miR-139/PDE2A-Notch1 feedback circuit represses stemness of gliomas by inhibiting Wnt/β-catenin signaling

Rationale: The malignant phenotypes of glioblastomas (GBMs) are primarily attributed to glioma stem cells (GSCs). Our previous study and other reports have suggested that both miR-139 and its host gene PDE2A are putative antitumor genes in various cancers. The aim of this study was to investigate the roles and mechanisms of miR-139/PDE2A in GSC modulation.Methods: Clinical samples were used to determine miR-139/PDE2A expression. Patient-derived glioma stem-like cells (PD-GSCs) were stimulated for immunofluorescent staining, sphere formation assays and orthotopic GBM xenograft models. Bioinformatic analysis and further in vitro experiments demonstrated the downstream molecular mechanisms of miR-139 and PDE2A. OX26/CTX-conjugated PEGylated liposome (OCP) was constructed to deliver miR-139 or PDE2A into glioma tissue specifically.Results: We demonstrated that miR-139 was concomitantly transcribed with its host gene PDE2A. Both PDE2A and miR-139 indicated better prognosis of gliomas and were inversely correlated with GSC stemness. PDE2A or miR-139 overexpression suppressed the stemness of PD-GSCs. FZD3 and β-catenin, which induced Wnt/β-catenin signaling activation, were identified as targets of miR-139 and mediated the effects of miR-139 on GSCs. Meanwhile, PDE2A suppressed Wnt/β-catenin signaling by inhibiting cAMP accumulation and GSK-3β phosphorylation, thereby modulating the self-renewal of PD-GSCs. Notably, Notch1, which is also a target of miR-139, suppressed PDE2A/miR-139 expression directly via downstream Hes1, indicating that miR-139 promoted its own expression by the miR-139-Notch1/Hes1 feedback circuit. Expectedly, targeted overexpression miR-139 or PDE2A in glioma with OCP system significantly repressed the stemness and decelerated glioma progression.Conclusions: Our findings elaborate on the inhibitory functions of PDE2A and miR-139 on GSC stemness and tumorigenesis, which may provide new prognostic markers and therapeutic targets for GBMs.     

Amplification of RAD54B promotes progression of hepatocellular carcinoma via activating the Wnt/β-catenin signaling

Liver cancer was reported to be the sixth most frequently diagnosed cancer, and hepatocellular carcinoma (HCC) accounts for 75%-85% of primary liver cancer. Nevertheless, the concrete molecular mechanisms of HCC progression remain obscure, which is essential to elucidate. The expression profile of RAD54B in HCC was measured using qPCR and western blotting. Moreover, the levels of RAD54B in paraffin-embedded samples were evaluated using immunohistochemistry (IHC). The effect of RAD54B on HCC progression was testified by in vitro experiments, and in vivo orthotopic xenograft tumor experiments. The mechanisms of RAD54B promoting HCC progression were investigated through molecular and function experiments. Herein, RAD54B are dramatically upregulated in HCC tissues and cell lines both on mRNA and protein levels, and RAD54B can servers as an independent prognostic parameter of 5-year overall survival and 5-year disease-free survival for patients with HCC. Moreover, up-regulation of RAD54B dramatically increases the capacity for in vitro cell viability and motility, and in vivo intrahepatic metastasis of HCC cells. Mechanistically, RAD54B promotes the HCC progression through modulating the wnt/β-catenin signaling. Notably, blocking the wnt/β-catenin signaling axis can counteract the activating effects of RAD54B on motility of HCC cells. Besides, further analysis illustrates that DNA amplification is one of the mechanisms leading to mRNA overexpression of RAD54B in HCC. Our findings indicate that RAD54B might be a promising potential prognostic marker and a candidate therapeutic target to therapy HCC.     

