Cytoplasmic Β-catenin Expression Research Articles

β‑catenin expression in endometrioid type endometrial cancer: Expression patterns and impact on disease outcomes.

Determination of nuclear and/or cytoplasmic expression of β-catenin by immunohistochemistry in patients with endometrial cancer (EC) may constitute a potential diagnostic method for identifying patients with a catenin β1 (CTNNB1) gene mutation and those at risk of disease recurrence. The present study aimed to investigate β-catenin expression patterns in hysterectomy specimens of patients with endometrioid type EC using immunohistochemistry, and to examine the prognostic impact of β-catenin. The study was a single-institutional, retrospective cohort trial enrolling consecutive patients with a postoperative histopathological diagnosis of endometrioid EC who underwent hysterectomy between January 2015 and December 2018. Histopathology slides from 75 patients were stained with a monoclonal antibody targeting the β-catenin protein. Any percentage of nuclear staining, whether focal or diffuse, was considered 'β-catenin nuclear-positive'. The cytoplasmic staining reaction of β-catenin was assessed based on the percentage of stained cells and staining intensity. Immune-reactivity score (IRS) values were determined by multiplying the scores for the percentage of staining and staining intensity. IRS values 0 to 2 were regarded as negative expression, 3 to 4 as low expression, 6 to 8 as moderate expression, and 9 to 12 as high expression. Recurrence-free survival (RFS) was used as the prognostic endpoint. Only 2 out of 75 tissue samples (2.7%) exhibited nuclear β-catenin expression, with a low staining percentage of 5%. By contrast, cytoplasmic staining was observed in all samples (100%). According to the IRS findings, 1.3% of the samples exhibited negative cytoplasmic expression, 42.7% low expression, 38.7% moderate expression and 17.3% high expression. Cox regression analysis revealed that staining with β-catenin, either nuclear or cytoplasmic, had no impact on RFS, and stage was the sole independent prognostic factor. In conclusion, based on these results, β-catenin expression in endometrioid EC was revealed to be mostly cytoplasmic, with only 2.7% of tissue samples exhibiting nuclear expression. Overall, β-catenin expression has no impact on RFS.

Histogenetic insights and genetic landscape of fibromatosis-like undifferentiated gastric carcinoma: a focused study

BackgroundThe aim of this study was to elucidate the histogenesis and genetic underpinnings of fibromatosis-like undifferentiated gastric carcinoma (FLUGC), a rare pathological entity.MethodThrough a detailed analysis of seven cases, including histopathological evaluation, CTNNB1 gene mutation screening, human epidermal growth factor receptor 2 (HER2) protein level quantification, and HER2 gene amplification assessment to identify the pathological and molecular characteristics of FLUGC.ResultsOf the seven patients in this study, five were male and two were female (age: 39–73 years). Four patients presented with lesions in the gastric antrum and three had lesions in the lateral curvature of the stomach. Histopathologically, over 90% of the tumor consisted of aggressive fibromatosis-like tissue, including proliferating spindle fibroblasts and myofibroblasts and varying amounts of collagenous fibrous tissues. Undifferentiated cancer cells, accounting for less than 10%, were dispersed among the aggressive fibromatosis-like tissues. These cells were characterized by their small size and were relatively sparse without glandular ducts or nested mass-like structures. Immunophenotyping results showed positive expression of CKpan, CDX2, villin, and p53 in undifferentiated cancer cells; positive expression of vimentin in aggressive fibromatosis-like tissue; positive cytoplasmic expression of β-catenin; and focal cytoplasmic positive expression of smooth muscle actin (SMA). Genetic analysis did not reveal any mutations in the CTNNB1 gene test, nor was there amplification in the HER2 gene fluorescence in situ hybridization (FISH) test. Additionally, the Epstein-Barr encoding region (EBER) of in situ hybridization was negative; and the mismatch repair (MMR) protein was positive. Programmed cell death-1 (PD-1) was < 1–5%; programmed cell death ligand 1 (PD-L1): TPS = 1–4%, CPS = 3–8.ConclusionThe study highlights the significance of CTNNB1, HER2, EBER, and MMR as pivotal genetic markers in FLUGC, underscoring their relevance for diagnosis and clinical management. The rarity and distinct pathological features of FLUGC emphasize the importance of accurate diagnosis to prevent underdiagnosis or misdiagnosis and to raise awareness within the medical community.

Survivin, β-catenin, and ki-67 immunohistochemical expression in canine perivascular wall tumors: Preliminary assessment of prognostic significance.

