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Transcriptional coactivator with PDZ-binding motif (TAZ) and yes-associated protein (YAP) are equivalently placed downstream effectors of the Hippo pathway with oncogenic roles in human cancers. However, the expression profiles of TAZ/YAP differ depending on the cancer cell type, suggesting that these proteins have different roles during cancer progression, yet no studies have examined the biologic significance of the balance between TAZ and YAP expression levels. Here we examined the functional roles of TAZ/YAP in hepatocellular carcinoma progression. We found that TAZ, but not YAP, was predominantly expressed in HCC. TAZ knockdown under normal conditions attenuated cell growth in HCC cells; however, TAZ knockdown combined with 5-fluorouracil treatment significantly increased chemoresistance compared with control cells. Notably, TAZ knockdown induced compensatory YAP expression and was accompanied by upregulation of CD90, a HCC–specific cancer stem cell marker. Continuous treatment with 5-fluorouracil also induced YAP expression and promoted tumor formation in vivo. Conversely, double knockdown of TAZ/YAP reduced chemoresistance and tumorigenicity. Moreover, YAP knockdown aggravated HCC cell growth to a greater degree than TAZ knockdown, and YAP overexpression was strongly associated with poor prognoses in patients with HCC. Collectively, these studies demonstrate that TAZ and YAP exhibit different functional roles in cancer progression, and a shift to predominant YAP expression upon TAZ depletion conferred cancer stem cell–like properties including chemoresistance and tumorigenicity in HCC. Therefore, targeting of both TAZ/YAP will be required for a complete antitumor response in HCC. Cancer Res; 75(22); 4985–97. ©2015 AACR.

Introduction: The contact inhibition of proliferation is crucial for well-controlled organogenesis. Its disruption results in sustained cell proliferation, which is a hallmark of solid tumors. The Hippo signaling pathway has been implicated in contact inhibition of proliferation. Here, we investigated the role of TAZ (WWTR1), a key transcription co-activator of hippo-pathway, in HCC progression. Methods and Results: In HCC patients, the high TAZmRNA expression examined by real time PCR showed worsen survival outcomes compared with low expression group. Additionally, the tumor size in High TAZmRNA expression group was significantly larger than that in low expression group, and its high protein expression in HCC was deeply associated with the cell proliferative activity. A knockdown of TAZ expression in HCC cell lines attenuated the cell growth accompanying the downstream gene expressions (CTGF and CYR61). As the TAZ-mediated signaling pathway in cell growth, a knockdown of TAZ expression using RNAi inactivated the PI3K/Akt/mTOR pathway. Conclusion: Thus, TAZ plays a crucial role in tumor progression by accelerating cell growth via a PI3K/Akt/mTOR pathway, and tumoral TAZmRNA expression level may be a useful prognostic biomarker in HCC. Citation Format: Hiromitsu Hayashi, Hideyuki Kuroki, Shigeki Nakagawa, Takaaki Higashi, Keita Sakamoto, Naomi Yokoyama, Takatoshi Ishiko, Toru Beppu, Hideo Baba. TAZ (WWTR1), a key transcription co-activator of hippo-pathway, promotes hepatocellular carcinoma progression via PI3K/Akt/mTOR pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3520. doi:10.1158/1538-7445.AM2014-3520

[Background] P-cadherin belongs to the family of classic cadherin, and accumulating evidences shows hat P-cadherin is strongly associated with carcinogenesis and confers alignant phenotype of various cancers. Pancreatic cancer has a poor prognosis, with an overall 5-year survival rate of only 5%. Despite advances in therapy recently, its survival rate still remains poor. This study aimed to analyze P-cadherin expression and its prognostic value in pancreatic carcinoma and to validate the P-cadherin gene (CDH3) promoter methylation status as the mechanism of this gene regulation. [Methods] Seventy-three patients diagnosed with pancreatic cancer were