Pantothenate Kinase 1 Inhibits the Progression of Hepatocellular Carcinoma by Negatively Regulating Wnt/β-catenin Signaling

Hyperactivation of Wnt/β-catenin signaling has been reported in hepatocellular carcinoma (HCC). However, the mechanisms underlying the hyperactivation of Wnt/β-catenin signaling are incompletely understood. In this study, Pantothenate kinase 1 (PANK1) is shown to be a negative regulator of Wnt/β-catenin signaling. Downregulation of PANK1 in HCC correlates with clinical features. Knockdown of PANK1 promotes the proliferation, growth and invasion of HCC cells, while overexpression of PANK1 inhibits the proliferation, growth, invasion and tumorigenicity of HCC cells. Mechanistically, PANK1 binds to CK1α, exerts protein kinase activity and cooperates with CK1α to phosphorylate N-terminal serine and threonine residues in β-catenin both in vitro and in vivo. Additionally, the expression levels of PANK1 and β-catenin can be used to predict the prognosis of HCC. Collectively, the results of this study highlight the crucial roles of PANK1 protein kinase activity in inhibiting Wnt/β-catenin signaling, suggesting that PANK1 is a potential therapeutic target for HCC.     

CTHRC1 promotes growth,migration and invasion of trophoblasts via reciprocal Wnt/β-catenin regulation

Preeclampsia (PE) is a pregnancy complication that is characterized by high blood pressure and is associated with high maternal and fetal morbidities. At a mechanistic level, PE is characterized by reduced invasion ability of trophoblasts. Collagen triple helix repeat containing-1 (CTHRC1) is a well-known tumor-promoting factor in several malignant tumors, but its role in trophoblasts remains unknown. In this study, we characterized the expression of CTHRC1 in placenta tissue samples from PE pregnancies and from normal pregnancies. We used the trophoblasts cell lines HTR-8/SVneo and JEG-3 to investigate the role of CTHRC1 in cell migration, invasion and proliferation. Western blot, PCR and TOP/FOP luciferase activity assays were used to investigate the molecular mechanisms underlying these cell behaviors. Placenta tissue samples obtained from pregnant women with PE expressed lower levels of CTHRC1 than those of placenta tissues from women with normal pregnancies. Down-regulation of CTHRC1 impaired cell proliferation, migration and invasion of trophoblasts, while CTHRC1 overexpression promoted nuclear translocation of β-catenin, a result that was further confirmed by TOP/FOP luciferase activity assay. Our findings suggest that CTHRC1 promotes migration and invasion of trophoblasts via reciprocal Wnt/β-catenin signaling pathway. Down-regulation of CTHRC1 may be a potential mechanism underpinning the development of preeclampsia.     

KIF2C: a novel link between Wnt/β-catenin and mTORC1 signaling in the pathogenesis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and is the fourth-leading cause of cancer-related deaths worldwide. HCC is refractory to many standard cancer treatments and the prognosis is often poor, highlighting a pressing need to identify biomarkers of aggressiveness and potential targets for future treatments. Kinesin family member 2C (KIF2C) is reported to be highly expressed in several human tumors. Nevertheless, the molecular mechanisms underlying the role of KIF2C in tumor development and progression have not been investigated. In this study, we found that KIF2C expression was significantly upregulated in HCC, and that KIF2C up-regulation was associated with a poor prognosis. Utilizing both gain and loss of function assays, we showed that KIF2C promoted HCC cell proliferation, migration, invasion, and metastasis both in vitro and in vivo. Mechanistically, we identified TBC1D7 as a binding partner of KIF2C, and this interaction disrupts the formation of the TSC complex, resulting in the enhancement of mammalian target of rapamycin complex1 (mTORC1) signal transduction. Additionally, we found that KIF2C is a direct target of the Wnt/β-catenin pathway, and acts as a key factor in mediating the crosstalk between Wnt/β-catenin and mTORC1 signaling. Thus, the results of our study establish a link between Wnt/β-catenin and mTORC1 signaling, which highlights the potential of KIF2C as a therapeutic target for the treatment of HCC.Electronic supplementary materialThe online version of this article (10.1007/s13238-020-00766-y) contains supplementary material, which is available to authorized users.     

