Cytoplasmic Accumulation Of Beta-catenin Research Articles

Unusual Thyroid Tumors in a Young Woman

Question: A 24-year-old woman was referred to our hospital because of thyroid nodules. Physical examination revealed a painless nodule in the right lobe of the thyroid but no abnormal findings in the abdomen. The patient has no remarkable past history and laboratory studies, including blood count, chemistry, and thyroid function tests, were all within normal limits. Computed tomography revealed 2 thyroid tumors (Figure A) and fine needle aspiration from the right tumor suggested undifferentiated carcinoma. The patient underwent total thyroidectomy, which disclosed that both nodules were cancers with identical and unique histologic findings. They were composed of cribriform, morular, and papillary growth patterns (Figure B–D). Immunohistochemistry showed cytoplasmic and nuclear accumulation of beta-catenin (Figure E). The final diagnosis was cribriform-morular variant, a rare subtype of papillary thyroid carcinoma.View Large Image Figure ViewerDownload Hi-res image Download (PPT) What is a possible underlying gastrointestinal disease in this case? See the Gastroenterology web site (www.gastrojournal.org) for more information on submitting your favorite image to Clinical Challenges and Images in GI. Patients with familial adenomatous polyposis (FAP) are at risk for extracolonic malignancies, including duodenal ampullary cancer, neurologic tumors, and papillary thyroid cancer. Approximately 1%–2% of FAP patients develop thyroid carcinoma, which is observed almost exclusively in young, female patients (male:female ratio, 1:20).1Groen E.J. Roos A. Muntinghe F.L. et al.Extra-intestinal manifestations of familial adenomatous polyposis.Ann Surg Oncol. 2008; 15: 2439-2450Crossref PubMed Scopus (174) Google Scholar The diagnosis of thyroid cancer precedes polyposis in one third of FAP cases.2Tomoda C. Miyauchi A. Uruno T. et al.Cribriform-morular variant of papillary thyroid carcinoma: clue to early detection of familial adenomatous polyposis-associated colon cancer.World J Surg. 2004; 28: 886-889Crossref PubMed Scopus (78) Google Scholar Thyroid cancer in FAP is often multiple and shows the unique histology known as cribriform-morular variant (CMV) of papillary carcinoma.2Tomoda C. Miyauchi A. Uruno T. et al.Cribriform-morular variant of papillary thyroid carcinoma: clue to early detection of familial adenomatous polyposis-associated colon cancer.World J Surg. 2004; 28: 886-889Crossref PubMed Scopus (78) Google Scholar CMV accounts for only 0.1%–0.2% of all papillary thyroid carcinoma, whereas >50% of CMV is associated with FAP.2Tomoda C. Miyauchi A. Uruno T. et al.Cribriform-morular variant of papillary thyroid carcinoma: clue to early detection of familial adenomatous polyposis-associated colon cancer.World J Surg. 2004; 28: 886-889Crossref PubMed Scopus (78) Google Scholar The histologic feature of CMV is a mixture of cribriform, morular, trabecular, and papillary growth pattern. Immunohistochemically, CMV invariably demonstrates aberrant nuclear and cytoplasmic accumulation of beta-catenin, which is not observed in other types of thyroid cancer.3Kurihara K. Shimizu S. Chong J.M. et al.Nuclear localization of immunoreactive β-catenin is specific to familial adenomatous polyposis in papillary thyroid carcinoma.Jpn J Cancer Res. 2000; 91: 1100-1102Crossref PubMed Scopus (34) Google Scholar In this case, the pathologic findings of thyroid cancer suggested underlying FAP, although the patient did not complain any polyposis-associated symptoms. Colonoscopy after thyroidectomy revealed hundreds of colorectal polyps (Figure F) without colorectal cancer. Polypectomy was performed for large polyps, all of which were low-grade tubular adenoma. The patient's father has also suffered from FAP and underwent total colectomy at the age of 35, although she did not know this until the diagnosis of her FAP. Germline mutation analysis by direct sequence revealed a nonsense mutation of APC exon 7.

