Β-catenin Gene Mutations Research Articles

Nuclear translocation of β-catenin correlates with CD44 upregulation in Helicobacter pylori-infected gastric carcinoma

Infection with Helicobacter pylori CagA-positive strains is associated with gastric adenocarcinoma. CagA H. pylori activates the β-catenin signal by translocation into nucleus which promotes carcinogenesis. Deregulated accumulation of nuclear β-catenin enhances transcription of β-catenin target genes including CD44 and promotes malignant transformation. The aim of this study was to investigate whether nuclear translocation of β-catenin correlates with CD44 expression in CagA H. pylori-infected gastric carcinoma. To address these issues, we examined 140 gastric biopsy specimens by using immunohistochemical and immunofluorescence staining, Western blot, and mutational analysis of the exon 3 β-catenin gene. The nuclear localization of β-catenin was significantly (χ(2) = 21.175; P < 0.001) increased in advanced gastric carcinoma and also correlated (χ(2) = 22.857; P < 0.001) with the CagA H. pylori positive specimens. We also observed that tyrosine-phosphorylated β-catenin was significantly (χ(2) = 14.207; P < 0.001) increased in samples showing nuclear localization of β-catenin and also it correlated (χ(2) = 43.69; P < 0.03) with the CagA H. pylori positive specimens. Exon 3 β-catenin gene mutation was not detected in H. pylori-infected gastric carcinoma. CD44 up regulation was significantly associated with tyrosine-phosphorylated β-catenin (χ(2) = 22.5; P < 0.001), and this change was closely associated with nuclear translocation of β-catenin (χ(2) = 13.393; P < 0.001) in CagA H. pylori-infected gastric carcinoma. In conclusion, our data suggest that CagA H. pylori infection is responsible for the tyrosine phosphorylation of β-catenin and its nuclear translocation, which upregulates β-catenin target gene CD44 in gastric carcinoma.

Characterization of Differential Gene Expression in Adrenocortical Tumors Harboring β-Catenin (CTNNB1) Mutations

Mutations of β-catenin gene (CTNNB1) are frequent in adrenocortical adenomas (AA) and adrenocortical carcinomas (ACC). However, the target genes of β-catenin have not yet been identified in adrenocortical tumors. Our objective was to identify genes deregulated in adrenocortical tumors harboring CTNNB1 genetic alterations and nuclear accumulation of β-catenin. Microarray analysis identified a dataset of genes that were differently expressed between AA with CTNNB1 mutations and wild-type (WT) tumors. Within this dataset, the expression profiles of five genes were validated by real time-PCR (RT-PCR) in a cohort of 34 adrenocortical tissues (six AA and one ACC with CTNNB1 mutations, 13 AA and four ACC with WT CTNNB1, and 10 normal adrenal glands) and two human ACC cell lines. We then studied the effects of suppressing β-catenin transcriptional activity with the T-cell factor/β-catenin inhibitors PKF115-584 and PNU74654 on gene expression in H295R and SW13 cells. RT-PCR analysis confirmed the overexpression of ISM1, RALBP1, and PDE2A and the down-regulation of PHYHIP in five of six AA harboring CTNNB1 mutations compared with WT AA (n = 13) and normal adrenal glands (n = 10). RALBP1 and PDE2A overexpression was also confirmed at the protein level by Western blotting analysis in mutated tumors. ENC1 was specifically overexpressed in three of three AA harboring CTNNB1 point mutations. mRNA expression and protein levels of RALBP1, PDE2A, and ENC1 were decreased in a dose-dependent manner in H295R cells after treatment with PKF115-584 or PNU74654. This study identified candidate genes deregulated in CTNNB1-mutated adrenocortical tumors that may lead to a better understanding of the role of the Wnt-β-catenin pathway in adrenocortical tumorigenesis.

Study on β-catenin gene expression and mutation in breast carcinoma

Objective To discuss the expression and mutation of β-catenin gene in breast carcinoma.Methods 119 breast carcinomas, 72 adenosis of breast tissues and 40 normal breast tissues were studied.Using immunohistochemistry studies to detect the expressions of β-catenin, then analyzing their relationship with the clinicopathological features of breast carcinomas. Gene mutation of β-catenin (exon 3) in 44 cases of breast carcinoma were analyzed by polymerase chain reaction (PCR) and then by direct sequencing.Results In 40 cases of normal breast tissues, epithelial cells showed equally strong membranous of β-catenin protein at the cell-cell boundaries. The abnormal rate of β-catenin in breast carcinomas (78.15 %) was much higher than that of adenosis of breast tissues (44.44 %), the significant correlation was found (P＜0.01). The abnormal expression of β-catenin was associated with lymph node metastasis (P=0.031). Mutation of β-catenin gene was not detected in 44 cases of breast carcinoma, while the abnormal rate of β-catenin in 44 cases of breast carcinoma was 77.30 %. Conclusion β-catenin abnormal expression could be considered as an useful marker for determining metastasis and prognosis of human breast carcinoma and for guiding treatment. β-catenin abnormal expression is not caused by β-catenin gene mutation, maybe by other mechanisms. It needs further study. Key words: Breast neoplasms; β-catenin; Mutation