Dok5 regulates proliferation and differentiation of osteoblast via canonical Wnt/β-catenin signaling

Objective:In bone tissue engineering, the use of osteoblastic seed cells has been widely adopted to mediate the osteogenic differentiation so as to prompt bone regeneration and repair. It is hypothesized that Dok5 can regulate the proliferation and differentiation of osteoblasts. In this study, the role of Dok5 in osteoblast proliferation and differentiation was investigated.Methods:A lentiviral vector to silence Dok5 was transferred to C3H10, 293T and C2C12 cells. CCK-8 assay was used to detect the cell proliferation. Cells were stained by ALP and AR-S staining. Western blot and RT-PCR were used to detect the expression levels of related factors.Results:Dok5 expression level was gradually up-regulated during the osteoblast differentiation. Dok5 silencing down-regulated the expression levels of osteogenic biosignatures OPN, OCN, and Runx2 and suppressed the osteogenesis. Additionally, the osteoblast proliferation and canonical Wnt/β-catenin signaling were suppressed upon Dok5 knockdown, β-catenin expression level was significantly down-regulated in the knockdown group, while the expression levels of GSK3-β and Axin, negative regulators in the Wnt signaling pathway, were up-regulated. Furthermore, overexpression of Dok5 promoted the proliferation and osteogenesis and activated the canonical Wnt/β-catenin signaling pathway.Conclusion:Dok5 may regulate the osteogenic proliferation and differentiation via the canonical Wnt/β-catenin signaling pathway.     

Neuron specific enolase promotes tumor metastasis by activating the Wnt/β-catenin pathway in small cell lung cancer

Neuron-specific enolase (NSE) has been used as a specific biomarker for small cell lung cancer (SCLC) patients. Nevertheless, the biological function and mechanism of NSE in SCLC are still unclear. In this study, we clarified the role of NSE in the progression of SCLC and found that NSE expression was positively correlated with distant metastasis. Functional analysis showed that overexpression of NSE promoted migration and invasion of SCLC cells. Mechanism analysis showed that NSE overexpression induced epithelial-mesenchymal transition (EMT) of SCLC cells. Moreover, overexpression of NSE increased the protein expression of β-catenin and its downstream target genes, and silencing β-catenin eliminated NSE-mediated cell migration, invasion and EMT process. Furthermore, NSE interacted with β-catenin and inhibited the degradation of β-catenin. Besides, the animal experiments also indicated that NSE could promote the EMT process and distant metastasis of SCLC cells in vivo. In summary, our results revealed that NSE could promote the EMT process of SCLC cells by activating the Wnt/β-catenin signaling pathway, thereby promoting cell migration, invasion and distant metastasis, which might serve as a potential target for the therapy of SCLC patients.     

Inhibition of ClC-5 suppresses proliferation and induces apoptosis in cholangiocarcinoma cells through the Wnt/β-catenin signaling pathway

Chloride channel-5 (ClC-5), an important branch of the ClC family, is involved in the regulation of the proliferation and cell-fate of a variety of cells, including tumor cells. However, its function in cholangiocarcinoma (CCA) cells remains enigmatic. Here, we discovered that ClC-5 was up-regulated in CCA tissues and CCA cell lines, while ClC-5 silencing inhibited CCA cell proliferation and induced apoptosis. Further mechanism studies revealed that ClC-5 inhibition could inhibit Wnt/β-catenin signaling activity and further activate the mitochondria apoptotic pathway in CCA cells. Furthermore, rescuing Wnt/β-catenin signaling activation eliminated the anti-tumor function of ClC-5 knockdown. Together, our research findings illustrated that ClC-5 inhibition plays an anti-tumor role in CCA cells via inhibiting the activity of the Wnt/β-catenin pathway, which in turn activates the mitochondrial apoptotic pathway.     