Stromal CD10 expression and relationship to the E‐cadherin/β‐catenin complex in breast carcinoma

Previous investigations have indicated that stromal CD10 expression, and altered levels of both E-cadherin and beta-catenin, are associated with the biological aggressiveness of human carcinoma. The aim was to evaluate stromal CD10 expression and the association of stromal CD10 with E-cadherin and beta-catenin in breast carcinoma. The expression of CD10, E-cadherin and beta-catenin was immunohistochemically analysed in tissue microarrays containing 104 cases of invasive ductal carcinoma (IDC) and 10 cases of ductal carcinoma in situ (DCIS). Stromal CD10 was detected in 49.5% (50/101) of the IDC. No immunoreactivity was identified in the stromal cells of normal breast, DCIS or intraductal components of IDC. Accumulation of the cytoplasmic beta-catenin was found in 87.0% (87/100) of the IDC. Stromal CD10 expression in IDC was significantly correlated with tumour size (P = 0.027), stage (P < 0.001) and histological grade (P = 0.006), the presence of nodal (P = 0.048) and distant (P = 0.015) metastases, oestrogen receptor-negative status (P = 0.016), cytoplasmic beta-catenin accumulation (P = 0.031) and lower overall survival rate (P = 0.041). Stromal CD10 expression in IDC may constitute an important prognostic marker. Stromal CD10 expression with associated aggressive features might be related to aberrant beta-catenin expression.

Downregulation of HDPR1 is associated with poor prognosis and affects expression levels of p120‐catenin and β‐catenin in nonsmall cell lung cancer

HDPR1 (human homologue of Dapper) is considered as a Dishevelled (DVL) antagonist in WNT signaling. We recently reported that DVL was associated with cytoplasmic accumulation of beta-catenin in nonsmall cell lung cancer (NSCLC). Whether cytoplasmic accumulation of beta-catenin is correlated with HDPR1 is unclear. Xenopus Dapper (XDpr) was found to stabilize p120-catenin (p120ctn) in Xenopus embryogenesis. However, whether HDPR1 can regulate p120ctn expression level is not reported. Furthermore, how HDPR1 influences invasiveness in lung carcinogenesis is also not well understood. In this study, our aims were to explore the effects of HDPR1 on the lung carcinogenesis and to examine the relationship among HDPR1, beta-catenin, and p120ctn. Immunohistochemical analysis in 120 NSCLC tissues showed that HDPR1 was significantly lower in 82 specimens (68.3%). Reverse transcription (RT)-polymerase chain reaction (PCR) and Western blotting analysis showed that the mRNA and protein expression of HDPR1 were lower in tumor tissues as compared to corresponding nontumorous tissues. Moreover, reduced HDPR1 expression was related to the clinicopathological factors and was an independent risk factor for prognosis of the patients with NSCLC. In addition, HDPR1 expression was also associated with the expression of p120ctn and beta-catenin in lung cancer tissues. Knockdown of HDPR1 gene enhanced the invasive ability of lung cancer cells, which was dependent on p120ctn and independent of beta-catenin. In conclusion, the function of HDPR1 on regulating p120ctn may play an important role in human lung carcinogenesis. Restoration of HDPR1 gene may be a new therapeutic target of lung cancer.

Hypermethylation of APC promoter 1A is associated with moderate activation of Wnt signalling pathway in a subset of colorectal serrated adenomas

The role of Wnt signalling pathway in serrated adenomas (SAs) remains to be identified. The aim of this study was to determine whether Wnt signalling plays a role in the pathogenesis of SAs, and to clarify the mechanism of Wnt signalling activation in SAs. This study investigated immunoreactivities of adenomatous polyposis coli (APC) and beta-catenin, mutations of APC and beta-catenin genes, methylation status of APC promoter 1A in 12 SAs, and compared the findings with normal colorectal mucosa, hyperplastic polyps, traditional adenomas (TAs) and colorectal cancers (CRCs). APC expression was moderately decreased in SAs. Cytoplasmic accumulation of beta-catenin was demonstrated in 41.7% (5/12) of SAs, but membranous immunoreactivity of beta-catenin was lost in only 8.3% (1/12) of SAs. No beta-catenin mutation was detected in any of 12 SAs, and only one SA was found to be positive for APC gene mutation. Complete methylation of APC promoter 1A was found in 41.7% (5/12) of SAs, but in no TAs or CRCs. Hypermethylation of APC promoter 1A, instead of mutations involving APC and beta-catenin, contributes to moderate activation of Wnt signalling in a subset of SAs.

