Decreased expression of WNT2 in villi of unexplained recurrent spontaneous abortion patients may cause trophoblast cell dysfunction via downregulated Wnt/ß-catenin signaling pathway.

WNT2 has been reported to be important for placental development, especially for the proper vascularization of the placenta. However, its precise role in first-trimester trophoblast cells is still unknown. WNT2 expression in the villous tissues of unexplained recurrent spontaneous abortion (URSA) patients was compared with that of healthy women by Western blot. The function of WNT2 in HTR-8/SVneo trophoblast cells was evaluated by altering the cellular WNT2 level through overexpression and shRNA knockdown. The molecular mechanism of the effect of WNT2 on trophoblast cells was investigated. The association of WNT2 with the Wnt/ß-catenin signaling pathway was studied through Western blot and immunofluorescence. Results showed that WNT2 protein expression was significantly decreased in villi of the URSA group compared with the control group. In vitro studies showed that WNT2 could promote human trophoblast cell proliferation and migration through activating the Wnt/ß-catenin signaling pathway. Moreover, upon the knockdown of WNT2, trophoblast cell proliferation and migration were significantly suppressed. In conclusion, our study indicated that WNT2 plays an important role in trophoblast function. WNT2 insufficiency might cause impaired trophoblast cell proliferation and migration via downregulation of Wnt/ß-catenin signaling pathway.

Wnt/ß-catenin pathway is regulated by PITX2 homeodomain protein and thus contributes to the proliferation of human ovarian adenocarcinoma cell, SKOV-3.

Pituitary homeobox-2 (PITX2) plays a substantial role in the development of pituitary, heart, and brain. Although the role of PITX2 isoforms in embryonic development has been extensively studied, its possible involvement in regulating the Wnt signaling pathway has not been reported. Because the Wnt pathway is strongly involved in ovarian development and cancer, we focused on the possible association between PITX2 and Wnt pathway in ovarian carcinoma cells. Remarkably, we found that PITX2 interacts and regulates WNT2/5A/9A/6/2B genes of the canonical, noncanonical, or other pathways in the human ovarian cancer cell SKOV-3. Chromatin immunoprecipitation and promoter-reporter assays further indicated the significant association of PITX2 with WNT2 and WNT5A promoters. Detailed study further reveals that the PITX2 isoform specifically activates the canonical Wnt signaling pathway either directly or through Wnt ligands. Thus, the activated Wnt pathway subsequently enhances cell proliferation. Moreover, we found the activation of Wnt pathway reduces the expression of different FZD receptors that limit further Wnt activation, demonstrating the existence of an auto-regulatory feedback loop. In contrast, PITX2 could not activate the noncanonical pathway as the Wnt5A-specific ROR2 receptor does not express in SKOV-3 cells. Collectively, our findings demonstrated that, despite being a target of the canonical Wnt signaling pathway, PITX2 itself induces the same, thus leading to the activation of the cell cycle regulating genes as well as the proliferation of SKOV-3 cells. Collectively, we highlighted that the PITX2 and Wnt pathway exerts a positive feedback regulation, whereas frizzled receptors generate a negative feedback in this pathway. Our findings will help to understand the molecular mechanism of proliferation in ovarian cancer cells.

Cancer-associated fibroblasts secrete Wnt2 to promote cancer progression in colorectal cancer.

Recent studies have shown that the tumor microenvironment plays a significant role in the progression of solid tumors. As an abundant component of the tumor microenvironment, cancer-associated fibroblasts (CAFs) have been shown to promote tumorigenesis and cancer aggressiveness, but their molecular characteristics remain poorly understood. In the present study, paired CAFs and normal fibroblasts (NFs) were isolated from five colorectal cancer (CRC) tissues from patients who underwent surgical resection. The gene expression profiles of CAFs and NFs identified by RNA sequencing were compared to understand the complex role of CAFs in cancer progression. Gene Set Enrichment Analysis revealed that the gene sets related to the Wnt signaling pathway were highly enriched in CAFs, as well as TGFß signaling, which is considered to be a regulator of CAFs. Among the components of this pathway, Wnt2 was specifically expressed. The observations led us to speculate that Wnt2 is extremely involved in regulating CRC progression by CAFs. Thus, we performed immunohistochemical analysis on Wnt2 in 171 patients who underwent surgery for colorectal adenocarcinoma. Positive staining for Wnt2 was mainly observed in cancer stroma, although the immunoreactivity was weak in cancer cells. Wnt2 expression in CAFs was significantly correlated with depth of tumor (P < .001), lymph node metastasis (P = .044), TNM stage (P = .010), venous invasion (P < .001), and recurrence (P = .013). Subsequent in vitro analyses were conducted using conditioned medium (CM) from immortalized CAFs transfected with siRNA targeting Wnt2. As a result, cancer cell invasion and migration were significantly decreased in the CM from immortalized CAFs transfected with siRNA targeting Wnt2. Our findings indicated that Wnt2 protein released from CAFs enhances CRC cell invasion and migration. In conclusion, Wnt2 secreted by CAFs plays a key role in cancer progression and is a potential therapeutic target for CRC.

