Hidetoshi Nitta

Enhancement of Human Cancer Cell Motility and Invasiveness by Anaphylatoxin C5a via Aberrantly Expressed C5a Receptor (CD88)

Purpose: The anaphylatoxin C5a is a chemoattractant that induces leukocyte migration via C5a receptor (C5aR). There is emerging evidence that C5a is generated in the cancer microenvironment. We therefore sought C5aR expression and a direct influence of the C5a–C5aR axis on cancer cells. Experimental Design: C5aR expression was investigated in human cancer tissues and cell lines. Effects of C5a stimulation on cancer cells were studied by cytoskeletal rearrangement, time-lapse analysis, Matrigel chamber assay, and invasion in nude mouse in a comparison of C5aR-expressing cancer cells with control cells. Results: C5aR was aberrantly expressed in various human cancers. Several cancer cell lines also expressed C5aR. C5a triggered cytoskeletal rearrangement and enhanced cell motility three-fold and invasiveness 13-fold of C5aR-expressing cancer cells. Such enhancement by C5a was not observed in control cells. Cancer cell invasion was still enhanced in the absence of C5a concentration gradient and even after the removal of C5a stimulation, suggesting that random cell locomotion plays an important role in C5a-triggered cancer cell invasion. C5a increased the release of matrix metalloproteinases (MMP) from cancer cells by two- to 11-fold, and inhibition of MMP activity abolished the C5a-enhancing effect on cancer cell invasion. Compared with control cells, C5aR-expressing cells spread 1.8-fold more broadly at implanted nude mouse skin sites only when stimulated with C5a. Conclusions: These results illustrate a novel activity of the C5a–C5aR axis that promotes cancer cell invasion through motility activation and MMP release. Targeting this signaling pathway may provide a useful therapeutic option for cancer treatment. Clin Cancer Res; 19(8); 2004–13. ©2013 AACR.

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.

Induction of vascular leakage and blood pressure lowering through kinin release by a serine proteinase from Aeromonas sobria.

Aeromonas sobria causes septic shock, a condition associated with high mortality. To study the mechanism of septic shock by A. sobria infection, we examined the vascular leakage (VL) activity of A. sobria serine proteinase (ASP), a serine proteinase secreted by this pathogen. Proteolytically active ASP induced VL mainly in a bradykinin (BK) B2 receptor-, and partially in a histamine-H1 receptor-dependent manner in guinea pig skin. The ASP VL activity peaked at 10 min to 1.8-fold of the initial activity with an increased BK B2 receptor dependency, and attenuated almost completely within 30 min. ASP produced VL activity from human plasma apparently through kallikrein/kinin system activation, suggesting that ASP can generate kinin in humans. Consistent with the finding that a major part of the ASP-induced VL was reduced by a potent kallikrein inhibitor, soybean trypsin inhibitor that does not affect ASP enzymatic activity, ASP activated prekallikrein but not factor XII to generate kallikrein in a dose- and incubation time-dependent manner. ASP produced more VL activity directly from human low m.w. kininogen than high m.w. kininogen when both were used at their normal plasma concentrations. Intra-arterial injection of ASP into guinea pigs lowered blood pressure specifically via the BK B2 receptor. These data suggest that ASP induces VL through prekallikrein activation and direct kinin release from kininogens, which is a previously undescribed mechanism of A. sobria virulence and could be associated with the induction of septic shock by infection with this bacterium. ASP-specific inhibitors, and kinin receptor antagonists, might prove useful for the treatment or prevention of this fatal disease.

Comparison between hepatic resection and radiofrequency ablation as first-line treatment for solitary small-sized hepatocellular carcinoma of 3 cm or less.

It is a matter of debate whether hepatic resection (HR) or radiofrequency ablation (RFA) should be preferred for the treatment of patients with hepatocellular carcinoma (HCC). The aim of this study is to compare the long‐term outcome between HR and RFA in patients with solitary small‐sized HCC.

