Genetic alterations of Wnt signaling pathway-associated genes in hepatocellular carcinoma

Background and Aim: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Recently, abnormal activation of the Wnt pathway has been found to be involved in the carcinogenesis of HCC. However, the relationship between genetic changes in the Wnt pathway–associated genes and its protein expression has not been studied in patients with HCC and cirrhotic nodules. The purpose of this study is to explore the contribution of inappropriate activation of the Wnt pathway in liver carcinogenesis. Methods: Somatic mutation in exons 3–5 of AXIN1 and exon 3 of β‐catenin were analyzed by direct sequencing and expression of axin and β‐catenin proteins by immunohistochemistry in a series of 36 patients with HCC and cirrhosis. Results: The AXIN1 and β‐catenin gene mutations were observed in 25% (9/36) and 2.8% (1/36) of HCCs, respectively. All mutations detected in AXIN1 and β‐catenin genes were missense point mutations. Abnormal nuclear expression of β‐catenin was observed in 11 of 36 cases of HCCs (30.6%), but not in cirrhotic nodules. Reduced or absent expression of axin was seen in 24 of 36 HCCs (66.7%). The abnormal expression of β‐catenin and axin proteins was closely correlated with mutations of AXIN1 and β‐catenin (P < 0.0001 and P = 0.008, respectively). Conclusions: These data suggest that mutation of AXIN1 gene is a frequent and late event for HCC associated with cirrhosis, and is correlated significantly with abnormal expression of axin and β‐catenin. Therefore, activation of Wnt signaling through AXIN1 rather than β‐catenin mutation might play an important role in liver carcinogenesis.     

Detection of somatic beta-catenin mutations in primary pigmented nodular adrenocortical disease (PPNAD)

Background Primary pigmented nodular adrenocortical disease (PPNAD) leads to Cushing syndrome (CS) and is often associated with Carney complex (CNC). Genetic alterations of the type 1‐α regulatory subunit of cAMP‐dependent protein kinase A (PRKAR1A) and phosphodiesterase 11A4 (PDE11A) genes have been found in PPNAD. Recent studies have demonstrated that β‐catenin mutations are frequent in adrenocortical adenomas and carcinomas and that the Wnt‐signalling pathway is involved in PPNAD tumorigenesis. We hypothesized that adrenocortical adenomas that form in the context of PPNAD may harbour β‐catenin mutations. Methods We studied 18 patients with CS secondary to PPNAD who were screened for germline PRKAR1A and PDE11A mutations. Tumor DNA was extracted from pigmented adrenocortical adenoma and nodular adrenal hyperplasia. Mutation analysis of exons 3 and 5 of β‐catenin was performed using polymerase chain reaction and direct sequencing. Sections from formalin‐fixed, paraffin‐embedded tumour samples were studied by immunohistochemistry with an antibody against β‐catenin. Results Nine patients were carrying germline PRKAR1A mutations and one patient had a PDE11A mutation. We found somatic β‐catenin mutations in 2 of 18 patients (11%). In both cases, the mutations occurred in relatively large adenomas that had formed in the background of PPNAD. Tumor DNA analysis revealed a heterozygous ACC‐to‐GCC missense mutation in codon 41 (T41A) and a TCT‐to‐CCT missense mutation in codon 45 (S45P) of exon 3 of the β‐catenin gene that was confirmed at the cDNA level. There were no alterations in the DNA of PPNAD‐adjacent tissues and lymphocytes from the patients, indicating somatic events. Immunohistochemistry showed nuclear accumulation of β‐catenin in more than 90% of cells in adenomatous tissue whereas no nuclear immunoreactivity was detected in adjacent PPNAD nodular cells. Nuclear translocation of β‐catenin protein in the PPNAD adenoma suggests activation of the Wnt–β‐catenin pathway in PPNAD. Conclusions We report, for the first time, β‐catenin mutations in adenomas associated with PPNAD, further implicating Wnt–β‐catenin signalling in tumorigenesis linked to bilateral adrenal hyperplasias.     