Adhesion molecules α, β and γ‐catenin as prognostic factors of tumour progression in upper urinary tract urothelial tumours: the role of AKT‐P/GSK‐3β/β‐catenin pathway

OBJECTIVES To evaluate alpha, beta and gamma-catenin expression in upper urinary tract urothelial tumours (UUTC) and determine their value as prognostic factors; to investigate the correlation between the catenin complex and the AKT pathway. PATIENTS AND METHODS We retrospectively analysed 114 consecutive patients treated at our institution from 1990 to 2004; the mean follow-up was 54 months. Tumour samples were available from 70 patients, and included in tissue microarrays for immunohistochemical analysis. The antibodies used were anti-alpha, -beta and gamma-catenin, and antiphospho-AKT. The prognostic value of the expression of these molecules was analysed using tumour progression and cancer-specific survival as end-points. RESULTS Of the 114 patients, 27% developed tumour progression; the cancer-specific and overall survival were 77% and 60.6%, respectively. Abnormal alpha, beta and gamma-catenin expression was found in 44 (63%), 22 (31%) and 28 (41%) patients, respectively; the abnormal catenin expression patterns correlated with each other. Positive cytoplasm phospho-AKT expression was found in 27 (39%) patients. Three of them were found to have cytoplasmic beta-catenin accumulation and none of them nuclear expression. beta-catenin expression was the only one that was an independent marker of tumour progression, with a hazard ratio (95% confidence interval) of 3.1(1.2-8.6), together with grade (7.1, 1.2-55.8) and stage (4.6, 2.1-10). In the cancer-specific survival analysis, again beta-catenin was an independent prognostic factor (3.4, 1-11.5) together with stage (4.6, 2.2-9.8). CONCLUSIONS The loss of the normal membrane beta-catenin expression constitutes an independent factor of tumour progression and cancer-specific survival. Our data suggest that the AKT/GSK3beta/beta-catenin signalling pathway is not activated in the UUTC carcinogenesis.

High frequency of down-regulation of E-cadherin detected in benign sporadic insulinomas by multiplex ligation-dependent probe amplification

Insulinomas are the most common functioning pancreatic endocrine tumors, and the previous studies showed that the chromosomal aberrations of Chr.9q, 11q, and 22q were associated with the development and progression of insulinoma. To analyze the genetic alterations in sporadic insulinoma, we tested 23 tumor samples using multiplex ligation-dependent probe amplification. The results showed that 20 (87%) of the 23 patients had lost CDH1, a tumor suppressor gene. Immunofluorescence analysis of the E-cadherin and beta-catenin proteins further confirmed the impaired expression of E-cadherin and the translocation of beta-catenin in sporadic insulinomas. It was found that the cytoplasmic accumulation of beta-catenin coincided with the decrease or loss of E-cadherin synthesis during the tumorigenesis of sporadic insulinomas. Our study suggests that the inactivation of CDH1 is an important and early event in the development of these tumor types.

The structural and functional determinants of the Axin and Dishevelled DIX domains

BackgroundThe dishevelled and axin genes encode multi-domain proteins that play key roles in WNT signalling. Dishevelled prevents β-catenin degradation by interfering with the interaction of β-catenin with the degradation-mediating Axin-APC-GSK3β complex. This interference leads to an accumulation of cytoplasmic β-catenin, which enters the nucleus and interacts with transcription factors that induce expression of Wnt-target genes. Axin, as a component of the degradation-mediating complex, is a potent negative regulator of Wnt signalling, whereas Dishevelled is a potent activator. Both Dishevelled and Axin possess a DIX (Dishevelled/Axin) domain, which mediates protein-protein interactions, specifically homodimerization.ResultsAn evolutionary trace analysis of DIX domains identified conserved residues which, when mapped onto the crystal structure of the Axin DIX domain and a comparative model of the Dishevelled DIX domain, allow their categorization as residues of either structural or functional importance. We identify residues that are structural and functional determinants of the DIX domain fold, as well as those that are specific to homodimerization of Axin and Dishevelled.ConclusionThis report provides the first explanation of the mutant phenotypes caused by non-synonymous substitutions in the Dishevelled and Axin DIX domain by correlating their presumed functional significance with molecular structure.

