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.     

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.     

MicroRNA‐301b‐3p contributes to tumour growth of human hepatocellular carcinoma by repressing vestigial like family member 4

MicroRNAs (miRNAs) are key regulators in the tumour growth and metastasis of human hepatocellular carcinoma (HCC). Increasing evidence suggests that miR‐301b‐3p functions as a driver in various types of human cancer. However, the expression pattern of miR‐301b‐3p and its functional role as well as underlying molecular mechanism in HCC remain poorly known. Our study found that miR‐301b‐3p expression was significantly up‐regulated in HCC tissues compared to adjacent non‐tumour tissues. Clinical association analysis revealed that the high level of miR‐301b‐3p closely correlated with large tumour size and advanced tumour‐node‐metastasis stages. Importantly, the high miR‐301b‐3p level predicted a prominent poorer overall survival of HCC patients. Knockdown of miR‐301b‐3p suppressed cell proliferation, led to cell cycle arrest at G2/M phase and induced apoptosis of Huh7 and Hep3B cells. Furthermore, miR‐301b‐3p knockdown suppressed tumour growth of HCC in mice. Mechanistically, miR‐301b‐3p directly bond to 3′UTR of vestigial like family member 4 (VGLL4) and negatively regulated its expression. The expression of VGLL4 mRNA was down‐regulated and inversely correlated with miR‐301b‐3p level in HCC tissues. Notably, VGLL4 knockdown markedly repressed cell proliferation, resulted in G2/M phase arrest and promoted apoptosis of HCC cells. Accordingly, VGLL4 silencing rescued miR‐301b‐3p knockdown attenuated HCC cell proliferation, cell cycle progression and apoptosis resistance. Collectively, our results suggest that miR‐301b‐3p is highly expressed in HCC. miR‐301b‐3p facilitates cell proliferation, promotes cell cycle progression and inhibits apoptosis of HCC cells by repressing VGLL4.     

microRNA‐128‐3p overexpression inhibits breast cancer stem cell characteristics through suppression of Wnt signalling pathway by down‐regulating NEK2

Emerging evidence has reported that dysregulation of microRNAs (miRNAs) participated in the development of diverse types of cancers. Our initial microarray‐based analysis identified differentially expressed NEK2 related to breast cancer and predicted the regulatory microRNA‐128‐3p (miR‐128‐3p). Herein, this study aimed to characterize the tumour‐suppressive role of miR‐128‐3p in regulating the biological characteristics of breast cancer stem cells (BCSCs). CD44^＋CD24^?/low cells were selected for subsequent experiments. After verification of the target relationship between miR‐128‐3p and NEK2, the relationship among miR‐128‐3p, NEK2 and BCSCs was further investigated with the involvement of the Wnt signalling pathway. The regulatory effects of miR‐128‐3p on proliferation, migration, invasion and self‐renewal in vitro as well as tumorigenicity in vivo of BCSCs were examined via gain‐ and loss‐of‐function approaches. Highly expressed NEK2 was found in breast cancer based on GSE61304 expression profile. Breast cancer stem cells and breast cancer cells showed a down‐regulation of miR‐128‐3p. Overexpression of miR‐128‐3p was found to inhibit proliferation, migration, invasion, self‐renewal in vitro and tumorigenicity in vivo of BCSCs, which was further validated to be achieved through inhibition of Wnt signalling pathway by down‐regulating NEK2. In summary, this study indicates that miR‐128‐3p inhibits the stem‐like cell features of BCSCs via inhibition of the Wnt signalling pathway by down‐regulating NEK2, which provides a new target for breast cancer treatment.     

Propofol-induced miR-219-5p inhibits growth and invasion of hepatocellular carcinoma through suppression of GPC3-mediated Wnt/β-catenin signalling activation

