Toru Beppu

Epithelial–mesenchymal transition in cancer development and its clinical significance

The epithelial–mesenchymal transition (EMT) plays a critical role in embryonic development. EMT is also involved in cancer progression and metastasis and it is probable that a common molecular mechanism is shared by these processes. Cancer cells undergoing EMT can acquire invasive properties and enter the surrounding stroma, resulting in the creation of a favorable microenvironment for cancer progression and metastasis. Furthermore, the acquisition of EMT features has been associated with chemoresistance which could give rise to recurrence and metastasis after standard chemotherapeutic treatment. Thus, EMT could be closely involved in carcinogenesis, invasion, metastasis, recurrence, and chemoresistance. Research into EMT and its role in cancer pathogenesis has progressed rapidly and it is now hypothesized that novel concepts such as cancer stem cells and microRNA could be involved in EMT. However, the involvement of EMT varies greatly among cancer types, and much remains to be learned. In this review, we present recent findings regarding the involvement of EMT in cancer progression and metastasis and provide a perspective from clinical and translational viewpoints. (Cancer Sci 2009)

Glypican-3, overexpressed specifically in human hepatocellular carcinoma, is a novel tumor marker

With the global pandemic of hepatitis B and C infections, the incidence of Hepatocellular carcinoma (HCC) is rapidly increasing world wide. We identified glypican-3 (GPC3), a novel oncofetal gene over-expressed specifically in human HCC, as based on data of cDNA microarrays. As GPC3 is a GPI-anchored membrane protein and could be secreted, we attempted to detect secreted GPC3 protein in sera from HCC patients using Western blotting and ELISA. GPC3 protein was positive in sera of 40.0% (16/40) of HCC patients, and negative in sera from subjects with liver cirrhosis (LC) (0/13), chronic hepatitis (CH) (0/34), and healthy donors (0/60). All subjects were Japanese. Although 12 of 40 HCC patients were negative for both alpha-fetoprotein (AFP) and PIVKA-II well known tumor markers of HCC, four of these were GPC3-positive in the sera. We also observed vanishing GPC3 protein in the sera of three patients after the surgical treatment for HCC. On the other hand, immunohistochemical analysis revealed that HCC expressed GPC3 protein in all 14 HCC patients tested. In conclusion, GPC3, as defined in this study was shown to be a useful tumor marker for cancer-diagnosis for large numbers of patients with HCC.

Induction of haem oxygenase-1 by nitric oxide and ischaemia in experimental solid tumours and implications for tumour growth

SummaryInduction of haem oxygenase-1 (HO-1) as well as nitric oxide (NO) biosynthesis during tumour growth was investigated in an experimental solid tumour model (AH136B hepatoma) in rats. An immunohistochemical study showed that the inducible isoform of NO synthase (iNOS) was localized in monocyte-derived macrophages, which infiltrated interstitial spaces of solid tumour, but not in the tumour cells. Excessive production of NO in the tumour tissue was unequivocally verified by electron spin resonance spectroscopy. Tumour growth was moderately suppressed by treatment with either Nω-nitro-L-arginine methyl ester (L-NAME) or S-methylisothiourea sulphate (SMT). In contrast, HO-1 was found only in tumour cells, not in macrophages, by in situ hybridization for HO-1 mRNA. HO-1 expression in AH136B cells in culture was strongly enhanced by an NO (NO+) donor S-nitroso-N-acetyl penicillamine. HO-1 mRNA expression in the solid tumour in vivo decreased significantly after treatment with low doses of NOS inhibitors such as L-NAME and SMT (6–20 mg kg–1). However, the level of HO-1 mRNA in the solid tumour treated with higher doses of NOS inhibitor was similar to that of the solid tumour without NOS inhibitor treatment. Strong induction of HO-1 was also observed in solid tumours after occlusion or embolization of the tumour-feeding artery, indicating that ischaemic stress which may involve oxidative stress triggers HO-1 induction in the solid tumour. Lastly, it is of great importance that an HO inhibitor, zinc protoporphyrin IX injected intra-arterially to the solid tumour suppressed the tumour growth to a great extent. In conclusion, HO-1 expression in the solid tumour may confer resistance of tumour cells to hypoxic stress as well as to NO-mediated cytotoxicity.