enrolled in this study. P-cadherin expression was examined by immunohistochemistry, and correlated with various clinicopathological features and prognosis. Furthermore, P-cadherin expression and its promoter methylation status were analyzed in pancreatic cancer cell lines. [Results] In pancreatic tissues, 50.7% (37/73) of cases shows a high expression of P-cadherin by immunohistochemical analysis, and high expression of P-cadherin was significantly correlated with gender, posterior tissue invasion and other organs invasion. In multivariate analyses, P-cadherin expression was the independent prognostic factors for disease-free survival (relative risk:2.68, p=0.005). P-cadherin expression was found to be related to its promoter methylation status in pancreatic cancer cell lines. Furthermore, when P-cadherin was down-regulated by specific siRNA, cell migration and invasion were significantly reduced. [Conclusion] These results suggested that expression of P-cadherin is a useful prognostic marker and seems to be important in the malignant behavior in pancreatic cancer. We are now conducting experiments to validate the CDH3 methylation status in the cancerous tissues and their adjacent normal counterpart, and investigating the biological role of P-cadherin in pancreatic cancer. Note: This abstract was not presented at the meeting. Citation Format: Keita Sakamoto, Katsunori Imai, Shigeki Nakagawa, Hidetoshi Nitta, Hiromitsu Hayashi, Akira Chikamoto, Takatoshi Ishiko, Toru Beppu, Hideo BaBa. P-cadherin overexpression is associated with poor prognosis in pancreatic carcinoma and is regulated by its promoter hypomethylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 376. doi:10.1158/1538-7445.AM2014-376

Background: Intrahepatic cholangiocellular carcinoma (ICC) is characterized as a poor prognostic disease represented by the highly invasiveness. Epithelial-mesenchymal transition (EMT) plays a pivotal role in cancer invasion and metastasis, and then TGF-β signaling is a potent inducible factor of EMT. Integrin-β6, which is an epithelial restricted trans-membrane protein, promotes binding to extracellular matrix proteins (fibronectin, vitronectin and tenascin) facilitating cell migration and activates TGFβ1 via binding to the latency associated peptide (LAP) of the TGF-β complex during embryogenesis and wound healing. In this study, we investigated the role of integrin-β6 during TGF-β-mediated EMT using ICC cell lines, and examined the clinic-pathological significance of Integrinβ6 expression in ICC patients. Material and Methods: We measured integrin-β6 mRNA expression by real time PCR using 7 ICC cell lines (Hucct1, HuH28, OZ, RBE, Ssp25, TFK1, Ysccc). To examine the role of integrin-β6 during TGF-β-mediated EMT in ICC cell lines, we downregulated integrin-β6 expression by RNAi during TGF-β-medaited EMT. Furthermore, using 55 resected ICC specimens, we evaluated integrin-β6 expression by immunohistochemical staining. Results: Of 7 ICC cell lines, Ysccc and OZ cell lines revealed high integrin-β6 expressions. Addition of TGF-β to Ysccc cell successfully induced EMT, characterized by the upregulated vimentin expression, downregulated E-cadherin expression, spindle-shaped morphology, and high invasiveness, with the integrin-β6 induction. The inhibition of integrin-β6 expression using RNAi abolished TGF-β-mediated EMT induction. In clinical setting, high integrin-β6 expression was significantly associated with Stage IV, lymph node metastasis, and blood vessel invasion, and then resulted in worse prognosis compared with low expression group. Conclusion : Integrin-β6 is required for TGF-β-mediated EMT and promotes the disease progression of ICC. Citation Format: Takaaki Higashi, Hiromitu Hayashi, Hideyuki Kuroki, Shigeki Nakagawa, Keita Sakamoto, Hidetoshi Nitta, Akira Chikamoto, Toru Toru, Hideo Baba. Integrin-β6 is required for transforming growth factor (TGF)-β-mediated epithelial-mesenchymal transition (EMT) and promotes the disease progression of intrahepatic cholangiocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3468. doi:10.1158/1538-7445.AM2014-3468