Pegylated interferon alpha targets Wnt signaling by inducing nuclear export of β-catenin

Pegylated-Interferon-α2a (peg-IFN), a first line therapy for Hepatitis C virus (HCV) patients, also impacts the recurrence of hepatocellular carcinoma (HCC). The activation of the Wnt pathway due to β-catenin gene mutations contributes to the development of a significant subset of HCC. Herein, we explored the effect of peg-IFN on Wnt/β-catenin signaling in vitro and in vivo. Multiple human hepatoma cell lines were treated with Peg-IFN to assess its effect on the Wnt pathway and the mechanisms involved. Transgenic (TG) mice expressing stable β-catenin mutant in the liver were exposed to diethylnitrosamine (DEN) and treated with peg-IFN. In vitro, peg-IFN decreased the transcriptional activity of β-catenin/Tcf and did so independently of JAK/Stat signaling. Peg-IFN treatment led to increased mRNA and protein expression of RanBP3, a known β-catenin nuclear export factor, in all hepatoma cells. Co-precipitation studies showed an increased association between RanBP3 and β-catenin after peg-IFN treatment. The siRNA-mediated RanBP3 knockdown abrogated Peg-IFN-induced decrease in TOPFlash reporter activity. In vivo, Peg-IFN treatment led to increased nuclear RanBP3, decreased nuclear β-catenin and cyclin D1, and decreased cytoplasmic glutamine synthetase. Increased association of RanBP3 and β-catenin was also observed in vivo in response to Peg-IFN that led to decreased hepatocyte proliferation. Peg-IFN inhibits β-catenin signaling through the up-regulation of RanBP3, which may be a contributory mechanism for the delayed HCC and improved survival in treated HCV patients. This observation might have chemo-preventive or chemo-therapeutic implications in tumor with aberrant Wnt pathway activation.

Immunogenetics: achieving new heights by immunohistochemistry; newer immunohistochemical markers of value in surgical pathology

Immunogenetics refers to the use of immunohistochemical staining to reflect the underlying genetic alterations. The potential applications are: (1) understanding basic mechanism of disease or tumour; (2) aid in diagnosis; and (3) aid in prognostication. This is a simple and inexpensive way to infer the presence of specific molecular alterations. <b>Specific chromosomal translocations in tumours</b>: Examples include follicular lymphoma (BCL2); mantle cell lymphoma (Cyclin D1); tumours with ALK translocation (anaplastic large cell lymphoma, inflammatory myofibroblastic tumour); tumours with TFE-3 translocation (alveolar soft part sarcoma, and translocation carcinoma of kidney). <b>Specific mutations in tumours</b>: Examples include β-catenin gene mutation (such as solid-pseudopapillary tumour of pancreas), as reflected by abnormal nuclear translocation of the protein; p53, with extensive strong staining indicating presence of <i>P53</i> mutation, such as serous carcinoma of uterus. <b>Gene deletion or loss of function, as reflected by loss of staining</b>: Examples include mismatch repair gene proteins (colorectal cancer with microsatellite instability); E-cadherin (lobular neoplasm of breast); INI-1 (rhabdoid tumour). <b>Gene amplification</b>: Example: HER2 in breast cancer. <b>Surrogate markers</b>: Examples include expression of p16 in cervical epithelium as marker for high-risk HPV; ZAP-70 as marker for the unmutated group of CLL (worse prognosis).

