Endothelin Promotes Colorectal Tumorigenesis by Activating YAP/TAZ

Abstract

Endothelin receptor A (ETAR) promotes tumorigenesis by stimulating cell proliferation, migration, and survival. However, the mechanism of ETAR in promoting tumor growth is largely unknown. In this study, we demonstrate that ETAR stimulates colon cell proliferation, migration, and tumorigenesis through the activation of YAP/TAZ, two transcription coactivators of the Hippo tumor suppressor pathway. Endothelin-1 treatment induced YAP/TAZ dephosphorylation, nuclear accumulation, and transcriptional activation in multiple colon cancer cells. ETAR stimulation acted via downstream G-protein Gαq/11 and Rho GTPase to suppress the Hippo pathway, thus leading to YAP/TAZ activation, which was required for ETAR-induced tumorigenesis. Overall, these results indicate a critical role of the YAP/TAZ axis in ETAR signaling. Cancer Res; 77(9); 2413-23. ©2017 AACR.

Figure 1.

ET-1 activates YAP/TAZ in colon cancer cell lines. A and B, ET-1 activates YAP/TAZ in HCT116 cells. ET-1 induces the dephosphorylation of YAP/TAZ in time- and dose- dependent manners. HCT116 cells were serum starved for 14 hours and stimulated with ET-1 for the duration (A) or dose (B) indicated. Immunoblotting was performed with indicated antibodies. Phos-tag gels were used for assessment of YAP and TAZ phosphorylation status. C, ET-1 activates YAP/TAZ in SW480 and HT29 cells. The two cell lines were serum starved for 14 hours and were treated with ET-1 for the dose indicated for 1 hour. Immunoblotting was performed with indicated antibodies. D, ET-1 promotes YAP translocation from cytoplasm to nucleus. HCT116 cells were serum starved for 14 hours and stimulated with 100 nmol/L of ET-1 for 1 hour. Endogenous YAP(green) and nuclei (blue) were stained with specific antibody and DAPI, respectively; scale bar, 20 μm. E, Quantifications of YAP subcellular localization from at least 100 randomly selected cells in D. C, cytoplasm; N, nucleus. F, ET-1 induces TAZ translocation from cytoplasm to nucleus. HCT116 cells were serum starved for 14 hours and stimulated with 100 nmol/L of ET-1 for 1 hour. HCT116 cells were serum-starved for 14 hours and stimulated with 100 nmol/L of ET-1 for 1 hour. Endogenous TAZ (green) and nuclei (blue) were stained with specific antibody and DAPI, respectively; scale bar, 20 μm. G, Quantifications of TAZ subcellular localization from at least 100 randomly selected cells in F. C, cytoplasm; N, nucleus.

Figure 2.

YAP/TAZ and TEAD mediate the physiologic functions of ET-1 in target gene induction. A, ET-1 induces expression of YAP/TAZ target genes. HCT116 cells were serum starved for 14 hours and stimulated with 100 nmol/L of ET-1 for 3 hours. The mRNA levels of CTGF, CYR61, ANKRD1,and INHBA were quantified by real-time PCR. Data, mean ± SD. B, YAP/TAZ are required for ET-1 to induce downstream gene expression. HCT116 cells were transfected with control or YAP/TAZ siRNAs. After 48 hours, HCT116 cells were serum starved for 14 hours and stimulated with 100 nmol/L of ET-1. Protein levels of CTGF and the knockdown efficiency of YAP/TAZ were determined by immunoblotting. C, YAP/TAZ are required for ET-1 to induce downstream gene expression. HCT116 cells were transfected with control or YAP/TAZ siRNAs. After 48 hours, HCT116 cells were serum starved for 14 hours and stimulated with 100 nmol/L of ET-1 for 3 hours. The mRNA levels of indicated target genes were measured by real-time PCR. Data, mean ± SD. D, TEAD is indispensable for ET-1 to induce target gene expression. HCT116 cells were transfected with control or TEAD1/3/4 siRNAs. Serum-starved cells were treated with 100 nmol/L of ET-1. Protein levels of CTGF and the knockdown efficiency of TEAD1 were determined by immunoblotting.

Figure 3.

ET-1 stimulation activates YAP/TAZ through ETAR and Gα_q/11. A, ET-1 activates YAP/TAZ through ETAR. HCT116 cells were transfected with control or ETAR siRNAs. Serum-starved cells were treated with 100 nmol/L of ET-1 for 1 hour. YAP/TAZ and knockdown efficiency of ETAR were determined by immunoblotting. B, BQ123 blocks ET-1-induced YAP/TAZ activation. Serum-starved HCT116 cells were pretreated with 2 μmol/L of BQ123 for 4 hours, and then were treated with 100 nmol/L of ET-1 for indicated time. Protein levels of CTGF and YAP/TAZ were determined by immunoblotting. C, BQ123 blocks YAP nuclear localization induced by ET-1 stimulation. HCT116 cells were pretreated with 2 μmol/L of BQ123 for 4 hours. Serum-starved cells were treated with 100 nmol/L of ET-1 for 1 hour. Immunofluorescence staining was performed for endogenous YAP (green) and nuclei (DAPI, blue); scale bar, 20 μm. D, Quantifications of YAP subcellular localization from at least 100 randomly selected cells in C. C, cytoplasm; N, nucleus. E, ET-1 signals through Gα_q/11 to induce YAP/TAZ activation. HCT116 cells were transiently transfected with control, Gα_q/11, or Gα_s siRNAs. Serum-starved cells were treated with 100 nmol/L of ET-1 for 1 hour. YAP/TAZ and knockdown efficiency of Gα_q/11 and Gα_s were determined by immunoblotting. F, ET-1 stimulation induces YAP/TAZ nuclear localization through Gα_q/11. HCT116 cells were transiently transfected with control, Gα_q/11 or Gα_s siRNAs. Serum-starved cells were treated with 100 nmol/L of ET-1. Endogenous YAP (green) and nuclei (blue) were stained with specific antibody and DAPI, respectively; scale bar, 20 μm. G, Quantifications of YAP subcellular localization from at least 100 randomly selected cells in F. C, cytoplasm; N, nucleus.

