The Expression of Yes-Associated Protein (YAP) Maintains Putative Cancer Stemness and Is Associated with Poor Prognosis in Intrahepatic Cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC) is resistant to most chemotherapeutic agents. Yes-associated protein (YAP) is related to tumor progression; however, its role in ICC remains unknown. We investigated the mechanism underlying YAP-mediated cancer progression by focusing on the property of cancer stem cells (CSCs) in ICC. Immunohistochemistry results revealed the positive YAP expression in 37 of 52 resected ICC cases. Those with positive YAP expression showed poor prognosis in Kaplan-Meier analysis (P = 0.023). YAP expression was associated with vimentin and the putative CSC marker, hepatic oval cell marker 6 (OV-6). The knockdown of YAP expression using specific siRNAs in ICC cells decreased octamer-binding transcription factor 4 (OCT4) expression in Western blot analyses and OV-6 and CD133 expression in flow cytometry analysis. Verteporfin, a YAP inhibitor, decreased N-cadherin and OCT4 expression in Western blot analyses. In vitro sphere formation and anoikis resistance assays revealed the impairment in CSC property and anoikis resistance in response to the decrease in YAP expression. Verteporfin treatment activated the protein kinase B/mechanistic target of rapamycin signaling pathway and dramatically impaired IL-6–stimulated STAT3 phosphorylation in ICC cells. The combination of verteporfin and rapamycin, an inhibitor of mechanistic target of rapamycin phosphorylation, inhibited cell proliferation and tumor growth. In conclusion, verteporfin regulates multiple signaling pathways and, in combination with rapamycin, might be a promising therapeutic strategy for ICC treatment. Intrahepatic cholangiocarcinoma (ICC) is resistant to most chemotherapeutic agents. Yes-associated protein (YAP) is related to tumor progression; however, its role in ICC remains unknown. We investigated the mechanism underlying YAP-mediated cancer progression by focusing on the property of cancer stem cells (CSCs) in ICC. Immunohistochemistry results revealed the positive YAP expression in 37 of 52 resected ICC cases. Those with positive YAP expression showed poor prognosis in Kaplan-Meier analysis (P = 0.023). YAP expression was associated with vimentin and the putative CSC marker, hepatic oval cell marker 6 (OV-6). The knockdown of YAP expression using specific siRNAs in ICC cells decreased octamer-binding transcription factor 4 (OCT4) expression in Western blot analyses and OV-6 and CD133 expression in flow cytometry analysis. Verteporfin, a YAP inhibitor, decreased N-cadherin and OCT4 expression in Western blot analyses. In vitro sphere formation and anoikis resistance assays revealed the impairment in CSC property and anoikis resistance in response to the decrease in YAP expression. Verteporfin treatment activated the protein kinase B/mechanistic target of rapamycin signaling pathway and dramatically impaired IL-6–stimulated STAT3 phosphorylation in ICC cells. The combination of verteporfin and rapamycin, an inhibitor of mechanistic target of rapamycin phosphorylation, inhibited cell proliferation and tumor growth. In conclusion, verteporfin regulates multiple signaling pathways and, in combination with rapamycin, might be a promising therapeutic strategy for ICC treatment. Intrahepatic cholangiocarcinoma (ICC) is the second most frequent primary liver cancer after hepatocellular carcinoma and originates from the epithelium of the intrahepatic bile duct.1Malhi H. Gores G.J. Cholangiocarcinoma: modern advances in understanding a deadly old disease.J Hepatol. 2006; 45: 856-867Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 2Rizvi S. Gores G.J. Pathogenesis, diagnosis, and management of cholangiocarcinoma.Gastroenterology. 