Mutations in the AXIN1 Gene in Advanced Prostate Cancer

BackgroundThe Wnt signalling pathway directs aspects of embryogenesis and is thought to contribute to maintenance of certain stem cell populations. Disruption of the pathway has been observed in many different tumour types. In bowel, stomach, and endometrial cancer, this is usually due to mutation of genes encoding Wnt pathway components APC or β-catenin. Such mutations are rare in hepatocellular carcinomas and medulloblastomas with Wnt pathway dysfunction, and there, mutation in genes for other Wnt molecules, such as Axin, is more frequently found. ObjectiveAlthough evidence of abnormal activation of the Wnt pathway in prostate cancer has been demonstrated by several groups, APC and β-catenin mutations are infrequent. We sought mutations in genes encoding Wnt pathway participants in a panel of prostate cancer clinical specimens and cell lines. Design, setting, and participantsDNA was obtained from 49 advanced prostate cancer specimens using laser microdissection followed by whole genome amplification and 8 prostate cancer cell lines. MeasurementsThe DNA samples were screened for mutations in the genes encoding APC, β-catenin, and Axin. The subcellular distribution of β-catenin expression was assessed in the clinical specimens using immunohistochemistry. Results and limitationsAbnormal patterns of β-catenin expression, suggesting Wnt pathway dysregulation, were observed in 71% of specimens. One APC mutation, two β-catenin gene mutations, and 7 DNA sequence variations in the Axin gene were detected. Four different Axin polymorphisms were also found in the cell lines. The study does not provide definite evidence that the observed sequence changes alter protein function, promoting neoplasia, but the potential functional relevance of these variants is discussed. ConclusionsThese data contribute to our understanding of the role of Wnt dysregulation in prostatic tumourigenesis and support the current interest in the pathway as a therapeutic target. Of particular interest, we identified three new potentially functionally relevant AXIN1 mutations.

PIK3CA mutations in the kinase domain (exon 20) of uterine endometrial adenocarcinomas are associated with adverse prognostic parameters

Mutations of the oncogene PIK3CA occur frequently in endometrial carcinomas, but their prognostic significance is unclear. To determine the clinicopathological and molecular implications of these mutations, PIK3CA status was investigated in 109 endometrial (102 endometrioid and 7 mixed) carcinomas and the results were compared with clinicopathological parameters associated with prognosis. Tumors were also investigated for microsatellite instability and PTEN, β-catenin gene (CTNNB1), K-RAS, and B-RAF mutations. We found 35 PIK3CA somatic missense mutations in 32 (29%) endometrial carcinomas. Eighteen mutations occurred in exon 20 (kinase domain), and 17 in exon 9 (helical domain). Almost all mutated tumors were pure endometrioid adenocarcinomas. All tumors with PIK3CA mutations exhibited myometrial invasion (P=0.032). Lymphovascular invasion was found more frequently in mutated (28%) than nonmutated carcinomas (18%). Histological grade varied significantly according to the location of the PIK3CA mutations whether in exon 9 or exon 20 (P=0.033). The frequency of exon 9 mutations was higher in grade 1 carcinomas (57%) than in grade 2 (29%) or grade 3 (14%) tumors. Conversely, mutations in exon 20 were more common in grade 3 (60%) than in grade 2 (20%) or grade 1 (20%) carcinomas. None of the tumors confined to the endometrium (stage IA) had PIK3CA mutations. Furthermore, whereas 64% of adenocarcinomas with exon 9 mutations had invaded ≤½ of the myometrial thickness (stage IB), 73% of tumors with exon 20 mutations had either deeper myometrial invasion (stage IC) or cervical involvement (stage II) (P=0.045). PIK3CA mutations coexisted with microsatellite instability and mutations in PTEN, CTNNB1, K-RAS, and B-RAF genes. These results favor that PIK3CA mutations are associated with myometrial invasion and, moreover, that tumors harboring PIK3CA mutations in exon 20 are frequently high-grade, deeply invasive endometrial carcinomas that tend to exhibit lymphovascular invasion.

Altered distribution of β-catenin and prognostic roles in colorectal carcinogenesis

Objective. Although β-catenin cytoplasmic stabilization and nuclear translocation play a key role in initiation of colorectal cancer (CRC), the mechanisms are far from clear. The aim of this study was to investigate the relation of expressions of cyclooxygenase (COX)-2 and E-cadherin, and the β-catenin gene exon 3 mutation to the altered distribution of β-catenin, and their roles in CRC progression and prognosis. Material and methods. Expressions of β-catenin, COX-2 and E-cadherin in 96 tissue specimens were detected by immunohistochemistry, and mutation of β-catenin gene exon 3 was screened by polymerase chain reaction (PCR) and denaturing high-performance liquid chromatography (DHPLC). Results. Cytoplasmic/nuclear expression of β-catenin and reduced membranous expression of E-cadherin were associated, respectively, with the earlier and later stages of sequential colorectal carcinogenesis (p<0.05). The altered distribution of β-catenin was significantly associated with both high Dukes’ stages and poor differentiation of CRC (p<0.05). It also had a parallel relationship with COX-2 overexpression (p<0.05, Spearman's rank analysis), but not with reduced E-cadherin expression. Kaplan-Meier analysis showed a significantly worse survival rate for CRC patients with altered expression of β-catenin (p<0.05, log-rank test). Nevertheless, we failed to find any exon 3 mutation of β-catenin gene in all 60 cases of CRC. Conclusions. Altered distribution of β-catenin occurs in the early stage of colorectal carcinogenesis and has a parallel relationship with COX-2 overexpression. It may serve as a potential marker for the progression and prognosis of CRC. The exon 3 mutation did not appear contributive to the abnormal expression of β-catenin in CRCs in a Chinese population.

