Kenji Yorita

Prognostic significance of circumferential cell surface immunoreactivity of glypican‐3 in hepatocellular carcinoma

Background: GC33 is a recently developed monoclonal antibody against human glypican‐3 (GPC3), which is significantly upregulated in hepatocellular carcinoma (HCC). GC33 recognizes a GPC3 ectodomain and shows significant antitumour activity in vivo. Thus, humanized GC33 antibody may be a promising tool for treating HCC having cell surface GPC3 expression.

Dickkopf-1 is overexpressed in human pancreatic ductal adenocarcinoma cells and is involved in invasive growth

The protein products of the Dickkopf (DKK) genes are antagonists of Wnt glycoproteins, which participate in tumor development and progression by binding to frizzled receptors. In this study, the expression of DKK‐1 was analyzed in a panel of 43 human cultured carcinoma cell lines. DKK‐1 expression was consistently and significantly upregulated in pancreatic carcinoma cell lines. Low level of DKK‐3 expression was also seen. In contrast, the expression of DKK‐2 and ‐4 was not detectable in most pancreatic carcinoma cell lines. The overexpression of DKK‐1 was confirmed in surgically resected human pancreatic cancer tissues, in which the mRNA level was evaluated in paired samples from cancerous and noncancerous pancreatic tissues. In ductal adenocarcinomas (23 cases), DKK‐1 mRNA levels were significantly upregulated compared to corresponding noncancerous tissues in a statistically significant level. To test the biological role of DKK‐1 in pancreatic carcinoma cells, we performed a knockdown of DKK‐1 in SUIT‐2 human pancreatic adenocarcinoma cell line and S2‐CP8, its metastatic subline, using a retroviral short hairpin RNA expression vector. DKK‐1 knockdown resulted in reduced migratory activity of SUIT‐2 in vitro. The in vitro growth rate and Matrigel invasion were also suppressed by DKK‐1 knockdown in S2‐CP8 cells. Collectively, the evidence suggests that, despite of its presumed antagonistic role in Wnt signaling, DKK‐1 may have a role in the aggressiveness of pancreatic carcinoma cells and could, therefore, serve as a novel biomarker of pancreatic cancer.

Intrapancreatic accessory spleen mimicking endocrine tumor of the pancreas: case report and review of the literature.

Accessory spleen is an anomaly that is observed in about 10% of individuals by the autopsy study, and most accessory spleens are located close to the splenic hilum. Although accessory spleen is a frequently encountered entity, intrapancreatic accessory spleen (IPAS) is rarely recognized radiologically and is sometimes mistaken for another type of pancreatic neoplasm. Only 10 IPAS cases surgically resected as solid pancreatic mass have been reported in the English literature. We herein report a case of IPAS mimicking an endocrine tumor of the pancreas and review of the literature.

Membrane-Bound Serine Protease Inhibitor HAI-1 Is Required for Maintenance of Intestinal Epithelial Integrity

Hepatocyte growth factor activator inhibitor type 1 (HAI-1), encoded by the serine protease inhibitor Kunitz type 1 (SPINT1) gene, is a membrane-bound serine protease inhibitor expressed in epithelial tissues. Mutant mouse models revealed that HAI-1/SPINT1 is essential for placental labyrinth formation and is critically involved in regulating epidermal keratinization through interaction with its cognate cell surface protease, matriptase. HAI-1/SPINT1 is abundantly expressed in both human and mouse intestinal epithelium; therefore, we analyzed its role in intestinal function using mice with intestinal epithelial cell-specific deletion of Spint1 generated by interbreeding mice carrying Spint1(LoxP) homozygous alleles with transgenic mice carrying the Cre recombinase gene controlled by the intestine-specific Villin promoter. Although the resulting mice had normal development and appearance, crypts in the proximal aspect of the colon, including the cecum, exhibited histologic abnormalities and increased apoptosis and epithelial cell turnover accompanied by increased intestinal permeability. Distended endoplasmic reticula were observed ultrastructurally in some crypt epithelial cells, indicative of endoplasmic reticular stress. To study the role of HAI-1/SPINT1 in mucosal injury, we induced colitis by adding dextran sodium sulfate to the drinking water. After dextran sodium sulfate treatment, intestine-specific HAI-1/SPINT1-deficient mice had more severe symptoms and a significantly lower survival rate relative to control mice. These results suggest that HAI-1/SPINT1 plays an important role in maintaining colonic epithelium integrity.