2013; 145: 1215-1229Abstract Full Text Full Text PDF PubMed Scopus (857) Google Scholar Surgical resection is the first-line treatment for ICC. However, most patients with ICC are diagnosed at the advanced stage. The resectable rate of ICC is only 15%, whereas the median survival time is ≤3 years.3Buettner S. van Vugt J.L. IJzermans J.N. Groot Koerkamp B. Intrahepatic cholangiocarcinoma: current perspectives.Onco Targets Ther. 2017; 10: 1131-1142Crossref PubMed Scopus (90) Google Scholar Most ICC cases are resistant to chemotherapeutic agents.4Lee K. Lee K.B. Jung H.Y. Yi N.J. Lee K.W. Suh K.S. Jang J.J. The correlation between poor prognosis and increased yes-associated protein 1 expression in keratin 19 expressing hepatocellular carcinomas and cholangiocarcinomas.BMC Cancer. 2017; 17: 441Crossref PubMed Scopus (22) Google Scholar Hence, it is crucial to elucidate the mechanism underlying ICC progression and discover novel therapeutic targets for ICC treatment. Yes-associated protein (YAP) is a major downstream effector of the Hippo signaling pathway. YAP plays important roles in the regulation of organ size and tissue development, regeneration, and differentiation; and it is associated with the self-renewal ability of stem cells in normal tissues.5Dong J. Feldmann G. Huang J. Wu S. Zhang N. Comerford S.A. Gayyed M.F. Anders R.A. Maitra A. Pan D. Elucidation of a universal size-control mechanism in Drosophila and mammals.Cell. 2007; 130: 1120-1133Abstract Full Text Full Text PDF PubMed Scopus (1769) Google Scholar, 6Pan D. The hippo signaling pathway in development and cancer.Dev Cell. 2010; 19: 491-505Abstract Full Text Full Text PDF PubMed Scopus (1695) Google Scholar, 7Gao T. Zhou D. Yang C. Singh T. Penzo-Mendez A. Maddipati R. Tzatsos A. Bardeesy N. Avruch J. Stanger B.Z. Hippo signaling regulates differentiation and maintenance in the exocrine pancreas.Gastroenterology. 2013; 144 (1553.e1): 1543-1553Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 8Mo J.S. Park H.W. Guan K.L. The Hippo signaling pathway in stem cell biology and cancer.EMBO Rep. 2014; 15: 642-656Crossref PubMed Scopus (430) Google Scholar Activation of the Hippo pathway induces the inactivation of YAP through the phosphorylation of Ser127 by a large tumor suppressor kinase 1/2. The phosphorylated YAP is bound by a 14-3-3 protein in the cytoplasm.9Zhao B. Wei X. Li W. Udan R.S. Yang Q. Kim J. Xie J. Ikenoue T. Yu J. Li L. Zheng P. Ye K. Chinnaiyan A. Halder G. Lai Z.C. Guan K.L. Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control.Genes Dev. 2007; 21: 2747-2761Crossref PubMed Scopus (2102) Google Scholar On the contrary, the translocation of the unphosphorylated YAP into the nucleus leads to its binding to the TEA domain (TEAD) family of transcription factors, in turn leading to cell proliferation and apoptosis inhibition in normal tissues.9Zhao B. Wei X. Li W. Udan R.S. Yang Q. Kim J. Xie J. Ikenoue T. Yu J. Li L. Zheng P. Ye K. Chinnaiyan A. Halder G. Lai Z.C. Guan K.L. Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control.Genes Dev. 2007; 21: 2747-2761Crossref PubMed Scopus (2102) Google Scholar The functions of YAP have been observed in various malignancies, such as hepatocellular carcinoma,10Xu M.Z. Yao T.J. Lee N.P. Ng I.O. Chan Y.T. Zender L. Lowe S.W. Poon R.T. Luk J.M. Yes-associated protein is an independent prognostic marker in hepatocellular carcinoma.Cancer. 2009; 115: 4576-4585Crossref PubMed Scopus (414) Google Scholar ovarian cancer,11Hall C.A. Wang R. Miao J. Oliva E. Shen X. Wheeler T. Hilsenbeck S.G. Orsulic S. Goode S. Hippo pathway effector Yap is an ovarian cancer oncogene.Cancer Res. 2010; 70: 8517-8525Crossref PubMed Scopus (197) Google Scholar colorectal cancer,12Wang L. Shi S. Guo Z. Zhang X. Han S. Yang A. Wen W. Zhu Q. Overexpression of YAP and TAZ is an independent predictor of prognosis in colorectal cancer and related to the proliferation and metastasis of colon cancer cells.PLoS One. 2013; 8: e65539Crossref PubMed Scopus (221) Google Scholar gastric cancer,13Kang W. Tong J.H. Chan A.W. Lee T.L. Lung R.W. Leung P.P. So K.K. Wu K. Fan D. Yu J. Sung J.J. To K.F. Yes-associated protein 1 exhibits oncogenic property