MicroRNA profiling in hepatocellular tumors is associated with clinical features and oncogene/tumor suppressor gene mutations

Molecular classifications defining new tumor subtypes have been recently refined with genetic and transcriptomic analyses of benign and malignant hepatocellular tumors. Here, we performed microRNA (miRNA) profiling in two series of fully annotated liver tumors to uncover associations between oncogene/tumor suppressor mutations and clinical and pathological features. Expression levels of 250 miRNAs in 46 benign and malignant hepatocellular tumors were compared to those of 4 normal liver samples with quantitative reverse-transcriptase polymerase chain reaction. miRNAs associated with genetic and clinical characteristics were validated in a second series of 43 liver tumor samples and 16 nontumor samples. miRNA profiling unsupervised analysis classified samples in unique clusters characterized by histological features (tumor/nontumor, P < 0.001; benign/malignant tumors, P < 0.01; inflammatory adenoma and focal nodular hyperplasia, P < 0.01), clinical characteristics [hepatitis B virus (HBV) infection, P < 0.001; alcohol consumption, P < 0.05], and oncogene/tumor suppressor gene mutations [beta-catenin, P < 0.01; hepatocyte nuclear factor 1alpha (HNF1alpha), P < 0.01]. Our study identified and validated miR-224 overexpression in all tumors and miR-200c, miR-200, miR-21, miR-224, miR-10b, and miR-222 specific deregulation in benign or malignant tumors. Moreover, miR-96 was overexpressed in HBV tumors, and miR-126* was down-regulated in alcohol-related hepatocellular carcinoma. Down-regulations of miR-107 and miR-375 were specifically associated with HNF1alpha and beta-catenin gene mutations, respectively. miR-375 expression was highly correlated to that of beta-catenin-targeted genes as miR-107 expression was correlated to that of HNF1alpha in a small interfering RNA cell line model. Thus, this strongly suggests that beta-catenin and HNF1alpha could regulate miR-375 and miR-107 expression levels, respectively. Hepatocellular tumors may have a distinct miRNA expression fingerprint according to malignancy, risk factors, and oncogene/tumor suppressor gene alterations. Dissecting these relationships provides a new hypothesis to understand the functional impact of miRNA deregulation in liver tumorigenesis and the promising use of miRNAs as diagnostic markers.

Multiple Giant Pilomatricoma in Familial Sotos Syndrome

Cerebral giantism or Sotos syndrome consists of a pre- and postnatal overgrowth whose genetic basis are mutations and deletions of the nuclear receptor-binding SET domain containing protein gene. These patients have an increased risk of developing neoplasms, especially in adulthood. We report a 9-year-old boy, diagnosed with familial Sotos syndrome, who had two pilomatrixoma, symmetrically located on both sides of the neck, measuring 4 cm in diameter. Genetic study of the tumor tissue showed deletion of exon 22 of the NSD1 gene, whereas beta-catenin gene mutations were not detected. To the best of our knowledge, presentation of multiple pilomatricomas with Sotos syndrome has never been reported. Therefore their association probably is incidental. Nevertheless, the unusual size of our patient's pilomatricomas could be due to deletion of the NSD1 gene, which characterizes Sotos syndrome.

Loss of cell-adhesion molecule complexes in solid pseudopapillary tumor of pancreas

Solid pseudopapillary tumor of pancreas (SPT) is a rare neoplasm that occurs most often in young females with the two distinct features, the ‘solid-cystic' gross appearance, and the ‘solid-pseudopapillary' microscopic pattern. It has been reported that almost all SPT tumors contain a mutation in the β-catenin gene; however, the histogenetic origin of this tumor remains largely a mystery. E-cadherin is a cell adhesion molecule that links to catenins to form cell adhesion junctions, which is associated with the cytoskeleton formation. In this study, we examined the expression of E-cadherin and β-catenin from SPT in an attempt to determine the molecular basis for the unusual morphology of this tumor. Nine cases of SPT were retrieved from Surgical Pathologic Archives of three institutions, including one male and eight females. H&E slides of each case were reviewed to confirm the diagnosis. The β-catenin gene was sequenced in one case. E-cadherin and β-catenin immunostains, were performed on all nine cases. Sequencing analysis on one case showed a point mutation of the β-catenin gene, confirming previous findings that almost all SPT tumors contain mutation in the β-catenin gene. Immunostains showed that, in both solid and pseudopapillary areas, all the tumor cells lost expression of E-cadherin, and β-catenin nuclear expression was observed in all cases. Our findings suggest that loss of cytoplasmic β-catenin protein in the cell adhesion complex due to β-catenin gene mutation, results in instability of the complex, loss of E-cadherin in cell membrane, and eventually dissociation of the tumor cells to form the pseudopapillary pattern.