Disruption of the basal body compromises proteasomal function and perturbs intracellular Wnt response

Primary cilia and basal bodies are evolutionarily conserved organelles that mediate communication between the intracellular and extracellular environments. Here we show that bbs1, bbs4 and mkks (also known as bbs6), which encode basal body proteins, are required for convergence and extension in zebrafish and interact with wnt11 and wnt5b. Suppression of bbs1, bbs4 and mkks transcripts results in stabilization of beta-catenin with concomitant upregulation of T-cell factor (TCF)-dependent transcription in both zebrafish embryos and mammalian ciliated cells, a defect phenocopied by the silencing of the axonemal kinesin subunit KIF3A but not by chemical disruption of the cytoplasmic microtubule network. These observations are attributable partly to defective degradation by the proteasome; suppression of BBS4 leads to perturbed proteasomal targeting and concomitant accumulation of cytoplasmic beta-catenin. Cumulatively, our data indicate that the basal body is an important regulator of Wnt signal interpretation through selective proteolysis and suggest that defects in this system may contribute to phenotypes pathognomonic of human ciliopathies.

Inappropriate activation of androgen receptor by relaxin via β-catenin pathway

We have previously demonstrated that human H2-relaxin can mediate androgen-independent growth of LNCaP through a mechanism that involves the activation of the androgen receptor (AR) signaling pathway. The goal of the current study is to elucidate the mechanism(s) by which H2-relaxin causes activation of the AR pathway. Our data indicate that there is cross-talk between AR and components of the Wnt signaling pathway. Addition of H2-relaxin to LNCaP cells resulted in increased phosphorylation of protein kinase B (Akt) and inhibitory phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) with subsequent cytoplasmic accumulation of beta-catenin. Immunoprecipitation and immunocytochemical studies demonstrated that the stabilized beta-catenin formed a complex with AR, which was then translocated into the nucleus. Chromatin immunoprecipitation analysis determined that the AR/beta-catenin complex binds to the proximal region of the prostate-specific antigen promoter. Inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, using LY294002, prevented both H2-relaxin-mediated phosphorylation of Akt and GSK-3beta and translocation of beta-catenin/AR into the nucleus. Knockdown of beta-catenin levels using a beta-catenin-specific small interfering RNA inhibited H2-relaxin-induced AR activity. The combined data demonstrate that PI3K/Akt and components of the Wnt pathway can facilitate H2-relaxin-mediated activation of the AR pathway.

Human scribble accumulates in colorectal neoplasia in association with an altered distribution of β-catenin

The loss of epithelial polarity and tissue architecture is a diagnostic feature of malignant tumors. In Drosophila, genetic studies identified 3 neoplastic tumor suppressor genes (nTSGs), and a loss of nTSGs has been shown to result in a disruption of apical-basal polarity and neoplastic growth in epithelial cells. Scribble is one type of the Drosophila nTSGs, which encodes a membrane-associated cytoplasmic protein containing the multi-PDZ domain. In contrast to Drosophila scribble, the oncogenic roles of its mammalian homologues have not yet been established. We herein immunohistochemically examined the distributions of hScrib protein in human colorectal neoplasia using affinity-purified antibody. In 50 cases of colorectal adenomas and adenocarcinomas, the accumulation of hScrib protein was commonly observed in comparison with the adjacent normal epithelia. Furthermore, the overexpression and distribution of hScrib was observed to extensively overlap with the cytoplasmic accumulation of beta-catenin. Like beta-catenin, the intense immunoreactivity of hScrib was often observed in small adenomas, thus, suggesting that hScrib could be involved in an early step of colon carcinogenesis. Five corresponding liver metastases showed a comparable immunoreactivity for anti-hScrib in comparison with their primary sites. In an immunofluorescence analysis on cultured cell lines, the loss of membranous staining of hScrib was observed according to the cytoplasmic translocation of beta-catenin. We herein demonstrate that the accumulation of hScrib protein might therefore be involved in colon carcinogenesis while also providing a possible link between hScrib and beta-catenin.

