Published OnlineFirst March 1, 2017; DOI: 10.1158/0008-5472.CAN-16-3229 Cancer Molecular and Cellular Pathobiology Research Endothelin Promotes Colorectal Tumorigenesis by Activating YAP/TAZ Zhen Wang1,2, Peng Liu1,2, Xin Zhou1,2, Tianxiang Wang1, Xu Feng1,2, Yi-Ping Sun1, Yue Xiong1,3, Hai-Xin Yuan1, and Kun-Liang Guan1,4 Abstract Endothelin receptor A (ETAR) promotes tumorigenesis by induced YAP/TAZ dephosphorylation, nuclear accumulation, and stimulating cell proliferation, migration, and survival. However, transcriptional activation in multiple colon cancer cells. ETAR the mechanism of ETAR in promoting tumor growth is largely stimulation acted via downstream G-protein Gaq/11 and Rho unknown. In this study, we demonstrate that ETAR stimulates GTPase to suppress the Hippo pathway, thus leading to YAP/TAZ colon cell proliferation, migration, and tumorigenesis through activation, which was required for ETAR-induced tumorigenesis. the activation of YAP/TAZ, two transcription coactivators of Overall, these results indicate a critical role of the YAP/TAZ axis in the Hippo tumor suppressor pathway. Endothelin-1 treatment ETAR signaling. Cancer Res; 77(9); 2413–23. Ó2017 AACR. Introduction tumor growth and progression, angiogenesis, and metastatic spread(4,5,8).TheimportanceofETARincancerindicates The endothelin family consists of a group of 21-amino acid that ETAR-specific antagonists (e.g., BQ123, atrasentan, and peptides produced primarily in the endothelium, epithelium, and ZD4054) are potential cancer therapeutic drugs. In fact, ETAR smooth muscle cells (1, 2). Among the three isoforms of endothe- activation induces the activity of tumor-related proteinases and lins, endothelin-1 (ET-1) is the most abundant and widely subsequently promotes cell migration and cell invasion, which expressed. Extensive studies have revealed a wide range of bio- could be blocked by BQ123 (9, 10). logical functions of ET-1, including vasoconstriction, cell prolif- Consistent with a prominent role of ET-1 in tumorigenesis, eration, and cell migration (3). In addition, ET-1 signaling has several ETAR antagonists have been tested in clinical trials. been involved in tumorigenesis in multiple tissues (4). It acts as a Atrasentan showed significant positive effects in brief pain growth factor in several cancers, including carcinoma of the inventory, PSA progression, and bone markers on patients in prostate, ovary, colon, cervix, breast, kidney, lung, central nervous phase I and II trials (11). In a phase III prostate cancer trial, system tumors as well as melanoma, Kaposi sarcoma, and bone atrasentan treatment showed improvement in the median time metastasis (5, 6). to disease progression in patients, although the primary end- ET-1 signals through plasma membrane–localized endothe- point was not achieved (12, 13). Atrasentan is also under phase lin receptor A (ETAR) and endothelin receptor B (ETBR), two II trials for other types of cancer, including renal, ovarian, non– classical G-protein–coupled receptors (GPCR). ETAR has been small cell lung, and brain cancers (11). ZD4054 (also known as reported to be a primary vasoconstrictor and growth-promoting zibotentan) is a specific ETAR antagonist and has been studied receptor, whereas ETBR inhibits cell growth and vascular con- in a multicenter phase II, randomized, double blind, placebo- striction (1, 7). Upon activation by ET-1, ETAR contributes controlled trial. Patients that received ZD4054 experienced a to cell proliferation, migration, and survival. It also promotes 35% reduction in the risk of death, which has showed improved overall survival (14). ZD4054 is being evaluated in 1Key Laboratory of Molecular Medicine of Ministry of Education and Institutes of hormone-resistant prostate cancer in phase III clinical trials Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, (15, 16). In addition, Bosentan and YM598, two nonselective 2 3 China. School of Life Sciences, Fudan University, Shanghai, China. Department antagonists for both ETAR and ETBR, have shown promising of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, therapeutic effect on prostate cancer in clinical trials (17). University of North Carolina at Chapel Hill, Chapel Hill, North Carolina. 4Depart- ment of Pharmacology and Moores Cancer Center, University of California, San Previous studies have shown higher expression of ETAR and ET- Diego, La Jolla, California. 