Identification of HLA-A2- or HLA-A24-Restricted CTL Epitopes Possibly Useful for Glypican-3-Specific Immunotherapy of Hepatocellular Carcinoma

Purpose and Experimental Design: We previously reported that glypican-3 (GPC3) was overexpressed, specifically in hepatocellular carcinoma (HCC) and melanoma in humans, and it was useful as a novel tumor marker. We also reported that the preimmunization of BALB/c mice with dendritic cells pulsed with the H-2Kd-restricted mouse GPC3298-306 (EYILSLEEL) peptide prevented the growth of tumor-expressing mouse GPC3. Because of similarities in the peptide binding motifs between H-2Kd and HLA-A24 (A*2402), the GPC3298-306 peptide therefore seemed to be useful for the immunotherapy of HLA-A24+ patients with HCC and melanoma. In this report, we investigated whether the GPC3298-306 peptide could induce GPC3-reactive CTLs from the peripheral blood mononuclear cells (PBMC) of HLA-A24 (A*2402)+ HCC patients. In addition, we used HLA-A2.1 (HHD) transgenic mice to identify the HLA-A2 (A*0201)–restricted GPC3 epitopes to expand the applications of GPC3-based immunotherapy to the HLA-A2+ HCC patients. Results: We found that the GPC3144-152 (FVGEFFTDV) peptide could induce peptide-reactive CTLs in HLA-A2.1 (HHD) transgenic mice without inducing autoimmunity. In five out of eight HLA-A2+ GPC3+ HCC patients, the GPC3144-152 peptide-reactive CTLs were generated from PBMCs by in vitro stimulation with the peptide and the GPC3298-306 peptide-reactive CTLs were also generated from PBMCs in four of six HLA-A24+ GPC3+ HCC patients. The inoculation of these CTLs reduced the human HCC tumor mass implanted into nonobese diabetic/severe combined immunodeficiency mice. Conclusion: Our study raises the possibility that these GPC3 peptides may therefore be applicable to cancer immunotherapy for a large number of HCC patients.

Antiapoptotic effect of haem oxygenase-1 induced by nitric oxide in experimental solid tumour

Induction of haem oxygenase-1 (HO-1) may provide an important protective effect for cells against oxidative stress. Here, we investigated the mechanism of cytoprotection of HO-1 in solid tumour with a focus on the antiapoptotic activity of HO-1. Treatment of rat hepatoma AH136B cells with the HO inhibitor zinc protoporphyrin IX (ZnPP IX) or tin protoporphyrin IX resulted in extensive apoptotic changes of tumour cells both in vivo and in vitro. Caspase-3 activity of the ZnPP IX-treated hepatoma cells increased significantly. Moreover, ZnPP IX-induced apoptosis was completely inhibited by simultaneous incubation with a specific caspase-3 inhibitor and was partially abrogated by bilirubin, a reaction product of HO. In vivo ZnPP IX treatment did not affect nitric oxide (NO) production and tumour blood flow. Western blot analyses showed that HO-1 expression in AH136B cells was strongly upregulated by NO donors, for example, S-nitroso-N-acetyl penicillamine and propylamine NONOate in vitro; conversely, it was remarkably reduced in vivo by pharmacological blockade of NOS. We conclude that HO-1 may function in antiapoptotic defense of the tumour, and thus it may have important protective and beneficial effects for tumour cells against oxidative stress induced by NO, which is produced in excess during solid tumour growth in vivo.

Systemic inflammatory response syndrome and organ dysfunction following gastrointestinal surgery.

OBJECTIVES Progression from systemic inflammatory response syndrome (SIRS) to sepsis, severe sepsis, and septic shock has been demonstrated in a variety of patients. However, the presence of SIRS alone was not helpful in predicting the development of multiple organ dysfunction syndrome (MODS) since SIRS includes many nonprogressive conditions. This study was conducted to investigate the clinical significance of SIRS in postoperative patients. DESIGN Retrospective study. SETTING The surgical department of a university hospital. PATIENTS Two hundred ninety-two consecutive patients who received elective common gastrointestinal surgery (esophagectomy, pancreatoduodenectomy, hepatectomy, gastrectomy, colorectal resection, and laparoscopic cholecystectomy) between 1992 and 1995. INTERVENTIONS Patients were analyzed for preoperative physiologic status, surgical stress parameters, and postoperative status of SIRS, complications, and end-organ dysfunction. MEASUREMENTS AND MAIN RESULTS Duration of SIRS or positive criterias number of SIRS after surgery significantly correlated with surgical stress parameters (blood loss/body weight and operation time) and peak serum C-reactive protein concentrations. SIRS that continued or reappeared after postoperative day 3 was an early sign of postoperative complications. SIRS continuing consecutively for 2 days after postoperative day 3 had a 70.6% positive predictive value and a 92.5% negative predictive value for postoperative complications. Septic complications and prolongation of SIRS were associated with MODS. Five of six patients who met the SIRS criteria for >30 days developed severe MODS, and three of them died. CONCLUSIONS SIRS is a useful criterion for the recognition of postoperative complications and end-organ dysfunctions. Early recovery from SIRS may arrest the progression of organ dysfunction.

Significance of alternatively activated macrophages in patients with intrahepatic cholangiocarcinoma.