Expression of klotho and β‐catenin in esophageal squamous cell carcinoma,and their clinicopathological and prognostic significance

Esophageal carcinoma is one of the most common types of cancers in the world; the molecular mechanism underlying its tumorigenesis is still not well understood. This study was aimed at investigating the expression of klotho and β‐catenin in patients with esophageal squamous cell carcinoma (ESCC) and analyzing their association with clinicopathological variables and their effects on prognosis. The expression patterns of klotho and β‐catenin were determined by tissue microarray and immunohistochemical technique in ESCC and normal tissues, and their correlations with clinicopathological characteristics were investigated using univariate and multivariate analysis. The serum klotho levels in 40 ESCC patients and controls were measured by sandwich enzyme‐linked immunosorbent assay system (ELISA). The expression level of klotho was significantly lower in ESCC than in the adjacent noncancerous tissues (30 vs. 50%, P < 0.000), and the protein level was negative correlated with clinical staging, histological grade, lymph node metastasis, and invasion depth (P < 0.05). Whereas, the expression of β‐catenin was much higher in ESCC than their corresponding normal mucosa tissues (78.3 vs. 11.5%, P < 0.000), and the level of protein correlated only with histological grade and invasion depth (P < 0.05). Correlation analysis showed the expression level of klotho inversely correlated with that of β‐catenin (r = ?0.214, P < 0.01). Patients with klotho‐positive tumors had longer survival than those with klotho‐negative tumors (P < 0.01). Cox proportional hazards model analysis demonstrated that positive expression of klotho was an important factor indicating good prognosis (hazard ratio, 0.371; 95% confidence interval, 0.201–0.685; P < 0.01). ELISA showed that the level of serum klotho was markedly higher (461.50 ± 43.30 pg/mL) than control group (239.37 ± 20.65 pg/mL) (P < 0.001). Receiver operating characteristic analysis gave a cut‐off value of 327.031 of serum klotho with a sensitivity of 81.3% and specificity of 81.2% (P < 0.000). Our present study demonstrated for the first time that klotho might be a novel biomarker candidate for predicting progression and prognosis in patients with ESCC.     

Frequent mutations of beta-catenin gene in sporadic secreting adrenocortical adenomas

Objective Molecular alterations remain largely unknown in most sporadic adrenocortical tumours and hyperplasias. In our previous work, we demonstrated the differential expression of several Wnt/β‐catenin signalling‐related genes implicated in ACTH‐independent macronodular adrenal hyperplasias (AIMAH). To better understand the role of Wnt/β‐catenin signalling in adrenocortical tumours, we performed mutational analysis of the β‐catenin gene. Methods We studied 53 human adrenocortical samples (33 adenomas, 4 carcinomas, 13 AIMAH, 3 ACTH‐dependent adrenal hyperplasias) and the human adrenocortical cancer cell line NCI‐H295R. All samples were screened for somatic mutations in exons 3 and 5 of the β‐catenin gene. Eleven and six samples were analysed for β‐catenin protein expression by Western blotting and immunohistochemistry, respectively. Results No mutations were detected in adrenocortical carcinomas, AIMAH and ACTH‐dependent hyperplasias. Genetic alterations were found in 5 (15%) out of 33 adenomas: three cortisol‐secreting adenomas, one aldosterone‐secreting adenoma and one nonfunctional adenoma. Two‐point mutations occurred at serine residues of codons 37 and 45 (S37C and S45F). The remaining three tumours contained deletions of 6, 55 and 271 bp. H295R cells carry an activating S45P mutation. Western blot analysis of samples with 55‐ and 271‐bp deletions showed an additional shorter and more intense band representing an accumulation of the mutated form of β‐catenin protein. In addition, cytoplasmic and/or nuclear accumulation of β‐catenin was observed in mutated adenomas by immunohistochemistry. Conclusions Activating mutations of exon 3 of the β‐catenin gene are frequent in adrenocortical adenomas, and further characterization of the Wnt/β‐catenin signalling pathway should lead to a better understanding of adrenal tumourigenesis.     

Wnt/β‐catenin signaling is hyperactivated in systemic sclerosis and induces Smad‐dependent fibrotic responses in mesenchymal cells

Objective

Fibrosis in human diseases and animal models is associated with aberrant Wnt/β‐catenin pathway activation. The aim of this study was to characterize the regulation, activity, mechanism of action, and significance of Wnt/β‐catenin signaling in the context of systemic sclerosis (SSc).

Methods

The expression of Wnt signaling pathway components in SSc skin biopsy specimens was analyzed. The regulation of profibrotic responses by canonical Wnt/β‐catenin was examined in explanted human mesenchymal cells. Fibrotic responses were studied using proliferation, migration, and gel contraction assays. The cell fate specification of subcutaneous preadipocytes by canonical Wnt signaling was evaluated.