Propofol is one of the most extensively used intravenous anaesthetic agents, which has been found to improve the surgical intervention outcome of several types of cancer, including hepatocellular carcinoma (HCC). Additionally, in vitro and in vivo experiments have also indicated that propofol affects the biological behaviour of HCC. However, the underlying mechanisms of the surgical resection of HCC with propofol have not been fully understood. In the present study, we aimed to investigate the underlying mechanism of propofol inhibition of the growth and invasion of HCC cells. Our results showed that treatment with propofol suppressed the proliferation, invasion and migration of HCC in vitro. The subcutaneous xenograft tumour and orthotopic xenograft tumour experiments in nude mice showed that propofol significantly decreased tumour volumes, growth rates and the liver orthotopic xenograft tumour in vivo. Furthermore, the underlying mechanism investigations of the suppressive effects of propofol on HCC cells revealed that propofol treatment upregulated the expression levels of the candidate tumour suppressor miR-219-5p. Silencing of propofol-induced miR-219-5p using anti-miR-219-5p abrogated the inhibitory effects on the proliferation, migration and invasion of HCC cells exerted by propofol treatment. Additionally, we demonstrated that propofol reversed the epithelial-mesenchymal transition of Huh7 and SMMC7721 cells via miR-219-5p induction. The molecular mechanism behind these findings is that propofol-induced miR-219-5p inhibits HCC cell progression by targeting glypican-3 and subsequently results in the inhibition of Wnt/β-catenin signalling. Taken together, our study provides new insights into the advantages of the surgical intervention of HCC with propofol anaesthetization.     

Regulation of the EGFR/ErbB signalling by clathrin in response to various ligands in hepatocellular carcinoma cell lines

Membrane receptor intracellular trafficking and signalling are frequently altered in cancers. Our aim was to investigate whether clathrin‐dependent trafficking modulates signalling of the ErbB receptor family in response to amphiregulin (AR), EGF, heparin‐binding EGF‐like growth factor (HB‐EGF) and heregulin‐1β (HRG). Experiments were performed using three hepatocellular carcinoma (HCC) cell lines, Hep3B, HepG2 and PLC/PRF/5, expressing various levels of EGFR, ErbB2 and ErbB3. Inhibition of clathrin‐mediated endocytosis (CME), by down‐regulating clathrin heavy chain expression, resulted in a cell‐ and ligand‐specific pattern of phosphorylation of the ErbB receptors and their downstream effectors. Clathrin down‐regulation significantly decreased the ratio between phosphorylated EGFR (pEGFR) and total EGFR in all cell lines when stimulated with AR, EGF, HB‐EGF or HRG, except in HRG‐stimulated Hep3B cells in which pEGFR was not detectable. The ratio between phosphorylated ErbB2 and total ErbB2 was significantly decreased in clathrin down‐regulated Hep3B cells stimulated with any of the ligands, and in HRG‐stimulated PLC/PRF/5 cells. The ratio between phosphorylated ErbB3 and total ErbB3 significantly decreased in clathrin down‐regulated cell lines upon stimulation with EGF or HB‐EGF. STAT3 phosphorylation levels significantly increased in all cell lines irrespective of stimulation, while that of AKT remained unchanged, except in AR‐stimulated Hep3B and HepG2 cells in which pAKT was significantly decreased. Finally, ERK phosphorylation was insensitive to clathrin inhibition. Altogether, our observations indicate that clathrin regulation of ErbB signalling in HCC is a complex process that likely depends on the expression of ErbB family members and on the autocrine/paracrine secretion of their ligands in the tumour environment.     

Management of intrauterine adhesions using human amniotic mesenchymal stromal cells to promote endometrial regeneration and repair through Notch signalling

Intrauterine adhesions (IUAs) severely hamper women's reproductive functions. Human amniotic mesenchymal stromal cell (hAMSC) transplantation is effective in treating IUAs. Here, we examined the function of Notch signalling in IUA treatment with hAMSC transplantation. Forty-five Sprague-Dawley female rats were randomly divided into the sham operation, IUA, IUA + E2, IUA + hAMSCs and IUA + hAMSCs + E2 groups. After IUA induction in the rats, hAMSCs promoted endometrial regeneration and repair via differentiation into endometrial epithelial cells. In all groups, the expression of key proteins in Notch signalling was detected in the uterus by immunohistochemistry. The results indicated Notch signalling activation in the hAMSCs and hAMSCs + E2 groups. We could also induce hAMSC differentiation to generate endometrial epithelial cells in vitro. Furthermore, the inhibition of Notch signalling using the AdR-dnNotch1 vector suppressed hAMSC differentiation (assessed by epithelial and mesenchymal marker levels), whereas its activation using the AdR-Jagged1 vector increased differentiation. The above findings indicate Notch signalling mediates the differentiation of hAMSCs into endometrial epithelial cells, thus promoting endometrial regeneration and repair; Notch signalling could have an important function in IUA treatment.     

