Cancer Letters

Cancer Letters

Volume 434, 10 October 2018, Pages 120-129
Cancer Letters

Original Articles
Angiotensin receptor blockade attenuates cholangiocarcinoma cell growth by inhibiting the oncogenic activity of Yes-associated protein

https://doi.org/10.1016/j.canlet.2018.07.021Get rights and content

Highlights

  • Angiotensin receptor blocker suppresses human cholangiocarcinoma cell growth.

  • Angiotensin receptor blocker inhibits YAP oncogenic activity.

  • Angiotensin receptor blocker shows anti-angiogenic effect in cholangiocarcinoma.

Abstract

Cholangiocarcinoma (CCA) is a destructive malignancy with limited responsiveness to conventional chemotherapy. Although angiotensin receptor blockers (ARBs) have gained attention for their potential anticancer activity, little is known about their effects on CCA. The transcriptional co-activator, Yes-associated protein (YAP) is a critical oncogene in several cancers, including CCA. Following recent evidence showing that YAP is regulated by angiotensin II (AT-II), we investigated the effects of an ARB, losartan, on two human CCA cell lines (KKU-M213 and HuCCT-1) with regards to YAP oncogenic regulation. Losartan suppressed AT-II-induced CCA cell proliferation in a dose-dependent manner, induced apoptosis, decreased YAP (Ser127), and downregulated the YAP target genes CTGF, CYR61, ANKRD1, and MFAP5. However, losartan did not affect epithelial–mesenchymal transition, differentiation, or stemness in the CCA cells. Xenograft tumor growth assay showed that oral administration of a low clinical dose of losartan considerably reduced subcutaneous tumor burden and attenuated intratumor vascularization in CCA cell-derived xenograft tumors in BALB/c nude mice. These results indicate that ARB therapy could serve as a potential novel strategy for CCA treatment.

Introduction

Cholangiocarcinoma (CCA) is a hepatobiliary cancer with a steadily increasing annual incidence [1,2]; it is associated with a poor 5-year survival rate and limited therapeutic options [1,2]. Although surgical resection is the first-line treatment of choice for intrahepatic CCA, 20%–40% of patients are not eligible for surgery as their tumors are locally advanced, have metastasized, involve blood vessels, or have extended into both hepatic lobes [3]. This results in a median survival of approximately 24 months for most patients diagnosed with CCA. According to a phase III randomized controlled trial for biliary tract cancers performed in the UK, the treatment of choice for unresectable CCA comprises a cisplatin/gemcitabine combination [4]. However, the success of these treatment agents remains limited, and their long-term usage is often associated with severe side effects. Alternative approaches are therefore needed. Further, identifying a clinically available compound, which exerts antitumor activity against CCA through a novel molecular target and with proven safety for long-term administration, would be extremely important.

Yes-associated protein (YAP) is a primary effector of the Hippo tumor-suppressor pathway and has been identified as a transcriptional co-activator that interacts with p73, Runx2, Tbx5, SMAD, ErbB4, Pax3, and TEAD family proteins [[5], [6], [7]]. Hippo/YAP signaling is an evolutionarily conserved pathway that regulates tissue growth and organ size by modulating cell proliferation, apoptosis, and self-renewal of progenitor cells [8,9]. Central to the Hippo pathway is a kinase cascade, wherein MST1/2 kinases (ortholog of Drosophila Hippo) and SAV1 form a complex to phosphorylate LATS1/2 [[5], [6], [7]]. Phosphorylated LATS1/2 kinases sequentially phosphorylate and inhibit two major downstream effectors of the Hippo pathway: the transcription co-activators YAP and TAZ [[5], [6], [7], [8], [9]]. Recent studies have indicated that YAP is a critical oncogene whose dysregulation can lead to tumorigenesis [[10], [11], [12]]. When the Hippo enzymatic cascade collapses, YAP is dislodged from its cytoplasmic anchorage and shuttles into the nuclei, where it acts as a transcription co-activator stimulating target downstream genes and acquiring oncogenicity by binding with TEAD family transcription factors [[10], [11], [12]]. Oncogenic YAP has been shown to occur both in human CCA cell lines and patient specimens; further, nuclear YAP can be potentially used as an independent prognostic marker for overall survival in CCA [13]. Thus, pharmacological YAP inhibitors may comprise novel therapeutic targets.