High survivin expression has been correlated with poor outcomes in several canine tumors but not in soft tissue tumors (STTs). Survivin is a target gene of the Wnt/β-catenin pathway, which is involved in human STT oncogenesis. Immunohistochemistry for survivin, β-catenin, and Ki-67 was performed on 41 canine perivascular wall tumors (cPWTs), and statistical associations of protein expression and histopathologic and clinical variables with clinical outcomes were investigated. Immunohistochemically, there was nuclear positivity (0.9%-12.2% of tumor cells) for survivin in 41/41 (100%), cytoplasmic positivity (0 to > 75% of tumor cells) for survivin in 31/41 (76%), nuclear positivity (2.9%-67.2% of tumor cells) for β-catenin in 24/41 (59%), and cytoplasmic positivity (0% to > 75% of tumor cells) for β-catenin in 23/41 (56%) of cPWTs. All tumors expressed nuclear Ki-67 (2.2%-23.5%). In univariate analysis and multivariate analysis (UA and MA, respectively), every 1% increase of nuclear survivin was associated with an increase of the instantaneous death risk by a factor of 1.15 [hazard ratio (HR) = 1.15; P = .007]. Higher nuclear survivin was associated with grade II/III neoplasms (P = .043). Expression of cytoplasmic survivin, nuclear and cytoplasmic β-catenin, and nuclear Ki-67 were not significantly associated with prognosis in UA nor MA. Tumor size was a significant prognostic factor for local recurrence in UA [subdistribution HR (SDHR) = 1.19; P = .02] and for reduced overall survival time in MA. According to UA and MA, a unitary increase of mitotic count was associated with an increase of the instantaneous death risk by a factor of 1.05 (HR = 1.05; P = .014). Nuclear survivin, mitotic count, and tumor size seem to be potential prognostic factors for cPWTs. In addition, survivin and β-catenin may represent promising therapeutic targets for cPWTs.

Association of the combined parameters including the frequency of primary cilia, PD-L1, Smoothened protein, membranous β-catenin and cytoplasmic β-catenin expression with the outcome of patients with clear cell renal cell carcinoma.

The objective of this study was to investigate the association and combined prognostic significance of the PD-L1, Smoothened protein and β-catenin expressions in patients with clear cell renal cell carcinoma (ccRCC). The PD-L1, Smoothened protein and β-catenin expression were evaluated in 104 ccRCC patients. All studied tumor samples were acquired from nephrectomy specimens of primary tumors and not from biopsies or metastases. An indirect immunohistochemistry using polyclonal rabbit anti-Smoothened antibody, monoclonal mouse anti-human β-catenin-1 antibody, immunohistochemical assay PD-L1 28-8 pharmDx using monoclonal rabbit anti-PD-L1 antibody and anti-VHL (C- terminal) rabbit antibody was used. Immunohistochemistry was scored semiquantitavely. Median overall survival (OS) was significantly better in patients with lower PD-L1 expression (≤5%), Smoothened protein (SMO) expression (<5%) or cytoplasmic β-catenin expression (≤75%) than in patients with higher expressions of these biomarkers (P<0.001, P=0.047, and P<0.001, respectively). Membranous β-catenin showed an opposite effect with its lower expression (≤75%) being associated with longer OS (P=0.020). There was significant association between PD-1 and PD-L1 expression (P=0.007) and significant association of tumor grade (WHO 2016) with membranous β-catenin (P<0.001), cytoplasmic β-catenin (P=0.005), pVHL (P=0.042), PD-L1 (P=0.049) and PD-1 (P=0.028) expression. The present study provides the first data on the potential association and combined prognostic significance of frequency of primary cilia, PD-L1, Smoothened protein and β-catenin expression with the outcome in clear cell renal cell carcinoma.

Favorable prognostic significance of membranous β-catenin expression and negative prognostic significance of cytoplasmic β-catenin expression in pancreatic cancer.

The aim of this study was to investigate the prognostic significance of membranous β-catenin and cytoplasmic β-catenin expression in pancreatic cancer patients (pts). One hundred pts with histologically verified exocrine pancreatic ductal adenocarcinoma were retrospectively studied. The membranous β-catenin, cytoplasmic β-catenin, and cell nucleus β-catenin expression were immunohistochemically evaluated. The expression of membranous β-catenin was <5% in none of the pts, 5-25% in one patient, 26-50% in 2 pts, 51-75% in 14 pts, and >75% in 81 pts. The expression of cytoplasmic β-catenin was <5% in 34 pts, 5-25% in 42 pts, 26-50% in 18 pts, 51-75% in 3 pts, and >75% in one patient. The expression of β-catenin in the cell nucleus was negative in all pts. At the time of the last follow-up, 21 pts were alive and 79 pts had died. Median OS was 1.3 (0.4-2.3) years in pts with membranous β-catenin expression ≤75% and 1.7 (1.3-2.1) years in pts with membranous β-catenin expression >75% (p=0.045). Median OS was (1.3-2.0) 1.6 years in pts with cytoplasmic β-catenin expression ≤25% and 0.9 (0.5-1.2) years in pts with cytoplasmic β-catenin expression >25% (p=0.040). In the univariate Cox proportional hazard models HR (95% CI) was 0.556 (0.311-0.995) in pts with membranous β-catenin expression >75% (p=0.048) and 2.200 (1.216-3.980) in pts with cytoplasmic β-catenin expression >25% (p=0.009). The present results indicate a favorable prognostic significance of membranous β-catenin expression in pancreatic cancer.

