DKK3 blocked translocation of β‐catenin/EMT induced by hypoxia and improved gemcitabine therapeutic effect in pancreatic cancer Bxpc‐3 cell

The Wnt/β‐catenin signalling pathway is activated in pancreatic cancer initiation and progression. Dickkopf‐related protein 3 (DKK3) is a member of the human Dickkopf family and an antagonist of Wnt ligand activity. However, the function of DKK3 in this pathway in pancreatic cancer is rarely known. We examined the expression of DKK3 in six human pancreatic cancer cell lines, 75 pancreatic cancer and 75 adjacent non‐cancerous tissues. Dickkopf‐related protein 3 was frequently silenced and methylation in pancreatic cancer cell lines (3/6). The expression of DKK3 was significantly lower in pancreatic cancer tissues than in adjacent normal pancreas tissues. Further, ectopic expression of DKK3 inhibits nuclear translocation of β‐catenin induced by hypoxia in pancreatic cancer Bxpc‐3 cell. The forced expression of DKK3 markedly suppressed migration and the stem cell‐like phenotype of pancreatic cancer Bxpc‐3 cell in hypoxic conditions through reversing epithelial–mesenchymal transition (EMT). The stable expression of DKK3 sensitizes pancreatic cancer Bxpc‐3 cell to gemcitabine, delays tumour growth and augments gemcitabine therapeutic effect in pancreatic cancer xenotransplantation model. Thus, we conclude from our finding that DKK3 is a tumour suppressor and improved gemcitabine therapeutic effect through inducing apoptosis and regulating β‐catenin/EMT signalling in pancreatic cancer Bxpc‐3 cell.     

Aberrant methylation of WD‐repeat protein 41 contributes to tumour progression in triple‐negative breast cancer

WD‐repeat proteins are implicated in a variety of biological functions, most recently in oncogenesis. However, the underlying function of WD‐repeat protein 41 (WDR41) in tumorigenesis remains elusive. The present study was aimed to explore the role of WDR41 in breast cancer. Combined with Western blotting and immunohistochemistry, the results showed that WDR41 was expressed at low levels in breast cancer, especially in triple‐negative breast cancer (TNBC). Using methylation‐specific PCR (MSP), we observed that WDR41 presented hypermethylation in MDA‐MB‐231 cells. Methylation inhibitor 5‐aza‐2′‐deoxycytidine (5‐aza‐dC) management increased the expression of WDR41 in MDA‐MB‐231 cells, but not in MCF‐10A (normal mammary epithelial cells) or oestrogen receptor‐positive MCF‐7 breast cancer cells. WDR41‐down‐regulation promoted, while WDR41‐up‐regulation inhibited the tumour characteristics of TNBC cells including cell viability, cell cycle and migration. Further, WDR41‐up‐regulation dramatically suppressed tumour growth in vivo. Mechanistically, WDR41 protein ablation activated, while WDR41‐up‐regulation repressed the AKT/GSK‐3β pathway and the subsequent nuclear activation of β‐catenin in MDA‐MB‐231 cells, and 5‐aza‐dC treatment enhanced this effect. After treatment with the AKT inhibitor MK‐2206, WDR41‐down‐regulation‐mediated activation of the GSK‐3β/β‐catenin signalling was robustly abolished. Collectively, methylated WDR41 in MDA‐MB‐231 cells promotes tumorigenesis through positively regulating the AKT/GSK‐3β/β‐catenin pathway, thus providing an important foundation for treating TNBC.     

MicroRNA‐300 promotes apoptosis and inhibits proliferation,migration, invasion and epithelial‐mesenchymal transition via the Wnt/β‐catenin signaling pathway by targeting CUL4B in pancreatic cancer cells