Figure 4.

ET-1 acts through LATS, Rho/ROCK, and actin cytoskeleton to activate YAP/TAZ. A, ET-1 treatment decreases LATS1 activity. HCT116 cells were serum-starved for 14 hours and stimulated with 100 nmol/L of ET-1 for 1 hour. LATS1 was immunoprecipitated and in vitro kinase assay was performed using GST-YAP as a substrate. Phosphorylation of GST-YAP by LATS1 was determined by phospho-YAP antibody. B, Ectopic expression of LATS2 blocks ET-1-induced YAP/TAZ activation. HCT116 cells were transiently transfected with control, LATS2 wild-type (WT), or kinase dead mutant (K/R). Serum-starved cells were treated with 100 nmol/L of ET-1 for 1 hour. Phosphorylationand protein levels of YAP/TAZ were determined by immunoblotting. C, Rho GTPase is involved in YAP/TAZ activation by ET-1. HCT116 cells were transiently transfected with control, Myc-Rho-L63, or Rho-GDI-GFP mutant. Serum-starved cells were treated with 100 nmol/L of ET-1 for 1 hour. Phosphorylation and protein levels of YAP/TAZ were determined by immunoblotting. D, Inactivation of Rho GTPase prevents YAP/TAZ dephosphorylation by ET-1 stimulation. Serum-starved HCT116 cells were pretreated with C3 for 4 hours, followed by treatment with 100 nmol/L of ET-1 for 1 hour. Phosphorylation and protein levels of YAP/TAZ were determined by immunoblotting. E, ROCK is required for ETAR-induced YAP/TAZ activation. Serum-starved HCT116 cells were pretreated with GSK429286 (0.5 μmol/L) or Y27632 (1 μmol/L) for 4 hours, followed by treatment with 100 nmol/L of ET-1 for 1 hour. Phosphorylation and protein levels of YAP/TAZ were determined by immunoblotting. F, Disruption of actin cytoskeleton blocks ET-1-induced YAP/TAZ activation. Serum-starved HCT116 cells were pretreated with latrunculin B (Lat B; 1 mg/mL) for 20 minutes, followed by treatment with 100 nmol/L of ET-1 for 1 hour. Phosphorylation and protein levels of YAP/TAZ were determined by immunoblotting.

Figure 5.

ETAR promotes cell migration and cell proliferation through YAP/TAZ. A, Loss of YAP/TAZ impairs ETAR-induced cell migration. HCT116 cells were transfected with siRNAs targeting YAP and TAZ. After 48 hours, cells were serum starved for 14 hours and stimulated with ET-1 for 4 hours. Cell migration was determined by Transwell cell migration assay as described in Materials and Methods. Cells were stained by crystal violet (left) and quantified (right). The P value of <0.001 was calculated by Student t test and shown. Data, mean ± SD. B, Verification of YAP/TAZ knockdown in HCT116 cells. Cells were transfected and treated in parallel as that in A. Protein levels of YAP/TAZ was determined by immunoblotting. C, Establishment of HCT116 stable cells with ETAR overexpression, YAP/TAZ knockdown, or the combination of both. Expressions of ETAR and YAP/TAZ were determined by immunoblotting. D, YAP/TAZ mediate ETAR-induced cell proliferation. Growth of HCT116 stable cells described in C was determined by cell growth assay. Stable cells were cultured in triplicate in McCoy’s 5A medium supplemented with 5% FBS. Cells were counted daily by Countstar IC1000. The asterisks labeled at day 5 indicate P value was 0.006 and 0.004 between groups of shCtr+ETAR and shCtr+Vec or shYAP/TAZ+ETAR, respectively. The P value of < 0.01 indicates significant difference. The P value was calculated by paired Student t test and is shown. Error bars represent cell numbers ± SD from triplicate experiments.

Figure 6.

ETAR promotes xenograft tumor growth through YAP/TAZ. A, ETAR is overexpressed in colon carcinoma samples. BRCA, breast carcinoma; COAD, colon adenocarcinoma; LIHC, liver hepatocellular carcinoma; PRAD, prostate adenocarcinoma. Log₂ mRNA expression from RNA Seq V2 RSEM was downloaded from TCGA data portal shown by log₂ RSEM. The P value was calculated by Wilcoxon test. The corresponding P value is shown. B, YAP/TAZ are required for ETAR to promote tumor growth in a xenograft mouse model. Xenograft was performed in male nude mice using cells described in Fig. 5C. Three weeks after injection, mice were sacrificed and tumors were separated and photographed. C, Tumor weight was measured for indicated groups. The P value of <0.001 indicates significant difference. P value was calculated by Student t test and is shown.