PRMT5 acts as a tumor suppressor by inhibiting Wnt/β-catenin signaling in murine gastric tumorigenesis

Previous studies have demonstrated the in vitro oncogenic role of protein arginine methyltransferase 5 (PRMT5) in gastric cancer cell lines. The in vivo function of PRMT5 in gastric tumorigenesis, however, is still unexplored. Here, we showed that Prmt5 deletion in mouse gastric epithelium resulted in spontaneous tumorigenesis in gastric antrum. All Prmt5-deficient mice displayed intestinal-type gastric cancer within 4 months of age. Of note, 20% (2/10) of Prmt5 mutants finally developed into invasive gastric cancer by 8 months of age. Gastric cancer caused by PRMT5 loss exhibited the increase in Lgr5⁺ stem cells, which are proposed to contribute to both the gastric tumorigenesis and progression in mouse models. Consistent with the notion that Lgr5 is the target of Wnt/β-catenin signaling, whose activation is the most predominant driver for gastric tumorigenesis, Prmt5 mutant gastric cancer showed the activation of Wnt/β-Catenin signaling. Furthermore, in human gastric cancer samples, PRMT5 deletion and downregulation were frequently observed and associated with the poor prognosis. We propose that as opposed to the tumor-promoting role of PRMT5 well-established in the progression of various cancer types, PRMT5 functions as a tumor suppressor in vivo, at least during gastric tumor formation.     

GALNT1 Enhances Malignant Phenotype of Gastric Cancer via Modulating CD44 Glycosylation to Activate the Wnt/β-catenin Signaling Pathway

O-glycosylation is a widespread post-translational modification of proteins. Aberrant O-glycosylation is a hallmark of cancer. Here, we show that the polypeptide N-acetylgalactosamine-transferase 1 (GALNT1) is frequently upregulated in gastric cancer and is correlated with poor survival. Overexpression of GALNT1 promoted, whereas knockdown suppressed proliferation, migration, and invasion of gastric cancer cells in vitro and in vivo. Mechanistically, GALNT1 enhances aberrant initiation of O-glycosylation and results in CD44 glycoproteins modified with abundant Tn antigens, thereby activating the Wnt/β-catenin signaling pathway. Collectively, this study demonstrates that GALNT1 overexpression in gastric cancer promotes the Wnt/β-catenin signaling pathway via abnormal O-glycosylation of CD44 to enhance malignancy, providing a novel strategy for the development of therapeutic reagents against gastric cancer.     

Endocytosis of Peptidase Inhibitor SerpinE2 promotes Myocardial Fibrosis through activating ERK1/2 and β-catenin Signaling Pathways

Cardiac fibrosis is one of the common pathological processes in many cardiovascular diseases characterized by excessive extracellular matrix deposition. SerpinE2 is a kind of protein that inhibits peptidase in extracellular matrix and up-regulated tremendously in mouse model of cardiac fibrosis induced by pressure-overloaded via transverse aortic constriction (TAC) surgery. However, its effect on cardiac fibroblasts (CFs), collagen secretion and the underlying mechanism remains unclear. In this study, DyLight® 488 green fluorescent dye or His-tagged proteins were used to label the exogenous serpinE2 protein. It was showed that extracellular serpinE2 translocated into CFs by low-density lipoprotein receptor-related protein 1 (LRP1) and urokinase plasminogen activator receptor (uPAR) of cell membrane through endocytosis. Knockdown of LRP1 or uPAR reduced the level of serpinE2 in CFs and down-regulated the collagen expression. Inhibition of the endocytosis of serpinE2 could inhibit ERK1/2 and β-catenin signaling pathways and subsequently attenuated collagen secretion. Knockdown of serpinE2 attenuates cardiac fibrosis in TAC mouse. We conclude that serpinE2 could be translocated into cardiac fibroblasts due to endocytosis through directly interact with the membrane protein LRP1 and uPAR, and this process activated the ERK1/2, β-catenin signaling pathways, consequently promoting collagen production.     

