Shuho Semba

To determine the etiology of combined high-grade squamous intraepithelial lesion (HSIL) and adenocarcinoma in situ (AIS) of the uterine cervix, we examined human papillomavirus (HPV) subtypes, methylation status of the HPV-16 L1 gene, and immunohistochemical staining pattern of Krt7 in 8 cases of combined HSIL and AIS. Overall, 6 (75%) of 8 patients with combined HSIL and AIS were infected by the same subtype of HPV in both HSIL and AIS (cases 1-5, HPV-16; and case 6, HPV-18), whereas 2 (25%) patients showed infection with different subtypes of HPV (case 7, HPV-31 and -18; and case 8, HPV-52 and -16, in HSIL and AIS, respectively). The degrees of methylation at CpG islands within the HPV-16 L1 gene were almost equivalent between HSIL and AIS in cases 1-4, whereas a great difference in CpG methylation patterns between two was seen in only 1 case (case 5). In addition, both patients infected with different subtypes of HPV between HSIL and AIS were positive for Krt7 only within the AIS component. Based on these results, we propose two distinct developmental pathways of combined HSIL and AIS of the uterine cervix, the common pathway and the individual pathway.

At the invasive front of colorectal carcinoma (CRC), the existence of tumor budding (TB), the detachment and migration of small clusters of tumor cells from the neoplastic epithelium, correlates with high incidence of local invasion and distant metastasis; however, the molecular background of TB is still unknown. In human CRC-derived SW480 cells, CD133+ cells showed cancer stem cell (CSC)-like properties, high tumorigenicity and pluripotency. By a comparative study of gene expression between CD133+ and CD133- SW480 cells, high sensitivity against transforming growth factor-β (TGF-β) was suggested in CD133+ SW480 cells. Interestingly, treatment with recombinant TGF-β1 increased the numbers of cells expressing CD133 and SNAI1. Furthermore, in CD133- SW480 cells, the SNAI1-induced epithelial-mesenchymal transition (EMT) restored the population of CD133+ cells and increased tumorigenicity, cell motility/invasiveness and matrix metalloproteinase 2 (MMP2) expression. In stage II CRC tissues, TB was asso­ciated with increased levels of SNAI1 expression as well as high incidence of metachronous lymph node metastasis post-surgical resection. These findings suggest that TGF-β regulates not only the induction of EMT but also the restoration of CSCs in CRC. The tumor microenvironment at the invasive front is important for the formation of tumor buds in CRC.

Aberrant expression of Brahma-related gene-1 (BRG1), a core component of the SWI/SNF chromatin-remodelling complex, has been implicated in cancer development; however, the biological significance of BRG1 in colorectal carcinoma (CRC) remains unknown. In CRC tissues, expression of BRG1 and Brahma (BRM) was investigated immunohistochemically. Colorectal carcinoma-derived DLD-1 cells were used for knockdown of BRG1 and PTEN with small interfering RNA (siRNA) and transduction of Akt. Complementary DNA (cDNA) microarray analysis was performed to explore the genes affected by BRG1. Expression of BRG1, but not BRM, was frequently elevated in CRC specimens, and knockdown of BRG1 suppressed cell proliferation of DLD-1 cells. By cDNA microarray, we determined that PTEN expression was negatively regulated by BRG1 in DLD-1 cells, which subsequently influenced the cyclin D1 levels via the phosphoinositide 3-OH kinase (PI3K)–Akt signalling pathway. The interplay of BRG1 on cyclin D1 expression was confirmed by the introduction of Akt and knockdown of PTEN in the BRG1 siRNA-transduced DLD-1 cells. Interestingly, this positive correlation between BRG1 and cyclin D1 expression was also observed in CRC specimens. Brahma-related gene-1 has an important role in the process of CRC development by activating the PI3K–Akt signalling pathway and resultant upregulation of cyclin D1 levels.