Selective blockade of B7‐H3 enhances antitumour immune activity by reducing immature myeloid cells in head and neck squamous cell carcinoma

Immature myeloid cells including myeloid‐derived suppressor cells (MDSCs) and tumour‐associated macrophages (TAMs) promote tumour growth and metastasis by facilitating tumour transformation and angiogenesis, as well as by suppressing antitumour effector immune responses. Therefore, strategies designed to reduce MDSCs and TAMs accumulation and their activities are potentially valuable therapeutic goals. In this study, we show that negative immune checkpoint molecule B7‐H3 is significantly overexpressed in human head and neck squamous cell carcinoma (HNSCC) specimen as compared with normal oral mucosa. Using immunocompetent transgenic HNSCC models, we observed that targeting inhibition of B7‐H3 reduced tumour size. Flow cytometry analysis revealed that targeting inhibition of B7‐H3 increases antitumour immune response by decreasing immunosuppressive cells and promoting cytotoxic T cell activation in both tumour microenvironment and macroenvironment. Our study provides direct in vivo evidence for a rationale for B7‐H3 blockade as a future therapeutic strategy to treat patients with HNSCC.     

A literature review on function and regulation mechanism of DKK4

Dickkopf-related protein 4 (DKK4) is a member of the dickkopf family and an inhibitor of the Wnt/β-catenin signalling pathway. This review surveyed the single nucleotide polymorphisms (SNPs), copy number variations (CNVs), hypermethylation, regulation mechanism, correlation with clinicopathological parameters and chemotherapeutic resistance of DKK4. The signal pathways involved in DKK4 mainly include Wnt/β-catenin pathway and Wnt-JNK pathway independent β-catenin. DKK4 expression was upregulated in Renal Cell Carcinoma (RCC), Colorectal Cancer, Gastric Cancer (GC), Non-small Cell Lung Cancer (NSCLC) and Epithelial Ovarian Cancer (EOC), while downregulated in Hepatocellular Carcinoma (HCC). DKK4 is not only involved in tumour growth, invasion, migration and chemotherapy resistance, but also in osteoblastogenesis and secondary hair or meibomian gland formation. DKK4 has also been linked to schizophrenia.     

Hinokiflavone induces apoptosis via activating mitochondrial ROS/JNK/caspase pathway and inhibiting NF‐κB activity in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the sixth most common malignancy with limited treatment options. Hinokiflavone (HF), a natural biflavonoid, has shown to inhibit the proliferation of melanoma, whereas its antitumour effect against HCC and the underlying mechanisms remain elusive. Here, we aimed at evaluating its antitumour effect against HCC in both in vitro and in vivo. Cell counting kit 8, colony formation assay, PI/RNase staining and Western blotting revealed that HF inhibited the proliferation of HCC cells via G0/G1 cell cycle arrest with p21/p53 up‐regulation. DAPI staining, Annexin V‐FITC/PI staining and Western blotting confirmed that HF triggered caspase‐dependent apoptosis. Moreover, HF increased the levels of mitochondrial reactive oxygen species (mtROS) and activated c‐Jun N‐terminal kinase (JNK) pathway, as measured by MitoSOX Red staining and Western blotting. After respectively inhibiting mtROS (Mito‐TEMPO) and JNK (SP600125), HF‐induced apoptosis was reversed. Additionally, Western blotting documented that HF suppressed nuclear factor kappa B (NF‐κB) activity and the anti‐apoptotic genes downstream, contributing to cell apoptosis. Finally, in vivo studies demonstrated that HF significantly impaired tumour growth in HCC xenograft. Collectively, these findings suggested that HF induced apoptosis through activating mtROS/JNK/caspase pathway and inhibiting NF‐κB signalling, which may represent a novel therapeutic agent for treating HCC.     