G protein-coupled receptors (GPCRs) are thought to modulate the Hippo/YAP pathway [14]. Such is the case of angiotensin II type 1 receptor (AT1R), a central regulator of the renin-angiotensin-aldosterone system (RAAS). Its ligand, angiotensin II (AT-II), plays a key role in many pathophysiological activities, including vascular hormone secretion, tissue growth, and cancer [15,16]. Currently, AT1R blockers (ARBs) are clinically used to treat hypertension and heart failure, because of their effects on the cardiovascular system, and we previously reported that a highly hydrophilic ARB, losartan, showed suppressive effects against the growth of several experimental tumors including hepatocellular carcinoma and pancreatic cancer [17,18]. Recent bench research has also revealed that AT-II binding to the AT1R can activate YAP by inhibiting the Hippo pathway in podocytes [19]. However, it remains unknown whether this process is associated with CCA development and whether losartan can inhibit CCA growth in conjunction with YAP/TEAD regulation.

The present study investigated the impact of AT-II on CCA cell growth, considering Hippo/YAP regulation and the anticancer properties of the ARB losartan against human CCA cells. We show that losartan suppresses AT-II-stimulated CCA cell proliferation via YAP inactivation and inhibits intratumor angiogenesis in CCA-derived murine xenograft models. On the basis of these results, we propose that losartan should be evaluated as a novel treatment modality for CCA.

Section snippets

Compounds and cell culture

Human AT-II acetate salt was obtained from BACHEM (Bubendorf, Switzerland), and losartan potassium was supplied by Merck & Co., Inc. (New Jersey, USA). Verteporfin (Merck KGaA., Darmstadt, Germany) was used as a YAP inhibitor. Two human CCA cell lines, KKU-M213 and HuCCT1, and human umbilical vascular endothelial cells (HUVECs) were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). HepG2 were obtained from the RIKEN BRC CELL BANK (Ibaraki, Japan).

Losartan suppressed human CCA cell growth in vitro

To investigate the antitumor effect of losartan on human CCA, we first evaluated mRNA expression level of AT-II type1 receptor (AGTR1) on two human CCA lines (KKU-M213, HuCCT-1). As shown in Fig. 1A, both CCA lines exhibited higher mRNA levels of AGTR1 than HepG2, a human liver cancer line, and lower levels than HUVECs, which are recognized models of AGTR1 expression [20,21]. These findings demonstrate that both CCA lines express AGTR1.

Given the high AGTR1 expression levels, we next assessed

Discussion

ARBs are widely used to manage cardiovascular diseases and chronic kidney disease, and available epidemiologic evidence suggests that they affect cancer incidence. An increasing number of studies have also elucidated the involvement of RAAS signaling, especially the AT1R/AT-II axis, in certain cancers, including breast, lung, prostate, pancreas, and liver cancers [18,[26], [27], [28], [29]]. It is noteworthy that some preclinical studies have showed an impact of AT-II on CCA development and

Author contribution

S.S performed most of the in vitro and in vivo experiments, analyzed and interpreted all data, and drafted the manuscript. K.K designed the study, orchestrated all the experimental data, and drafted the manuscript. N.N, K.S, S.S, K.N, K.K, H.K, M.K, K.M, T.N, and A.M offered technical/material support.

H.Y supervised the study, revised the article, and finally approved its submission.

Disclosure of conflicts of interest

No potential conflicts of interest were disclosed by all authors.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Acknowledgements

The authors would like to thank Enago (www.enago.jp) for the English language review.

References (48)

  • A. Miyamoto et al.

    Binding of MAGP2 to microfibrils is regulated by proprotein convertase cleavage

    Matrix Biol.

    (2014)
  • H.W. Park et al.

    Alternative Wnt signaling activates YAP/TAZ

    Cell

    (2015)
  • S. Rizvi et al.