The von Hippel-Lindau tumor suppressor gene product represses oncogenic beta-catenin signaling in renal carcinoma cells.

Loss of von Hippel-Lindau (VHL) tumor suppressor gene function occurs in familial and most sporadic clear cell renal cell carcinoma (RCC), resulting in the aberrant expression of genes that control cell proliferation, invasion, and angiogenesis. The molecular mechanisms by which VHL loss leads to tumorigenesis are not yet fully defined. VHL loss has been shown to allow robust RCC cell motility, invasiveness, and morphogenesis in response to hepatocyte growth factor (HGF) stimulation, processes that are known to contribute to tumor invasiveness and metastatic potential. Among the most likely intracellular mediators of these HGF-driven activities is beta-catenin, a structural link between cadherens and the actin cytoskeleton, as well as a gene transactivator. We show that reconstitution of VHL expression in RCC cells repressed HGF-stimulated beta-catenin tyrosyl phosphorylation, adherens junction disruption, cytoplasmic beta-catenin accumulation, and reporter gene transactivation in RCC cells. Ectopic expression of a ubiquitination-resistant beta-catenin mutant specifically restored HGF-stimulated invasion and morphogenesis in VHL-transfected RCC cells. VHL gene silencing in non-RCC renal epithelial cells phenotypically mimicked VHL loss in RCC, and HGF-driven invasiveness was blocked by the expression of a dominant-negative mutant of Tcf. We conclude that, unlike many other cancers, where HGF pathway activation contributes to malignancy through the acquisition of autocrine signaling, receptor overexpression, or mutation, in RCC cells VHL loss enables HGF-driven oncogenic beta-catenin signaling. These findings identify beta-catenin as a potential target in biomarker and drug development for RCC.

Mutations of the APC, beta-catenin, and axin 1 genes and cytoplasmic accumulation of beta-catenin in oral squamous cell carcinoma

The Wnt pathway is involved in carcinogenesis and three regulatory genes of the Wnt pathway, APC, beta-catenin and Axin are mutated in some primary human cancers. Mutations in these genes can impair the down regulation of beta-catenin, which results in the stabilization of beta-catenin, accumulation of free beta-catenin and subsequent activation of the Wnt pathway. To clarify the genetic alterations of components of the Wnt pathway in oral squamous cell carcinoma (SCC), we examined mutations in the APC, beta-catenin and Axin genes and subcellular localization of beta-catenin. 20 oral SCC tissues and four cell lines derived from oral SCC were used. Mutational analysis was performed by a single-strand conformation polymorphism (SSCP) method and direct sequencing analysis. The samples were also examined by immunohistochemical staining and immunoblot analysis. In 3 of 4 cell lines, mutations were observed in the APC and Axin1 genes without amino acid substitutions. In a clinical sample, a mutation in the Axin1 gene was detected; a T insertion at codon 250 resulted in the formation of a stop codon at codon 259. In addition, cytoplasmic accumulation of beta-catenin was observed in 3 (75%) of 4 cell lines and 18 (90%) of 20 cancer tissue samples. The Axin1 gene may be one of the mutational target in oral SCC. In addition, the cytoplasmic accumulation of beta-catenin is a common characteristic of oral SCC, but is not closely associated with mutational alterations in the APC, beta-catenin and Axin1 genes.