EphB4 forward signalling mediates angiogenesis caused by CCM3/PDCD10‐ablation

CCM3, also named as PDCD10, is a ubiquitous protein expressed in nearly all tissues and in various types of cells. It is essential for vascular development and post‐natal vessel maturation. Loss‐of‐function mutation of CCM3 predisposes for the familial form of cerebral cavernous malformation (CCM). We have previously shown that knock‐down of CCM3 stimulated endothelial angiogenesis via impairing DLL4‐Notch signalling; moreover, loss of endothelial CCM3 stimulated tumour angiogenesis and promoted tumour growth. The present study was designed to further elucidate the inside signalling pathway involved in CCM3‐ablation‐mediated angiogenesis. Here we report for the first time that silencing endothelial CCM3 led to a significant up‐regulation of EphB4 mRNA and protein expression and to an increased kinase activity of EphB4, concomitantly accompanied by an activation of Erk1/2, which was reversed by treatment with the specific EphB4 kinase inhibitor NVP‐BHG712 (NVP), indicating that silencing CCM3 activates EphB4 kinase forward signalling. Furthermore, treatment with NVP rescued the hyper‐angiogenic phenotype induced by knock‐down of endothelial CCM3 in vitro and in vivo. Additional study demonstrated that the activation of EphB4 forward signalling in endothelial cells under basal condition and after CCM3‐silence was modulated by DLL4/Notch signalling, relying EphB4 at downstream of DLL4/Notch signalling. We conclude that angiogenesis induced by CCM3‐silence is mediated by the activation of EphB4 forward signalling. The identified endothelial signalling pathway of CCM3‐DLL4/Notch‐EphB4‐Erk1/2 may provide an insight into mechanism of CCM3‐ablation‐mediated angiogenesis and could potentially contribute to novel therapeutic concepts for disrupting aberrant angiogenesis in CCM and in hyper‐vascularized tumours.     

Scutellarin inhibits proliferation and invasion of hepatocellular carcinoma cells via down‐regulation of JAK2/STAT3 pathway

The prognosis of hepatocellular carcinoma (HCC) is poor because of high incidence of recurrence and metastasis. JAK/STAT signalling pathway regulates cell proliferation, apoptosis, differentiation and migration and epithelial‐mesenchymal transition (EMT) is also considered to contribute to invasion and metastasis of epithelial malignant tumours. Scutellarin is an active component found in many traditional Chinese herbs and has been regularly used in anti‐inflammatory and antitumour medicine. This study aimed to identify the effect of scutellarin and its possible mechanism of action in HCC cells. Proliferation, colony‐forming, apoptosis and cell migration assays were used to examine the effect of scutellarin on HCC cells. Quantitative real‐time PCR and Western blotting were performed to study the molecular mechanisms of action of scutellarin. Light and electron microscopy and immunofluorescence analysis were performed to study the effect of scutellarin on cellular mechanics. We show that scutellarin potentially suppresses invasiveness of HepG2 and MHCC97‐H cells in vitro by remodelling their cytoskeleton. The molecular mechanism behind it might be the inhibition of the EMT process, which could be attributed to the down‐regulation of the JAK2/STAT3 pathway. These findings may provide new clinical ideas for the treatment of liver cancer.     

Hepatoma‐derived growth factor promotes growth and metastasis of hepatocellular carcinoma cells

We aimed to elucidate the effects of hepatoma‐derived growth factor (HDGF) on growth and metastasis of hepatocellular carcinoma (HCC) cells. Tissue microarrays with 236 HCC specimens and 18 extrahepatic metastases were utilized to detect the HDGF expression by immunohistochemistry. Meanwhile, HDGF expressions in HCC cell lines with different metastatic potentials were examined using immunofluorescence staining, real‐time PCR and western blotting. After HDGF silencing, the growth and metastatic potentials of HCC cells were evaluated by soft agar assay, invasion assay, together with tumorigenicity assay in nude mice. The gelatin zymography was performed by detecting MMP‐2 and MMP‐9 levels. Additionally, western blotting was conducted to determine the levels of total and phosphorylated ERK1/2, JNK, p38 and Akt. The results showed that HDGF was overexpressed in HCC metastasis tumour, and the expression increased with the differentiation degree of tumours (Grade I 44.0%, Grade II 48.4% and Grade III 65.6%). Consistently, HDGF levels were positively associated with the metastatic capability of HCC cells (MHCC97L < MHCC97H < HCCLM3). The growth and metastasis were suppressed by HDGF‐siRNA. Gelatinolytic activities were enhanced in the three metastatic HCC cell lines, but had no significant difference among them. The tumourigenicity and metastatic capability of HCCLM3 cells in nude mice were inhibited after silencing HDGF. Meanwhile, HDGF‐siRNA specifically suppressed the total and phosphorylated protein levels of ERK1/2, while not JNK, p38 and Akt. In conclusion, HDGF was overexpressed in HCC patients and cells, and HDGF might be closely correlated with HCC metastasis via regulating ERK signalling pathway. Copyright © 2016 John Wiley & Sons, Ltd.     