    Cholangiocarcinoma - evolving concepts and therapeutic strategies

    Nat. Rev. Clin. Oncol.

    (2018)
  • J.M. Banales et al.

    Expert consensus document: cholangiocarcinoma: current knowledge and future perspectives consensus statement from the European Network for the Study of Cholangiocarcinoma (ENS-CCA)

    Nat. Rev. Gastroenterol. Hepatol.

    (2016)
  • J. Valle et al.

    Cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer

    N. Engl. J. Med.

    (2010)
  • F. Cottini et al.

    Rescue of Hippo coactivator YAP1 triggers DNA damage-induced apoptosis in hematological cancers

    Nat. Med.

    (2014)
  • J.S. Mo et al.

    The Hippo signaling pathway in stem cell biology and cancer

    EMBO Rep.

    (2014)
  • Y. Liu-Chittenden et al.

    Genetic and pharmacological disruption of the TEAD-YAP complex suppresses the oncogenic activity of YAP

    Genes Dev.

    (2012)
  • B. Zhao et al.

    The Hippo pathway in organ size control, tissue regeneration and stem cell self-renewal

    Nat. Cell Biol.

    (2011)
  • K.F. Harvey et al.

    The Hippo pathway and human cancer

    Nat. Rev. Canc.

    (2013)
  • T. Pei et al.

    YAP is a critical oncogene in human cholangiocarcinoma

    Oncotarget

    (2015)
  • K. Egami et al.

    Role of host angiotensin II type 1 receptor in tumor angiogenesis and growth

    J. Clin. Invest.

    (2003)
  • H. Yoshiji et al.

    Combination of sorafenib and angiotensin-II receptor blocker attenuates preneoplastic lesion development in a non-diabetic rat model of steatohepatitis

    J. Gastroenterol.

    (2014)
  • R. Noguchi et al.

    Synergistic inhibitory effect of gemcitabine and angiotensin type-1 receptor blocker, losartan, on murine pancreatic tumor growth via anti-angiogenic activities

    Oncol. Rep.

    (2009)
  • Cited by (25)

    • Prognosis for intrahepatic cholangiocarcinoma patients treated with postoperative adjuvant transcatheter hepatic artery chemoembolization

      2020, Current Problems in Cancer
      Citation Excerpt :

      The incidence of ICC is only about 10% but is on the rise.8-11 The seriousness of the situation is shown by the cumulative mortality rate having increased by 39% between 1979 and 2004.12-15 Although there has been some progress in the study of ICC in recent years, therapy and prognosis for the disease are still far from satisfactory.16-18

    • Whole-Transcriptome Sequencing Identifies Key Differentially Expressed mRNAs, miRNAs, lncRNAs, and circRNAs Associated with CHOL

      2020, Molecular Therapy Nucleic Acids
      Citation Excerpt :

      Cholangiocarcinoma (CHOL) is a tumor arising from biliary tract epithelia with features of cholangiocyte differentiation.1,2,1–3 CHOL is commonly categorized by anatomic location: intrahepatic, perihilar, and distal CHOL.4–6 It is the subordinate most extensive hepatic malignancy after hepatocellular carcinoma, accounting for 15%–20% of primary hepatobiliary malignancies, and the overall incidence of CHOL seems to be increasing worldwide.7–10

    • Emerging roles of YAP/TAZ in lung physiology and diseases

      2018, Life Sciences
      Citation Excerpt :

      For example, inhibiting YAP expression suppresses pancreatic cancer progression by disrupting tumor stromal interactions [91]. Intriguingly, angiotensin receptor blocker attenuates cholangiocarcinoma cell growth by inhibiting the oncogenic activity of Yes-associated protein [92]. SRC family kinase inhibition with dasatinib, a drug widely used in chronic myelogenous leukemia, resulted in loss of YAPY357 phosphorylation [12].

    • Control of stem cell renewal and fate by YAP and TAZ

      2023, Nature Reviews Molecular Cell Biology
    View all citing articles on Scopus
    View full text