Long non‐coding RNA LINC01225 promotes proliferation,invasion and migration of gastric cancer via Wnt/β‐catenin signalling pathway

Emerging evidence has classified the aberrant expression of long non‐coding RNAs (lncRNAs) as a basic signature of various malignancies including gastric cancer (GC). LINC01225 has been shown to act as a hepatocellular carcinoma‐related gene, with its expression pattern and biological function not clarified in GC. Here, we verified that LINC01225 was up‐regulated in tumour tissues and plasma of GC. Analysis with clinicopathological information suggested that up‐regulation of LINC01225 was associated with advanced disease and poorer overall survival. Receiver operating characteristic (ROC) analysis showed that plasma LINC01225 had a moderate accuracy for diagnosis of GC. In addition, knockdown of LINC01225 led to retardation of cell proliferation, invasion and migration, and overexpression of LINC01225 showed the opposite effects. Mechanistic investigations showed that LINC01225 silencing inhibited epithelial‐mesenchymal transition (EMT) process and attenuated Wnt/β‐catenin signalling of GC. Furthermore, ectopic expression of Wnt1 or suppression of GSK‐3β abolished the si‐LINC01225‐mediated suppression against EMT, thereby promoting cell proliferation, invasion and migration of GC. In conclusion, LINC01225 promotes the progression of GC through Wnt/β‐catenin signalling pathway, and it may serve as a potential target or strategy for diagnosis or treatment of GC.     

Zbed3 promotes proliferation and invasion of lung cancer partly through regulating the function of Axin‐Gsk3β complex

Our previous work showed that Zbed3 is overexpressed in nonsmall cell lung cancer and that down‐regulation of Zbed3 inhibited β‐catenin expression and cancer cell proliferation and invasiveness. Here, we investigated Zbed3's ability to promote lung cancer cell proliferation and invasion and the involvement of the Axin/TPC/glycogen synthase kinase 3β (Gsk‐3β) complex to the response. Coimmunoprecipitation assays showed that wild‐type Zbed3 bound to Axin but a Zbed3 mutant lacking the Axin binding site did not. In A549 and H1299 lung cancer cells, Zbed3 overexpression promoted cancer cell proliferation and invasiveness, as well as Wnt signalling and expression of downstream mediators, including β‐catenin, cyclin D1 and MMP7 (P < 0.05). In contrast, the Zbed3 mutant failed to enhance β‐catenin expression (P > 0.05), and its ability to promote cancer cell proliferation and invasiveness was much less than wild‐type Zbed3 (P < 0.05). The ability of Zbed3 to increase β‐catenin levels was abolished by Axin knockdown in A549 cells (P > 0.05). Similarly, treating the cells with a GSK‐3β inhibitor abolished Zbed3's ability to increase β‐catenin levels and Wnt signalling. These results indicate that Zbed3 enhances lung cancer cell proliferation and invasiveness at least in part by inhibiting Axin/adenomatous polyposis coli/GSK‐3β‐mediated negative regulation of β‐catenin levels.     

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.     

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.     

E‐cadherin can limit the transforming properties of activating β‐catenin mutations

Wnt pathway deregulation is a common characteristic of many cancers. Only colorectal cancer predominantly harbours mutations in APC, whereas other cancer types (hepatocellular carcinoma, solid pseudopapillary tumours of the pancreas) have activating mutations in β‐catenin (CTNNB1). We have compared the dynamics and the potency of β‐catenin mutations in vivo. Within the murine small intestine (SI), an activating mutation of β‐catenin took much longer to achieve Wnt deregulation and acquire a crypt‐progenitor cell (CPC) phenotype than Apc or Gsk3 loss. Within the colon, a single activating mutation of β‐catenin was unable to drive Wnt deregulation or induce the CPC phenotype. This ability of β‐catenin mutation to differentially transform the SI versus the colon correlated with higher expression of E‐cadherin and a higher number of E‐cadherin:β‐catenin complexes at the membrane. Reduction in E‐cadherin synergised with an activating mutation of β‐catenin resulting in a rapid CPC phenotype within the SI and colon. Thus, there is a threshold of β‐catenin that is required to drive transformation, and E‐cadherin can act as a buffer to sequester mutated β‐catenin.     

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.     