The study aims to verify the hypothesis that up‐regulation of microRNA‐300 (miR‐300) targeting CUL4B promotes apoptosis and suppresses proliferation, migration, invasion, and epithelial‐mesenchymal transition (EMT) of pancreatic cancer cells by regulating the Wnt/β‐catenin signaling pathway. Pancreatic cancer tissues and adjacent tissues were collected from 110 pancreatic cancer patients. Expression of miR‐300, CUL4B, Wnt, β‐catenin, E‐cadherin, N‐cadherin, Snail, GSK‐3β, and CyclinD1 were detected using qRT‐PCR and Western blot. CFPAC‐1, Capan‐1, and PANC‐1 were classified into blank, negative control (NC), miR‐300 mimics, miR‐300 inhibitors, siRNA‐CUL4B, and miR‐300 inhibitors + siRNA‐CUL4B groups. The proliferation, migration, invasion abilities, the cell cycle distribution, and apoptosis rates were measured in CCK‐8 and Transwell assays. Pancreatic cancer tissues showed increased CUL4B expression but decreased miR‐300 expression. When miR‐300 was lowly expressed, CUL4B was upregulated which in‐turn activated the Wnt/β‐catenin pathway to protect the β‐catenin expression and thus induce EMT. When miR‐300 was highly expressed, CUL4B was downregulated which in‐turn inhibited the Wnt/β‐catenin pathway to prevent EMT. Weakened cell migration and invasion abilities and enhanced apoptosis were observed in the CUL4B group. The miR‐300 inhibitors group exhibited an evident increase in growth rate accompanied the largest tumor volume. Smaller tumor volume and slower growth rate were observed in the miR‐300 mimics and siRNA‐CUL4B group. Our study concludes that lowly expressed miR‐300 may contribute to highly expressed CUL4B activating the Wnt/β‐catenin signaling pathway and further stimulating EMT, thus promoting proliferation and migration but suppressing apoptosis of pancreatic cancer cells.     

Suppression of epidermal growth factor receptor‐mediated β‐catenin nuclear accumulation enhances the anti‐tumor activity of phosphoinositide 3‐kinase inhibitor in breast cancer

Phosphoinositide 3‐kinase (PI3K) signaling is frequently deregulated in breast cancer and plays a critical role in tumor progression. However, resistance to PI3K inhibitors in breast cancer has emerged, which is due to the enhanced β‐catenin nuclear accumulation. Until now, the mechanisms underlying PI3K inhibition‐induced β‐catenin nuclear accumulation remains largely unknown. In the present study, we found inhibition of PI3K with LY294002 promoted β‐catenin nuclear accumulation in MCF‐7 and MDA‐MB‐231 breast cancer cells. Combining PI3K inhibitor LY294002 with XAV‐939, an inhibitor against β‐catenin nuclear accumulation, produced an additive anti‐proliferation effect against breast cancer cells. Subsequent experiments suggested β‐catenin nuclear accumulation induced by PI3K inhibition depended on the feedback activation of epidermal growth factor receptor (EGFR) signaling pathway in breast cancer cells. Inhibition of EGFR phosphorylation with Gefitinib enhanced anti‐proliferation effect of PI3K inhibitor LY294002 in MCF‐7 and MDA‐MB‐231 cells. Taken together, our findings may elucidate a possible mechanism explaining the poor outcome of PI3K inhibitors in breast cancer treatment.     

Oestrogen induces epithelial‐mesenchymal transition in endometriosis via circ_0004712/miR‐148a‐3p sponge function

Endometriosis is a common, chronic gynaecologic disease affecting up to 10% of women in their reproductive age and leading to pain and infertility. Oestrogen (E₂)‐induced epithelial‐mesenchymal transition (EMT) process has been considered as a key factor of endometriosis development. Recently, the dysregulated circular RNAs (circRNAs) have been discovered in endometriosis tissues. However, the molecular mechanism of circRNAs on the E₂‐induced EMT process in endometriosis is still unknown. Here, we demonstrated that circ_0004712 up‐regulated by E₂ treatment in endometrial epithelial cells. Knock‐down the expression of circ_0004712 significantly suppressed E₂‐induced cell migration activity. Meanwhile, we identified miR‐148a‐3p as a potential target miRNA of circ_0004712. Inhibited the expression of miR‐148a‐3p could recovered the effect of circ_0004712 knock‐down in E₂‐treated endometrial epithelial. Furthermore, Western blot assay showed that E₂ treatment could increase the expression and activity of β‐catenin, snail and N‐cadherin and reduce the expression of E‐cadherin. The expression and activity of β‐catenin pathway were recovered by circ_0004712 knock‐down or miR‐148a‐3p overexpression. Altogether, the results demonstrate that circ_0004712/miR‐148a‐3p plays an important role in E₂‐induced EMT process in the development of endometriosis, and the molecular mechanism may be associated with the β‐catenin pathway. This work highlighted the importance of circRNAs in the development of endometriosis and provide a new biomarker for diagnosis and therapies.     

β‐catenin regulates IRF3‐mediated innate immune signalling in colorectal cancer

Objective

β‐catenin is one of the most critical oncogenes associated with many kinds of human cancers, especially in the human CRC. Innate immunity recognizes tumour derived damage‐associated molecular patterns (DAMPs) and primes the anti‐tumour adaptive responses. While the function of β‐catenin in CRC tumourigenesis is well established, its impact on innate immune evasion is largely unknown. The aim of this study is to characterize the role of β‐catenin in inhibiting RIG‐I‐like receptor (RLR)‐mediated IFN‐β signalling in colorectal cancer.