Background: Vasohibin-1 (VASH1) is isolated as an endogenous angiogenesis inhibitor produced by the vascular endothelium, and the status of VASH1 expression has been documented in various cancer prognosis. Some reports shows that VASH1 was negatively regulated by enhancer of zeste homolog 2 (EZH2) epigenetically. The aim of this study was to assess the relationship between EZH2 and VASH1, and the prognostic value of this two molecules in intrahepatic cholangiocarcinoma (ICC) patients. Method: We examine 47 cases of intrahepatic cholangiocarcinoma to which the R0 liver resection had been performed by our department between 1993 years and 2010. Immunohistochemical staining of VASH1 and EZH2 was performed using paraffin-embedded block, and we examined the relation among the expression of VASH1 and a clinicopathological factor (sex, age, tumor diameter, tumor number, differentiation level, microscopic vascular invasion, and metastasis in lymph node), EZH2 expression, recurrence-free survival (RFS) and overall survival (OS). Result: VASH1 and EZH2 positivity was observed in 24 (51.1%) and 24 (51.1%) patients respectively, and VASH1 positivity was inversely correlated with EZH2 high expression (R=0.3870, p=0.0018). VASH1 positivity was not significantly correlate with RFS or OS. On the other hands, VASH1 negative and EZH2 positive was observed in 17 (36.2%) patients, and was a significant disadvantage to both RFS (relative risk (RR) 3.39, 95% confidence interval (CI) 1.28-8.67, p=0.0157) and OS (RR 3.69, 95% CI 1.20-11.2, 0.0237) in univariate Cox hazard analysis. In multivariate analysis, VASH1 negative and EZH2 positive group was an independent risk factor for both RFS (RR 4.19, 95% CI 1.33-13.13, p=0.0150), and OS (RR 8.11, 95% CI 2.01-42.1, p=0.0030). Conclusion: The expression of VASH1 was inversely correlate with EZH2 expression. VASH1 negative and EZH2 positive was an independent risk factor for OS and DFS in ICC patients. Citation Format: Shigeki Nakagawa, Yasuo Sakamoto, Ryuma Tokunaga, Takaaki Higashi, Keita Sakamoto, Akira Chikamoto, Takatoshi Ishiko, Toru Beppu, Hideo Baba. Vasohibine-1 negative and EZH2 positive may predict the prognosis of cholangiocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4732. doi:10.1158/1538-7445.AM2014-4732

Background Worldwide, while a hepatectomy has been performed as the curative procedure for liver cancers, a failure in liver regeneration following hepatectomy can result in functional liver disorders and hepatic failure. Therefore, a novel therapeutic strategy to accelerate liver regeneration enables to undergo safety curative hepatectomy for liver cancers. Transforming growth factor-β1 (TGF-β1) is a potent inhibitor of mitogen-stimulated DNA synthesis in hepatocyte. Since TGF-β is synthesized and secreted as a latent complex, an important step in regulating its biological activity is the conversion of the latent form into the active one. We reported that matricellular protein thrombospondin-1 (TSP-1), a major activator of latent TGF-β1 is a negative regulator of liver regeneration after hepatectomy using TSP-1 null mouse. In response to partial hepatectomy, TSP-1 production is acutely and transiently induced in the endothelial cells of the regenerative liver. These findings indicate the possibility of a novel therapeutic strategy for accelerating liver regeneration by targeting TSP-1 mediated TGF-β1 activation. This study aimed to clarify whether LSKL peptide, inhibiting TSP-1-mediated TGF-β activation, promoted liver regeneration after hepatectomy in mice. Method We performed 70% hepatectomy in mice receiving normal saline, LSKL peptide, and sham operation. LSKL peptide was administered intraperitoneally at abdominal closure and 6 h after hepatectomy. LSKL peptide (30 mg/6 mL/kg) was intraperitoneally administered before abdominal wall closure and at 6 h after 70% hepatectomy. Mice received an intraperitoneal injection of 5-bromo-2-deoxyuridine (BrdU), a marker for the S-phase of the cell cycle ,2 h prior to sacrifice. Result LSKL peptide administration attenuated