Results

Analysis of published genome‐wide expression data revealed elevated expression of the Wnt receptor FZD2 and the Wnt target LEF1 and decreased expression of Wnt antagonists DKK2 and WIF1 in skin biopsy specimens from subsets of patients with diffuse cutaneous SSc compared to the other distinct subsets. Immunohistochemical analysis showed increased nuclear β‐catenin expression in these biopsy specimens. In vitro, Wnt‐3a induced β‐catenin activation, stimulated fibroblast proliferation and migration, collagen gel contraction, and myofibroblast differentiation, and enhanced profibrotic gene expression. Genetic and pharmacologic approaches were used to demonstrate that these profibrotic responses involved autocrine transforming growth factor β signaling via Smads. In contrast, in explanted subcutaneous preadipocytes, Wnt‐3a repressed adipogenesis and promoted myofibroblast differentiation.

Conclusion

Canonical Wnt signaling was hyperactivated in SSc skin biopsy specimens. In explanted mesenchymal cells, Wnt‐3a stimulated fibrogenic responses while suppressing adipogenesis. Taken together, these results indicate that Wnts have potent profibrotic effects, and that canonical Wnt signaling plays an important role in the pathogenesis of fibrosis and lipoatrophy in SSc.

     

β‐catenin regulates parathyroid hormone/parathyroid hormone–related protein receptor signals and chondrocyte hypertrophy through binding to the intracellular C‐terminal region of the receptor

Objective

To investigate the underlying mechanisms of action and functional relevance of β‐catenin in chondrocytes, by examining the role of β‐catenin as a novel protein that interacts with the intracellular C‐terminal portion of the parathyroid hormone (PTH)/PTH‐related protein (PTHrP) receptor type 1 (PTHR‐1).

Methods

The β‐catenin–PTHR‐1 binding region was determined with deletion and mutagenesis analyses of the PTHR1 C‐terminus, using a mammalian two‐hybrid assay. Physical interactions between these 2 molecules were examined with an in situ proximity ligation assay and immunostaining. To assess the effects of gain‐ and loss‐of‐function of β‐catenin, transfection experiments were performed to induce overexpression of the constitutively active form of β‐catenin (ca‐β‐catenin) and to block β‐catenin activity with small interfering RNA, in cells cotransfected with either wild‐type PTHR1 or mutant forms (lacking binding to β‐catenin). Activation of the G protein α subunits G_αs and G_αq in the cells was determined by measurement of the intracellular cAMP accumulation and intracellular Ca²⁺ concentration, while activation of canonical Wnt pathways was assessed using a TOPflash reporter assay.

Results

In differentiated chondrocytes, β‐catenin physically interacted and colocalized with the cell membrane–specific region of PTHR‐1 (584–589). Binding of β‐catenin to PTHR‐1 caused suppression of the G_αs/cAMP pathway and enhancement of the G_αq/Ca²⁺ pathway, without affecting the canonical Wnt pathway. Inhibition of Col10a1 messenger RNA (mRNA) expression by PTH was restored by overexpression of ca‐β‐catenin, even after blockade of the canonical Wnt pathway, and Col10a1 mRNA expression was further decreased by knockout of β‐catenin (via the Cre recombinase) in chondrocytes from β‐catenin–floxed mice. Mutagenesis analyses to block the binding of β‐catenin to PTHR1 caused an inhibition of chondrocyte hypertrophy markers.

Conclusion

β‐catenin binds to the PTHR‐1 C‐tail and switches the downstream signaling pathway from G_αs/cAMP to G_αq/Ca²⁺, which is a possible mechanism by which chondrocyte hypertrophy may be regulated through the PTH/PTHrP signal independent of the canonical Wnt pathway.

     

Upregulation of beta-catenin levels in superior frontal cortex of chronic alcoholics