Wnt2 knock down by RNAi inhibits the proliferation of in vitro-cultured human keloid fibroblasts.

To study the effect of knocking down wingless-related MMTV integration site 2 (Wnt2) expression by RNAi on the growth and signaling pathways of ex vitro-cultured keloid fibroblasts (KFB).Human KFB were isolated from 10 keloid patient specimens. The KFB cells were then transfected with 4 pairs of small interfering RNA (siRNA) targeting human Wnt2, respectively. Reverse transcriptase-polymerase chain reaction and Western blot analysis were conducted to verify the knock down of Wnt2, and the expression of ß-catenin glycogen synthase kinase-3ß (GSK-3ß) and cyclin D1 were examined.siRNA Wnt2 transfection (siWnt2) resulted in the significant inhibition of Wnt2 expression at both the mRNA and protein levels. The expression of ß-catenin, GSK-3ß, p-GSK-3ß, and cyclin D1 at the protein level also decreased in siWnt2 cells. siWnt2 resulted in a substantially slower growth and significant delay in cell doubling time of the KFB cells compared with control groups. Further, the siRNA knock down of GSK-3ß and ß-catenin resulted in slower proliferation rates, respectively.Wnt2 siRNA has an inhibitive effect on keloid fibroblast proliferation, which may be a potential therapeutic approach for keloid and other human fibrotic diseases.

Alterations of the Wnt signaling pathway during the neoplastic progression of Barrett's esophagus.

Aberrant activation of the Wnt signaling pathway has been reported during neoplastic progression in Barrett's esophagus (BE). However, mutations in APC and CTNNB1 genes were rarely observed. In this study, expression pattern of Wnt ligands, Frizzled receptors and APC, as well as the methylation status of the APC, SFRP1 and SFRP2 promoter genes were investigated in normal esophageal mucosa and in preneoplastic and neoplastic lesions of BE patients. Promoter methylation of APC was found in all BE samples and in 95% of esophageal adenocarcinomas (EAC). Full methylation of APC correlated with lack of expression. In EAC, nuclear translocation of beta-catenin was observed regardless of the expression of APC. WNT2 expression was higher in dysplasia and EAC than in BE, with 20/26 (77%) of the EAC showing high expression of WNT2. SFRP1 methylation occurred in all BE samples and in 96% of EAC, while SFRP2 was methylated in 73% of the normal squamous esophageal mucosa samples. In conclusion, (1) alterations of key regulators of the Wnt signaling are frequent in the pathogenesis of BE; (2) the APC and SFRP1 genes are inactivated by promoter methylation in BE; (3) the WNT2 gene is upregulated along the progression from low-grade dysplasia to EAC.

Twist relates to tubular epithelial-mesenchymal transition and interstitial fibrogenesis in the obstructed kidney.

Epithelial-mesenchymal transition (EMT) is a critical step in renal fibrosis. It has been recently reported that a transcription factor, Twist, plays a pivotal role in metastasis of breast tumors by inducing EMT. In this study, we examined whether Twist relates to renal fibrogenesis including EMT of tubular epithelia, evaluating Twist expression level in the unilateral ureteral obstruction (UUO) model. Kidneys of mice subjected to UUO were harvested 1, 3, 7, and 10 days after obstruction. Compared with control kidneys, Twist mRNA-level significantly increased 3 days after UUO (UUO day 3 kidney) and further augmented until 10 days after UUO. Twist expression increased in tubular epithelia of the dilated tubules and the expanded interstitial areas of UUO kidneys, where cell-proliferating appearances were frequently found in a time-dependent manner. Although a part of tubular cells in whole nephron segment were immunopositive for Twist in UUO day 7 kidneys, tubular epithelia downstream of nephron more frequently expressed Twist than upstream of nephron. In UUO day 7 kidneys, some tubular epithelia were confirmed to coexpress Twist and fibroblast-specific protein-1, a marker for EMT, indicating that Twist is involved in tubular EMT under pathological state. Twist was expressed also in a number of alpha-smooth muscle actin-positive myofibroblasts located in the expanded interstitial area of UUO kidneys. From these findings, the present investigation suggests that Twist is associated with tubular EMT, proliferation of myofibroblasts, and subsequent renal fibrosis in obstructed kidneys.