Materials and Methods

Immunohistochemical staining and western blotting were conducted to study the expression of β‐catenin, IRF3 and phospho‐IRF3 (p‐IRF3) in CRC samples and cell lines. Plaque assay determining virus replication was performed to assess the regulation of β‐catenin on IFN‐β signalling. The inhibition of β‐catenin on RLR‐mediated IFN‐β signalling was further studied by real‐time analyses and reporter assays in the context of lentiviral‐mediated β‐catenin stably knocking down. Lastly, co‐immunoprecipitation and nuclear fractionation assay were conducted to monitor the interaction between β‐catenin and IRF3.

Results

We found that high expression of β‐catenin positively correlated with the expression of IRF3 in CRC cells. Overexpression of β‐catenin increased the viral replication. Conversely knocking down of β‐catenin inhibited viral replication. Furthermore, our data demonstrated that β‐catenin could inhibit the expression of IFN‐β and interferon‐stimulated gene 56 (ISG56). Mechanistically, we found that β‐catenin interacted with IRF3 and blocked its nuclear translocation.

Conclusion

Our study reveals an unprecedented role of β‐catenin in enabling innate immune evasion in CRC.

     

Blockade of JAK2 activity suppressed accumulation of β‐catenin in leukemic cells

The Wnt/β‐catenin pathway has been implicated in leukemogenesis. We found β‐catenin abnormally accumulated in both human acute T cell leukemia Jurkat cells and human erythroleukemia HEL cells. β‐Catenin can be significantly down‐regulated by the Janus kinase 2 specific inhibitor AG490 in these two cells. AG490 also reduces the luciferase activity of a reporter plasmid driven by LEF/β‐catenin promoter. Similar results were observed in HEL cells infected with lentivirus containing shRNA against JAK2 gene. After treatment with 50 µM AG490 or shRNA, the mRNA expression levels of β‐catenin, APC, Axin, β‐Trcp, GSK3α, and GSK3β were up‐regulated within 12–16 h. However, only the protein levels of GSK3β and β‐Trcp were found to have increased relative to untreated cells. Knockdown experiments revealed that the AG490‐induced inhibition of β‐catenin can be attenuated by shRNA targeting β‐TrCP. Taken together; these results suggest that β‐Trcp plays a key role in the cross‐talk between JAK/STAT and Wnt/β‐catenin signaling in leukemia cells. J. Cell. Biochem. 111: 402–411, 2010. © 2010 Wiley‐Liss, Inc.     

BMP‐2 modulates β‐catenin signaling through stimulation of Lrp5 expression and inhibition of β‐TrCP expression in osteoblasts

Canonical BMP and Wnt signaling pathways play critical roles in regulation of osteoblast function and bone formation. Recent studies demonstrate that BMP‐2 acts synergistically with β‐catenin to promote osteoblast differentiation. To determine the molecular mechanisms of the signaling cross‐talk between canonical BMP and Wnt signaling pathways, we have used primary osteoblasts and osteoblast precursor cell lines 2T3 and MC3T3‐E1 cells to investigate the effect of BMP‐2 on β‐catenin signaling. We found that BMP‐2 stimulates Lrp5 expression and inhibits the expression of β‐TrCP, the F‐box E3 ligase responsible for β‐catenin degradation and subsequently increases β‐catenin protein levels in osteoblasts. In vitro deletion of the β‐catenin gene inhibits osteoblast proliferation and alters osteoblast differentiation and reduces the responsiveness of osteoblasts to the BMP‐2 treatment. These findings suggest that BMP‐2 may regulate osteoblast function in part through modulation of the β‐catenin signaling. J. Cell. Biochem. 108: 896–905, 2009. © 2009 Wiley‐Liss, Inc.     

Epigenetic silencing of the WNT antagonist Dickkopf 3 disrupts normal Wnt/β‐catenin signalling and apoptosis regulation in breast cancer cells