in gastric cancer and its nuclear accumulation associates with poor prognosis.Clin Cancer Res. 2011; 17: 2130-2139Crossref PubMed Scopus (203) Google Scholar and gallbladder cancer.14Li M. Lu J. Zhang F. Li H. Zhang B. Wu X. Tan Z. Zhang L. Gao G. Mu J. Shu Y. Bao R. Ding Q. Wu W. Dong P. Gu J. Liu Y. Yes-associated protein 1 (YAP1) promotes human gallbladder tumor growth via activation of the AXL/MAPK pathway.Cancer Lett. 2014; 355: 201-209Crossref PubMed Scopus (52) Google Scholar YAP overexpression is associated with angiogenesis, invasion, and epithelial-to-mesenchymal transition (EMT) in malignancies, including ICC, resulting in an unfavorable prognosis.15Avruch J. Zhou D. Fitamant J. Bardeesy N. Mou F. Barrufet L.R. Protein kinases of the Hippo pathway: regulation and substrates.Semin Cell Dev Biol. 2012; 23: 770-784Crossref PubMed Scopus (166) Google Scholar, 16Marti P. Stein C. Blumer T. Abraham Y. Dill M.T. Pikiolek M. Orsini V. Jurisic G. Megel P. Makowska Z. Agarinis C. Tornillo L. Bouwmeester T. Ruffner H. Bauer A. Parker C.N. Schmelzle T. Terracciano L.M. Heim M.H. Tchorz J.S. YAP promotes proliferation, chemoresistance, and angiogenesis in human cholangiocarcinoma through TEAD transcription factors.Hepatology. 2015; 62: 1497-1510Crossref PubMed Scopus (156) Google Scholar, 17Pei T. Li Y. Wang J. Wang H. Liang Y. Shi H. Sun B. Yin D. Sun J. Song R. Pan S. Sun Y. Jiang H. Zheng T. Liu L. YAP is a critical oncogene in human cholangiocarcinoma.Oncotarget. 2015; 6: 17206-17220Crossref PubMed Scopus (105) Google Scholar A premalignant lesion in the liver tumor is stimulated by bile acid, and YAP and the upstream target IQ motif-containing GTPase-activating protein 1 are related to bile acid–mediated regulation of liver growth and tumorigenesis via the Hippo pathway.18Anakk S. Bhosale M. Schmidt V.A. Johnson R.L. Finegold M.J. Moore D.D. Bile acids activate YAP to promote liver carcinogenesis.Cell Rep. 2013; 5: 1060-1069Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar Putative cancer stem cells (CSCs) exhibit self-renewal capacity and the ability of multilineage differentiation in neoplastic tumors.19Du L. Li Y.J. Fakih M. Wiatrek R.L. Duldulao M. Chen Z. Chu P. Garcia-Aguilar J. Chen Y. Role of SUMO activating enzyme in cancer stem cell maintenance and self-renewal.Nat Commun. 2016; 7: 12326Crossref PubMed Scopus (60) Google Scholar The treatment of CSCs may serve as a novel targeting therapy because CSCs are resistant to various therapeutic agents that may result in tumor relapse after chemoradiotherapy or surgical resection. Recent studies have demonstrated that YAP maintains the CSC-like property in urinary bladder cancer20Zhao A.Y. Dai Y.J. Lian J.F. Huang Y. Lin J.G. Dai Y.B. Xu T.W. YAP regulates ALDH1A1 expression and stem cell property of bladder cancer cells.Onco Targets Ther. 2018; 11: 6657-6663Crossref PubMed Scopus (18) Google Scholar and breast cancer.21Kim T. Yang S.J. Hwang D. Song J. Kim M. Kyum Kim S. Kang K. Ahn J. Lee D. Kim M.Y. Kim S. Seung Koo J. Seok Koh S. Kim S.Y. Lim D.S. A basal-like breast cancer-specific role for SRF-IL6 in YAP-induced cancer stemness.Nat Commun. 2015; 6: 10186Crossref PubMed Scopus (123) Google Scholar However, its role in ICC remains unclear. Herein, we describe the clinical significance of YAP expression by focusing on the relationship between YAP expression and putative CSC property in ICC. The inhibition of YAP activation resulted in the impairment in CSC property, proliferation ability of ICC in vitro, and tumor growth in vivo. The data provide insights into effects of YAP on CSC property and a new platform for translational therapeutics in ICC. The clinical samples and data were obtained from 52 patients who underwent curative surgical resection for the mass-forming type of ICC in the Department of General Surgery, Chiba University Hospital (Chiba, Japan) from April 2004 to June 2016. All patients were histologically diagnosed with primary ICC. Nine patients were administrated