Knockdown Of Beta-catenin Research Articles

FOXC1 induces cancer stem cell-like properties through upregulation of beta-catenin in NSCLC

BackgroundAccumulating evidence suggests that cancer stem cells (CSCs) play a critical role in tumor initiation, progression and therapy, and recent studies have indicated that Forkhead box C1 (FOXC1) is strongly associated with CSCs. This study investigates the regulatory effects of FOXC1 on CSC-like properties in non-small cell lung cancer (NSCLC).MethodsWe analyzed FOXC1 expression in NSCLC using the Cancer Genome Atlas (TCGA) database on UALCANC and performed survival analyses of NSCLC patients on Human Protein Atlas. CSC-like properties were analyzed based on CSC marker-positive cell population, self-renewal ability, stemness-related gene expression, tumorigenicity and drug resistance. The percentage of CD133+ cells was analyzed by flow cytometric analysis. Self-renewal ability was detected by sphere-formation analysis. Real-time PCR, western blotting and immunohistochemical staining were employed to detect mRNA and protein levels. Tumorigenicity was determined based on a xenograft formation assay, and effects of FOXC1 on drug resistance were assessed by cell viability and apoptosis assays. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays were used to investigate the binding of FOXC1 to beta-catenin promoter.ResultsFOXC1 expression was found to be elevated in NSCLC tissues and negatively correlated with patient survival. FOXC1 knockdown reduced CD133+ cell percentage, suppressed self-renewal ability, decreased expression of stemness-related genes (Oct4, NANOG, SOX2 and ABCG2) and inhibited NSCLC cell tumorigenicity in vivo. Moreover, FOXC1 knockdown increased cisplatin and docetaxel sensitivity and reduced gefitinib resistance, whereas FOXC1 overexpression enhanced CSC-like properties. Luciferase reporter and ChIP assays showed beta-catenin to be a direct transcriptional target of FOXC1. Furthermore, overexpression of beta-catenin reversed the CSC-like property inhibition induced by FOXC1 knockdown, and knockdown of beta-catenin attenuated the CSC-like properties induced by FOXC1 overexpression.ConclusionsThis study demonstrates that FOXC1 induces CSC-like properties in NSCLC by promoting beta-catenin expression. The findings indicate that FOXC1 is a potential molecular target for anti-CSC-based therapies in NSCLC.

Abstract 4896: Role of CDK8 in colon cancer hepatic metastasis

Abstract About half of all colon cancer patients will develop liver metastases and the 5-year survival for these patients is less than 13%, making colon cancer the second most lethal cancer worldwide. Cyclin dependent kinase 8 (CDK8), which regulates transcription but not cell cycle progression, has been identified as an oncogene amplified in many colon cancers. CDK8 acts as a positive mediator of oncogenic transcription pathways regulated by Wnt/beta-catenin and TGF-beta, both of which are strongly associated with tumor metastasis. In a colon cancer liver metastasis model based on splenic injection of CT26 murine colon carcinoma cells, treatment with Senexin B, a highly selective small-molecule inhibitor of CDK8 and its paralog CDK19, strongly inhibited metastatic growth in the liver and prolonged the survival of mice with hepatic metastases. In contrast to the effect on liver metastasis, CDK8 inhibition had little or no effect on cell growth in culture or at primary tumor sites. Hepatic metastasis was inhibited to the same extent when Senexin B was administered starting from the time of tumor injection or only during a later part of the study, suggesting that the drug affected metastatic growth in the liver rather than just initial colonization. Liver metastasis was similarly decreased by CDK8 knockdown in CT26 cells, suggesting that the anti-metastatic activity of Senexin B was due at least in part to its effect on tumor cells. Transcription profiling indicated that CDK8 inhibition by shRNA or Senexin B strongly decreased the expression of metastasis-associated metalloproteinases MMP 13, MMP10, and MMP3 and at the same time drastically increased the expression of TIMP3, a metalloproteinase inhibitor. TIMP3 overexpression in CT26 cells, like CDK8 inhibition, decreased hepatic metastasis. CDK8 inhibition also blocked TGF-beta or Wnt3a-stimulated transwell Matrigel invasion of CT26 cells in vitro. Knockdown of beta-catenin in CT26 cells decreased the expression of MMP3 and MMP13, while knockdown of SMAD4 (transcriptional mediator of TGF-beta pathway) induced TIMP3 expression. Beta-catenin knockdown decreased both the primary tumor growth in the spleen and metastatic growth in the liver, whereas SMAD4 knockdown, like that of CDK8, selectively inhibited liver metastasis. Senexin B also suppressed hepatic metastasis after splenic injection of human HCT116 colon carcinoma cells. In summary, our data identified CDK8 as a key transcriptional regulator of colon cancer metastatic growth in the liver, interacting with TGF-beta and Wnt/beta-catenin pathways and regulating the expression of MMPs and Timp3. CDK8/19 inhibitors, which are now entering clinical trials, may be effective for the treatment of hepatic metastasis of colon cancer. Citation Format: Jiaxin Liang, Mengqian Chen, Mythreye Karthikeyan, M. Marjorette Pena, Daniel Hughes, Vimala Kaza, Chang-Uk Lim, Eugenia Broude, Igor B. Roninson. Role of CDK8 in colon cancer hepatic metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4896. doi:10.1158/1538-7445.AM2017-4896