Many studies have shown that tumor‐associated macrophages (TAMs) contribute to tumor development and poor prognosis in various cancers. In this study, we investigated the macrophage populations and phenotypes, and their correlation to angiogenesis, immunosuppression, and clinical prognosis in intrahepatic cholangiocarcinoma (ICC). CD68 (+) and CD163 (+) macrophage infiltration was analyzed in paraffin‐embedded tissue samples from 39 patients. CD163 is used as a marker of M2 macrophages. Neovascularization and infiltration of forkhead box P3 (FOXP3) (+) regulatory T cells were also evaluated. The number of CD68 (+) and CD163 (+) macrophages was positively correlated with the numbers of vessels and regulatory T cells. The number of CD163 (+) cells was more closely associated with them. Intrahepatic cholangiocarcinoma (ICC) patients with high counts of CD163 (+) macrophages showed poor disease‐free survival (P = 0.0426). The macrophage density was not correlated with overall survival. In an in vitro study using ICC cell lines (HuCCT1, RBE, and MEC) and human macrophages, tumor cell supernatant (TCS) from cell lines induced an activation of signal transducers and activators of transcription‐3 (Stat3) and macrophage polarization toward the M2 phenotype. Tumor cell supernatant (TCS) from HuCCT1 most strongly induced Stat3 activation and production of cytokines and other bioactive molecules such as interleukin (IL)‐10, vascular endothelial growth factor (VEGF)‐A, transforming growth factor (TGF)‐β, and matrix metalloproteinase (MMP)‐2. Down‐regulation of Stat3 by siRNA significantly suppressed the production of IL‐10 and VEGF‐A. These results provide suggestive evidence that TAMs contribute to cancer progression via Stat3 activation, and CD163 is useful for evaluating M2 TAMs and predicting the clinical prognosis of ICC patients. (Cancer Sci)

Relationship between plasma cytokine concentration and multiple organ failure in patients with acute pancreatitis.

The dynamic aspects of circulating cytokines and cytokine modulators and their relationship with development of multiple organ failure (MOF) in patients with acute pancreatitis were analyzed. All cytokine and C-reactive protein levels in the circulation were higher than those in the MOF group. In particular, plasma concentrations of soluble tumor necrosis factor receptors (sTNF-RI and sTNF-RII) were significantly higher in patients with MOF than in those without even at admission. Furthermore, plasma concentrations of sTNF-Rs and interleukin-1 (IL-1) receptor antagonist (IL-1ra) were much higher than those of their counterparts, TNF-&agr; and IL-1&bgr;, respectively. These results suggest that the plasma concentrations of sTNF-Rs are useful predictors for the development of MOF, and actions of TNF-&agr; and IL-1&bgr; could be regulated by their modulators (soluble receptor and receptor antagonist, respectively) in the pathologic condition of severe acute pancreatitis.

CD44s regulates the TGF-β-mediated mesenchymal phenotype and is associated with poor prognosis in patients with hepatocellular carcinoma

The prognosis for individuals diagnosed with hepatocellular carcinoma (HCC) remains poor because of the high frequency of invasive tumor growth, intrahepatic spread, and extrahepatic metastasis. Here, we investigated the role of the standard isoform of CD44 (CD44s), a major adhesion molecule of the extracellular matrix and a cancer stem cell marker, in the TGF-β-mediated mesenchymal phenotype of HCC. We found that CD44s was the dominant form of CD44 mRNA expressed in HCC cells. Overexpression of CD44s promoted tumor invasiveness and increased the expression of vimentin, a mesenchymal marker, in HCC cells. Loss of CD44s abrogated these changes. Also in the setting of CD44s overexpression, treatment with TGF-β1 induced the mesenchymal phenotype of HCC cells, which was characterized by low E-cadherin and high vimentin expression. Loss of CD44s inhibited TGF-β-mediated vimentin expression, mesenchymal spindle-like morphology, and tumor invasiveness. Clinically, overexpression of CD44s was associated with low expression of E-cadherin, high expression of vimentin, a high percentage of phospho-Smad2-positive nuclei, and poor prognosis in HCC patients, including reduced disease-free and overall survival. Together, our findings suggest that CD44s plays a critical role in the TGF-β-mediated mesenchymal phenotype and therefore represents a potential therapeutic target for HCC.

Complications and management of microwave coagulation therapy for primary and metastatic liver tumors

P = 0.006) and higher (P = 0.032), respectively, than those of patients without complications. The incidence of complications increased significantly when the tumor size was more than 4 cm (P = 0.008). Abscesses and bleeding were successfully treated using percutaneous drainage and interventional angiography, respectively, but as the other serious complications were not able to be treated effectively once induced, prophylaxis is important to facilitate MCT. Transcatheter cooling of the intrahepatic bile duct during MCT and the administration of an anticancer agent into the abdominal cavity are recommended to prevent biloma and dissemination, respectively. MCT is indicated for tumors less than 4 cm in diameter to reduce the risk of complications. The prophylaxis and treatment of these complications enhance the safety of MCT.