Primary Mutation of Exon 3 of β-catenin (CTNNB1) Gene in Colorectal Tumors: Correlation with Mutation of Adenomatous Polyposis Coli Gene, Microsatellite Instability, and Prognostic Significance

Objective: We explore the mutational patterns of the exon 3 of the β-catenin (CTNNB1) gene in colorectal tumors and assess whether primary mutation in this region is associated with mutation of the adenomatous polyposis coli (APC) gene, highfrequency microsatellite instability (MSI-H), and prognostic significance. Methods: The mutational patterns of the exon 3 of the β-catenin gene in 464 colorectal tumors were assessed by direct DNA sequencing of polymerase chain reaction-amplified products. Mutation of APC gene was first analyzed by topographic Denaturing High Performance Liquid Chromatography, followed by direct DNA sequencing and protein truncation test. The subcellular localization of β-catenin was done by immunocytochemistry. MSI-H was defined as the appearance of MSI in at least 2 of the 5 examined chromosomal loci (BAT-25, BAT-26, D5S346, D2S123, D17S250). Results: We found that primary mutation of the β-catenin gene in colorectal tumors was very low (n=12, 2.59%) in incidence. Besides previously reported conserved mutational sites including codon 34,37,41 and 45, two novel mutations in codon 59 (threonine→alanine) and 60 (serine→alanine)were detected. The colorectal tumors with mutation in codon 59 or 60 seem to be similar to those with mutation in codon 34, 37, 41, or 45 in terms of nuclear translocation of β-catenin, mutational status of the APC gene, and other clinicopathologic features. Compared to the control group (n=48), randomly selected from the 452 tumors with wild-type β-catenin, tumors with primary β-catenin mutation tended to present with nuclear accumulation of β-catenin protein (p=0.015). Primary β-catenin mutation was mutually exclusive with the mutation of APC gene in advanced colorectal cancer (p＜0.001). Moreover, the mutational rate of β-catenin was significantly higher in tumors with MSI-H (p＜0.001). Multivariate analysis indicated that MSI-H was a significantly (p=0.045) independent favorable prognostic factor with a relative risk of 0.112 (95% Confidence Interval: 0.013-0.954). However, primary mutation in exon 3 of the β-catenin gene, nuclear β-catenin expression, and APC mutation were without prognostic significance. Conclusions: DNA sequences encoding the regulatory domain of β-catenin protein may extend from codon 29 to codon 60 in the exon 3 of this gene. Primary mutation of exon 3 of the β-catenin gene was very rare in colorectal tumors, mutually exclusive with APC mutation only in advanced disease, and associated with MSI-H.

Effective Treatment of Tumors with Strong β-Catenin/T-Cell Factor Activity by Transcriptionally Targeted Oncolytic Herpes Simplex Virus Vector

The Wnt/beta-catenin/T-cell factor (Tcf) pathway is aberrantly up-regulated in the majority of colorectal cancers (CRC) and hepatoblastomas due to either an APC or beta-catenin gene mutation. We constructed synthetic promoters, T and TE, which contain tandem repeats of a Tcf responsive element without and with the human 4F2 gene intronic enhancer, respectively. Although the T and TE promoters showed higher transcriptional activity than a control promoter in all CRC and hepatoblastoma cell lines tested, with low activities in most other tumor cell lines, the level of transcription varied considerably among the CRC and hepatoblastoma cell lines. In some CRC cell lines, the TE promoter displayed higher levels of transcription than even the human CMV(IE) promoter. In those CRC cells, the APC gene mutations were located within a small segment between the first and second 20-amino-acid repeats in the mutation cluster region of the APC protein. We created a transcriptionally targeted oncolytic herpes simplex virus vector (bM24-TE) in which replication is driven by the TE promoter. This vector efficiently and specifically replicated in and killed tumor cells with strong beta-catenin/Tcf signaling. Intratumoral injection of bM24-TE significantly reduced the growth of highly beta-catenin active SW480 CRC tumors and induced a complete response in half of them, whereas it had no effect on the growth of beta-catenin-inactive A549 tumors. Our results suggest that a transcriptionally regulated oncolytic herpes vector targeting beta-catenin/Tcf signal is very efficacious against CRC tumors carrying an APC gene mutation between the first and second 20-amino-acid repeats.