Mechanisms of Cytoplasmic β-Catenin Accumulation and Its Involvement in Tumorigenic Activities Mediated by Oncogenic Splicing Variant of the Receptor Originated from Nantes Tyrosine Kinase

The beta-catenin pathway plays a critical role in the pathogenesis of certain types of cancers. To gain insight into mechanisms by which altered receptor tyrosine kinases regulate cytoplasmic beta-catenin accumulation, the effect of an oncogenic receptor originated from Nantes (RON) variant on beta-catenin accumulation and the role of beta-catenin in RON-mediated tumorigenic activities were studied. In NIH3T3 cells harboring oncogenic variant RONDelta160, increased beta-catenin accumulation with tyrosine phosphorylation and nuclear translocation was observed. Overexpression of RONDelta160 also resulted in increased expression of beta-catenin target genes c-myc and cyclin D1. By analyzing cellular proteins that regulate beta-catenin stabilities, it was found that RONDelta160 activates the protein disheveled (DVL) and inactivates glycogen synthase kinase-3beta by Ser-9 residue phosphorylation. These effects were channeled by RONDelta160-activated PI 3-kinase-AKT pathways that are sensitive to specific inhibitors, such as wortmannin, but not to other chemical inhibitors. Silencing RONDelta160 expression by specific small interfering RNA blocked not only beta-catenin expression but also c-myc and cyclin D1 expression, suggesting that RON expression is required for the activation of the beta-catenin signaling pathway. Moreover, it was found that knockdown of the beta-catenin gene expression by small interfering RNA techniques reduces significantly the RONDelta160-mediated NIH3T3 cell proliferation, focus-forming activities and anchorage-independent growth. Thus, the oncogenic RON variant regulates beta-catenin stabilities through activation of DVL and inactivation of glycogen synthase kinase-3beta. The activated beta-catenin cascade is one of the pathways involved in tumorigenic activities mediated by the oncogenic RON variant.

Accumulation of cytoplasmic β‐catenin correlates with reduced expression of E‐cadherin, but not with phosphorylated Akt in esophageal squamous cell carcinoma: Immunohistochemical study

Accumulation of beta-catenin in cytoplasm occurs frequently during the pathogenesis of esophageal squamous cell carcinoma (ESCC). The mechanism leading to this alteration, however, is largely unknown. In the present study, immunohistochemistry was performed for beta-catenin, E-cadherin and Ser473 phosphorylated Akt (P-Akt) in 44 tissue samples of ESCC and corresponding normal esophageal epithelium. Exon 3 of the beta-catenin gene was analyzed by using single-strand conformation polymorphism and direct sequencing. In addition to the reduced expression of E-cadherin and membranous beta-catenin observed in 65.9% and 68% of ESCC tested, respectively, cytoplasmic accumulation of beta-catenin was also detected in 68% (30/44) cases. However, only two cases were found to have the same beta-catenin gene mutation. The data showed that cytoplasmic accumulation of beta-catenin was significantly associated with reduced expression of E-cadherin (P < 0.05) and that of membranous beta-catenin (P < 0.05). Furthermore, cytoplasmic beta-catenin was correlated significantly with lymph node metastasis (P < 0.05). In contrast, although strong staining of P-Akt occurred in 14 of 44 cases (32%), there was no significant correlation between the positive staining of P-Akt and cytoplasmic beta-catenin. Taken together these results suggest that the lost membranous beta-catenin might translocate to cytoplasm depending on reduced expression of E-cadherin, while Akt seems unlikely to play a role in this process.

Cytoplasmic expression of E‐cadherin and β‐Catenin correlated with LOH and hypermethylation of the APC gene in oral squamous cell carcinomas

Inactivation of the adenomatous polyposis coli (APC) gene results in accumulation and translocation of beta-Catenin, which are important for malignant development. The aim of the present study is to investigate the possible role of APC/beta-Catenin pathway in oral squamous cell carcinomas. The DNA from 34 patients was examined for loss of heterozygosity (LOH) at two markers surrounding the APC, and for hypermethylation of the APC promoter by using methylation-specific polymerase chain reaction (MS-PCR). Fifteen of 34 samples were stained immunohistochemically to show the expression of E-cadherin and beta-Catenin. We found that cytoplasmic rather than membrane staining of E-cadherin and beta-Catenin was a prominent aberrant tumour-related alteration, and that this expression was mainly present in moderately and poorly differentiated tumours. LOH and hypermethylation of the APC promoter was found in four of 31 and five of 34 carcinoma samples, respectively. Four of five cases presenting LOH/hypermethylation showed cytoplasmic expression of E-cadherin and beta-Catenin by immunohistochemical (IHC) staining. The present results indicate that LOH at the APC locus or hypermethylation of the APC promoter 1a may lead to free beta-Catenin accumulation in cytoplasm of oral carcinoma cells and thereby to oral malignant progression.