The purpose of this study is to investigate the role of the WW domain-containing oxidoreductase (WWOX) tumor suppressor that maps to the common fragile site FRA16D (16q23.3-24.1) during the development of gastric carcinoma (GC), we examined the altered expression of WWOX in GC cell lines and tissue samples as well as the effects of restoration of the WWOX gene into WWOX-deficient GC cells. All GC cell lines (HSC-45, HSC-57, HSC-59, MKN-7, and MKN-74) showed reduced WWOX expression at the mRNA and protein levels and hypermethylation at the WWOX regulatory site was detected in HSC-45 and HSC-59 cells. Interestingly, treatment with the deacetylating agent trichostatin A and the demethylating agent 5-aza-2′-deoxycytidine restored endogenous WWOX expression levels in HSC-59 cells. Restoration of the WWOX gene with Ad-WWOX into HSC-59 cells effectively suppressed cell growth and increased the population of cells in subG₁ DNA content. In GC tissue samples, the loss of WWOX expression was detected in 24 (33%) of 73 GC cases in accordance with the hypermethylation at the WWOX regulatory site. Surprisingly, negative immunoreactivity against WWOX showed a significant relationship with several clinicopathologic findings, including histology (P = 0.0001), depth of invasion (P = 0.0004), lymph node metastasis (P = 0.0003), vessel infiltration (lymphatic vessels, P = 0.0167 and venous vessels, P = 0.0005), and clinicopathologic stage (P = 0.001). These findings suggest that repression of WWOX expression may play an important role in stomach carcinogenesis. WWOX thus appears to be a good biomarker for molecular diagnosis of the grade of malignancy of GCs.

Phosphatase of regenerating liver‐3 (PRL‐3) is a protein tyrosine phosphatase (PTP) that is frequently overexpressed in liver metastases of colorectal carcinomas (CRCs). The PTP activity of the PRL‐3 protein is indispensable for the promotion of distant metastasis of CRC; however, little is known about the effect of PRL‐3 on cell growth. In this study, we investigated a novel protein that can connect to PRL‐3 to modulate the proliferation of CRC cells. In CRC‐derived SW480 cells, transduction of ectopic wild‐type PRL‐3, but not the C104S catalytic “dead” mutant, up‐regulated cell proliferation and increased the population of cells at the S and G₂/M phases. Also, inhibition of PTP activity of the PRL‐3 protein by treatment with the PRL‐3 inhibitor suppressed cell proliferation in a dose‐dependent manner as well as PRL‐3 knockdown by RNA interference. Using a comparative study of monodimensional gel electrophoresis of immunoprecipitates from PRL‐3‐transfected SW480 cells and subsequent mass spectrometry analysis, nucleolar‐specific protein nucleolin (NCL) was identified as a novel PRL‐3‐binding protein. We confirmed physiological interaction between PRL‐3 and NCL, and found that PRL‐3 phosphatase activity was associated with the suppression of the phospho‐NCL levels and nucleolar assembly of NCL protein. In CRC cases, nucleolar NCL expression was correlated not only with higher levels of PRL‐3 expression but also with frequent incidence of lymph node metastasis and a higher clinicopathologic stage. These findings suggest that NCL is involved in PRL‐3‐mediated cancer progression/metastasis signaling, which plays an important role in the acceleration of CRC growth. (Cancer Sci 2010)

We have previously shown that WW domain-containing oxidoreductase (WWOX) has tumour-suppressing effects and that its expression is frequently reduced in pancreatic carcinoma. In this study, we examined WWOX expression in intraductal papillary mucinous neoplasm of the pancreas (IPMN) to assess the function of WWOX in pancreatic duct tumourigenesis using immunohistochemistry and methylation-specific polymerase chain reaction analysis. Among 41 IPMNs including intraductal papillary mucinous adenomas (IPMAs) and intraductal papillary mucinous carcinomas (IPMCs), loss or reduced WWOX immunoreactivity was detected in 3 (15%) of 20 IPMAs and 17 (81%) of 21 IPMCs. In addition, hypermethylation of the WWOX regulatory site was detected in 1 (33%) of 3 WWOX(−) IPMAs and 9 (53%) of 17 WWOX(−) IPMCs, suggesting that hypermethylation may possibly be important in the suppression of WWOX expression. Reduction of WWOX expression was significantly correlated with a higher Ki-67 labelling index but was not correlated with the ssDNA apoptotic body index. Interestingly, decreased WWOX expression was significantly correlated with loss of SMAD4 expression in these IPMNs. The results indicate that downregulation of WWOX expression by the WWOX regulatory region hypermethylation is critical for transformation of pancreatic duct.