chemotherapy before surgery, and 16 patients were administrated adjuvant chemotherapy after surgery. The Ethics Committees of our institute approved the protocol of this study (approval number 2965), and written informed consent was obtained from each patient before surgery. Slides were deparaffinized in xylene and rehydrated through a graded alcohol series. For antigen retrieval, the slides were microwaved in 10 mmol/L citrate buffer (pH 6.0) for 12 minutes. Endogenous peroxidase activity was blocked with 3% H2O2 in methanol. After blocking any non-specific protein binding, the fixed tissues were incubated overnight at 4°C with primary antibodies specific for YAP (Santa Cruz Biotechnology, Inc., Dallas, TX; dilution 1:1000), E-cadherin (Cell Signaling Technology, Beverly, MA; dilution 1:400), vimentin (Santa Cruz Biotechnology, Inc.; dilution 1:100), matrix metalloproteinase (MMP)-9 (Santa Cruz Biotechnology, Inc.; dilution 1:100), OV-6 (R&D Systems, Minneapolis, MN; dilution 1:100), or phosphorylated STAT3 (p-STAT3; Cell Signaling Technology; dilution 1:100). The slides were washed three times with phosphate-buffered saline (PBS) and treated with a biotinylated secondary antibody for 30 minutes at room temperature. An EnVision kit (Dako, Glostrup, Denmark) and 3,3′-diaminobenzidine chromogen were used for visualization. Immunohistochemistry (IHC) staining was evaluated and independently scored by two pathologists (K.S. and T.M.) blinded to the clinical characteristics of the patients. YAP expression in the nucleus was evaluated and categorized into two groups (positive and negative expression). IHC staining for E-cadherin (membranous staining of cancer cells), vimentin (cytoplasm staining of cancer cells), MMP-9 (nucleus staining of cancer cells), and OV-6 (cytoplasm staining of cancer cells) was also evaluated. The staining patterns of these markers were scored as low expression (0% to 30% of the cancer cells had positive staining) or high expression (>30% of the tumor cells had positive staining). The human ICC cell lines HuCCT1 and HuH-28 were obtained from the Japanese Collection of Research Bioresources Cell Bank. TKKK cells were provided by the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). HuCCT1 and HuH-28 cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum. TKKK cells were cultured on a collagen plate (Sumitomo Bakelite Co, Ltd, Tokyo, Japan) in low-glucose Dulbecco's modified Eagle's medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal bovine serum at 37°C and 5% CO2. Double-stranded siRNAs used to knockdown YAP expression were as follows: siRNA1, Hs_YAP1_6 (catalog number SI04438637); siRNA2, Hs_YAP1_8 (catalog number SI04438651); and control siRNA (AllStars negative-control siRNA; Qiagen Inc., Valencia, CA). These siRNAs (final concentration, 20 nmol/L) were transfected into HuCCT1 and TKKK cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific). The cells were harvested 72 hours after transfection for Western blot analysis, sphere formation assay, and anoikis resistance assay. Total cellular proteins were purified with radioimmunoprecipitation assay buffer (Sigma-Aldrich). Protein (30 μg) was loaded onto a 7.5% to 15% XV PANTERA Gel (DRC; Tama, Tokyo, Japan), and the separated protein bands were transferred onto a polyvinylidene difluoride membrane. The membrane was blocked with 5% milk and incubated at 4°C overnight with the following primary antibodies: YAP (Santa Cruz Biotechnology, Inc.; dilution 1:1000), phosphorylated YAP (Cell Signaling Technology; dilution 1:1000), E-cadherin (Cell Signaling Technology; dilution 1:1000), N-cadherin (Santa Cruz Biotechnology, Inc.; dilution 1:1000), vimentin (Santa Cruz Biotechnology, Inc.; dilution 1:1000), OV-6 (R&D Systems; dilution 1:1000), OCT4 (Abcam, Cambridge, UK; dilution 1:1000), Akt (Cell Signaling Technology; dilution 1:1000), phosphorylated Akt (p-Akt; Cell Signaling Technology; dilution 1:1000), mechanistic target of rapamycin (mTOR; Cell Signaling