Activation of Wnt/β‐catenin signalling is required for TGF‐β/Smad2/3 signalling during myofibroblast proliferation

Fibrosis in animal models and human diseases is associated with aberrant activation of the Wnt/β‐catenin pathway. Despite extensive research efforts, effective therapies are still not available. Myofibroblasts are major effectors, responsible for extracellular matrix deposition. Inhibiting the proliferation of the myofibroblast is crucial for treatment of fibrosis. Proliferation of myofibroblasts can have many triggering effects that result in fibrosis. In recent years, the Wnt pathway has been studied as an underlying factor as a primary contributor to fibrotic diseases. These efforts notwithstanding, the specific mechanisms by which Wnt‐mediated promotes fibrosis reaction remain obscure. The central role of the transforming growth factor‐β (TGF‐β) and myofibroblast activity in the pathogenesis of fibrosis has become generally accepted. The details of interaction between these two processes are not obvious. The present investigation was conducted to evaluate the level of sustained expression of fibrosis iconic proteins (vimentin, α‐SMA and collagen I) and the TGF‐β signalling pathway that include smad2/3 and its phosphorylated form p‐smad2/3. Detailed analysis of the possible molecular mechanisms mediated by β‐catenin revealed epithelial–mesenchymal transition and additionally demonstrated transitions of fibroblasts to myofibroblast cell forms, along with increased activity of β‐catenin in regulation of the signalling network, which acts to counteract autocrine TGF‐β/smad2/3 signalling. A major outcome of this study is improved insight into the mechanisms by which epithelial and mesenchymal cells activated by TGFβ1‐smad2/3 signalling through Wnt/β‐catenin contribute to lung fibrosis.     

Paired box 5 is a frequently methylated lung cancer tumour suppressor gene interfering β‐catenin signalling and GADD45G expression

Recent studies suggest that paired box 5 (PAX5) is down‐regulated in multiple tumours through its promoter methylation. However, the role of PAX5 in non‐small cell lung cancer (NSCLC) pathogenesis remains unclear. The aim of this study is to examine PAX5 expression, its methylation status, biological functions and related molecular mechanism in NSCLC. We found that PAX5 was widely expressed in normal adult tissues but silenced or down‐regulated in 88% (7/8) of NSCLC cell lines. PAX5 expression level was significantly lower in NSCLC than that in adjacent non‐cancerous tissues (P = 0.0201). PAX5 down‐regulation was closely associated with its promoter hypermethylation status and PAX5 expression could be restored by demethylation treatment. Frequent PAX5 promoter methylation in primary tumours (70%) was correlated with lung tumour histological types (P = 0.006). Ectopic expression of PAX5 in silenced lung cancer cell lines (A549 and H1975) inhibited their colony formation and cell viability, arrested cell cycle at G2 phase and suppressed cell migration/invasion as well as tumorigenicity in nude mice. Restoration of PAX5 expression resulted in the down‐regulation of β‐catenin and up‐regulation of tissue inhibitors of metalloproteinase 2, GADD45G in lung tumour cells. In summary, PAX5 was found to be an epigenetically inactivated tumour suppressor that inhibits NSCLC cell proliferation and metastasis, through down‐regulating the β‐catenin pathway and up‐regulating GADD45G expression.     

URG11 promotes gastric cancer growth and invasion by activation of β‐catenin signalling pathway

Upregulated gene 11 (URG11), a new gene upregulated by Heptatitis B Virus X protein (HBx), was previously shown to activate β‐catenin and promote hepatocellular growth and tumourigenesis. Although the oncogenic role of URG11 in the development of hepatocellular carcinoma has been well documented, its relevance to other human malignancies and the underlying molecular mechanisms remain largely unknown. Here we reported a novel function of URG11 to promote gastric cancer growth and metastasis. URG11 was found to be highly expressed in gastric cancer tissues compared with adjacent nontumourous ones by immunohistochemical staining and western blot. Knockdown of URG11 expression by small interfering RNA (siRNA) effectively attenuated the proliferation, anchorage‐independent growth, invasiveness and metastatic potential of gastric cancer cells. URG11 inhibition led to decreased expression of β‐catenin and its nuclear accumulation in gastric cancer cells and extensive costaining between URG11 and β‐catenin was observed in gastric cancer tissues. Transient transfection assays with the β‐catenin promoter showed that it was inhibited by URG11‐specific small inhibitory RNA. Moreover, suppression of endogenous URG11 expression results in decreased activation of β‐catenin/TCF and its downstream effector genes, cyclinD1 and membrane type 1 matrix metallopeptidase (MT1‐MMP), which are known to be involved in cell proliferation and invasion, respectively. Taken together, our data suggest that URG11 contributes to gastric cancer growth and metastasis at least partially through activation of β‐catenin signalling pathway. These findings also propose a promising target for gene therapy in gastric cancer.     