Loss of membrane‐bound serine protease inhibitor HAI‐1 induces oral squamous cell carcinoma cells' invasiveness

A loss of balance between cell membrane‐associated proteases and their inhibitors may underlie cancer invasion and metastasis. We analysed the roles of a membrane‐ associated serine protease inhibitor, HAI‐1, in oral squamous cell carcinoma (OSCC). While membranous HAI‐1 was widely observed in cancer cells of human OSCC tissues, this was significantly reduced at the infiltrative invasion front. In vitro, HAI‐1 was detected in all eight OSCC cell lines examined, in which its cognate membrane protease, matriptase was also expressed. HAI‐1 expression knock‐down (KD) in OSCC lines, SAS and HSC‐3, reduced the growth of both lines in vitro but significantly enhanced SAS tumourigenicity in vivo, which was accompanied by histological changes suggestive of the epithelial‐mesenchymal transition. Both HAI‐1‐KD lines also exhibited significantly enhanced migratory capability, and membrane‐associated but not truncated HAI‐1 was required to rescue this phenotype. Other OSCC lines (HSC‐2, Sa3, Ca9‐22) also showed enhanced migration in response to HAI‐1 KD. The enhanced migration is partly attributed to dysregulation of matriptase, as simultaneous matriptase KD alleviated the migration of HAI‐1‐KD cells. HAI‐1 deficiency also altered the expression of CD24, S100A4, CCND2 and DUSP6, all of which are involved in tumour progression. While matriptase was involved in the increased CD24 expression associated with HAI‐1 deficiency, the protease appeared to be not responsible for the altered expression of other genes. Therefore, a matriptase‐independent mechanism for the invasiveness associated with HAI‐1 KD is also present. Together, these observations suggest that HAI‐1 has a crucial suppressive role in OSCC cell invasiveness. Copyright

Hepatocyte Growth Factor Activator Inhibitor Type 1 Is a Suppressor of Intestinal Tumorigenesis

Hepatocyte growth factor activator inhibitor type 1 (HAI-1/SPINT1) is a membrane-bound serine protease inhibitor expressed on the surface of epithelial cells. Although HAI-1/SPINT1 is abundantly expressed in the intestinal epithelium, its role in intestinal tumorigenesis is not known. In this study, we investigated the role of Hai-1/Spint1 in intestinal tumorigenesis using mouse models. The membranous Hai-1/Spint1 immunoreactivity was decreased in murine Apc(Min/+) tumors and also in carcinogen (azoxymethane treatment followed by dextran sodium sulfate administration)-induced colon tumors compared with the adjacent non-neoplastic epithelium. The decreased immunoreactivity appeared to be due to sheddase activity of membrane-type 1 matrix metalloprotease. Then, we examined the effect of intestine-specific deletion of Spint1 gene on Apc(Min/+) mice. The loss of Hai-1/Spint1 significantly accelerated tumor formation in Apc(Min/+) mice and shortened their survival periods. Activation of HGF was enhanced in Hai-1/Spint1-deficient Apc(Min/+) intestine. Gene expression profiling revealed upregulation of the Wnt/β-catenin signaling circuit, claudin-2 expression, and angiogenesis not only in tumor tissue but also in the background mucosa without macroscopic tumors in Hai-1/Spint1-deficient Apc(Min/+) intestine. Intestinal deletion of Spint1 also enhanced the susceptibility to carcinogen-induced colon tumorigenicity of wild-type Apc mice. Our findings suggest that HAI-1/SPINT1 has a crucial role in suppressing intestinal tumorigenesis, which implies a novel link between epithelial cell surface serine protease inhibitors and protection from carcinogenic stimuli.