CD44s signals the acquisition of the mesenchymal phenotype required for anchorage-independent cell survival in hepatocellular carcinoma

Background:Circulating tumour cells (CTCs) have an important role in metastatic processes, but details of their basic characteristics remain elusive. We hypothesised that CD44-expressing CTCs show a mesenchymal phenotype and high potential for survival in hepatocellular carcinoma (HCC).Methods:Circulating CD44+CD90+ cells, previously shown to be tumour-initiating cells, were sorted from human blood and their genetic characteristics were compared with those of tumour cells from primary tissues. The mechanism underlying the high survival potential of CD44-expressing cells in the circulatory system was investigated in vitro.Results:CD44+CD90+ cells in the blood acquired epithelial–mesenchymal transition, and CD44 expression remarkably increased from the tissue to the blood. In Li7 and HLE cells, the CD44high population showed higher anoikis resistance and sphere-forming ability than did the CD44low population. This difference was found to be attributed to the upregulation of Twist1 and Akt signal in the CD44high population. Twist1 knockdown showed remarkable reduction in anoikis resistance, sphere formation, and Akt signal in HLE cells. In addition, mesenchymal markers and CD44s expression were downregulated in the Twist1 knockdown.Conclusions:CD44s symbolises the acquisition of a mesenchymal phenotype regulating anchorage-independent capacity. CD44s-expressing tumour cells in peripheral blood are clinically important therapeutic targets in HCC.

Cancer cells release anaphylatoxin C5a from C5 by serine protease to enhance invasiveness

Anaphylatoxin C5a indirectly fosters cancer cells through recruitment of myeloid-derived suppressor cells (MDS) for inhibiting antitumor CD8+ T cells and induction of neovascularization. We recently found activation of cancer cells by C5a directly via the C5a-receptor (C5aR; CD88) to enhance invasiveness. Thus, C5a possibly contributes to cancer progression rather than elimination. C5a generation in cancer tissues has been reported; however, the mechanism is not fully elucidated. Cancer cell expression of complement regulatory molecules suggests inefficient C5a generation through activation of the complement system in response to cancer cells. To explore another C5a generation mechanism in cancer tissues, we examined cancer cells for C5a-releasing activity from C5. C5a was present in C5-supplemented culture media of cancer cells including C5aR-expressing cells, and the media enhanced C5aR-expressing cancer cell invasion, which was abolished by anti-C5a antibody. The C5a-releasing activity was absent in the supernatants of the media and was inhibited by aprotinin, a serine protease inhibitor, and decanoyl-Arg-Val-Lys-Arg-chloromethylketone but not by inhibitors specific for cysteine, acid, or metal proteases. These results indicated C5a release from C5 by a cancer cell membrane-bound serine protease that can cleave peptide bonds at the carboxy-terminal site of paired basic amino acid residues. Cancer cell C5a release from the complement-immobilized plasma supported feasibility of this cancer cell protease-dependent C5a generation in cancer tissues. The new mechanism of C5a generation suggests self-activation of C5aR-expressing cancer cells to enhance invasiveness and induction of MDS recruitment and neovascularization to create a microenvironment favorable for cancer progression.

Functional assessment versus conventional volumetric assessment in the prediction of operative outcomes after major hepatectomy