Technology; dilution 1:1000), phosphorylated mTOR (p-mTOR; Cell Signaling Technology; dilution 1:1000), STAT3 (Cell Signaling Technology; dilution 1:1000), p-STAT3 (Cell Signaling Technology; dilution 1:1000), and β-actin (Cell Signaling Technology; dilution 1:2000). After washing three times with tris-buffered saline with Tween 20, a horseradish peroxidase–conjugated anti-rabbit antibody (Santa Cruz Biotechnology, Inc.; dilution 1:2000) was added to the membrane as the secondary antibody at room temperature for 1 hour. Protein bands were detected by enhanced chemiluminescence (GE Healthcare, Buckinghamshire, UK), and images were obtained with the LAS 4000 camera system (Fujifilm, Tokyo, Japan). Quantification was performed with ImageJ software version 1.51 (NIH, Bethesda, MD; http://imagej.nih.gov/ij). One million cells were suspended in 100 μL PBS and incubated with mouse IgG1 allophycocyanin-conjugated antibody (R&D Systems; dilution 1:100) and anti-human OV-6 allophycocyanin-conjugated antibody (R&D Systems; dilution 1:20) for 10 minutes at room temperature and anti-human CD133 allophycocyanin-conjugated antibody (Miltenyi Biotec, San Diego, CA; dilution 1:11) for 30 minutes on ice in the dark. After washing with PBS, cells were resuspended in 1 mL PBS and examined using the CANTO II fluorescence-activated cell sorting system (Beckton-Dickinson, Franklin Lakes, NJ). Data were analyzed using Kaluza software version 2.1 (Beckman Coulter, Brea, CA). Verteporfin (Sigma-Aldrich) was dissolved in dimethyl sulfoxide (Sigma-Aldrich) and diluted in PBS. The solution was added to the medium at a final concentration of 10 or 20 μmol/L and incubated with cells for 72 hours. An equal concentration of dimethyl sulfoxide was added to the control cells. HuCCT1 and TKKK cells were seeded in 96-well ultralow attachment plates (Corning Inc., New York, NY) at a density of 10 cells/well in sphere medium. The sphere medium was prepared as previously described.22Rovira M. Scott S.G. Liss A.S. Jensen J. Thayer S.P. Leach S.D. Isolation and characterization of centroacinar/terminal ductal progenitor cells in adult mouse pancreas.Proc Natl Acad Sci U S A. 2010; 107: 75-80Crossref PubMed Scopus (230) Google Scholar After culturing cells for 7 days, the number of sphere cells, defined as cell clusters with a diameter >50 μm, was counted. The sphere formation rate was assessed as the ratio of the number of spheres on day 7/the number of spheres on day 1.23Takano S. Reichert M. Bakir B. Das K.K. Nishida T. Miyazaki M. Heeg S. Collins M.A. Marchand B. Hicks P.D. Maitra A. Rustgi A.K. Prrx1 isoform switching regulates pancreatic cancer invasion and metastatic colonization.Genes Dev. 2016; 30: 233-247Crossref PubMed Scopus (76) Google Scholar To evaluate the ability of anoikis resistance, HuCCT1 and TKKK cells were incubated with growth factor–free medium for 24 hours at 37°C, and the colony formation assay was performed.23Takano S. Reichert M. Bakir B. Das K.K. Nishida T. Miyazaki M. Heeg S. Collins M.A. Marchand B. Hicks P.D. Maitra A. Rustgi A.K. Prrx1 isoform switching regulates pancreatic cancer invasion and metastatic colonization.Genes Dev. 2016; 30: 233-247Crossref PubMed Scopus (76) Google Scholar A total of 3000 cells/well suspended in a medium with 0.3% agar were seeded onto 24-well culture plates coated with a medium containing 1% agar (bottom layer). The number of colonies was counted 14 days after seeding cells. HuCCT1, TKKK, and HuH-28 cells (3000 cells/well) were seeded onto 96-well plates and precultured for 24 hours. Cell number was quantified after treatment of cells with PBS (as control), verteporfin (2 or 5 μmol/L), mTOR inhibitor rapamycin (25 nmol/L; FUJIFILM Wako Pure Chemical Corp., Osaka, Japan), and the combination of 2 or 5 μmol/L verteporfin and 25 nmol/L rapamycin using Cell Counting Kit- 8 (Dojindo Laboratories, Kumamoto, Japan) at days 0, 1, 3, and 5. Protein expression of STAT3 and p-STAT3 was assessed by Western blot analysis in ICC cell