Upregulated TC1 and downregulated Chibby were correlated with the aberrant β-catenin expression in laryngeal squamous cell carcinoma.

As an important member of Wnt/β-catenin signaling pathway, the aberrant expression of β-catenin has been implicated in many cancers. Chibby, a β-catenin binding partner, is an antagonist involved in this pathway. In contrast, thyroid cancer 1 (TC1) as an activator of this pathway can relieve the antagonistic activity of Chibby on the β-catenin-mediated transcription and is high expressed in human tumors. The objectives of this study were to examine the expression of TC1, Chibby, and β-catenin and investigate the association among them in laryngeal squamous cell carcinoma (LSCC). The expression of TC1, Chibby, β-catenin, c-Myc, Cyclin D1, and matrix metalloproteinase-7 (MMP-7) were examined by immunohistochemistry in samples from 53 LSCC patients. Compared with normal laryngeal squamous epithelium (NLSE), there were upregulated expression of TC1, downregulated expression of Chibby, and aberrant cytoplasmic expression of β-catenin in the LSCC tissues (P < .001). The high expression of TC1 was correlated with the tumor site, advanced TNM and T stage, lymphovascular invasion, and poor differentiation in LSCC tissues (P < .050). There were correlations between the aberrant expression of β-catenin and the tumor site, advanced TNM and T stage, lymphovascular invasion, perineurial invasion, and poor differentiation in LSCC tissues (P < .050). Upregulated TC1 and downregulated Chibby were correlated with aberrant expression of β-catenin (P < .001), but no correlation between them (P = .076). The percent of abnormal expression of β-catenin in LSCC was 96.00% in TC1+/Chibby-, 73.68% in TC1+/Chibby+, 0.00% in TC1-/Chibby-, and 0.00% in TC1-/Chibby + group (P < .001). High expression of c-Myc, Cyclin D1, and MMP-7 was observed in LSCC tissues (P < .001). There was statistically significant about the expression of Cyclin D1 and MMP-7 among the groups of TC1+/Chibby-, TC1+/Chibby+, TC1-/Chibby-, and TC1-/Chibby + (P < .001), but was not significance about the expression of c-Myc among them (P = .339). No association was found between overall survival and the expression of TC1, Chibby, and β-catenin (P > .05). The upregulated expression of TC1 and downregulated expression of Chibby were correlated with the aberrant expression of β-catenin and the high expression of Cyclin D1 and MMP-7 in LSCC tissues.

Altered E-cadherin/β-catenin expression in feline cutaneous squamous cell carcinomas

Cutaneous squamous cell carcinoma (CSCC) is the most common malignant skin tumour in cats and the nature of the molecular mechanisms involved is poorly defined. Included among the molecular mechanisms in human and canine CSCCs is altered expression of E-cadherin/β-catenin. This study aimed to explore the immunohistochemical expression pattern of E-cadherin and β-catenin in 43 samples of feline CSCC by using a tissue microarray to elucidate whether expression of these molecules is dysregulated. Membrane expression of E-cadherin and membrane and cytoplasmic expression of β-catenin were significantly reduced in the CSCCs. Cytoplasmic expression of E-cadherin and nuclear expression of β-catenin were also found in some CSCCs. These findings indicate that altered expression of E-cadherin and β-catenin is a frequent event in feline CSCCs, suggesting that these molecules play an important role in acquisition of the malignant phenotype in feline patients with CSCC. The results also suggest the existence of a subpopulation of feline patients with CSCC in which the Wnt pathway may contribute to epidermal carcinogenesis.

Vulvo-vaginal epithelial tumors in mares: A preliminary investigation on epithelial-mesenchymal transition and tumor-immune microenvironment.

Vulvo-vaginal epithelial tumors are uncommon in mares, and data on the epithelial-to-mesenchymal transition (EMT) and the tumor-immune microenvironment (TIME) are still lacking. This is a study investigating the equus caballus papillomavirus type 2 (EcPV2) infection state as well as the EMT process and the tumor microenvironment in vulvo-vaginal preneoplastic/ benign (8/22) or malignant (14/22) epithelial lesions in mares. To do this, histopathological, immunohistochemical, transcriptomic, in situ hybridization, and correlation analyses were carried out. Immunohistochemistry quantification showed that cytoplasmic E-cadherin and β-catenin expression as well as nuclear β-catenin expression were features of malignant lesions, while benign/preneoplastic lesions were mainly characterized by membranous E-cadherin and β-catenin expression. Despite this, there were no differences between benign and malignant equine vulvo-vaginal lesions in the expression of downstream genes involved in the canonical and noncanonical wnt/β-catenin pathways. In addition, malignant lesions were characterized by a lower number of cells with cytoplasmic cytokeratin expression as well as a slightly higher cytoplasmic vimentin immunolabeling. The TIME of malignant lesions was characterized by more numerous CD204+ M2-polarized macrophages. Altogether, our results support the hypothesis that some actors in TIME such as CD204+ M2-polarized macrophages may favor the EMT process in equine vulvo-vaginal malignant lesions providing new insights for future investigations in the field of equine EcPV2-induced genital neoplastic lesions.