Phosphatase of regenerating liver‐3 (PRL‐3) is a member of the PRL protein tyrosine phosphatase family and has been proposed to promote the invasiveness and metastastic capability of colorectal cancers (CRCs); however, the underlying mechanisms and target molecules of PRL‐3 protein remain unknown. On the basis of the biological significance of PRL‐3 phosphatase activity confirmed by the catalytically inactive PRL‐3 mutant (C104S) and a PRL‐3 inhibitor in CRC‐derived SW480 cells, we performed protein expression profiling to search for PRL‐3‐mediated effector proteins. By a comparative study of phosphorylated proteins that differentially expressed in wild type and C104S mutant PRL‐3‐transfected SW480 cells; the cytoskeletal intermediate filament keratin 8 (KRT8) was identified as a physiological PRL‐3‐interacting protein. Indeed, treatment with the PRL‐3 inhibitor effectively suppressed the phosphorylation of KRT8 at S73 and S431. Moreover, we detected the physiological interaction between PRL‐3 and KRT8 and their colocalization at cellular lamellipodias and ruffles in vivo. In CRC tissue samples, tumor cells with high PRL‐3 expression showed reduction or loss of phosphorylated KRT8 expression, particularly at the invasive front and in the liver metastases. In conclusion, our results indicate that PRL‐3 may play an important role for the promotion of CRC cell migration and metastatic potential through direct KRT8 dephosphorylation.

Carnitine palmitoyltransferase-II (CPT-II) deficiency is an autosomal recessive disease involving mitochondrial long-chain fatty acid oxidation that results in a distinct clinical phenotype. Reported herein is an autopsy case of the neonatal form of CPT-II deficiency in a 2-day-old Japanese boy who died due to a severe hepatocardiomuscular disease with an extremely early onset. Autopsy examination indicated massive pulmonary atelectasis with intra-alveolar hemorrhage, and the patient had marked cardiomegaly and hepatomegaly, both of which demonstrated the presence of abundant intracytoplasmic steatosis. Three years after the autopsy examination, CPT-II deficiency was suggested by acylcarnitine analysis of dried-blood on filter paper from the patient's younger sister at the age of 1. The younger sister also died due to sudden onset of cardiopulmonary arrest; a remarkable increase of long-chain (C16-18) acylcarnitines was detected on tandem mass spectrometry (TMS). Decreased CPT-II expression was detected in the liver, heart and kidney of the patient. Furthermore, del1737C, a novel mutation of the CPT-II gene, was detected as well as a known GA transition at codon 174. Eventually, laboratory and autopsy findings led to diagnosis of the neonatal form of CPT-II deficiency. TMS can be expected to be widely used to detect metabolic disorders in neonates.

The WW‐domain‐containing oxidoreductase (WWOX) gene spans the common chromosomal fragile site FRA16D (16q23.2) and is believed to be a tumor suppressor in various human malignancies. We have previously shown frequent down‐modulation of Wwox expression in pancreatic carcinoma (PC); however, biological function of Wwox in pancreatic duct carcinogenesis remains unknown. In PANC‐1 (Wwox‐negative) PC‐derived cells, restoration of recombinant WWOX gene expression with adenoviral gene delivery (Ad‐WWOX) effectively increased the number of cells with subG₁ DNA contents in a multiplicity of infection‐dependent manners: Ad‐WWOX infection up‐regulated caspase‐3 activity and reduced procaspase‐3 and procaspase‐8 levels. We also confirmed that restoration of WWOX gene suppressed cell growth in vitro and tumorigenicity in vivo. In addition, transduction of wild‐type WWOX‐expressing vector inhibited PANC‐1 colony formation; however, substitution of Y33 of Wwox with arginine did not lead to inhibition of colony formation, suggesting the biological significance of the WW1 domain of Wwox for its tumor‐suppressing activity. In PC tissue samples, abundant cytoplasmic Wwox expression was detected in the normal pancreatic duct epithelium, whereas Wwox expression was frequently reduced not only in a large fraction of PC but also in precancerous lesions in accord with the pancreatic intraepithelial neoplasia (PanIN) grade, which was closely correlated with patients’ poorer outcome. Interestingly, the existence of Wwox expression was associated with elevated mothers against decapentaplegic homolog 4 (Smad4) protein levels in vitro and in vivo. These findings suggest that down‐modulation of Wwox expression is an early event and may be associated with the down‐regulation of Smad4 protein levels during pancreatic duct carcinogenesis. (Cancer Sci 2008; 99: 1370–1376)