Overexpression of poly(ADP-ribose) polymerase-1 (PARP-1) in the early stage of colorectal carcinogenesis

Colorectal carcinogenesis is initiated mainly by aberrant activation of the Wnt signaling pathway, caused by mutation of either APC or β-catenin (CTNNB1) gene. Poly(ADP-ribose) polymerase-1 (PARP-1) is a highly conserved nuclear enzyme, which binds tightly to DNA and plays a role in DNA repair, recombination, proliferation and genomic stability. It has recently been shown that PARP-1 is a novel co-activator of TCF-4/β-catenin-evoked gene transactivation and may play a role in colorectal carcinogenesis. The aim of this study was to examine the PARP-1 expression and determine whether it is correlated with the expression of β-catenin and its target genes such as c-myc, cyclin D1 and matrix metalloproteinase (MMP)-7 in the early stage of sporadic colorectal carcinogenesis. Using the semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), 91 colorectal tumours, including 65 adenomas and 26 submucosal (pT1) cancers, were analysed for the expression of PARP-1, β-catenin, c-myc, cyclin D1 and MMP-7. Immunohistochemical analysis of PARP-1 and β-catenin was also performed. PARP-1 mRNA overexpression was detected in 64 (70.3%) of the 91 tumours. PARP-1 overexpression was significantly correlated with tumour size and histopathology. Overexpression of β-catenin, c-myc, cyclin D1 and MMP-7 mRNA expression was observed in 39.6%, 78.0%, 83.5% and 72.5% of the 91 tumours, respectively. PARP-1 overexpression was correlated significantly with overexpression of β-catenin, c-myc, cyclin D1 and MMP-7. Correlation of PARP-1 expression with β-catenin overexpression was also demonstrated by immunohistochemistry. The results suggest that PARP-1, in conjunction with β-catenin, c-myc, cyclin D1 and MMP-7, plays an important role in the early stage of colorectal carcinogenesis.

Identification of genes involved in endometrial cancer etiopathogenesis by the mean of MacroArray technique

20102 Endometrial cancer is one of the most common neoplasms of reproductive system. According to data accumulated in last decades it has been concluded that microsatellite instability and PTEN, K-ras, β-catenin gene mutations are crucial for endometrial cancer etiopathogenesis. Moreover, it is well known that all genes encoded proteins involved in cell cycle regulation, cell differentiation or surrounding tissues infiltration contribute to tumor and metastases development. It has been stressed that different factors such as VEGF, which play a key role in neoplastic angiogenesis are also very important for tumor growth. Complexity of molecular mechanisms leading to endometrial cancer development implies that only experiments utilizing array technique can collect enough data to understand all relationships between different molecular pathways during tumor development and to identify new clinically usable markers of neoplasm. Using MacroArray technique we analyzed expression profile of neoplastic tissue derived from 40 patients diagnosed with endometrial cancer. After data collection all genes present on chip were assigned to different groups according to their role in oncogenesis and artificial neuronal network analysis were performed in order to individuate genes those expression was significantly different in neoplastic tissue regarding to controls. Such procedure allowed us to give shape to preliminary gene expression profile typical for neoplastic tissue and to estimate protein expression of the most significant predictors of neoplastic transformation. Comparison of obtained data with tumor grade can reveal new markers of endometrial cancer useful in routine diagnostic procedures. No significant financial relationships to disclose.

Aberrant Wnt/β-Catenin Signaling in Pancreatic Adenocarcinoma

Wnt/β-catenin signaling plays an important role in normal development. However, its aberrant activation is associated with several cancers. The aim of this study is to examine the Wnt/β-catenin pathway in patients with advanced pancreatic adenocarcinoma (n = 31). Paraffin sections from tumors (n = 16) and normal pancreata (n = 3) were used to determine the localization of β-catenin. An additional 15 frozen tumors, adjacent normal pancreata (n = 5), or normal pancreata (n = 4) were utilized for protein isolation. Tumors were also examined for mutations in exon 3 of the CTNNB1 gene. More than 65% of the tumors showed an increase in total β-catenin, consistent with its enhanced membranous, cytoplasmic, and nuclear localization, but only two showed mutations in CTNNB1. The majority of the remaining tumors demonstrated concurrent increases in Wnt-1 and frizzled-2 (positive regulators) and a decrease in Ser45/Thr41-phospho-β-catenin. Electrophoretic mobility shift assay demonstrated β-cateninT-cell factor binding in tumors only. Adenomatous polyposis coli and axin, which are both negative regulators, remained unchanged. Unexpectedly, total glycogen synthase kinase-3β protein was elevated in these tumors. Elevated levels of E-cadherin were also observed, although E-cadherin-β-catenin association in tumors remained unaffected. Thus, Wnt/β-catenin activation was observed in 65% of pancreatic adenocarcinomas, independently of β-catenin gene mutations in most tumors.