Lipid nanoparticles containing PlncRNA-1 promote apoptosis of liver cancer cells by down-regulating Wnt/beta-catenin signaling

PlncRNA-1 has an anti-cancer property. Liposome nanoparticles (LNPs) are used as carriers of targeted drugs and contribute to tumor-targeted drug delivery. However, the interaction between LNPs containing PlncRNA-1 and β-catenin, a key component of Wnt signaling pathway, and its role in liver cancer have not yet been confirmed. Human liver cancer cells were exposed to LNPs containing PlncRNA-1 at different concentrations followed by analysis of cell proliferation, cell cycle distribution and apoptosis. Wnt/β-catenin pathway-related gene expression was determined. LNPs containing PlncRNA-1 dose-dependently suppressed the proliferation of liver cancer HepG2 cells with IC50 at 24 h, 48 h and 72 h of 12.8±0.67 μmol/L, 8.8±0.43 μmol/L and 4.6±0.42 μmol/L, respectively. PlncRNA-1-loaded LNPs (5, 10, and 20 μmol/L) dose-dependently arrested HepG2 cells in G2/M phase and induced apoptosis (p &gt; 0.05). Administration of LNPs carrying PlncRNA-1 decreased the expression of overall, cytoplasmic, and nuclear β-catenin, thereby suppressing Wnt/β-catenin pathway. Protein expression levels of GSK-3 (p-tyr216) and Axin-2 were increased, while protein expression levels of Dvl-2, Dvl-3, GSK-3β, c-myc and survivin were significantly decreased. Invasion and metastasis of malignant tumors is an important factor. Inhibiting invasion of cancer cells effectively might improve the prognosis of cancer patients. LNPs carrying PlncRNA-1 promoted cell cycle transition and cell apoptosis by decreasing the expression of Dvl-2, Dvl-3, intracellular β-catenin, cytoplasmic β-catenin, and total β-catenin protein, which may relate to the inhibition of Wnt/β-catenin pathway.

Developmental competence of IVF and SCNT goat embryos is improved by inhibition of canonical WNT signaling.

The specific role of the canonical WNT/β-catenin signaling pathway during the preimplantation development of goat remains unclear. Our objective was to investigate the expression of β-CATENIN, one of the critical components of Wnt signaling pathway, in IVF embryos and compare it with SCNT embryos in goat. In addition, we evaluated the consequence of inhibition of β-catenin using IWR1. Initially, we observed cytoplasmic expression of β-CATENIN in 2 and 8-16 cell stage embryos and membranous expression of β-CATENIN in compact morula and blastocyst stages. Furthermore, while we observed exclusively membranous localization of β-catenin in IVF blastocysts, we observed both membranous and cytoplasmic localization in SCNT blastocysts. We observed that Inhibition of WNT signaling by IWR1 during compact morula to blastocyst transition (from day 4 till day 7 of in vitro culture) increased blastocyst formation rate in both IVF and SCNT embryos. In conclusion, it seems that WNT signaling system has functional role in the preimplantation goat embryos, and inhibition of this pathway during the period of compact morula to blastocyst transition (D4-D7) can improve preimplantation embryonic development.

2-Deoxy-D-glucose simultaneously targets glycolysis and Wnt/β-catenin signaling to inhibit cervical cancer progression.

Cervical cancer is one of the most common female malignant tumors, with typical cancer metabolism characteristics of increased glycolysis flux and lactate accumulation. 2-Deoxy-D-glucose (2-DG) is a glycolysis inhibitor that acts on hexokinase, the first rate-limiting enzyme in the glycolysis pathway. In this research, we demonstrated that 2-DG effectively reduced glycolysis and impaired mitochondrial function in cervical cancer cell lines HeLa and SiHa. Cell function experiments revealed that 2-DG significantly inhibited cell growth, migration, and invasion, and induced G0/G1 phase arrest at non-cytotoxic concentrations. In addition, we found that 2-DG down-regulated Wingless-type (Wnt)/β-catenin signaling. Mechanistically, 2-DG accelerated the degradation of β-catenin protein, which resulted in the decrease of β-catenin expression in both nucleus and cytoplasm. The Wnt agonist lithium chloride and β-catenin overexpression vector could partially reverse the inhibition of malignant phenotype by 2-DG. These data suggested that 2-DG exerted its anti-cancer effects on cervical cancer by co-targeting glycolysis and Wnt/β-catenin signaling. As expected, the combination of 2-DG and Wnt inhibitor synergistically inhibited cell growth. It is noteworthy that, down-regulation of Wnt/β-catenin signaling also inhibited glycolysis, indicating a similar positive feedback regulation between glycolysis and Wnt/β-catenin signaling. In conclusion, we investigated the molecular mechanism by which 2-DG inhibits the progression of cervical cancer in vitro, elucidated the interregulation between glycolysis and Wnt/β-catenin signaling, and preliminarily explored the effect of combined targeting of glycolysis and Wnt/β-catenin signaling on cell proliferation, which provides more possibilities for the formulation of subsequent clinical treatment strategies.