Smad2 phosphorylation at 6 h after hepatectomy and thereafter accelerated S-phase entry of hepatocytes at 24 and 48 h. The accelerated hepatocyte proliferation by LSKL peptide administration resulted in faster recovery of the residual liver weight at 48 h and body weight at 168 h. H&E staining and blood biochemical examinations revealed no significant adverse effects occurred following the two LSKL peptide administrations during the early period after hepatectomy. Conclusion Only two doses of LSKL peptide during the early period after partial hepatectomy can promote liver regeneration and body weight recovery without any significant side effects. The transient inhibition of TGF-β signal activation in the early period after hepatectomy was sufficient for accelerating hepatocyte. Inhibiting TSP-1-medaited TGF-β activation using LSKL peptide may thus be a promising strategy to promote liver regeneration following hepatectomy. Citation Format: Hideyuki Kuroki, Hiromitsu Hayashi, Shigeki Nakagawa, Keita Sakamoto, Takaaki Higashi, Hidetoshi Nitta, Daisuke Hashimoto, Akira Chikamoto, Toru Beppu, Hideo Baba. LSKL peptide inhibits thrombospondin-1-mediated TGF-β signal activation and accelerates liver regeneration after hepatectomy in mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2700. doi:10.1158/1538-7445.AM2014-2700

Background. P-cadherin belongs to the family of classic cadherin, and accumulating evidences shows that P-cadherin is strongly associated with carcinogenesis and confers malignant phenotype of various cancers including breast, pancreatic, and colon carcinomas. Intrahepatic cholangiocarcinoma (ICC) is the second most common subtype of primary hepatobillary cancer. Despite advances in therapy, its survival rate remains poor. This study aimed to analyze P-cadherin expression and its prognostic value in Intrahepatic cholangiocarcinoma and to validate the P-cadherin gene (CDH3) promoter methylation status as the mechanism of this gene regulation. Methods. Fifty-nine patients diagnosed with ICC who underwent hepatic resection in our department were enrolled in this study. Using the paraffin-embedded tumor tissues, expression status of P-cadherin was evaluated and correlated with various clinicopathological features (age, gender, tumor markers, tumor diameter and number, stage, gross type, grade of tumor differentiation, vessel invasion, and lymph node metastases) and prognosis. Furthermore, P-cadherin expression and CDH3 promoter methylation status in 8 cholangiocarcinoma cell lines (HuCCT1, YSCCC, MEC, OZ, HuH28, RBE, SSP25, and TKKK) were analyzed. Results. In ICC tissues, 50.8% (30/59) of cases shows a high expression of P-cadherin by immunohistochemical analysis, and high expression of P-cadherin was significantly correlated with tumor diameter > 3.8 cm (p=0.0006). In multivariate analyses, P-cadherin expression was the independent prognostic factors for disease-free survival (relative risk:2.72, p=0.0138) and overall survival (relative risk:3.23 p=0.0012). Of the 8 cell lines, 3 (HuCCT1, YSCCC, OZ) were found to express P-cadherin protein and mRNA. These cell lines showed complete CDH3 promoter unmethylation. However, other cell lines showed partial methylation and unmetylation. From this result, the CDH3 promoter methylation was found to be related to the expression of P-cadherin. Conclusion. These results suggested that expression of P-cadherin is a useful prognostic marker and seems to be important in the malignant behavior in ICC. We are now conducting experiments to validate the CDH3 methylation status in the cancerous tissues and their adjacent normal counterpart, and investigating the biological role of P-cadherin in ICC. Citation Format: Keita Sakamoto, Katsunori Imai, Shigeki Nakagawa, Hirohisa Okabe, Hidetoshi Nitta, Hiromitsu Hayashi, Akira Chikamoto, Takatoshi Ishiko, Toru Beppu, Hideo Baba. Prognostic impact of P-cadherin overexpression associated with CDH3 promoter hypomethylation in intrahepatic cholangiocarcinoma (ICC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3044. doi:10.1158/1538-7445.AM2013-3044