Phosphorylation of human CEACAM1-LF by PKA and GSK3β promotes its interaction with β-catenin

CEACAM1-LF, a homotypic cell adhesion adhesion molecule, transduces intracellular signals via a 72 amino acid cytoplasmic domain that contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and a binding site for β-catenin. Phosphorylation of Ser503 by PKC in rodent CEACAM1 was shown to affect bile acid transport or hepatosteatosis via the level of ITIM phosphorylation, but the phosphorylation of the equivalent residue in human CEACAM1 (Ser508) was unclear. Here we studied this analogous phosphorylation by NMR analysis of the ¹⁵N labeled cytoplasmic domain peptide. Incubation with a variety of Ser/Thr kinases revealed phosphorylation of Ser508 by GSK3bβ but not by PKC. The lack of phosphorylation by PKC is likely due to evolutionary sequence changes between the rodent and human genes. Phosphorylation site assignment by mass spectrometry and NMR revealed phosphorylation of Ser472, Ser461 and Ser512 by PKA, of which Ser512 is part of a conserved consensus site for GSK3β binding. We showed here that only after phosphorylation of Ser512 by PKA was GSK3β able to phosphorylate Ser508. Phosphorylation of Ser512 by PKA promoted a tight association with the armadillo repeat domain of β-catenin at an extended region spanning the ITIMs of CEACAM1. The kinetics of phosphorylation of the ITIMs by Src, as well dephosphorylation by SHP2, were affected by the presence of Ser508/512 phosphorylation, suggesting that PKA and GSK3β may regulate the signal transduction activity of human CEACAM1-LF. The interaction of CEACAM1-LF with β-catenin promoted by PKA is suggestive of a tight association between the two ITIMs of CEACAM1-LF.     

SNHG1 represses the anti-cancer roles of baicalein in cervical cancer through regulating miR-3127-5p/FZD4/Wnt/β-catenin signaling

As a flavonoid, baicalein exhibits remarkable anti-cancer roles in several cancers. However, the factors regulating the antitumorigenic roles of baicalein in cervical cancer remain undefined. Here, we revealed that long noncoding RNA SNHG1 is implicated in the tumor-suppressive roles of baicalein. Functional assays demonstrated that ectopic expression of SNHG1 attenuates the roles of baicalein in repressing cervical cancer cell viability, inducing apoptosis, and repressing migration. SNHG1 silencing promotes the tumor-suppressive roles of baicalein in cervical cancer cell viability, apoptosis, and migration. Xenograft assays showed that SNHG1 reverses the tumor-suppressive roles of baicalein in repressing cervical cancer growth in vivo. Mechanistic investigations revealed that SNHG1 directly binds miR-3127-5p and up-regulates FZD4, a target of miR-3127-5p. Via regulating miR-3127-5p/FZD4, SNHG1 activates Wnt/β-catenin signaling. Moreover, SNHG1 reverses the repressive role of baicalein on Wnt/β-catenin signaling. The effect of SNHG1 on the antitumorigenic process of baicalein was abolished by Wnt/β-catenin signaling inhibitor ICG-001. Together, our observations demonstrated that SNHG1 represses the tumor-suppressive roles of baicalein in cervical cancer through regulating miR-3127-5p/FZD4/Wnt/β-catenin axis, and suggested that targeting SNHG1 represents a potential strategy to enhance the tumor-suppressive roles of baicalein in cervical cancer.Impact statementBaicalein exhibits anti-cancer roles in several cancers. However, the factors influencing the antitumorigenic efficiencies of baicalein in CC remain largely unclear. Here, we provide convincing evidences that lncRNA SNHG1 attenuates the tumor-suppressive roles of baicalein in CC cell viability, apoptosis, migration, and CC tumor growth. This study further demonstrates that the influences of SNHG1 in the antitumorigenic process of baicalein are achieved through modulating the miR-3127-5p/FZD4Wnt/β-catenin axis. SNHG1 attenuates the repressive role of baicalein on Wnt/β-catenin. Therefore, SNHG1 is a novel modulator of the tumor-suppressive roles of baicalein and SNHG1 represents a therapeutic intervention target to reinforce the tumor-suppressive roles of baicalein in CC.