Dickkopf‐related protein 3 (DKK3) is an antagonist of Wnt ligand activity. Reduced DKK3 expression has been reported in various types of cancers, but its functions and related molecular mechanisms in breast tumorigenesis remain unclear. We examined the expression and promoter methylation of DKK3 in 10 breast cancer cell lines, 96 primary breast tumours, 43 paired surgical margin tissues and 16 normal breast tissues. DKK3 was frequently silenced in breast cell lines (5/10) by promoter methylation, compared with human normal mammary epithelial cells and tissues. DKK3 methylation was detected in 78% of breast tumour samples, whereas only rarely methylated in normal breast and surgical margin tissues, suggesting tumour‐specific methylation of DKK3 in breast cancer. Ectopic expression of DKK3 suppressed cell colony formation through inducing G0/G1 cell cycle arrest and apoptosis of breast tumour cells. DKK3 also induced changes of cell morphology, and inhibited breast tumour cell migration through reversing epithelial‐mesenchymal transition (EMT) and down‐regulating stem cell markers. DKK3 inhibited canonical Wnt/β‐catenin signalling through mediating β‐catenin translocation from nucleus to cytoplasm and membrane, along with reduced active‐β‐catenin, further activating non‐canonical JNK signalling. Thus, our findings demonstrate that DKK3 could function as a tumour suppressor through inducing apoptosis and regulating Wnt signalling during breast tumorigenesis.     

MicroRNA‐1275 induces radiosensitization in oesophageal cancer by regulating epithelial‐to‐mesenchymal transition via Wnt/β‐catenin pathway

Acquired radioresistance is one of the main obstacles for the anti‐tumour efficacy of radiotherapy in oesophageal cancer (EC). Recent studies have proposed microRNAs (miRNAs) as important participators in the development of radioresistance in various cancers. Here, we investigated the role of miR‐1275 in acquired radioresistance and epithelial‐mesenchymal transition (EMT) in EC. Firstly, a radioresistant cell line KYSE‐150R was established, with an interesting discovery was observed that miR‐1275 was down‐regulated in KYSE‐150R cells compared to the parental cells. Functionally, miR‐1275 inhibition elevated radioresistance in KYSE‐150 cells via promoting EMT, whereas enforced expression of miR‐1275 increased radiosensitivity in KYSE‐150R cells by inhibiting EMT. Mechanically, we demonstrated that miR‐1275 directly targeted WNT1 and therefore inactivated Wnt/β‐catenin signalling pathway in EC cells. Furthermore, WNT1 depletion countervailed the promoting effect of miR‐1275 suppression on KYSE‐150 cell radioresistance through hampering EMT, whereas WNT1 overexpression rescued miR‐1275 up‐regulation‐impaired EMT to reduce the sensitivity of KYSE‐150R cells to radiation. Collectively, our findings suggested that miR‐1275 suppressed EMT to encourage radiosensitivity in EC cells via targeting WNT1‐activated Wnt/β‐catenin signalling, providing a new therapeutic outlet for overcoming radioresistance of patients with EC.     

IL‐8 promotes cell migration through regulating EMT by activating the Wnt/β‐catenin pathway in ovarian cancer

Interleukin‐8 (IL‐8), as an inflammatory chemokine, has been previously shown to contribute to tumorigenesis in several malignancies including the ovarian cancer. However, little is known about how IL‐8 promotes the metastasis and invasion of ovarian cancers cells. In this study, we found that IL‐8 and its receptors CXCR1 and CXCR2 were up‐regulated in advanced ovarian serous cancer tissues. Furthermore, the level of IL‐8 and its receptors CXCR1 and CXCR2 expression were associated with ovarian cancer stage, grade and lymph node metastasis. In vitro, IL‐8 promoted ovarian cancer cell migration, initiated the epithelial‐mesenchymal transition (EMT) program and activated Wnt/β‐catenin signalling. However, when treated with Reparixin (inhibitor of both IL‐8 receptors CXCR1 and CXCR2), effect of both endogenous and exogenous IL‐8 was reversed. Together, our results indicated that IL‐8 triggered ovarian cancer cells migration partly through Wnt/β‐catenin pathway mediated EMT, and IL‐8 may be an important molecule in the invasion and metastasis of ovarian cancer.     

miR‐150‐5p suppresses the stem cell‐like characteristics of glioma cells by targeting the Wnt/β‐catenin signaling pathway

Glioma is the most common brain tumor malignancy with high mortality and poor prognosis. Emerging evidence suggests that cancer stem cells are the key culprit in the development of cancer. MicroRNAs have been reported to be dysregulated in many cancers, while the mechanism underlying miR‐150‐5p in glioma progression and proportion of stem cells is unclear. The expression levels of miR‐150‐5p and catenin beta 1 (CTNNB1, which encodes β‐catenin) were measured by quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot. The expression levels of downstream genes of the Wnt/β‐catenin pathway and stem cell markers were detected by qRT‐PCR. Tumorigenesis was investigated by cell viability, colony formation, and tumor growth in vitro and in vivo. The interaction between miR‐150‐5p and β‐catenin was explored via bioinformatics analysis and luciferase activity assay. We found that miR‐150‐5p was downregulated in glioma and its overexpression inhibited cell proliferation, colony formation, and tumor growth. Moreover, miR‐150‐5p directly suppressed CTNNB1 and negatively regulated the abundances of downstream genes of the Wnt/β‐catenin pathway and stem cell markers. Furthermore, miR‐150‐5p expression was decreased and β‐catenin level was enhanced in CD133+ glioma stem cells. Knockdown of miR‐150‐5p contributed to CD133? cells with stem cell‐like phenotype, whereas overexpression of miR‐150‐5p suppressed CD133+ glioma stem cell‐like characteristics. In conclusion, miR‐150‐5p inhibited the progression of glioma by controlling stem cell‐like characteristics via regulating the Wnt/β‐catenin pathway, providing a novel target for glioma treatment.     