Infrequent COX-2 expression due to promoter hypermethylation in gastric cancers in Dalian, China.

Cyclooxygenase-2 (COX-2) has been shown to play oncogenic roles during stepwise gastrocarcinogenesis, and its expression is correlated with Helicobacter pylori infection, tumor necrosis factor alpha-mediated nuclear factor (NF)-kappaB activation, and Wnt signaling. To examine COX-2 expression and the status of its regulatory factors, we examined 49 gastric cancers (GCs), 21 premalignant tissues, and 10 noncancerous gastric mucosa from residents of Dalian, China. Unexpectedly, it was found that COX-2 expression was infrequent in the gastric samples (18.8%, 15/80) regardless of the type of lesion or morphological phenotype. H pylori infection was detected in 19 of 35 tested GC cases. Tumor necrosis factor alpha expression, NF-kappaB nuclear translocation, or Wnt2 overexpression was observed in 56 (82.3%) of 68, 40 (50.0%) of 80, and 62 (77.5%) of 80 of the gastric tissue samples, respectively. Methylation-sensitive restriction enzyme digestion followed by polymerase chain reaction of COX-2 promoter regions revealed a remarkably high hypermethylation rate (100%, 20/20) among the COX-2-negative GCs, which was associated with the overexpression of DNA methyltransferase (DNMT) 1 (r = 0.587, P < .01). These results indicate that (1) in contrast to previous findings using other GC sources, our results show that COX-2 activity may not be a critical molecular event during GC formation, (2) the tumor-promoting effects of H pylori infection and Wnt and NF-kappaB activities may be mediated by COX-2-independent pathways, and (3) promoter hypermethylation is the major cause of COX-2 silencing in Dalian GCs, apparently because of increased expression of DNMTs (especially DNMT1). Consequently, a COX-2-oriented preventive or therapeutic strategy is not practical for Dalian GCs. The frequent COX-2 hypermethylation observed in Dalian GCs could have insightful epigenetic and epidemiologic implications.

Clinicopathological significance of wnt/ß-catenin signaling pathway in esophageal squamous cell carcinoma.

BACKGROUND/AIM: Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors. It has been reported that Wnt signaling pathway plays an important role in Esophageal Cancer progression, metastasis and invasion. However the clinicopathological significance of Wnt2, GSK3ß, and ß-catenin in ESCC has been little reported. In the present study, the aim of this study was to investigate the clinicopathologic and prognosis roles of Wnt2, GSK3ß, and ß-catenin in ESCC tissue. METHODS: 265 ESCC samples were analyzed by immunohistochemistry using Wnt2, GSK3ß, and ß-catenin antibodies. Then, correlation of Wnt2, GSK3ß, and ß-catenin expression with clinicopathological features and prognosis of ESCC patients was statistically analyzed. RESULTS: Cytoplasmic Wnt2 overexpression was detected in 55.5% (147 of 265) ESCCs, which was significantly correlated with the degree of differentiation (P=0.031). Cytoplasmic GSK3ß overexpression was detected in 7.2% (19 of 265) ESCCs, and aberrant ß-catenin expression was identified in 54.3% (144 of 265) of ESCCs. The positive rate of Wnt2 significantly increased with the malignant degree of Kazak ESCC patients. The aberrant ß-catenin expression in GSK3ß-negative ESCC was significantly associated with the ethnic, tumor size, tumor location, degree of differentiation, AJCC stage, lymph node status. Furthermore, the expression of ß-catenin implicated the ethnic difference (P=0.019). In Kaplan-Meier curve analysis, no significant correlation was observed between the expression of Wnt2, GSK3ß, ß-catenin and the poor prognosis of ESCCs. CONCLUSION: The aberrant ß-catenin expression could be an adverse underlying factor in carcinogenesis and progression of ESCC. There was a different statistical signification for ß-catenin in Kazakhs to compare with Hans.

NGF/γ-IFN inhibits androgen-independent prostate cancer and reverses androgen receptor function through downregulation of FGFR2 and decrease in cancer stem cells.

