Takayoshi Kaida

An Imbalance in TAZ and YAP Expression in Hepatocellular Carcinoma Confers Cancer Stem Cell–like Behaviors Contributing to Disease Progression

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.

miR-9-3p plays a tumour-suppressor role by targeting TAZ (WWTR1) in hepatocellular carcinoma cells.

Background:The inactivation of the Hippo pathway lead to TAZ (PDZ-binding motif)/YAP (yes-associated protein) overexpression, and is associated with worse prognostic outcomes in various cancers including hepatocellular carcinoma (HCC). Although there are several reports of microRNA (miR) targeting for YAP, miR targeting for TAZ remains unclear. The aim of this study is to identify the miR targeting TAZ expression in HCC.Methods:MicroRNA expression was analysed using the Human miFinder 384HC miScript miR PCR array, and was compared between low and high TAZ expression cell lines. Then, we extracted miR-9-3p as a tumour-suppressor miR targeting TAZ. We examined the functional role of miR-9-3p using miR-9-3p mimic and inhibitor in HCC cell lines).Results:In HCC cell lines and HCC clinical samples, there was the inverse correlation between miR-9-3p and TAZ expressions. TAZ expression was induced by treatment of miR-9-3p inhibitor and was downregulated by treatment of miR-9-3p mimic. Treatment of miR-9-3p mimic inhibited cell proliferative ability with downregulated phosphorylations of Erk1/2, AKT, and β-catenin in HLF. Inversely, treatment of miR-9-3p inhibitor accelerated cell growth compared with control in HuH1.Conclusions:MicroRNA-9-3p was identified as the tumour-suppressor miR targetting TAZ expression in HCC cells.

CXCL12/CXCR4 activation by cancer‐associated fibroblasts promotes integrin β1 clustering and invasiveness in gastric cancer

Cancer‐associated fibroblasts (CAFs) are reportedly involved in invasion and metastasis in several types of cancer, including gastric cancer (GC), through the stimulation of CXCL12/CXCR4 signaling. However, the mechanisms underlying these tumor‐promoting effects are not well understood, which limits the potential to develop therapeutic targets against CAF‐mediated CXCL12/CXCR4 signaling. CXCL12 expression was analyzed in resected GC tissues from 110 patients by immunohistochemistry (IHC). We established primary cultures of normal fibroblasts (NFs) and CAFs from the GC tissues and examined the functional differences between these primary fibroblasts using co‐culture assays with GC cell lines. We evaluated the efficacy of a CXCR4 antagonist (AMD3100) and a FAK inhibitor (PF‐573,228) on the invasive ability of GC cells. High CXCL12 expression levels were significantly associated with larger tumor size, increased tumor depth, lymphatic invasion and poor prognosis in GC. CXCL12/CXCR4 activation by CAFs mediated integrin β1 clustering at the cell surface and promoted the invasive ability of GC cells. Notably, AMD3100 was more efficient than PF‐573,228 at inhibiting GC cell invasion through the suppression of integrin β1/FAK signaling. These results suggest that CXCL12 derived from CAFs promotes GC cell invasion by enhancing the clustering of integrin β1 in GC cells, resulting in GC progression. Taken together, the inhibition of CXCL12/CXCR4 signaling in GC cells may be a promising therapeutic strategy against GC cell invasion.

C5a receptor (CD88) promotes motility and invasiveness of gastric cancer by activating RhoA