Molecular mechanisms of resistance of endometrial hyperplasia to progestogen therapy based on the study of the expression of estrogen and progesterone receptors and paracrine cellular markers of cellular interaction

The problem of resistance of non atypical endometrial hyperplasia (NEH) to traditionally accepted, pathogenetically sound therapy with different types of progestins is currently an unsolved problem. In about 17-20% of cases there is a recurrence or even progression of atypical forms of endometrial hyperplasia (AEH), which required the use of surgical treatments. The aim of the study was to investigate the results of hormone therapy with different types of progestins for the treatment of endometrial hyperplasia in women with different types of expression of estrogen and progesterone receptors in combination with the expression of intercellular adhesion molecules E-cadherin and β-catenin to determine the cause of hormonal resistance, formation of groups of women with progestogen-sensitive endometrial hyperplasia (EH) non atypical type NEH (+), which can use progestogens for treatment, and progestogen-resistant forms of endometrial hyperplasia with non atypical NEH (-), which should be offered alternative therapy. The study was performed on the morphological material of the endometrium obtained by diagnostic biopsy in women with abnormal uterine bleeding who were diagnosed with NEH by histological examination. For immunohistochemical study, 80 endometrial samples were taken from women with abnormal uterine bleeding (AUВ) and in the same women after treatment of endometrial hyperplasia without atypia in 3 and 6 months of therapy. The control group (CG) consisted of a group of 20 women who were followed-up without treatment tactics. All women were divided into 3 groups in which different types of progestins were used for treatment: group I – continuous intake of 100 mg of micronized progesterone per os twice a day for 6 months, group II – 20 mg of dihydrogesterone per os twice a day for 6 months, group III – in which LNG-IUD was used. The state of proliferation and differentiation in the studied tissues was assessed by the expression of their key molecular participants – estrogen receptors (ERα) and progesterone (PGR), transmembrane glycoproteins of E-cadherin and β-catenin. ERα and PGR expression were determined by immu­nohistochemistry and calculated by the semi-quantitative H-index method. Evaluation of the expression of E-cadherin and β-catenin was performed by determining the percentage of IHH-positive cells to these antigens depending on the degree of their color. The criterion for the effectiveness of NEH treatment was considered to be a biopsy in 3 and 6 months of treatment in the absence of pathological changes in the endometrium. The results showed that after the use of progestogens in group I there was a change in the endometrium to the secretory type in 45% of cases, in group II with dydrogesterone therapy – in 55% there was a reduction of EH to normal histological picture. The intrauterine system with levonorgestrel (LNG-IUD) showed the greatest efficiency, with the use of which in 75% of cases normalization of the endometrial structure was observed. In the control group in 32% of cases the structure of the endometrium normalized. After 6 months of treatment with gestagens, both oral forms (micronized progesterone, dydrogesterone) and the use of LNG-IUDs showed a positive effect from their use; the therapeutic form of gestagens for therapy is not significant in reducing excessive endometrial proliferation. In the control group of patients who did not receive therapy or discontinued therapy for various reasons, it was shown that 47% of patients had spontaneous regression of EH. The overall percentage of no effect from treatment was 20% in groups I, II and III. Determination of ERα expression in all groups showed a pronounced expression in glands and stroma, which did not differ significantly in the group with NEH (+) and in NEH (-). Analysis of PGR expression of NEH in endometrium (-) showed that in glandular cells (50.82±0.73) and in the stroma (47.34±0.82) it was lower than in the endometrium of women with NEH (+) (gland 187±3.1; stroma 166.4±2.3; p&lt;0.05), as well as in the unchanged endometrium in the proliferative phase (glands 193.2±8.5; stroma 178.7±6.3; p&lt;0.05) and the secretory phase (glands 140.2±4.4; stroma 116.6±3.1; p&lt;0.05). A study of E-cadherin expression in women with EH (-) showed that in 86.4% of cases the expression was absent and in 13.6% it decreased. In NEH (+) women, 49.2% of E-cadherin expression was weak, in 34.4% – moderate, and in 16.4% – negative, indicating an association between PGR and E-cadherin expression. In women with NEH (-) there was marked cytoplasmic expression of β-catenin up to 80%, which can be interpreted as potentially threatening the progression of NEH in atypical forms and relapses. Thus, the study of molecular mechanisms of resistance of endometrial hyperplasia to progestogen therapy will help to develop a differential approach to its diagnosis and treatment.