HDPR1, a novel inhibitor of the WNT/β-catenin signaling, is frequently downregulated in hepatocellular carcinoma: involvement of methylation-mediated gene silencing

Oncogenic activation of the WNT/beta-catenin signaling pathway is common in hepatocellular carcinoma (HCC). Dishevelled (Dvl), a key activator of the pathway, inhibits the adenomatous polyposis coli complex, and this leads to the accumulation of beta-catenin and promotes tumorigenesis. Recently, a novel inhibitor of Dishevelled, namely Dapper (Dpr), was isolated in Xenopus. To explore whether HDPR1, the human homologue of Dpr, has an anti-oncogenic role in hepatocarcinogenesis, we studied the expression of this gene in HCCs. We found that there were two alternatively spliced transcripts of HDPR1, designated as alpha and beta forms, in human liver. Downregulation of the gene expression was observed in 31 (43%) of the 72 human HCC samples using the primer pair that amplified both transcripts. Furthermore, the HDPR1alpha was downregulated in 42 (58%) of 72 human HCCs and the downregulation significantly correlated with accumulation of beta-catenin. Also, downregulation of HDPR1 by RNA interference in HLE cells led to cytoplasmic accumulation of beta-catenin. Furthermore, a CpG island located at the promoter region and exon 1 of the HDPR1 gene was methylated in 22 (51%) of human HCCs. We showed that downregulation of HDPR1, in hepatoma cell lines, was associated with methylation of this CpG island using bisulfite sequencing and 5-aza-2'-deoxycytidine demethylation experiment. In addition to methylation-mediated downregulation of HDPR1, allelic loss (13-28% of informative cases) was detected using microsatellite markers flanking the HDPR1 locus. To conclude, downregulation of HDPR1 is common in HCCs, frequently involves hypermethylation of the promoter region, and allelic loss of the HDPR1 locus may also play a role.

Liver-targeted disruption ofApcin mice activates β-catenin signaling and leads to hepatocellular carcinomas

Although inappropriate activation of the Wnt/beta-catenin pathway has been implicated in the development of hepatocellular carcinoma (HCC), the role of this signaling in liver carcinogenesis remains unclear. To investigate this issue, we constructed a mutant mouse strain, Apc(lox/lox), in which exon 14 of the tumor-suppressor gene adenomatous polyposis coli (Apc) is flanked by loxP sequences. i.v. injection of adenovirus encoding Cre recombinase (AdCre) at high multiplicity [10(9) plaque-forming units (pfu) per mouse] inactivated the Apc gene in the liver and resulted in marked hepatomegaly, hepatocyte hyperplasia, and rapid mortality. beta-Catenin signaling activation was demonstrated by nuclear and cytoplasmic accumulation of beta-catenin in the hepatocytes and by the induction of beta-catenin target genes (glutamine synthetase, glutamate transporter 1, ornithine aminotransferase, and leukocyte cell-derived chemotaxin 2) in the liver. To test a long-term oncogenic effect, we inoculated mice with lower doses of AdCre (0.5 x 10(9) pfu per mouse), compatible with both survival and persistence of beta-catenin-activated cells. In these conditions, 67% of mice developed HCC. beta-Catenin signaling was strongly activated in these Apc-inactivated HCCs. The HCCs were well, moderately, or poorly differentiated. Indeed, their histological and molecular features mimicked human HCC. Thus, deletion of Apc in the liver provides a valuable model of human HCC, and, in this model, activation of the Wnt/beta-catenin pathway by invalidation of Apc is required for liver tumorigenesis.