PURPOSE In this study we examined whether conventional future remnant liver volume (FR volume) or function (FR function) better predicted the operative outcome after major hepatectomy. METHODS Of 510 patients who underwent hepatectomy for various indications, 133 patients with major hepatectomy were enrolled in this study. FR volume and the corresponding FR function routinely were measured with a 99mTc-GSA scintigraphy single-proton emission computed tomography fusion system. FR function was defined as the future remnant liver uptake ratio of 99mTc-GSA per whole liver by single-proton emission computed tomography. FR volume or function in cases with insufficient FR volume or function required for major hepatectomy were defined as marginal. RESULTS Morbidity, liver dysfunction-related morbidity, and mortality after major hepatectomy occurred in 40 (30%), 10 (7.5%), and 8 (5.7%) patients, respectively. Thirty-two of the 133 patients were diagnosed as marginal using FR volume, but only 11 patients were diagnosed as marginal using FR function. These results indicated that 21 patients (16%) were switched to the safe group using functional assessment. Operative outcomes in patients with safe FR function (n = 122) were equivalent to those of patients with safe FR volume (n = 101), but patients with marginal FR function (n = 11) had substantially worse outcomes than patients with marginal FR volumes (n = 32). Logistic regression analysis identified marginal FR function, but not volume, as a risk factor for worse operative outcome after major hepatectomy. Portal vein embolization induced the substantially greater FR function compared with FR volume. Although liver volume equally corresponds to liver function under normal conditions, liver cirrhosis was greatly associated with the major discrepancy (more than 10%) in patients without portal vein embolization. CONCLUSION Functional assessment for future remnant liver identified patients who were eligible for curative hepatectomy despite a marginal status based on conventional volumetric assessment. Thus, marginal FR function is a better predictor of operative outcome after major hepatectomy than FR volume, especially in patients with nonhealthy liver.

Activation of prothrombin by ASP, a serine protease released from Aeromonas sobria.

The effect of a serine protease (ASP) secreted from Aeromonas sobria on plasma coagulation was investigated. Proteolytically active ASP promoted human plasma coagulation in a dose‐dependent manner. Consistent with the preference for a factor Xa‐specific oligo‐peptide substrate, ASP produced enzymatic activity from human prothrombin but not from factors IX and X. ASP cleaved prothrombin to produce enzymatically active 37 kDa‐fragment displaying the same molecular mass as α‐thrombin. ASP is the first bacterial serine protease that produces α‐thrombin, through which ASP may contribute to the induction of thrombotic tendency in disseminated intravascular coagulation complicated with sepsis caused by A. sobria infections.

Production of C5a by ASP, a Serine Protease Released from Aeromonas sobria

Aeromonas sobria causes pus and edema at sites of infection. However, the mechanisms underlying these effects have not been elucidated. C5a, the amino-terminal fragment of the complement 5th component (C5), mimics these events. To investigate the involvement of C5a in the pathophysiology of A. sobria infection, we examined release of C5a from human C5 by a serine protease (ASP), a putative virulence factor secreted by this bacterium. C5 incubated with enzymatically active ASP induced neutrophil migration in a dose-dependent manner from an ASP concentration of 3 nM and in an incubation time-dependent manner in as little as 7 min, with neutrophil accumulation in guinea pigs at intradermal injection sites and neutrophil superoxide release. These effects on neutrophils were inhibited by a C5a-receptor antagonist. The ASP incubation mixture with C5 but not C3 elicited vascular leakage in a dose- and incubation time-dependent manner, which was inhibited by a histamine H1-receptor antagonist. Together with these C5a-like activities, ASP cleaved C5 to release only one C5a Ag, the m.w. of which was similar to that of C5a. Immunoblotting using an anti-C5a Ab revealed generation of a C5a-like fragment from human plasma incubated with ASP. These results suggest that ASP-elicited neutrophil migration and vascular leakage via C5a production from C5 could occur in vivo, which was supported by that ASP did not affect functions of C5a and neutrophil C5a receptor. Through C5a generation, ASP could be associated with the induction of pus and edema caused by infection with this bacterium.

Triple positive tumor markers predict recurrence and survival in early stage hepatocellular carcinoma

Hepatectomy is feasible for patients with hepatocellular carcinoma (HCC) with good hepatic function who meet the Milan criteria. Several studies have indicated that tumor markers of HCC, α‐fetoprotein (AFP), Lens culinaris agglutinin‐reactive fraction of AFP percentage and protein induced by vitamin K absence/antagonist‐II were good predictors of malignant potential. It is important to identify highly malignant cases of HCC, and the aim of this study was to clarify the impact of triple positive tumor markers as the prognostic factors for early stage HCC within the Milan criteria.