Abstract 5177: E7386 : First-in-class orally active CBP/beta-catenin modulator as an anticancer agent

Abstract Carcinogenesis is often accelerated by the aberrant activation of components molecules of Wnt signaling pathway, especially, APC and beta-catenin are frequently reported to be mutated in various cancers. Therefore, Wnt signal pathway is thought to be one of the attractive therapeutic targets. PRI-724 generated by PRISM Pharma is a small molecule inhibitor of beta-catenin and its transcriptional coactivator CREB binding protein (CBP) thereby specific modulating Wnt/beta-catenin signaling pathway by intravenous continuous infusion. Here we firstly generated orally active small molecular inhibitor, E7386. E7386 disrupted the interaction between beta-catenin and CBP in co-immunoprecipitation assay. E7386 inhibited canonical Wnt signaling pathway /TCF reporter gene activity in LiCl-stimulated HEK-293 and MDA-MB-231 in a dose dependent manner with IC50 values of 55 nmol/L and 73 nmol/L, respectively. E7386 modulated the expression of Wnt signaling pathway related genes including AXIN2 and other genes, which were down-regulated by artificial knockdown of beta-catenin. These results indicate that E7386 controls the expression of Wnt target genes through modulation of beta-catenin/CBP interaction. Next we investigated anti-polyposis effect in ApcMin/+ mice as an in vivo proof of mechanism model. ApcMin/+ mice develops polyps in the intestinal tract caused by the aberrant activation of Wnt/beta-catenin signaling pathway. Oral administration of E7386 significantly suppressed the number of polyposis in a dose dependent manner at the dose range from 8.5 to 50 mg/kg. In addition, E7386 significantly changed the expressions of Wnt related genes in whisker follicle of ApcMin/+mice model. Finally, we investigated anti-tumor activity of E7386 in vitro tumor proliferation panel against 28 human tumor cell lines. E7386 showed relatively potent anti-proliferative activity against cancer cell lines harboring exclusively mutated Wnt signaling pathway molecules such as APC or beta-catenin. E7386 also showed significant antitumor activity in a dose dependent manner on human tumor cell line xenograft harboring APC mutation. Taken together, E7386 is a first in class orally active CBP/beta-catenin modulator and showed potent inhibitory activity against aberrant activation of Wnt/beta-catenin signaling pathway. Citation Format: Kazuhiko Yamada, Yusaku Hori, Atsumi Yamaguchi, Masahiro Matsuki, Shuntaro Tsukamoto, Akira Yokoi, Taro Semba, Yoichi Ozawa, Satoshi Inoue, Yuji Yamamoto, Kentaro Iso, Kazutaka Nakamoto, Hitoshi Harada, Naoki Yoneda, Atsushi Takemura, Masayuki Matsukura,, Kenji Kubara, Takenao Odagami, Masao Iwata, Akihiko Tsuruoka, Toshimitsu Uenaka, Junji Matsui, Tomohiro Matsushima, Kenich Nomoto, Hiroyuki Kouji, Takashi Owa. E7386 : First-in-class orally active CBP/beta-catenin modulator as an anticancer agent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5177. doi:10.1158/1538-7445.AM2017-5177