Antitumor effect of dehydroxymethylepoxyquinomicin, a small molecule inhibitor of nuclear factor-κB, on glioblastoma

Glioblastoma is the most malignant type of brain tumor. Despite recent advances in therapeutic modalities, the prognosis of glioblastoma remains very poor. Recent studies have indicated that RelA/nuclear factor (NF)-κB is consistently activated in human glioblastoma. In this study, we searched for a new treatment modality for glioblastoma, by examining the effects of dehydroxymethylepoxyquinomicin (DHMEQ), a unique small molecule inhibitor of NF-κB. Addition of DHMEQ to cultured human glioblastoma cells inhibited the nuclear translocation of RelA. It also reduced the growth rate of human glioblastoma cells significantly in 6 cell lines and modestly in 3 among 10 cell lines examined. Then, we performed further analyses using 3 sensitive cell lines (U87, U251, and YKG-1). The growth retardation was accompanied by G2/M arrest in vitro. Increased apoptosis was observed in U87 and YKG-1, but not U251 cells after DHMEQ treatment. Then, we tested the efficacy of DHMEQ in chemoprevention through the use of a nude mouse model. Subcutaneous tumors formed by U87 or U251 cells were reduced by ∼40% in size by intraperitoneal administration of DHMEQ started immediately after implantation of the cells. DHMEQ treatment achieved statistically significant improvements in survival curves of mice intracranially implanted with U87 or U251 cells. Histological analysis revealed increased areas of necrosis, increased numbers of collapsed microvessels, decreased nuclear immunoreactivity of RelA, and decreased immunoreactivity of urokinase-type plasminogen activator in the DHMEQ-treated U87 tumor tissues. These results suggest that the targeting of NF-κB by DHMEQ may serve as a promising treatment modality in glioblastoma.

Hepatocyte growth factor activator inhibitor type 1 suppresses metastatic pulmonary colonization of pancreatic carcinoma cells

Hepatocyte growth factor activator inhibitor type 1 (HAI‐1) is a transmembrane protease inhibitor that regulates the activities of membrane‐bound and extracellular serine proteases. HAI‐1 has two Kunitz‐type inhibitor domains with the N‐terminal Kunitz domain (KD1) responsible for inhibiting known target proteases. Previously, we reported that knockdown of HAI‐1 in the human pancreatic carcinoma cell line SUIT‐2 resulted in epithelial to mesenchymal transition. To evaluate the role of HAI‐1 in metastasis, we examined the metastatic capability of SUIT‐2 cells that did or did not stably express HAI‐1 short‐hairpin RNA in an experimental pulmonary metastasis assay using nude mice. The extent of pulmonary metastasis was verified by histological examination and direct measurement of human cytokeratin 19 mRNA levels. One week after injecting SUIT‐2 cells into mouse tail veins, apparent metastatic colonization was observed in 36% (4/11) of mice injected with HAI‐1‐knockdown SUIT‐2, whereas none (0/11) of the control mice were positive for metastasis. After 2 weeks the metastasis positive ratios were 80% (4/5) and 40% (2/5), and after 4 weeks the ratios were 82% (9/11) and 45% (5/11) for HAI‐1‐knockdown and control SUIT‐2 cells, respectively. Thus, loss of HAI‐1 promoted pulmonary metastasis. Co‐injection of recombinant KD1 abolished metastasis produced by HAI‐1‐knockdown SUIT‐2 cells after 1 week. Moreover, recombinant KD1 restored E‐cadherin levels in HAI‐1 knockdown SUIT‐2 cells and reduced their invasiveness in vitro. These data indicate that HAI‐1 regulates pulmonary metastasis of SUIT‐2, and KD1 may have therapeutic application for inhibiting metastatic cancer cell spreading. (Cancer Sci 2011; 102: 407–413)

Aberrant expression of monocarboxylate transporter 4 in tumour cells predicts an unfavourable outcome in patients with hepatocellular carcinoma

The tumour cell microenvironment, which includes local oxygen saturation, pericellular pH and stromal cells, can modulate tumour progression. This study determined the prognostic impact of infiltrating tumour‐associated macrophages and the expression of monocarboxylate transporter 4 (MCT4) and glypican 3 (GPC3) in hepatocellular carcinoma (HCC) clinical specimens.

Activation of macrophage-stimulating protein by human airway trypsin-like protease.

HAT cleaves proMSP by protease assay (View interaction)