lines. Cells were pretreated with or without 10 μmol/L verteporfin for 2 hours, followed by treatment with or without IL-6 (10 ng/mL; PeproTech Inc., Rocky Hill, NJ) for 20 minutes. All experiments were performed in accordance with animal welfare and the local authorities. Eight-week–old KSN/Slc nude mice (Japan SLC Inc., Hamamatsu, Japan) were used for the in vivo experiments. The cell suspensions, including 1 × 105 HuCCT1 cells in 50 μL of Dulbecco's modified Eagle's medium, were mixed with 50 μL of Matrigel (Corning Inc.) and injected subcutaneously into the bilateral flanks of each mouse. Five weeks after cancer cell injection, all mice were blindly divided into three groups (K.S. and M.T.; control group, verteporfin group, and verteporfin and rapamycin group). Xenograft tumors were assessed 3 weeks after initiation of treatment. Animal studies were approved by the Committee on the Use of Live Animals for Teaching and Research of Chiba University. Data are expressed as the median ± SD for all clinical analyses and as the means ± SEM for the analysis of in vitro experiments. All in vitro experiments were independently assessed at least three times. Tumor volume was calculated using the following formula: π/6 × (L × W × W), where L is the tumor length and W is the tumor width.23Takano S. Reichert M. Bakir B. Das K.K. Nishida T. Miyazaki M. Heeg S. Collins M.A. Marchand B. Hicks P.D. Maitra A. Rustgi A.K. Prrx1 isoform switching regulates pancreatic cancer invasion and metastatic colonization.Genes Dev. 2016; 30: 233-247Crossref PubMed Scopus (76) Google Scholar Cumulative survival rates were calculated using the Kaplan-Meier method, and the significance of difference in survival rate was analyzed by the log-rank test. Statistical significance of the results was determined by U-test, χ2 test, or Welch t-test. P < 0.05 was considered significant in all analyses. Statistical calculations were performed using JMP 13 (SAS Institute Inc., Cary, NC). YAP expression was analyzed in human primary ICC tissues with IHC staining. YAP protein was predominantly localized in the nucleus, and weak expression was detected in the cytoplasm of ICC cells (Figure 1A). IHC evaluation revealed 37 cases (71.2%) with positive YAP expression in the cell nucleus and 15 cases (28.8%) with negative YAP expression among 52 ICC cases (Figure 1, A and B). Analysis of primary tumor volume revealed significantly larger tumor sizes in the positive YAP expression group than in the negative YAP expression group (P = 0.038) (Figure 1C). Clinicopathologic features of groups with positive/negative YAP expression are shown in Table 1. Among various clinical parameters, only primary tumor volume was significantly associated with positive YAP expression (P = 0.02). The Kaplan-Meier analysis results showed that the positive YAP group had a significantly shorter overall survival (P = 0.023) than the negative YAP group (Figure 1D). Univariate Cox proportional hazard regression analysis revealed significant associations of sex (male), serum carbohydrate antigen 19-9 (≥36.8 U/mL), primary tumor volume (≥34,000 mm3), poor differentiation, presence of para-aortic lymph node metastasis, and positive YAP expression with overall survival (Table 2). These results suggested that positive YAP expression is a good predictor of poor prognosis in patients with ICC after curative surgery.Table 1Relationships between YAP Expression and Clinicopathologic Features of Patients with ICCFeatureYAP expressionP valuePositive (n = 37)Negative (n = 15)Age in years, means ± SD66.4 ± 8.764.3 ± 8.40.70Male:female ratio21:1611:40.26Serum CEA (≥5/<5), ng/mL5:322:130.66Serum CA19-9 (≥36.8/<36.8), U/mL17:208:70.40Primary tumor volume (≥34,000/<34,000), mm325:125:100.02∗P < 0.05.Tumors, n (multiple/single)12:253:120.36Vascular invasion (+/−)29:811:40.70Differentiation (G1/G2 and G3)†According to the International Union Against Cancer Classification.27:1014:10.20ICGR15, means ± SD, %9.0 ± 5.410.0 ± 5.30.14Operation method (