SFRP4-Mediated Inhibition of Canonical Wnt-Signaling in DLBCL

Introduction: Wnt-signaling enhances proliferation in a variety of malignancies including diffuse large B-cell lymphoma (DLBCL) and exosomes, small secreted microvesicles, are known to carry active wnt-molecules to mediate wnt-signaling. We found consistent suppression of SFRP4, an inhibitor of canonical wnt-signaling in DLBCL, elucidated it's mechanism of action and addressed it´s impact on regulating proliferation of diffuse large B-cell lymphomas. We conducted a screen of chemical wnt-inhibitors and assessed wnt-addiction in DLBCL cell lines.Material & Methods: The Cancer Cell Line Encyclopedia (CCLE) was used to assess the transcript abundace of SFRP4 in 18 DLBCL cell lines. SFRP4-expression was confirmed using western blot and immunofluorescence in representative DLBCL cell lines. Purified exosomes of DLBCL cell lines, recombinant human SFRP4 and SFRP4-shRNA were used to address the biological mechanism of SFRP4 action. 5-Azacytidine was used to restore SFRP4-expression. Furthermore shRNA mediated knockdown of beta-catenin and pharmacological inhibition of canonical wnt-signaling using XAV-939, IWP-2, C59 and UM206 were used to address wnt-addiction in DLBCL cell lines.Results: We found SFRP4 to be significantly suppressed in a variety of malignancies, including DLBCL, with promotor methylation as a known mechanism of suppression. SFRP4 was able to suppress exosomal mediated canonical wnt-signaling. SFRP4 inhibited proliferation in DLBCL cell lines and enhanced cytotoxicity of doxorubicin. Treatment with 5-Azacytidine restored expression of SFRP4, induced decline of beta-catenin and suppressed cell proliferation. Knock down of beta-catenin and pharmacological perturbation of canonical wnt-signaling identified wnt-addiction in beta-catenin expressing cell lines in the absence of a MYC translocation or amplification.Conclusions: SFRP4 suppresses proliferation of DLBCL cell lines by antagonizing exosomal mediated canonical wnt-signaling. Pharmacological inhibition of canonical wnt-signaling significantly decreased proliferation of DLBCL cell lines in the absence of genomic alterations involving MYC. DisclosuresNo relevant conflicts of interest to declare.

358 Knockdown of beta-catenin with dicer-substrate siRNAs down-regulates Wnt/beta-catenin pathway signaling

358 Knockdown of beta-catenin with dicer-substrate siRNAs down-regulates Wnt/beta-catenin pathway signaling

Inhibition of WNT signalling interferes with HCV replication

Background and Aims: The tumor relevant wnt pathway activity is associated with higher HCV replication. HCV core and NS5 protein have recently been shown to induce wnt pathway activity, implicating a cause for progression to HCC. We now investigated if wnt pathway activity might affect HCV replication. Methods: For in vitro experiments the HCV replicon system expressing HCV genes NS3 to NS5B alone (Huh5–15) or in combination with firefly luciferase (LucUbiNeo-ET) was used. To promote wnt pathway activity the glycogen synthase kinase 3 beta (GSK3b) inhibitors Kenpaullone or SB216763 were used. Inhibition of the wnt pathway was achieved by use of either Quercetin or 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), an inhibitor of beta-catenin stabilizing kinase CK2, or by transient knockdown of beta-catenin by siRNA. Gene expression was measured by real time RT-PCR and Western Blot analysis. Wnt pathway activity was measured by a reporter assay for beta catenin related transcription (CRT assay). Results: Our results confirm that activity of the wnt pathway is increased in replicon cell lines compared to the replicon background cell line Huh7 and that the HCV protein NS5A is involved. Additionally we found that NS3B is able to increase Wnt pathway activity. Inhibition of the wnt signaling pathway by quercetin, TBB or siRNA directed against beta-catenin reduced HCV replication significantly without toxic effects on cultured cells. Moreover, activation of wnt pathway activity by inhibitors of GSK3b increased HCV replication. Conclusions: HCV replication induces wnt signaling which in turn seems to promote HCV replication. Therefore, inhibition of the wnt signaling pathway might not only represent a way to interfere with tumor growth but might also become a novel approach for HCV therapy.