Overexpression of human pituitary tumor transforming gene (hPTTG), is regulated by ?-catenin /TCF pathway in human esophageal squamous cell carcinoma

Overexpression of human pituitary tumor transforming gene (PTTG) is wildly detected in many tumors, including esophageal cancer. Besides overexpression of PTTG in esophageal squamous cell carcinoma (ESCC) tissues and cells, we detected accumulation of cytoplasmic beta-catenin in ESCC. In our study, a putative TCF4-binding element (TBE) was identified in PTTG promoter region. The activity of PTTG promoter containing the TBE was activated by S37Abeta-catenin and inhibited by dominant-negative TCF. Furthermore, the activation by S37Abeta-catenin was mostly abrogated among PTTG promoter region without the TBE or with a mutant one. By using biotin-streptavidin pull-down assay, we also found that the TBE among PTTG promoter bound to TCF-4 protein. Moreover, levels of PTTG mRNA and protein were increased by S37Abeta-catenin. Finally, it is noticeable that we detected a correlation between beta-catenin localization and PTTG expression in 69 primary ESCC (p<0.01). In brief, our study shows that overexpression of PTTG in ESCC is likely due to the activation of beta-catenin/WNT signaling. As a target gene of beta-catenin/TCF pathway, PTTG may play an important role in tumorigenesis of human ESCC.

β-Catenin Mutations Are Frequent in Calcifying Odontogenic Cysts, but Rare in Ameloblastomas

We have reported previously that alterations to beta-catenin occur frequently in adamantinomatous craniopharyngioma. Based on its histological resemblance to some odontogenic tumors, we suspected the presence of common genetic alterations among these tumors. To address this issue, 11 cases of calcifying odontogenic cyst (COC) and 20 cases of ameloblastoma were investigated for the presence of beta-catenin mutations and beta-catenin expression. Ten COCs were successfully analyzed by direct sequencing, and nine of them were found to harbor somatic beta-catenin mutations. Immunohistochemically, all of the COCs showed nuclear and cytoplasmic expression of beta-catenin with a heterogeneous pattern. No beta-catenin mutations were found in ameloblastomas, except for one case of the follicular type. All follicular ameloblastomas exhibited moderate nuclear and cytoplasmic accumulation of beta-catenin, in contrast to the predominantly membranous expression seen in the plexiform type. beta-Catenin mutation is considered to be a characteristic genetic feature of COC, and may play a critical role in its histogenesis. Although ameloblastoma closely resembles COC histologically, the two have genetically distinctive features.

Neutrophils induce sequential focal changes in endothelial adherens junction components: role of elastase.

In vitro studies have indicated that polymorphonuclear leukocytes (PMNs) traverse endothelial cell monolayers via the paracellular pathway (i.e., through endothelial cell-cell junctions. Herein, we assessed whether the adherens junctions (AJs) are disrupted during PMN transendothelial cell migration. Human umbilical vein endothelial cells (HUVECs) were grown to confluence on porous membranes and activated with interleukin-1beta, and PMN transendothelial migration was facilitated by formyl-methionyl-leucyl-phenylalanine. Using dual immunofluorescence staining and laser scanning confocal microscopy, we assessed the effects of PMN-endothelial cell adhesive interactions (i.e., adhesion to and emigration across monolayers) on the AJ components vascular endothelial (VE)-cadherin, beta-catenin, alpha-catenin, and gamma-catenin. In the AJ immediately adjacent to the adherent PMN, there was a loss of staining for some of the AJ components. AJ components further away from HUVEC-PMN adhesive interactions were unaffected. An iterative approach indicated that the four components were sequentially lost from the AJ. beta-catenin was lost first, followed by VE-cadherin, alpha-catenin, and, finally, gamma-catenin. In the absence of PMNs, the cross-linking of VE-cadherin, but not platelet endothelial cell adhesion molecule-1 or intercellular adhesion molecule-1, increased the cytoplasmic accumulation of beta-catenin. During PMN transendothelial migration, all of the junctional components under study were lost at the immediate site of monolayer penetration. Again, at regions removed from the actual site of PMN penetration of the monolayers, the AJ components were unaffected. PMN-induced disorganization of the AJs was partially prevented by an elastase inhibitor. These findings suggest that adherent PMNs induce a localized, sequential disassembly of AJs, which is partially mediated by PMN-derived elastase and involves the initial loss of an intracellular component of AJs (i.e., beta-catenin).