Tanshinone II A improves the chemosensitivity of breast cancer cells to doxorubicin by inhibiting β‐catenin nuclear translocation

Numerous evidence link aberrant nuclear β‐catenin accumulation to the development of breast cancer resistance, therefore, targeted inhibition of β‐catenin nuclear translocation may effectively improve the chemosensitivity of breast cancer. Doxorubicin (Dox) is the most commonly used chemotherapeutic drug for breast cancer. Here, we determined that tanshinone II A (Tan II A) could improve the sensitivity of Dox‐resistant breast cancer MCF‐7/dox cells to Dox, and evaluated whether the sensitization effect of Tan II A on Dox was targeted to inhibit β‐catenin nuclear translocation. The results showed that Tan II A not only significantly inhibited the nuclear translocation of β‐catenin in MCF‐7/dox cells treated by Dox but also inhibited the nuclear translocation of β‐catenin in MCF‐7 cells treated by Dox to a certain degree. Furthermore, when the above two cells treated by Dox combined with Tan II A were intervened with β‐catenin agonist WAY‐262611, with the re‐nuclear translocation of β‐catenin in the cells, the sensitization effect of Tan II A on Dox was greatly reduced. These results indicated that Tan II A could improve the chemosensitivity of breast cancer cells to Dox by inhibiting β‐catenin nuclear translocation. Therefore, Tan II A could be used as a potential chemosensitizer in combination with Dox for breast cancer chemotherapy.     

Suppression of MAPK signaling in BRAF‐activated PTEN‐deficient melanoma by blocking β‐catenin signaling in cancer‐associated fibroblasts

Cancer‐associated fibroblasts (CAFs) in the tumor microenvironment have been associated with formation of a dynamic and optimized niche for tumor cells to grow and evade cell death induced by therapeutic agents. We recently reported that ablation of β‐catenin expression in stromal fibroblasts and CAFs disrupted their biological activities in in vitro studies and in an in vivo B16F10 mouse melanoma model. Here, we show that the development of a BRAF‐activated PTEN‐deficient mouse melanoma was significantly suppressed in vivo after blocking β‐catenin signaling in CAFs. Further analysis revealed that expression of phospho‐Erk1/2 and phospho‐Akt was greatly reduced, effectively abrogating the activating effects and abnormal cell cycle progression induced by Braf and Pten mutations. In addition, the epithelial–mesenchymal transition (EMT)‐like process was also suppressed in melanoma cells. Taken together, our data highlight an important crosstalk between CAFs and the RAF‐MEK‐ERK signaling cascade in BRAF‐activated melanoma and may offer a new approach to abrogate host‐dependent drug resistance in targeted therapy.     

PCNP promotes ovarian cancer progression by accelerating β‐catenin nuclear accumulation and triggering EMT transition

Ever reports showed that PCNP is associated with human cancers including neuroblastoma and lung cancer. However, the role and underlying molecular mechanism of PCNP in ovarian cancer have not been plenty elucidated. Herein, we first investigated the expression of PCNP in ovarian cancer tissues and cells, the effects of PCNP in ovarian cancer proliferation, apoptosis, migration and invasion, and determined the molecular mechanism of PCNP in ovarian cancer progression. The results indicated that PCNP was significantly overexpressed in human ovarian cancer tissues and cells, and related to poor prognosis in ovarian cancer patients. In addition, we also detected that PCNP promoted ovarian cancer cells growth, migration and invasion, as well as inhibited ovarian cancer cells apoptosis. Mechanistically, PCNP binding to β‐catenin promoted β‐catenin nuclear translocation and further activated Wnt/β‐catenin signalling pathway. Moreover, PCNP regulated the expression of genes involved in EMT and further triggered EMT occurrence. Conclusionally, PCNP may promote ovarian cancer progression through activating Wnt/β‐catenin signalling pathway and EMT, acting as a novel and promising target for treating ovarian cancer.