Tau Overexpression Inhibits Cell Apoptosis with the Mechanisms Involving Multiple Viability-Related Factors

The formation of neurofibrillary tangles, mainly composed of hyperphosphorylated tau protein, is a hallmark in the brain of human tauopathies, including Alzheimer's disease (AD). Although neurons bearing neurofibrillary tangles are constantly exposed to various apoptotic stimuli, they do not appear to preferentially die by apoptosis. The underlying mechanism for such resistance to apoptosis remains elusive. Previously, we studied the role of tau phosphorylation in apoptosis and found that tau hyperphosphorylation by glycogen synthase kinase-3 (GSK-3) rendered cells more resistant to apoptosis. In this study, we show that the overexpression of tau without any exogenous activation of kinases also confers increased resistance to apoptosis in both N2a cells and in a tau transgenic mouse model. Mechanistically, the overexpression of tau was associated with a reduced p53 level, decreased release of cytochrome C from mitochondria, and inhibition of caspases-9/-3. Additionally, a decreased phosphorylation and increased nuclear translocation of beta-catenin were also detected in N2a/tau cells, and knockdown of beta-catenin eliminated the anti-apoptotic effect of tau. Furthermore, tau was spontaneously hyperphosphorylated upon overexpression and by staurosporine treatment. The phosphorylation level of p53 decreased upon tau overexpression, and a more profound reduction of the phosphorylated p53 was detected when the cells were treated with lithium and roscovitine, inhibitors of GSK-3 and cyclin-dependent kinase-5 (Cdk-5). These results suggest that the overexpression of tau, which may be hyperphosphorylated by endogenous GSK-3 and Cdk-5, is anti-apoptotic by mechanisms involving modulation of multiple anti-apoptotic factors, including beta-catenin and p53-mitochondria-caspase-mediated apoptotic pathways.

Conductin/axin2 and Wnt signalling regulates centrosome cohesion

Activated Wnt/beta-catenin signalling is a characteristic of many cancers and drives cell-cycle progression. Here, we report a mechanism linking Wnt/beta-catenin signalling to centrosome separation. We show that conductin/axin2, a negative regulator of beta-catenin, localizes at the centrosomes by binding to the centriole-associated component C-Nap1. Knockout or knockdown of conductin leads to premature centrosome separation--that is, splitting--which is abolished by knockdown of beta-catenin. Conductin promotes phosphorylation of the amino-terminal serine (Ser 33/37) and threonine (Thr 41) residues of centrosome-associated beta-catenin. Beta-catenin mutated at these residues causes centrosomal splitting, whereas a phospho-mimicking mutant of beta-catenin does not. Importantly, beta-catenin-induced splitting is not inhibited by blocking beta-catenin-dependent transcription. Treatment with Wnts and inhibition of glycogen synthase kinase 3 block beta-catenin phosphorylation and induce centrosomal splitting. These data indicate that Wnt/beta-catenin signalling and conductin regulate centrosomal cohesion by altering the phosphorylation status of beta-catenin at the centrosomes.

Global Regulator SATB1 Recruits β-Catenin and Regulates TH2 Differentiation in Wnt-Dependent Manner

Author SummaryIn vertebrates the canonical Wnt signalling culminates in β-catenin moving into the nucleus where it activates transcription of target genes. Wnt/β-catenin signalling is essential for the thymic maturation and differentiation of naïve T cells. Here we show that SATB1, a T cell lineage-enriched chromatin organizer and global regulator, binds to β-catenin and recruits it to SATB1's genomic binding sites so that genes formerly repressed by SATB1 are upregulated by Wnt signalling. Some of the genes known to be regulated by SATB1 (such as genes encoding cytokines and the transcription factor GATA3) are required for differentiation of Th2 cells, an important subset of helper T cells. Specifically we show that siRNA-mediated knockdown of SATB1 downregulated GATA-3 expression in differentiating human CD4+ T cells. Inhibiting Wnt signalling led to downregulation of GATA-3 and of signature TH2 cytokines such as IL-4, IL-10, and IL-13. Knockdown of β-catenin also produced similar results, thus together these data confirm the role of Wnt/β-catenin signalling in TH2 differentiation. Our data demonstrate that SATB1 orchestrates TH2 lineage commitment by modulating Wnt/β-catenin signalling.

THIS ARTICLE HAS BEEN RETRACTED Epstein–Barr virus‐encoded latent membrane protein 1 activates β‐catenin signaling in B lymphocytes