PRMT5 promotes epithelial‐mesenchymal transition via EGFR‐β‐catenin axis in pancreatic cancer cells

Protein arginine methyltransferase 5 (PRMT5) has been implicated in the development and progression of human cancers. However, few studies reveal its role in epithelial‐mesenchymal transition (EMT) of pancreatic cancer cells. In this study, we find that PRMT5 is up‐regulated in pancreatic cancer, and promotes proliferation, migration and invasion in pancreatic cancer cells, and promotes tumorigenesis. Silencing PRMT5 induces epithelial marker E‐cadherin expression and down‐regulates expression of mesenchymal markers including Vimentin, collagen I and β‐catenin in PaTu8988 and SW1990 cells, whereas ectopic PRMT5 re‐expression partially reverses these changes, indicating that PRMT5 promotes EMT in pancreatic cancer. More importantly, we find that PRMT5 knockdown decreases the phosphorylation level of EGFR at Y1068 and Y1172 and its downstream p‐AKT and p‐GSK3β, and then results in down‐regulation of β‐catenin. Expectedly, ectopic PRMT5 re‐expression also reverses the above changes. It is suggested that PRMT5 promotes EMT probably via EGFR/AKT/β‐catenin pathway. Taken together, our study demonstrates that PRMT5 plays oncogenic roles in the growth of pancreatic cancer cell and provides a potential candidate for pancreatic cancer treatment.     

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.

     

β‐Catenin and Rho GTPases as downstream targets of TGF‐β1 during pulp repair

TGF‐β1 (transforming growth factor‐β1) plays a central role in regulating proliferation, migration and differentiation of dental pulp cells during the repair process after tooth injury. Our previous study showed that p38 mitogen‐activated protein kinase may act downstream of TGF‐β1 signalling to effect the differentiation of dental pulp cells. However, the molecular mechanisms that trigger and regulate the process remain to be elucidated. TGF‐β1 interacts with signalling pathways such as Wnt/β‐catenin and Rho to induce diverse biological effects. TGF‐β1 activates β‐catenin signalling, increases β‐catenin nuclear translocation and interacts with LEF/TCF to regulate gene expression. Morphologic changes in response to TGF‐β1 are associated with activation of Rho GTPases, but are abrogated by inhibitors of Rho‐associated kinase, a major downstream target of Rho. These results suggest that the Wnt/β‐catenin and Rho pathways may mediate the downstream events of TGF‐β1 signalling.     

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.     

Resveratrol promotes trophoblast invasion in pre‐eclampsia by inducing epithelial‐mesenchymal transition

Impairment spiral arteries remodelling was considered to be the underlying cause of pathogenesis of pre‐eclampsia (PE). Resveratrol (RE) was reported that it could modulate cellar phenotype to ameliorate diverse human diseases. However, the biological function of RE in PE remains poorly understood. In this report, we investigated the effect of RE on trophoblast phenotype both in vivo and in vitro. We conducted MTT and transwell assays to explore cell proliferation and invasion events in HTR‐8/SVneo. In mice model, the clinical characteristics of PE were established through the injection of NG‐nitro‐l ‐arginine methyl ester (L‐NAME). Furthermore, related experiments were performed to detect cellar phenotype‐associated signalling pathway, including epithelial‐mesenchymal transition (EMT) and Wnt/β‐catenin. Cell assays indicated that RE could increase trophoblasts migration and invasion. In addition, hypertension and proteinuria were markedly ameliorated by RE compared with the controls in PE mice model. Moreover, treatment by RE in trophoblasts or in PE model, we found that RE activated EMT progress through the regulation of E‐cadherin, β‐catenin, N‐cadherin, vimentin expression, and further altered the WNT‐related gene expression, including WNT1, WNT3 and WNT5B. Our findings demonstrated that RE might stimulate the invasive capability of human trophoblasts by promoting EMT and mediating the Wnt/β‐catenin pathway in PE.     

MCM10 facilitates the invaded/migrated potentials of breast cancer cells via Wnt/β‐catenin signaling and is positively interlinked with poor prognosis in breast carcinoma

The minichromosome maintenance protein 10 (MCM10) is one of the MCM proteins that initiate DNA replication by interacting with CDC45‐MCM2–7. It has been reported that MCM10 has a role in breast cancer progression. However, MCM10 in breast cancer is still not comprehensively studied and further research is needed. This study was aimed at investigating the potential effects of MCM10 on metastasis, the prognosis of breast carcinoma, and its underlying mechanisms. Using the ONCOMINE database and the Kaplan‐Meier Plotter, MCM10 was significantly overexpressed in cancers, and high expression of MCM10 was involved in the poor prognosis of breast carcinoma. MCM10 can promote the proliferation, migration, and invasion of MDA‐MB‐231 cells. MCM10 knockdown brought about a radical reversal in cell behaviors. Meanwhile, decreased expression of β‐catenin and cyclin Dl was detected in MCM10 short hairpin RNA cells, implying that MCM10 might induce breast cancer metastasis via the Wnt/β‐catenin pathway.MCM10 can be defined as a potential diagnostic tool and a promising target for breast carcinoma.