Proto-Oncogenes β- and γ-Catenin in Leukemogenesis in Severe Congenital Neutropenia (CN).

Severe congenital neutropenia (CN) is characterized by a “maturation arrest” of myeloid progenitors at the promyelocytic stage with few or no mature neutrophils in the bone marrow and blood. Administration of granulocyte colony-stimulating factor (G-CSF) increases neutrophil numbers in most CN patients. Approximately 10–15 % of CN patients develop AML or MDS by mechanisms that are as yet unknown. Since AML/MDS are not observed in cyclic (CyN) or idiopathic neutropenia patients treated with G-CSF, an underlying defect of hematopoiesis rather than G-CSF therapy per se predisposes to malignant transformation in CN patients. Recently, activation of Wnt/β-catenin-/γ-catenin-signaling cascade has been considered as important mechanism in the pathogenesis of AML and CML by enhancement of self-renewal activity and by increase of leukemic potential of myeloid progenitors. Moreover, stabilization of β-catenin led to an increased formation of nuclear β-catenin-T-cell factor complexes and altered expression of Wnt-inducible target genes in a variety of human malignancies.In the present study we investigated the role of β-catenin/γ-catenin in leukemogenesis in CN patients. CD33+ progenitors from CN patients expressed 2.5 times higher levels of b-catenin and 4 times higher levels of γ-catenin mRNA and protein, as assessed by quantitative real-time PCR and Western Blot analysis. Most important, in CN patients this increase was paralleled by dramatically elevated levels of activated nuclear β-catenin and intracellular γ-catenin proteins in CD33+ cells, as compared to G-CSF-treated healthy controls and CyN patients. Moreover, mRNA and protein levels of β- and γ-catenins were further increased in CD33+ cells and leukemic blasts from 4 CN patients, who developed AML. In line with high β-/γ-catenins levels, expression of target genes c-jun, fra-1 and PPARD was also up-regulated. There was no correlation between activated Wnt/β-/γ-catenin signaling system and mutations in G-CSF receptor, or ELA2 gene.To investigate the mechanisms of stabilization and increased nuclear translocation of b-catenin, we analyzed the components of b-catenin-degradation multiprotein complex, which contains of Axin, GSK3β, and APC. No differences in expression of Axin, GSK3β and APC as well as in phosphorylation status of GSK3β in CD33+ cells from CN patients and controls were observed. Sequence analysis revealed no mutations in β-catenin gene. Furthermore we analysed the expression of E-cadherin, which forms the transmembrane core of adherent junctions by bridging to β-catenin and therefore modulates its subcellular localization and nuclear translocation. E-cadherin mRNA and protein expression was dramatically downregulated in CD33+ myeloid progenitors from CN patients, in comparison to G-CSF treated healthy controls. Moreover, confocal microscopy revealed very low levels of co-localized E-cadherin and β-catenin in CD33+ cells from CN patients. Therefore, we hypothesize that loss of E-cadherin expression results in nuclear accumulation of β-catenin and activation of its downstream signaling in CN. Taken together, high expression of the proto-oncogenes β- and γ-catenins and nuclear accumulation of β-catenin could contribute to the malignant transformation of myelopoiesis in CN.

