Takaaki Higashi

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.

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.

Effect of LSKL peptide on thrombospondin 1-mediated transforming growth factor β signal activation and liver regeneration after hepatectomy in an experimental model.

A strategy for accelerating liver regeneration after hepatectomy would offer great benefits in preventing postoperative liver failure and improving surgical outcomes. Transforming growth factor (TGF) β is a potent inhibitor of hepatocyte proliferation. Recently, thrombospondin (TSP) 1 has been identified as a negative regulator of liver regeneration by activation of local TGF‐β signals. This study aimed to clarify whether the LSKL (leucine–serine–lysine–leucine) peptide, which inhibits TSP‐1‐mediated TGF‐β activation, promotes liver regeneration after hepatectomy in mice.

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.

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

Diverse Basis of β-Catenin Activation in Human Hepatocellular Carcinoma: Implications in Biology and Prognosis

Aim β-catenin signaling is a major oncogenic pathway in hepatocellular carcinoma (HCC). Since β-catenin phosphorylation by glycogen synthase kinase 3β (GSK3β) and casein kinase 1ε (CK1ε) results in its degradation, mutations affecting these phosphorylation sites cause β-catenin stabilization. However, the relevance of missense mutations in non-phosphorylation sites in exon 3 remains unclear. The current study explores significance of such mutations in addition to addressing the clinical and biological implications of β-catenin activation in human HCC. Methods Gene alteration in exon3 of CTNNB1, gene expression of β-catenin targets such as glutamate synthetase (GS), axin2, lect2 and regucalcin (RGN), and protein expression of β-catenin were examined in 125 human HCC tissues. Results Sixteen patients (12.8%) showed conventional missense mutations affecting codons 33, 37, 41, and 45. Fifteen additional patients (12.0%) had other missense mutations in codon 32, 34, and 35. Induction of exon3 mutation caused described β-catenin target gene upregulation in HCC cell line. Interestingly, conventional and non-phosphorylation site mutations were equally associated with upregulation of β-catenin target genes. Nuclear localization of β-catenin was associated with poor overall survival (p = 0.0461). Of these patients with nuclear β-catenin localization, loss of described β-catenin target gene upregulation showed significant poorer overall survival than others (p = 0.0001). Conclusion This study suggests that both conventional and other missense mutations in exon 3 of CTNNB1 lead to β-catenin activation in human HCC. Additionally, the mechanism of nuclear β-catenin localization without upregulation of described β-catenin target genes might be of clinical importance depending on distinct mechanism.

In vitro modeling of hepatocellular carcinoma molecular subtypes for anti-cancer drug assessment

Tractable experimental model that accounts for inter-tumor molecular heterogeneity is a key element of anti-cancer drug development. Hepatocellular carcinoma is known to exhibit highly heterogeneous molecular aberrations across the tumors, including somatic genetic and epigenetic alterations. Previous studies showed that molecular tumor subtypes determined by transcriptome, as a comprehensive functional readout, are reproducibly observed across global patient populations irrespective of geographic and etiological variations. Here we demonstrate that transcriptomic hepatocellular carcinoma subtypes, S1 and S2, determined by our previous transcriptome meta-analysis of multiple clinical hepatocellular carcinoma cohorts, are presented in a panel of hepatoma cell lines widely used by the research community. Interestingly, cell line that resembles gene expression pattern of S3 subtype, representing less aggressive tumors, was not identified in the panel. MYC pathway-activated S2-like cell lines showed higher sensitivity to a small molecule BET bromodomain inhibitor, (+)-JQ1, which has anti-MYC activity. These results support the use of hepatoma cell lines as models to evaluate molecular subtype-specific drug response, which is expected to lead to development of tailored, precision care of the patients with hepatocellular carcinoma.