Numerical modelling of WNT/β-catenin signal pathway in characterization of EMT of colorectal carcinoma cell lines after treatment with Pt(IV) complexes

Background and objectiveColorectal cancer (CRC) is at the top of the most common cancer types in the world, with significant mortality rates among both men and women. Deregulation of Wnt/β-catenin pathway and cell-cell junctions’ components, acquisition of invasive phenotype, epithelial-mesenchymal transition (EMT) and invasion are important for development and progression of colorectal cancer. Numerical simulation presents method for estimation of the Wnt pathway via its individual components in cells, thus providing information about EMT, migratory and invasive potential. By using this numerical model, the effectiveness of treatment in EMT suppression can be assessed. Furthermore, the model can be adapted to ``every'' cell type, application time or duration of treatment can be also modified. MethodsWe characterized colorectal cancer (CRC) cell lines (HCT-116, SW-480) from the aspect of EMT, via markers β-catenin and E-cadherin using numerical modeling. To confirm the numerical model, cells were treated with sublethal concentrations of platinum(IV) complexes and their ligands. We confirmed β-catenin regulated expression of mesenchymal markers: N-cadherin, Vimentin and MMP-9, and decreased E-cadherin expression. Treatment-induced changes were determined in the protein expression of tested markers and results showed cell-specific responses. Molecular docking was performed to investigate exact effects of treatments on E-cadherin and β-catenin in cell-cell junctions and individually in tested cells. ResultsThe application of the numerical model via β-catenin and E-cadherin (experimentally measured), is largely valid for the categorization of EMT progression in cells. This numerical modeling better characterizes cells with single cell migration, higher expression of mesenchymal markers, and advanced mesenchymal phenotype like HCT-116 cell line. The model was validated for the treatments and results show HCT-116 cells as more sensitive to applied compounds, among which ligands were more potent in reducing migration and invasiveness. Anti-migratory/invasive effects were due to increased E-cadherin, cytoplasmic β-catenin expression and suppressed mesenchymal markers. In silico methods showed higher affinity of tested chemicals towards free β-catenin, which is the key for regulation of migratory/invasive potential. ConclusionsOur study shows that, no matter individual properties of cell lines and EMT degree, de novo formation of intercellular junctions stands in the basis of anti-migratory/invasive process.

Expression of β-Catenin in Thyroid Neoplasms (Histopathological and Immunohistochemical Study)

Background: Thyroid cancer is the most common malignant tumor of the endocrine system accounting for more than 90% of all endocrine cancer and 63% of all endocrine cancer deaths. β-catenin is a multifunctional protein that plays a key role in Wnt (wingless type) pathway and influences the expression of different genes and their proliferation, thus making it a potential therapeutic target.&#x0D; Aim of Work: This work aimed to examine immunohistochemical expression of β-catenin in different cases of thyroid neoplasms and to correlate between β-catenin expression and clinicopathological features of these thyroid neoplasms.&#x0D; Methods: This retrospective study was conducted on sixty cases of archived, formalin fixed, paraffin embedded tissue blocks that included different histologic types of thyroid neoplasms. Immunohistochemistry using β-catenin monoclonal antibody was performed using a standard avidin-biotin-peroxidase system. β-catenin expression was quantified both at membranous and cytoplasmic level. Immunostaining scores were based on the staining intensity (I) and the percentage of positive cells (P). β-catenin final score (H score) resulted by summation of I and P (ranging from 0 to 7). Cases with H score between 1–3 were considered with low score and cases with H score between 4–7 were considered with high score.&#x0D; Results: Of the malignant thyroid neoplasms in the studied cases, 81% showed positive β-catenin expression with the majority (86%) of the benign thyroid cases showing positive expression. Both membranous and cytoplasmic staining were both assessed in which the majority of the negative and high positive membranous cases also showed the same interpretation for cytoplasmic β-catenin expression. Positive correlations were proved between β-catenin expression of diagnosed malignant cases, (pvalue =0.042) where all hurthle cell, follicular and medullary carcinoma cases, 87.5% of studied papillary carcinoma cases and 50% of poorly differentiated carcinoma cases showed β-catenin positivity while all anaplastic carcinoma cases were negative. Furthermore, statistically significant findings were seen in cases with absence of extrathyroid extension (P value= 0.045) especially those displaying β-Catenin cytoplasmic expression with extrathyroid extension of studied malignant cases showing a P value= (0.011) . No significant correlation was found between β-catenin expression and patients' pathological diagnosis, gender, extent of primary tumor (pT), lymph node metastasis, multifocality and co-existing pathology among studied malignant cases.&#x0D; Conclusion: The present study suggests the prognostic role of β-catenin and its possible usage to identify patients who may benefit from adjuvant β-catenin targeted mono- or combined therapy for tumors expressing this protein, especially for thyroid cases that cannot be removed surgically or that do not respond to traditional treatment options.&#x0D; &#x0D; Key Words: β-catenin, immunohistochemistry, malignant thyroid neoplasms, benign thyroid neoplasms.&#x0D;

