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