Background: Chronic and excessive alcohol misuse results in neuroadaptive changes in the brain. The complex nature of behavioral, psychological, emotional, and neuropathological characteristics associated with alcoholism is likely a reflection of the network of proteins that are affected by alcohol‐induced gene expression patterns in specific brain regions. At the molecular level, however, knowledge remains limited regarding alterations in protein expression levels affected by chronic alcohol abuse. Thus, novel techniques that allow a comprehensive assessment of this complexity will enable the simultaneous assessment of changes across a group of proteins in the relevant neural circuitry. Methods: A proteomics analysis was performed using antibody microarrays to determine differential protein levels in superior frontal cortices between chronic alcoholics and age‐ and gender‐matched control subjects. Seventeen proteins related to the catenin signaling pathway were analyzed, including α‐, β‐, and δ‐catenins, their upstream activators cadherin‐3 (type I cadherin) and cadherin‐5 (type II cadherin), and 5 cytoplasmic regulators c‐Src, CK1ε, GSK‐3β, PP2A‐Cα, and APC, as well as the nuclear complex partner of β‐catenin CBP and 2 downstream genes Myc and cyclin D1. ILK, G_α1, G_β1, and G_β2, which are activity regulators of GSK‐3β, were also analyzed. Results: Both α‐ and β‐catenin showed significantly increased levels, while δ‐catenin did not change significantly, in chronic alcoholics. In addition, the level of the β‐catenin downstream gene product Myc was significantly increased. Average levels of the catenin regulators c‐Src, CK1ε, and APC were also increased in chronic alcoholics, but the changes were not statistically significant. Conclusion: Chronic and excessive alcohol consumption leads to an upregulation of α‐ and β‐catenin levels, which in turn increase downstream gene expressions such as Myc that is controlled by β‐catenin signaling. This study showed that the β‐catenin signal transduction pathway was upregulated by chronic alcohol abuse, and prompts further investigation of mechanisms underlying the upregulation of α‐ and β‐catenins in alcoholism, which may have considerable pathogenic and therapeutic relevance.     

Prognostic significance of β‐catenin and topoisomerase IIα in de novo acute myeloid leukemia

The Wnt/β‐catenin signaling is important for controlling self‐renewal of hematopoietic stem cells and its constitutive activation has recently been documented in a significant proportion of acute myeloid leukemia (AML) cases. Topoisomerase IIα (Topo IIα) is a marker of cell proliferation and a crucial target for anthracycline cytotoxicity, the mainstay of management employed in AML. We retrospectively investigated the prognostic roles of β‐catenin and topo IIα in a cohort of 59 patients with newly diagnosed AML by immunohistochemistry. Aberrant β‐catenin expression was demonstrated in 13 patients (22%), and it was more likely to occur in those with unfavorable karyotypes. Advanced age and poor performance status adversely influenced the achievement of complete remission, while neither aberrant β‐catenin expression nor enhanced topo IIα activity did. On multivariate survival analysis, four factors independently predicted a shortened overall survival: aberrant β‐catenin expression, high topo IIα activity, poor‐risk cytogenetics, and presence of at least one comorbidity factor. Our results suggest that both β‐catenin and topo IIα independently predicted an adverse prognosis and might serve as new markers for risk stratification in AML patients. Am. J. Hematol., 2009. © 2008 Wiley‐Liss, Inc.     

Effect of mutant β‐catenin on liver growth homeostasis and hepatocarcinogenesis in transgenic mice

Background: Mutations in the Wnt signalling pathway molecule β‐catenin are associated with liver cancer. Aims: Our aim was to confirm the effects of stabilized β‐catenin on liver growth, identify whether those effects were reversible and cell autonomous or non‐cell autonomous and to model β‐catenin‐induced liver cancer in mice. Methods: Using a liver‐specific inducible promoter, we generated transgenic mice in which the expression of mutant β‐catenin can be induced or repressed within hepatocytes in mice of different ages. Results: Similar to other models, the hepatic expression of mutant β‐catenin in our model beginning in utero or induced in quiescent adult liver resulted in a two‐fold liver enlargement and development of disease with a latency of 1–5 months, and mice displayed elevated blood ammonia and altered hepatic gene expression. Our model additionally allowed us to discover that molecular and phenotypic abnormalities were reversible following the inhibition of transgene expression. Hepatocyte transplant studies indicated that mutant β‐catenin could not increase the growth of transgene‐expressing foci in either growth‐permissive or ‐restrictive hepatic environments, but still directly altered hepatocyte gene expression. Mice with continuous but focal transgene expression developed hepatic neoplasms after the age of 1 year. Conclusions: Our findings indicate that hepatocyte gene expression is directly affected by mutant β‐catenin in a cell autonomous manner. However, hepatomegaly associated with diffuse hepatocyte‐specific expression of mutant β‐catenin is secondary to liver functional alteration or non‐cell autonomous. Both phenotypes are reversible. Nevertheless, some foci of transgene‐expressing cells progressed to carcinoma, confirming the association of mutant β‐catenin with liver cancer.     