MALT1 is a potential therapeutic target in glioblastoma and plays a crucial role in EGFR‐induced NF‐κB activation

Glioblastoma multiforme (GBM) is the most common malignant tumour in the adult brain and hard to treat. Nuclear factor κB (NF‐κB) signalling has a crucial role in the tumorigenesis of GBM. EGFR signalling is an important driver of NF‐κB activation in GBM; however, the correlation between EGFR and the NF‐κB pathway remains unclear. In this study, we investigated the role of mucosa‐associated lymphoma antigen 1 (MALT1) in glioma progression and evaluated the anti‐tumour activity and effectiveness of MI‐2, a MALT1 inhibitor in a pre‐clinical GBM model. We identified a paracaspase MALT1 that is involved in EGFR‐induced NF‐kB activation in GBM. MALT1 deficiency or inhibition significantly affected the proliferation, survival, migration and invasion of GBM cells both in vitro and in vivo. Moreover, MALT1 inhibition caused G1 cell cycle arrest by regulating multiple cell cycle–associated proteins. Mechanistically, MALTI inhibition blocks the degradation of IκBα and prevents the nuclear accumulation of the NF‐κB p65 subunit in GBM cells. This study found that MALT1, a key signal transduction cascade, can mediate EGFR‐induced NF‐kB activation in GBM and may be potentially used as a novel therapeutic target for GBM.     

Protein arginine methyltransferase 5 (PRMT5) activates WNT/β-catenin signalling in breast cancer cells via epigenetic silencing of DKK1 and DKK3

Protein arginine methyltransferase 5 (PRMT5) activity is dysregulated in many aggressive cancers and its enhanced levels are associated with increased tumour growth and survival. However, the role of PRMT5 in breast cancer remains underexplored. In this study, we show that PRMT5 is overexpressed in breast cancer cell lines, and that it promotes WNT/β-CATENIN proliferative signalling through epigenetic silencing of pathway antagonists, DKK1 and DKK3, leading to enhanced expression of c-MYC, CYCLIN D1 and SURVIVIN. Through chromatin immunoprecipitation (ChIP) studies, we found that PRMT5 binds to the promoter region of WNT antagonists, DKK1 and DKK3, and induces symmetric methylation of H3R8 and H4R3 histones. Our findings also show that PRMT5 inhibition using a specific small molecule inhibitor, compound 5 (CMP5), reduces PRMT5 recruitment as well as methylation of H3R8 and H4R3 histones in the promoter regions of DKK1 and DKK3, which consequently results in reduced expression CYCLIN D1 and SURVIVIN. Furthermore, CMP5 treatment either alone or in combination with 5-Azacytidine and Trichostatin A restored expression of DKK1 and DKK3 in TNBCs. PRMT5 inhibition also altered the growth characteristics of breast cancer cells and induced their death. Collectively, these results show that PRMT5 controls breast cancer cell growth through epigenetic silencing of WNT/β-CATENIN pathway antagonists, DKK1 and DKK3, resulting in up-regulation of WNT/β-CATENIN proliferative signalling.     