Purpose Anaphylatoxin C5a is a strong chemoattractant of the complement system that binds the C5a receptor (C5aR). The expression of C5aR is associated with poor prognosis in several cancers. However, the role of C5aR in gastric cancer (GC) is unknown. The aim of this study was to examine the role of C5aR on GC cell motility and invasion. Experimental Design The mechanism of invasion via C5aR was assessed by analyzing cytoskeletal rearrangement and RhoA activity after C5a treatment. Moreover, we investigated the relationship between C5aR expression and the prognosis of GC patients. Results Two human GC cell lines (MKN1 and MKN7) had high C5aR expression. An invasion assay revealed that C5a stimulation promoted the invasive ability of MKN1 and MKN7 cells and that this was suppressed by knockdown of C5aR using siRNA or a C5aR-antagonist. Moreover, overexpression of C5aR in GC cells enhanced the conversion of RhoA-guanosine diphosphate (RhoA-GDP) to RhoA-guanosine triphosphate (RhoA-GTP) after C5a stimulation and caused morphological changes, including increased expression of stress fibers and filopodia. Examination of tumor specimens from 100 patients with GC revealed that high C5aR expression (35 of 100 samples, 35.0%) was associated with increased invasion depth, vascular invasion and advanced stage. The 5-year overall survival of patients with high or low C5aR expression was 58.2% and 88.5%, respectively (p=0.008). Conclusions This study is the first to demonstrate that C5aR promotes GC cell invasion by activating RhoA and is associated with a poor prognosis in GC patients. Therefore, this study provides a biomarker for GC patients who require an advanced therapeutic strategy.

Intraoperative blood loss is not a predictor of prognosis for pancreatic cancer

PurposeThe relationship between intraoperative blood loss (IBL) and prognosis has been reported for some types of cancer, but not for pancreatic cancer, which has one of the highest mortality rates of any cancer. We conducted this study to analyze the relationship between IBL and clinical outcome for patients undergoing radical surgery for pancreatic cancer.MethodsThe subjects of this study were 144 patients who underwent curative pancreatectomy for invasive pancreatic cancer between 2002 and 2014. Clinicopathological characteristics were recorded and prognostic factors were identified by univariate and multivariate analyses.ResultsLarge IBL was significantly associated with advanced tumor stage, a long operation time, a large tumor, portal vein resection, and blood transfusion. According to univariate analysis, IBL was also significantly associated with overall survival (OS) and relapse-free survival (RFS); however, it was not an independent prognostic factor for OS and RFS in multivariate analysis. According to multivariate analysis, lymph node metastasis and R-status were independent prognostic factors for OS and RFS. A subgroup analysis of patients who received no blood transfusion showed similar results.ConclusionMinimizing IBL is very important; however, the present study found that positive lymph node metastasis and R-status were stronger independent prognostic factors for pancreatic cancer.

Heterogeneity of KRAS Mutations in Pancreatic Ductal Adenocarcinoma

Objectives Activating Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations are the most common and frequent changes observed in pancreatic cancer. This study aimed to determine the frequency and extent of intratumoral and metastatic lymph node KRAS mutation heterogeneity of resected pancreatic ductal adenocarcinoma. Methods Tumor tissues macrodissected from tumor centers, invasion fronts (n = 97), and lymph nodes (n = 11) were subjected to DNA extraction and mutation analysis of KRAS codons 12 and 13 by pyrosequencing. Results Activating mutations in codon 12 of KRAS were detected in 90 (92.8%) tumor centers. No mutations were detected in KRAS codon 13 in any patient. After a comparison of tumor centers and invasion fronts, intratumoral heterogeneity of KRAS was observed only in 4 (4.1%) cases. Additional invasion front tumor analysis revealed the same mutation status consistent with each tumor center. No heterogeneity was observed between primary tumors and metastatic lymph nodes. Conclusions Intratumoral heterogeneity of the KRAS mutational status is rare in pancreatic ductal adenocarcinoma. In addition, no KRAS heterogeneity between primary tumors and metastatic lymph nodes was detected in this study. This finding is consistent with the hypothesis that oncogenic activation of KRAS is the first driver mutation in pancreatic cancer.

Tumour-infiltrating inflammatory and immune cells in patients with extrahepatic cholangiocarcinoma