β-Catenin marks proliferating endothelial cells in glioblastoma

BackgroundAngiogenesis is a key process in the growth and maintenance of tumors. The Wnt signaling pathway is required for angiogenesis of the central nervous system though development of the blood–brain barrier and subsequent proliferation of endothelial cells during tumor growth. However, the specificity of the Wnt pathway in regulating endothelial cells of different central nervous systems remains to be investigated. Materials & methodsPatient-derived tissue samples from 35 paraffin-embedded tumors were used to assess β-catenin immunoexpression. Tumor samples consisted of the following pathologies: grade II diffuse astrocytoma, glioblastoma, hemangioblastoma, and metastatic adenocarcinoma (lung or breast primary). Average percent reactivity was recorded as a mean observed in ten high-power fields. The following scale was used to grade immunoreactivity: 0 = immunonegative, 1 = 1–25% reactive, 2 = 26–50% reactive, 3 = 51–75% reactive, 4 = 76–100% reactive. ResultsWhile we did not observe nuclear expression of β-catenin in any samples, there was uniform cytoplasmic expression of β-catenin within glial tumor cells. There was a clear distinction in tumor endothelial cells whereby diffuse staining was noted in areas of microvascular hyperplasia in GBM and a less immunoreactive profile in low-grade astrocytomas. By contrast, non-glial tumors, contained very minimal cytoplasmic β-catenin expression in tumor and stromal cells and were devoid of immunoreactivity in endothelial cells. Conclusionβ-catenin is unique marker of proliferating endothelial cells in GBM. Therapies targeting the spatial and structural heterogeneity inherent to GBM may prove to be efficacious and result in an improved survivorship.

BMI1 promotes spermatogonial stem cell maintenance by epigenetically repressing Wnt10b/β-catenin signaling.

The self-renewal of spermatogonial stem cells (SSCs) requires a special microenvironment and is strictly controlled. Previously, we identified BMI1 as a key regulator of spermatogenesis in a knock-out mouse model. However, the mechanisms by which BMI1 regulates SSC maintenance remain largely unknown. Herein, we show that BMI1 is essential for SSC maintenance. BMI1 directs the transcriptional repression of target genes by increasing H2AK119ub and reducing H3K4me3 in SSCs. Furthermore, BMI1 inhibition resulted in the transcriptional activation of Wnt10b and thereby promoted the nuclear translocation of β-catenin in SSCs. Importantly, the suppression of Wnt/β-catenin signaling restored both the cytoplasmic expression of β-catenin and SSC maintenance in BMI1-deficient SSCs. Finally, we demonstrated that Wnt/β-catenin signaling was also involved in BMI1-mediated SSC maintenance in vivo. Altogether, our study not only reveals a novel mechanism for BMI1 in the process of SSC maintenance, but also provides a potential new strategy for treating male infertility.

Altered Wnt5a expression affects radiosensitivity of non-small cell lung cancer via the Wnt/β-catenin pathway.

It has been reported that upregulation of wingless-type protein 5a (Wnt5a) is associated with poor prognosis in patients with non-small cell lung cancer (NSCLC). Wnt5a expression is often upregulated in radiation-resistant NSCLC cells. However, the biological functions or molecular mechanisms of radiosensitivity in NSCLC remain unknown. In the present study, MTT assay and flow cytometric analysis were performed to assess the effect of overexpression or knockdown of Wnt5a and/or radiation on the proliferation and apoptosis of NSCLC cells. Furthermore, western blot analysis was performed to detect canonical Wnt signaling (β-catenin) in H1650 and A549 cells. The results demonstrated that Wnt5a knockdown combined with irradiation inhibited proliferation and induced apoptosis in NSCLC cells compared with Wnt5a knockdown or radiotherapy alone. In addition, the combination of Wnt5a knockdown and irradiation decreased nuclear and increased cytoplasmic β-catenin expression in H1650 and A549 cells, the effects of which were reversed following overexpression of Wnt5a. The combination of overexpressing Wnt5a and irradiation resulted in significant tumor regression, while β-catenin knockdown reversed Wnt5a overexpression-induced NSCLC cell proliferation. Taken together, these results suggest that Wnt5a may be involved in the activation of β-catenin-dependent canonical Wnt signaling, and thus may influence the effectiveness of radiation therapy in NSCLC.

Prognostic Significance of Wnt1, Wnt2, E-Cadherin, and β-catenin Expression in Operable Non-small Cell Lung Cancer.