Androgen-independent prostate cancer (AIPC) is difficult to treat. Present study is to explore the inhibitory effect of a cytokine environment on AIPC and its mechanism. We utilized nerve growth factor (NGF)/γ-interferon (γ-IFN) to change the cytokine environment. Animal models and 2 androgen receptor (AR)-negative prostate cancer cell lines were used to evaluate the effect of NGF/γ-IFN. Flow cytometry, immunocytochemistry, western blotting, Tunel assay, colony formation efficiency, gene microarray, and in vivo bioluminescence were used to discern the mechanisms within NGF/γ-IFN that effect the environment. In vitro, NGF/γ-IFN effectively inhibited the proliferation of AIPC cell lines and promoted the apoptosis of the cancer cells. In vivo, NGF/γ-IFN suppressed the growth and metastasis of a tumor mass that arose from the AIPC cell line. After NGF/γ-IFN treatment, the AR-negative cell lines re-expressed AR and were then able to respond to the androgen. Contrary to expectations, the proliferation of cells was inhibited after dihydrotestosterone was added, and the results indicated that NGF/γ-IFN decreased the proportion of cancer stem cells. NGF/γ-IFN worked mainly through the downregulation of fibroblast growth factor receptor 2.

Embryonic Wnt gene expression in the nitrofen-induced hypoplastic lung using 3-dimensional imaging.

PURPOSE: Wnts have been reported to play a key role in the lung morphogenesis. We have previously reported that pulmonary gene expression of Wnt2 and Wnt7b is downregulated on day 15 of gestation in the nitrofen-induced congenital diaphragmatic hernia (CDH) model. However, the distribution pattern of gene expression of Wnts in the very early lung development remains unclear. Optical projection tomography (OPT) is a new technique for 3-dimensional imaging of small developing organs and gene distribution combined with whole-mount in situ hybridization. We designed this study to investigate the distribution pattern of Wnts gene expression in lung buds of nitrofen-induced CDH model using OPT. METHODS: Embryos from normal and nitrofen-treated dams were harvested on embryonic day 10 (E10), and divided into controls and nitrofen group, respectively. Whole-mount in situ hybridization to detect transcripts of Wnt2 and Wnt7b was performed, analyzed, and reconstructed using OPT. RESULTS: The expression of Wnt2 transcripts was detected in the lung bud mesenchyme and markedly diminished in nitrofen group compared to controls, whereas Wnt7b transcripts were expressed in the mesoderm of bronchi and the lung bud with no detectable difference between 2 groups. CONCLUSION: We provide evidence for the first time that Wnt2 expression is downregulated at lung bud stage in the nitrofen model. Optical projection tomography is potentially a useful approach to visualize both gene expression and morphology during very early stages of lung development.

Wnt2 expression and signaling is increased by different classes of antidepressant treatments.

BACKGROUND: Despite recent interest in glycogen synthase kinase-3beta (GSK-3beta) as a target for the treatment of mood disorders, there has been very little work related to these illnesses on the upstream signaling molecules that regulate this kinase as well as downstream targets. METHODS: With a focused microarray approach we examined the influence of different classes of antidepressants on Wnt signaling that controls GSK-3beta activity as well as the transcription factors that contribute to the actions of GSK-3beta. RESULTS: The results demonstrate that Wnt2 is a common target of different classes of antidepressants and also show differential regulation of Wnt-GSK-3beta signaling genes. Increased expression and function of Wnt2 was confirmed by secondary measures. Moreover, with a viral vector approach we demonstrate that increased expression of Wnt2 in the hippocampus is sufficient to produce antidepressant-like behavioral actions in well-established models of depression and treatment response. CONCLUSIONS: These findings demonstrate that Wnt2 expression and signaling is a common target of antidepressants and that increased Wnt2 is sufficient to produce antidepressant effects.

Downregulation of Wnt2 and beta-catenin by siRNA suppresses malignant glioma cell growth.

Increasing evidence suggests that aberrant activation of Wnt signaling is involved in tumor development and progression. Our earlier study on gene expression profile in human gliomas by microarray found that some members of Wnt family were overexpressed. To further investigate the involvement of Wnt signaling in gliomas, the expression of core components of Wnt signaling cascade in 45 astrocytic glioma specimens with different tumor grades was examined by reverse transcription-PCR and immunohistochemistry. Wnt2, Wnt5a, frizzled2 and beta-catenin were overexpressed in gliomas. Knockdown of Wnt2 and its key mediator beta-catenin in the canonical Wnt pathway by siRNA in human U251 glioma cells inhibited cell proliferation and invasive ability, and induced apoptotic cell death. Furthermore, treating the nude mice carrying established subcutaneous U251 gliomas with siRNA targeting Wnt2 and beta-catenin intratumorally also delayed the tumor growth. In both in vitro and in vivo studies, downregulation of Wnt2 and beta-catenin was associated with the decrease of PI3K/p-AKT expression, indicating the interplay between Wnt/beta-catenin and PI3K/AKT signaling cascades. In conclusion, the canonical Wnt pathway is of critical importance in the gliomagenesis and intervention of this pathway may provide a new therapeutic approach for malignant gliomas.