The Epstein-Barr virus-encoded latent membrane protein 1 is considered the Epstein-Barr virus oncogene based on its importance in Epstein-Barr virus-induced B-lymphocyte transformation. Beta-catenin is a potential oncogene, and its accumulation has been implicated in a variety of human cancers. Here, we found that beta-catenin protein was highly expressed in Epstein-Barr virus-immortalized B-cell lines compared with peripheral blood mononuclear cells from healthy donors. Beta-catenin expression in Epstein-Barr virus-immortalized B-cell line decreased following treatment with LY294002, an inhibitor of phosphatidylinositol 3-kinase. Treatment with LY294002 or knockdown of beta-catenin by small interfering RNA reduced the growth of Epstein-Barr virus-immortalized B-cell line. Transient transfection of latent membrane protein 1 expression plasmid increased beta-catenin protein expression and beta-catenin-dependent transcription. Latent membrane protein 1 deletions mutants lacking the carboxyl-terminal activating region 1 domain failed to enhance beta-catenin protein expression and beta-catenin-dependent transcriptional activity. They also failed to increase phosphorylated AKT expression. Dominant-negative AKT suppressed latent membrane protein 1-induced beta-catenin-dependent transcriptional activity. These results suggest that latent membrane protein 1 activates beta-catenin through the phosphatidylinositol 3-kinase/AKT signaling pathway. Activation of the beta-catenin pathway by Epstein-Barr virus may contribute to the lymphoproliferation characteristic of Epstein-Barr virus-infected B-cells.

Wnt Signaling Inhibits Forkhead Box O3a-induced Transcription and Apoptosis through Up-regulation of Serum- and Glucocorticoid-inducible Kinase 1

In human cancers, mutations in components of the Wnt signaling pathway lead to beta-catenin stabilization and result in augmented gene transcription. HCT116 colon cancer cells carry stabilizing mutations in beta-catenin and exhibit an elevated activation of Wnt signaling. To clarify the role of an overactive Wnt signaling, we used DNA microarray analysis to search for genes whose expression is up-regulated after knockdown of the wild type adenomatous polyposis coli (APC) tumor suppressor in HCT116 cells, which further enhances Wnt signaling activation. Serum and glucocorticoid-inducible kinase 1 (SGK1) was among the most up-regulated genes following APC knockdown through small interfering RNA. Up-regulation of SGK1 in response to small interfering RNA against APC was inhibited by concomitant knockdown of beta-catenin. Quantitative real time reverse transcription-PCR, Western blot, and chromatin immunoprecipitation analyses confirmed that SGK1 is a direct beta-catenin target gene. SGK1 negatively regulates the pro-apoptotic transcription factor Forkhead box O3a (FoxO3a) via phosphorylation and exclusion from the nucleus. We show that Wnt signaling activation results in FoxO3a exclusion from the nucleus and inhibits expression of FoxO3a target genes. Importantly, FoxO3a mutants that fail to be phosphorylated and therefore are regulated by SGK1 are not influenced by activation of Wnt signaling. In line, knockdown of SGK1 relieves the effects of Wnt signaling on FoxO3a localization and FoxO3a-dependent transcription. Finally, we show that induction of Wnt signaling inhibits FoxO3a-induced apoptosis. Collectively our results indicate that evasion of apoptosis is another feature employed by an overactive Wnt signaling.

β‐catenin signalling in mesenchymal islet‐derived precursor cells

Previously, we characterized human islet-derived precursor cells (hIPCs) as mesenchymal stem cells that migrate out from islets in vitro and can differentiate into functional islet-like structures following proliferative expansion. Here, we investigate the role of beta-catenin signalling in derivation and proliferation of hIPCs. Localization of beta-catenin was performed using confocal microscopy. Expression levels of beta-catenin target genes were measured by quantitative real-time polymerase chain reaction. Loss-of-function studies were performed using specific short interfering RNAs. Immunostaining of islet outgrowths revealed translocation of beta-catenin from plasma membranes in intact islets to the nucleus in cells migrating out. There were no nuclear beta-catenin-positive cells in intact islets whereas between 35% and 70% of cells in established hIPC cultures exhibited nuclear beta-catenin. Transcripts for beta-catenin target genes were increased in hIPCs compared to those in islets. Beta-catenin translocated to the cell membrane when hIPCs formed epithelial cell clusters. In proliferating hIPCs, there was a strong correlation between markers of proliferation and nuclear beta-catenin. Treatment of hIPCs with the glycogen synthase kinase-3beta inhibitor (2'Z,3'E)-6-Bromoindirubin-3'-oxime increased intracellular beta-catenin but reduced nuclear beta-catenin, and was associated with reduced cell proliferation. Finally, knockdown of beta-catenin decreased beta-catenin target gene expression and hIPC proliferation. These results support a functional role for beta-catenin during proliferation of hIPCs and suggest that activated beta-catenin signalling may also be important during hIPC derivation from islets.

Efficiency of si-RNA-mediated knock-down of beta-catenin in primary cultured rat Schwann cells

Efficiency of si-RNA-mediated knock-down of beta-catenin in primary cultured rat Schwann cells