Wnt inhibition leads to improved chemosensitivity in paediatric acute lymphoblastic leukaemia

While childhood acute lymphoblastic leukaemia (ALL) is now highly curable, the dismal prognosis for children who relapse warrants novel therapeutic approaches. Previously, using an integrated genomic analysis of matched diagnosis‐relapse paired samples, we identified overactivation of the Wnt pathway as a possible mechanism of recurrence. To validate these findings and document whether Wnt inhibition may sensitize cells to chemotherapy, we analysed the expression of activated β‐catenin (and its downstream target BIRC5) using multiparameter phosphoflow cytometry and tested the efficacy of a recently developed Wnt inhibitor, iCRT14, in ALL cell lines and patient samples. We observed increased activation of β‐catenin at relapse in 6/10 patients. Furthermore, treatment of leukaemic cell lines with iCRT14 led to significant downregulation of Wnt target genes and combination with traditional chemotherapeutic drugs resulted in a synergistic decrease in viability as well as a significant increase in apoptotic cell death. Finally, pre‐treatment of purified blasts from patients with relapsed leukaemia with the Wnt inhibitor followed by exposure to prednisolone, restored chemosensitivity in these cells. Our results demonstrate that overactivation of the Wnt pathway may contribute to chemoresistance in relapsed childhood ALL and that Wnt‐inhibition may be a promising therapeutic approach.     

Evidence for non‐functional Dickkopf‐1 (DKK‐1) signaling in chronic lymphocytic leukemia (CLL)

Purpose: Wnt signaling was demonstrated to be activated in chronic lymphocytic leukemia (CLL). It is thought to be responsible for the extended survival of CLL cells in vivo. Dickkopf1 (DKK1) is known to antagonize Wnt signaling by direct high‐affinity binding to the extracellular domain of WNT coreceptor lipoprotein receptor‐related protein 6 (LRP6). The purpose of this study was to investigate the effect of DKK1 in B‐CLL cells in vitro. Methods: Expression of DKK1 was estimated by Western blot and real‐time PCR. B cells from patients with CLL and healthy donors were incubated with recombinant DKK1. Survival was measured by flow cytometry. Primers for real‐time PCR were designed for extracellular domain of LRP6, responsible for DKK1 binding, and the intracellular region, essential for inhibiting GSK3 β. Results: Healthy and CLL cells express equivalent mRNA levels of DKK1 and LRP6. After treatment of CLL cells with recombinant DKK1 (1 μg/mL) for 3 h, there was no change in the levels of phosphorylated β‐catenin and total β‐catenin. Healthy B cells proved to have significantly higher levels of extracellular, DKK1 binding domain of LRP6. We estimated that in CLL cells every 6th LRP6 receptor is lacking the extracellular domain. Discussion: For the first time we show the expression of DKK1 in CLL cells. Unlike in similar tumors, the addition of DKK1 to culture of CLL cells does not inactivate WNT pathway. The reason for this could be the absence of the binding domain of LRP6. On the other hand, a truncated LRP6 without extracellular DKK1 binding domain could lead to an uncontrollable activation of WNT signaling.     

MTNR1B loss promotes chordoma recurrence by abrogating melatonin‐mediated β‐catenin signaling repression

Chordoma is an extremely rare malignant bone tumor with a high rate of relapse. While cancer stem cells (CSCs) are closely associated with tumor recurrence, which depend on its capacity to self‐renew and induce chemo‐/radioresistance, whether and how CSCs participate in chordoma recurrence remains unclear. The current study found that tumor cells in recurrent chordoma displayed more dedifferentiated CSC‐like properties than those in corresponding primary tumor tissues. Meanwhile, MTNR1B deletion along with melatonin receptor 1B (MTNR1B) down‐regulation was observed in recurrent chordoma. Further investigation revealed that activation of Gαi2 by MTNR1B upon melatonin stimulation could inhibit SRC kinase activity via recruiting CSK and SRC, increasing SRC Y530 phosphorylation, and decreasing SRC Y419 phosphorylation. This subsequently suppressed β‐catenin signaling and stemness via decreasing β‐catenin p‐Y86/Y333/Y654. However, MTNR1B loss in chordoma mediated increased CSC properties, chemoresistance, and tumor progression by releasing melatonin's repression of β‐catenin signaling. Clinically, MTNR1B deletion was found to correlate with patients’ survival. Together, our study establishes a novel convergence between melatonin and β‐catenin signaling pathways and reveals the significance of this cross talk in chordoma recurrence. Besides, we propose that MTNR1B is a potential biomarker for prediction of chordoma prognosis and selection of treatment options, and chordoma patients might benefit from targeting MTNR1B/Gαi2/SRC/β‐catenin axis.