Background:Inflammation and immune characteristics of the tumour microenvironment have therapeutic significance. The aim of this study was to investigate the clinical impact on disease progression in human extrahepatic cholangiocarcinoma (ECC).Methods:A total of 114 consecutive ECC patients with curative resection between 2000 and 2014 were enrolled. Tumour infiltrating CD66b+ neutrophils (TANs; tumour associated neutrophils), CD163+ M2 macrophages (TAMs; tumour associated macrophages), CD8+ T cells, and FOXP3+ regulatory T cells (Tregs) were assayed by immunohistochemistry, and their relationships with patient clinicopathological characteristics and prognosis were evaluated.Results:Tumour associated neutrophils were inversely correlated with CD8+ T cells (P=0.0001) and positively correlated with Tregs (P=0.001). High TANs (P=0.01), low CD8+ T cells (P=0.02), and high Tregs (P=0.04) were significantly associated with poor overall survival (OS). A high-risk signature, derived from integration of intratumoural inflammatory and immune cells, was significantly associated with poor recurrence-free survival (P=0.01) and OS (P=0.0008). A high-risk signature was correlated with postoperative distant metastases. Furthermore, a high-risk signature was related to the resistance to gemcitabine-based chemotherapy used after recurrence.Conclusions:Our data showed that tumour infiltrating inflammatory and immune cells may play a pivotal role in ECC progression and a high-risk signature predicted poor prognosis in ECC patients.

Preoperative platelet-to-lymphocyte ratio can predict recurrence beyond the Milan criteria after hepatectomy for patients with hepatocellular carcinoma.

Postoperative recurrence beyond the Milan criteria is a poor prognostic factor for patients with hepatocellular carcinoma (HCC) treated with various therapies. We investigated the most useful inflammation‐based prognostic score for predicting recurrence beyond the Milan criteria after initial liver resection.

Colon cancer metastasis mimicking intraductal papillary neoplasm of the extra-hepatic bile duct

Graphical abstract

Abstract 3125: miR-9-3p plays a tumor-suppressor role by targeting TAZ (WWTR1) in hepatocellular carcinoma cells

Background: Hepatocellular carcinoma is the sixth most prevalent cancer and the third most frequent cause of cancer-related death. Despite recent advance in cancer treatment including the development of molecular target drugs, the prognosis in patients with hepatocellular carcinoma (HCC) still remains poor. The inactivation of the Hippo-pathway lead to TAZ/YAP overexpression, and is associated with worse prognostic outcomes in various cancers including hepatocellular carcinoma (HCC). While there are several reports of miR targeting for YAP, miR targeting for TAZ remains unclear. The aim of this study is to identify the miR targeting TAZ expression in HCC. Methods: miR expression was analyzed using the Human miFinder 384HC miScript miR PCR Array and was compared between low and high TAZ expression cell lines. Then, we extracted miR-9-3p as a tumor-suppressor miR targeting TAZ using miRBase. We examined the functional role of miR-9-3p targeting TAZ using miR-9-3p mimic and inhibitor in HCC cell lines (HLF and HuH1 with high and low TAZ expressions, respectively). Results: In HCC cell lines and HCC clinical samples, there was the inverse correlation between miR-9-3p and TAZ expressions by real-time PCR. By western blotting, TAZ expression was induced by treatment of miR-9-3p inhibitor and was downregulated by treatment of miR-9-3p mimic. TAZ nuclear localization was indeed inhibited with a treatment with miR-9-3p mimics in HLF cells, and was inversely accelerated with a treatment of miR-9-3p inhibitors in HuH1 cells. Treatment of miR-9-3p mimic inhibited cell proliferative ability by downregulating the phosphorylation of Erk1/2 and Akt signals in HLF. Inversely, treatment of miR-9-3p inhibitor accelerated cell growth compared to control in HuH1. On the other hand, cell invasiveness was not affected by miR-9-3p. Conclusions: miR-9-3p was identified as the tumor-suppressor miR targeting TAZ expression in HCC cells. miR-9-3p played a crucial role in cell proliferation, but not in invasion, via Akt and Erk1/2 signals in HCC cells. Citation Format: Takaaki Higashi, Hiromitsu Hayashi, Hideaki Takeyama, Takayoshi Kaida, Kota Arima, Katsunobu Taki, Hirohisa Okabe, Hidetoshi Nitta, Daisuke Hashimoto, Akira Chikamoto, Toru Beppu, Hideo Baba. miR-9-3p plays a tumor-suppressor role by targeting TAZ (WWTR1) in hepatocellular carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3125. doi:10.1158/1538-7445.AM2015-3125