Gastric cancer: New genetic developments

Gastric cancer's (GC) incidence shows large geographic differences worldwide with the lowest rates occurring in most Western industrialized countries including the United States and the United Kingdom; in contrast, relatively high rates of GC occur in Japan, Korea, China, and South America, particularly Chile. The Laurén classification system classifies GC under two major histopathological variants: 1) an intestinal type and 2) a diffuse type. The intestinal type is more common in the general population, more likely to be sporadic and related to environmental factors such as diet, particularly salted fish and meat as well as smoked foods, cigarette smoking, and alcohol use. It exhibits components of glandular, solid, or intestinal architecture, as well as tubular structures. On the other hand, the diffuse type is more likely to have a primary genetic etiology, a subset of which, known as hereditary diffuse gastric cancer (HDGC), is due to the E-cadherin (CDH1) germline mutation. The diffuse type pathology is characterized by poorly cohesive clusters of cells which infiltrate the gastric wall, leading to its widespread thickening and rigidity of the gastric wall, known as linitis plastica. Helicobacter pylori infection is associated with risk for both the intestinal and diffuse varieties of gastric cancer. Germline truncating mutations of the CDH1 gene, which codes for the E-cadherin protein, were initially identified in three Maori families from New Zealand that were predisposed to diffuse GC. Since then, similar mutations have been described in more than 40 additional HDGC families of diverse ethnic backgrounds. It is noteworthy that two-thirds of HDGC families reported to date have proved negative for the CDH1 germline mutation. A number of candidate genes have been identified through analysis of the molecular biology of E-cadherin. Patients with evidence of the CDH1 germline mutation in the context of a family history of HDGC must be considered as candidates for prophylactic gastrectomy, given the extreme difficulty in its early diagnosis and its exceedingly poor prognosis when there is regional or distant spread. Specifically, the E-cadherin cytoplasmic tail interacts with catenins, assembling the cell-adhesion complex involved with E-cadherin mediated cell:cell adhesion. Beta-catenin and gamma-catenin compete for the same binding site on the E-cadherin cytoplasmic tail, directly linking the adhesion complex to the cytoskeleton through alpha-catenin. Beta-catenin gene (CTNNB1) mutations have been described predominantly in intestinal-type gastric cancers and CTNNB1 gene amplification and overexpression have recently been described in a mixed-type gastric cancer. This paper reviews the genetics of both intestinal and diffuse types of gastric carcinoma, their differential diagnosis, molecular genetics, pathology, and, when known, their mode of genetic transmission within families.

Immunohistochemical and Sequencing Analyses of the Wnt Signaling Components in Japanese Anaplastic Thyroid Cancers

We investigated the status of the components and target genes of the Wnt signaling pathway in Japanese anaplastic thyroid cancers (ATCs) in the present study. Nuclear and cytoplasmic positive staining of beta-catenin, which might indicate the existence of alterations in the Wnt signaling pathway, were found in 40.9% and 63.6% of the 22 ATC samples, respectively. The beta-catenin, adenomatous polyposis coli (APC) and Axin 1 gene mutations were observed in 4.5%, 9.0%, and 81.8% of the 22 ATC samples, respectively. Overexpression of cyclin D1 and c-myc, which are the target genes of the Wnt signaling pathway, was observed in 27.3% and 59.1% of the ATC samples, respectively. There was no significant correlation between nuclear or cytoplasmic positive staining of beta-catenin and nuclear positive staining of cyclin D1 or c-myc. Taken together, the results of beta-catenin immunohistochemistry suggest that alterations in the Wnt signaling pathway are associated with carcinogenesis of ATC, but the frequency of beta-catenin gene mutation in our series is lower than that previously reported. Furthermore, cyclin D1 and c-myc frequently accumulated in ATC, independently of dysfunction in the Wnt signaling pathway.

Aberrant nuclear/cytoplasmic localization and gene mutation of beta-catenin in classic pulmonary blastoma: beta-catenin immunostaining is useful for distinguishing between classic pulmonary blastoma and a blastomatoid variant of carcinosarcoma.

It is now known that gene mutation of beta-catenin with subsequent nuclear/cytoplasmic (N/C) overaccumulation of the protein plays an important role in tumorigenesis of various organs. We recently demonstrated that low-grade adenocarcinoma of the fetal lung type (L-FLAC)/well-differentiated fetal adenocarcinoma (WDFA), the epithelial prototype of classic pulmonary blastoma (CPB), shows N/C localization of beta-catenin with genetic mutation. This prompted us to further investigate the state of beta-catenin abnormality in CPB and related neoplasms. We studied 9 lung tumors previously diagnosed as biphasic pulmonary blastoma (PB). Histologically, 4 cases (median age 34 years) were CPB with l-FLAC/WDFA as the epithelial component, whereas 5 cases (median age 65 years) were a variant of carcinosarcoma with high-grade FLAC/clear cell adenocarcinoma with fetal lung features as the epithelial component, which we term the blastomatoid variant of carcinosarcoma (BCS). Immunohistochemically, all 4 CPBs showed aberrant N/C localization of beta-catenin both in the epithelial and mesenchymal components, with especially high staining intensity in the budding glands and morules. In contrast, all 5 BCSs showed preserved or diminished membranous expression and no significant N/C expression of beta-catenin in the epithelial component, and absent or focal N/C expression of beta-catenin in the mesenchymal component. Mutational analysis of exon 3 of the beta-catenin gene revealed that 3 CPBs harbored missense mutations (S29F, S37F, and S37F), whereas none of the 5 BCSs had this mutation. This study suggests that beta-catenin gene mutations may play a role in the tumorigenesis of CPB. Although CPB and BCS have often been grouped together as biphasic PB, they are different entities based on immunohistochemical and molecular analysis of beta-catenin. Immunostaining for beta-catenin is useful for the discrimination.