HSPA5 and HSPA6) in tumour tissues is associated with poor outcomes from HBV-related early-stage hepatocellular carcinoma. Int J Med Sci 12:256–263. https://doi.org/10.7150/ijms.10735
Yang Y-F, Pan Y-H, Tian Q-H, Wu D-C, Su S-G (2018) CBX1 indicates poor outcomes and exerts oncogenic activity in hepatocellular carcinoma. Transl Oncol 11:1110–1118. https://doi.org/10.1016/j.tranon.2018.07.002
Zhang J, Li B, Yang Q, Zhang P, Wang H (2015) Prognostic value of Aurora kinase A (AURKA) expression among solid tumor patients: a systematic review and meta-analysis. Jpn J Clin Oncol 45:629–636. https://doi.org/10.1093/jjco/hyv058
Zhu C-x, Xiong W, Wang M-l, Yang J, Shi H-j, Chen H-q, Niu G (2018) Nuclear G protein-coupled oestrogen receptor (GPR30) predicts poor survival in patients with ovarian cancer. J Int Med Res 46:723–731. https://doi.org/10.1177/0300060517717625
Acknowledgements
We thank Mrs. Bettina Federhofer for expert technical assistance. RNA sample processing and Affymetrix Clariom S microarray hybridization were carried out at the genomics core facility, the Center of Excellence for Fluorescent Bioanalytics (KFB, University of Regensburg, Germany).
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SST made substantial contributions in collection and interpretation of data and in manuscript preparation. MS made substantial contributions in interpretation of data and revising the manuscript critically. TI made substantial contributions in interpretation of data and preparation of the manuscript. AI made substantial contributions in interpretation of data and preparation of the manuscript. OO has been involved in revising the manuscript critically for important intellectual content. OT made substantial contributions to conception and design of the study, acquisition of data, analysis and interpretation of data, and preparation of the manuscript.
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Schüler-Toprak, S., Skrzypczak, M., Ignatov, T. et al. G protein-coupled estrogen receptor 1 (GPER-1) and agonist G-1 inhibit growth of ovarian cancer cells by activation of anti-tumoral transcriptome responses: impact of GPER-1 mRNA on survival. J Cancer Res Clin Oncol 146, 3175–3188 (2020). https://doi.org/10.1007/s00432-020-03333-4
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DOI: https://doi.org/10.1007/s00432-020-03333-4