1 in colon cancer tissue compared with normal tissue (18–20), whereas ETBR is expressed in a contrary pattern (21). In addition, Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). ETAR expression correlates with liver metastases of colorectal cancers (20, 21). ETAR may also promote colon cancer cell Corresponding Authors: Kun-Liang Guan, UCSD, RM 5333, 3855 Health Sciences Drive, La Jolla, CA 92093-0815. Phone: 858-822-7945; Fax: 858- survival in response to chemotherapy, such as cisplatin (20). A fi 534-7638; E-mail: [email protected]; Yue Xiong, [email protected]; and Hai-Xin preclinical study demonstrated that ZD4054 ef ciently sup- Yuan, Key Laboratory of Molecular Medicine of Ministry of Education and pressed colorectal cancer cell growth and progression, indicating Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, its potential role as adjuvant therapy in colorectal cancer (22). Shanghai 20032, China. E-mail: [email protected] However, the molecular mechanism of ETAR in promoting tumor doi: 10.1158/0008-5472.CAN-16-3229 cell survival, growth, or metastasis in colorectal cancer is not fully Ó2017 American Association for Cancer Research. understood. www.aacrjournals.org 2413 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst March 1, 2017; DOI: 10.1158/0008-5472.CAN-16-3229 Wang et al. The Hippo pathway was initially identified to be key player in Cell culture and transfection organ size control in Drosophila and lately found to be important HCT116 cells were cultured in McCoy's 5A medium (Sigma) in human cancer (23–26). The core components of the mamma- supplemented with 10% FBS (Gibco) and 50 mg/mL penicillin/ lian Hippo pathway contain a kinase cascade of MST1/2, MAP4Ks, streptomycin. Used in our current study, HCT116, HEK293T, and and LATS1/2, in which MST1/2 or MAP4Ks phosphorylate and ZR-75-30 cells were obtained from the ATCC in 2015, where they activate LATS. This kinase cascade regulates target genes expres- were characterized by Mycoplasma detection, isozyme detection, sion by inhibiting its downstream transcriptional coactivators DNA fingerprinting, and cell vitality detection. SW480, HT29, Yes-associated protein (YAP) and transcriptional coactivator SW620, SKBR3, MDA-MB-231, MCF7, and Du145 cells were with PDZ-binding motif (TAZ). LATS1/2 restrict the activity of purchased from cell bank of Chinese Academy of Sciences (Beij- YAP/TAZ by direct phosphorylation (27). Phosphorylated YAP/ ing, China) in 2014, where these cells were characterized by TAZ are sequestered in the cytoplasm by 14-3-3 binding (28–30). Mycoplasma detection, isozyme detection, DNA fingerprinting, Inhibition of LATS1/2 activity leads to dephosphorylation of YAP/ and cell vitality detection. These cells were maintained in DMEM TAZ, which then translocate to the nucleus, bind to the TEA medium (Invitrogen) supplemented with 10% FBS (Gibco) and domain family transcription factors (TEAD1-4), and promote the 50 mg/mL penicillin/streptomycin. PC3 cells were purchased in expression of target genes (31, 32). Generally, the Hippo pathway 2015 from Cell Center of the Institutes of Biomedical Sciences acts as an important inhibitor of tissue growth and organ size by (IBS), Fudan University (Shanghai, China), where these cells were controlling cell proliferation, apoptosis, and migration in characterized by Mycoplasma detection, isozyme detection, DNA response to a variety of extracellular and intracellular signals, fingerprinting, and cell vitality detection. PC3 cells were cultured including GPCR signaling (33, 34). in F12 medium (Sigma) supplemented with 10% FBS (Gibco) ETAR activation by ET-1 has been reported to induce the and 50 mg/mL penicillin/streptomycin. All cell lines were cultured expression of connective tissue growth factor (CTGF) and cyste- at 37 C with 5% CO2 and immediately stored in liquid nitrogen ine-rich protein 61 (CYR61), two well-defined target genes of the till use. A new frozen vial of the same batch of cells was restarted Hippo pathway (35–38), indicating that ET-1 may act upstream of every 2 to 3 months. For serum starvation, cells were incubated in the Hippo–YAP pathway. In this study, we revealed a mechanism DMEM medium, McCoy's 5A medium, or F12 medium, respec- of YAP/TAZ activation by ET-1/ETAR and its role in colorectal tively. Cells were transfected with plasmid DNA using Lipofecta- tumorigenesis. min2000 reagent (Invitrogen). siRNAs were transfected into cells following the manufacturer's instructions for Lipofectamine Materials and Methods RNAiMAX reagent (Invitrogen). To generate stable YAP/TAZ knocking-down cells, pMKO- Chemicals shScramble and pMKO-shTAZ retroviruses, which were produced Chemicals that were used in this study: Endothelin-1, in HEK293T cells using VSVG and GAG as packaging plasmids, BQ123, Tetramethylrhodamine B isothiocyanate-conjugated were used to infect HCT116 cells. pLKO-shYAP lentivirus was Phalloidin, and Latrunculin B