Role of the WWOX gene, encompassing fragile region FRA16D, in suppression of pancreatic carcinoma cells

Abstract

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 subG 1 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) P ancreatic carcinoma (PC) is among the most aggressive and lethal human diseases, with a very poor prognosis; even with complete surgical resection and adjuvant chemotherapy, the 5-year survival rate is less than 20%.(1) Infiltrating carcinomas of the exocrine pancreas arise from histologically identifiable intraductal precursors that undergo a series of architectural and cytologic changes. These intraductal lesions of the pancreas are also known as pancreatic intraepithelial neoplasias (PanIN), and they progress from flat to papillary without atypia to papillary with atypia to carcinoma in situ.(2,3) Multiple genetic and epigenetic alterations have been documented in PC: numerous alterations in KRAS,and SMAD4, (9) have been described in a variety of PanIN using both genetic and immunohistochemical analyses, and some in situ lesions eventually progress to infiltrating carcinoma.(3) But much remains unknown about development and progression of pancreatic duct lesions.Common chromosome fragile sites in the human genome are particularly susceptible to damage by environmental carcinogens. Common fragile sites have been observed at or near structural chromosome defects recognized ...

Hypermethylation-mediated reduction of WWOX expression in intraductal papillary mucinous neoplasms of the pancreas

“…In this study, we found frequent reduction of WWOX expression in IPMC, which was almost equivalent to the frequency of WWOX reduction in IDC (Nakayama et al, 2008). In turn, WWOX expression was relatively retained in most IPMAs, suggesting that reduced WWOX expression does not contribute to development of IPMA.…”

“…In pancreatic carcinoma cells and IDC tissue samples, a close correlation has been documented between hypermethylation of the WWOX regulatory region and low WWOX expression levels rather than LOH at the WWOX locus (Nakayama et al, 2008). In BxPC-3 (low WWOX expression) cells, frequent hypermethylation at the WWOX regulatory sites was detected by bisulphite sequencing (Figure 2A).…”

“…Because we have previously shown that increased SMAD4 expression at the protein level can be induced by the transfection of WWOX-expressing vector into pancreatic cancer-derived PANC-1 cells (Nakayama et al, 2008), we also examined the SMAD4 expression in these IPMN cases ( Figure 3A). In total, 13 Figure 3B).…”

“…Much as in other human malignancies, restoration of the WWOX gene effectively suppressed the cell growth and induced caspasedependent apoptosis in pancreatic cancer-derived cells in conjunction with an increase in SMAD4 expression (Nakayama et al, 2008). In this study, we assessed WWOX expression in IPMN (IPMA and IPMC) to know whether suppression of WWOX expression is common event during tumourigenesis of these pancreatic duct lesions, IDC and IPMN.…”

“…We previously showed that the WWOX gene promoter is frequently hypermethylated and WWOX expression frequently reduced in IDC cases (Kuroki et al, 2004;Nakayama et al, 2008). Much as in other human malignancies, restoration of the WWOX gene effectively suppressed the cell growth and induced caspasedependent apoptosis in pancreatic cancer-derived cells in conjunction with an increase in SMAD4 expression (Nakayama et al, 2008).…”