2‐phenylethynesulphonamide (PFT‐μ) enhances the anticancer effect of the novel hsp90 inhibitor NVP‐AUY922 in melanoma,by reducing GSH levels

Heat shock proteins (HSPs), are molecular chaperones that assist the proper folding of nascent proteins. This study aims to evaluate the antitumour effects of the hsp90 inhibitor NVP‐AUY922 in melanoma, both in vitro and in vivo. Our results show that NVP‐AUY922 inhibits melanoma cell growth in vitro, with down regulation of multiple signalling pathways involved in melanoma progression such as NF‐?B and MAPK/ERK. However, NVP‐AUY922 was unable to limit tumour growth in vivo. Cotreatment of A375M xenografts with NVP‐AUY922 and PFT‐μ, a dual inhibitor of both hsp70 and autophagy, induced a synergistic increase of cell death in vitro, and delayed tumour formation in A375M xenografts. PFT‐μ depleted cells from the reduced form of glutathione (GSH) and increased oxidative stress. The oxidative stress induced by PFT‐μ further enhanced NVP‐AUY922‐induced cytotoxic effects. These data suggest a potential therapeutic role for NVP‐AUY922 used in combination with PFT‐μ, in melanoma.     

Dickkopf‐1‐promoted vasculogenic mimicry in non‐small cell lung cancer is associated with EMT and development of a cancer stem‐like cell phenotype

To characterize the contributions of Dickkopf‐1 (DKK1) towards the induction of vasculogenic mimicry (VM) in non‐small cell lung cancer (NSCLC), we evaluated cohorts of primary tumours, performed in vitro functional studies and generated xenograft mouse models. Vasculogenic mimicry was observed in 28 of 205 NSCLC tumours, while DKK1 was detected in 133 cases. Notably, DKK1 was positively associated with VM. Statistical analysis showed that VM and DKK1 were both related to aggressive clinical course and thus were indicators of a poor prognosis. Moreover, expression of epithelial‐mesenchymal transition (EMT)‐related proteins (vimentin, Slug, and Twist), cancer stem‐like cell (CSC)‐related proteins (nestin and CD44), VM‐related proteins (MMP2, MMP9, and vascular endothelial‐cadherin), and β‐catenin‐nu were all elevated in VM‐positive and DKK1‐positive tumours, whereas the epithelial marker (E‐cadherin) was reduced in the VM‐positive and DKK1‐positive groups. Non‐small cell lung cancer cell lines with overexpressed or silenced DKK1 highlighted its role in the restoration of mesenchymal phenotypes and development of CSC characteristics. Moreover, DKK1 significantly promotes NSCLC tumour cells to migrate, invade and proliferate. In vivo animal studies demonstrated that DKK1 enhances the growth of transplanted human tumours cells, as well as increased VM formation, mesenthymal phenotypes and CSC properties. Our results suggest that DKK1 can promote VM formation via induction of the expression of EMT and CSC‐related proteins. As such, we feel that DKK1 may represent a novel target of NSCLC therapy.     

Temporal and contextual orchestration of cardiac fate by WNT‐BMP synergy and threshold

Cardiomyogenic development proceeds with a cascade of intricate signalling events that operate in a temporo‐spatial fashion to specify cardiac cell fate during early embryogenesis. In fact, conflicting reports exist regarding the role of Wnt/β‐catenin signalling during cardiomyogenesis. Here, we describe a dose‐dependent and temporal effect of Wnt/β‐catenin signalling on in vitro cardiomyogenesis using embryonic stem cells (ESCs) as a model system. We could demonstrate that canonical Wnt activation during early stage of differentiation either through ligand or by GSK3β inhibition helped in maintaining Oct4 and Nanog expressions, and in parallel, it promoted mesoderm and endoderm inductions. In contrast, it led to attenuation in cardiomyogenesis that was reversed by moderate concentration of DKK1, but not soluble Fz8. However, higher DKK1 could also block cardiomyogenesis, suggesting thereby governance of a particular signalling threshold underlying this developmental event. Interestingly, Wnt signalling activation at early stage modulated BMP4 expression in a stage‐specific manner. Wnt activation, synchronized with BMP4 and brachyury up‐regulation at early stage, correlated well with mesoderm induction. Conversely, Wnt activation led to BMP4 and Wnt5a down‐regulation at late stage culminating in cardiomyogenic attenuation. Our findings suggested the existence of precise regulatory machinery with context‐dependent role of Wnt for fine tuning mesoderm induction and its derivatives, through establishment of Wnt gradient during ESCs’ differentiation. Moreover, contrary to mere activation/inhibition, a specific threshold of Wnt and BMP and their synergy seemed necessary for providing the guiding cues in orchestrating mesoderm induction and subsequent cardiomyogenesis.