The role of Wnt family proteins, E-cadherin, and β-catenin in non-small cell lung cancer (NSCLC) is unclear. In this study, we assessed the expression of these proteins as well as their reciprocal interaction and clinical relevance in NSCLC. Immunohistochemical expression of Wnt1, Wnt2, E-cadherin, and β-catenin was assessed in 208 patients with NSCLC who underwent curative pulmonary resection. Expression of Wnt1, Wnt2, and E-cadherin was found in 49.5%, 22.3%, and 37.4% of the patients, respectively, whereas expression of membranous and cytoplasmic β-catenin was found in 23.7% and 34.8% of the patients, respectively. The expression of Wnt1 and E-cadherin was lower in squamous cell carcinoma than in adenocarcinoma and large cell carcinoma, and the expression of both Wnt proteins, E-cadherin, and membranous β-catenin was lower in poorly differentiated compared with well-differentiated tumors. None of the analyzed proteins was associated with relapse-free or overall survival. Expression of Wnt1, Wnt2, E-cadherin, and β-catenin is a common occurrence in NSCLC and is related to tumor histology and grade. However, these proteins have no prognostic role in operable NSCLC.

Wnt Signaling and Cadherin Expressions in Different Staging of Colorectal Cancer as Biomarkers for Metastasis: Study of SW480, COLO 320DM, and HCT116 Cellines

BACKGROUND: Early metastases is still unresolved problem in cancer management, eventually in colorectal cancer (CRC). In addition, many markers are useful just only in the late stage of CRC. AIM: This study evaluates the differences in the expression intensity of nuclear β-catenin, cytoplasmic β-catenin, E-cadherin, and N-cadherin between CRC SW480 cell line as control group and COLO320DM and HCT116 cell lines as case groups. MATERIALS AND METHODS: This study applied experimental research design with the different test methods. Culture growing and subcultures manufacturing for the CRC cell line models were done initially and followed by the immunofluorescence method by administering antibodies on β-catenin, E-cadherin, and N-cadherin, and continued with staining process using fluorescein-5-isothiocyanate and 4’, 6-diamidino-2-phenylindole. Observations were done using an immunofluorescence microscope. Calculation of area density in each cell to perceive the expressions of cytoplasmic and nuclear β-catenin, E-cadherin, and N-cadherin was conducted using ImageJ software, resulted in mean fluorescence intensity. RESULTS: There are significant differences in the expressions of cytoplasmic β-catenin, nuclear β-catenin, E-cadherin, and N-cadherin among SW480, COLO320DM, and HCT116 cell lines (p &lt; 0.05). Despite no significant differences in cytoplasmic and nuclear β-catenin expressions between SW480 and HCT116 cell lines, and in E-cadherin and N-cadherin expressions between COLO320DM and HCT116 cell lines (p &gt; 0.05). SW480 cell line has a higher expression of nuclear β-catenin than the cytoplasm (p &lt; 0.05). CONCLUSION: This study reveals differences in the expression of nucleic and cytoplasmic β-catenin, E-cadherin, and N-cadherin in three stages of CRC (Duke B, C, and D) refer to different activation invasion, migration, and metastatic processes. Furthermore, the high expression of nuclear β-catenin and N-cadherin in the early stage of CRC indicate there is a metastatic process in that stage, so nuclear β-catenin and cadherin can be considered as potential biomarkers in the early stage of this cancer.

SHP2 Potentiates the Oncogenic Activity of β-Catenin to Promote Triple-Negative Breast Cancer

Previous studies have reported dysregulated cytoplasmic and nuclear expression of the β-catenin protein in triple-negative breast cancer (TNBC) in the absence of Wnt signaling pathway dysregulation. However, the mechanism that sustains β-catenin protein dysregulation independent of Wnt signaling is not understood. In this study, we show that Src homology phosphotyrosyl phosphatase 2 (SHP2) is essential for β-catenin protein stability and for sustaining the cytoplasmic and nuclear pools in TNBC cells. The first evidence for this possibility came from immunofluorescence (IF) and immunoblotting (IB) studies that showed that inhibition of SHP2 induces E-cadherin expression and depletion of cytoplasmic and nuclear β-catenin, which in turn confers adherence junction mediated cell-cell adhesion. We further show that SHP2 promotes β-catenin protein stability by mediating the inactivation of GSK3β through its positive effect on Akt and ERK1/2 activation, which was confirmed by direct pharmacologic inhibition of the PI3K-Akt and the MEK-ERK signaling pathway. Finally, we show that SHP2-stabilized β-catenin contributes to TNBC cell growth, transformation, cancer stem cell (CSC) properties, and tumorigenesis and metastasis. Overall, the findings in this report show that SHP2 mediates β-catenin protein stability to promote TNBC. IMPLICATIONS: Data presented in this article demonstrates that SHP2 positively regulates β-catenin protein stability, which in turn promotes triple-negative breast cancer (TNBC) cell transformation, tumorigenesis, and metastasis.