Fbxw7 acts as an E3 ubiquitin ligase that targets c-Myb for nemo-like kinase (NLK)-induced degradation

The c-myb proto-oncogene product (c-Myb) is degraded in response to Wnt-1 signaling via a pathway involving TAK1 (transforming growth factor-beta-activated kinase 1), HIPK2 (homeodomain-interacting protein kinase 2), and NLK (Nemo-like kinase). NLK directly binds to c-Myb, which results in the phosphorylation of c-Myb at multiple sites, and induces its ubiquitination and proteasome-dependent degradation. Here, we report that Fbxw7, the F-box protein of an SCF complex, targets c-Myb for degradation in a Wnt-1- and NLK-dependent manner. Fbxw7alpha directly binds to c-Myb via its C-terminal WD40 domain and induces the ubiquitination of c-Myb in the presence of NLK in vivo and in vitro. The c-Myb phosphorylation site mutant failed to interact with Fbxw7alpha, suggesting that the c-Myb/Fbxw7alpha interaction is enhanced by NLK phosphorylation of c-Myb. Treatment of M1 cells with Fbxw7 small interfering RNA (siRNA) rescued the Wnt-induced c-Myb degradation and also the Wnt-induced inhibition of cell proliferation. NLK bound to Cul1, a component of the SCF complex, while HIPK2 interacted with both Fbxw7alpha and Cul1, suggesting that both kinases enhance the c-Myb/SCF interaction. In contrast to c-Myb, the v-myb gene product (v-Myb) encoded by the avian myeloblastosis virus was resistant to NLK/Fbxw7alpha-induced degradation. Thus, Fbxw7 is an E3 ubiquitin ligase of c-Myb, and the increased c-Myb levels may contribute, at least partly, to transformation induced by mutation of Fbxw7.     

Differential sensitivity of v-Myb and c-Myb to Wnt-1-induced protein degradation

Recently we have shown that the c-myb proto-oncogene product (c-Myb) is degraded in response to Wnt-1 signaling via the pathway involving TAK1 (transforming growth factor-beta-activated kinase), HIPK2 (homeodomain-interacting protein kinase 2), and NLK (Nemo-like kinase). NLK and HIPK2 bind directly to c-Myb, which results in the phosphorylation of c-Myb at multiple sites, followed by its ubiquitination and proteasome-dependent degradation. The v-myb gene carried by avian myeloblastosis virus has a transforming capacity, but the c-myb proto-oncogene does not. Here, we report that two characteristics of v-Myb make it relatively resistant to Wnt-1-induced protein degradation. First, HIPK2 binds with a lower affinity to the DNA-binding domain of v-Myb than to that of c-Myb. The mutations of three hydrophobic amino acids on the surface of the DNA-binding domain in v-Myb decrease the affinity to HIPK2. Second, a loss of multiple NLK phosphorylation sites by truncation of the C-terminal region of c-Myb increases its stability. Among 15 putative NLK phosphorylation sites in mouse c-Myb, the phosphorylation sites in the C-terminal region are more critical than other sites for Wnt-1-induced protein degradation. The relative resistance of v-Myb to Wnt-1-induced degradation may explain, at least in part, the differential transforming capacity of v-Myb versus c-Myb.     

The Wnt-NLK signaling pathway inhibits A-Myb activity by inhibiting the association with coactivator CBP and methylating histone H3

The c-myb proto-oncogene product (c-Myb) regulates proliferation and differentiation of hematopoietic cells. Recently we have shown that c-Myb is degraded in response to Wnt-1 stimulation via a pathway involving TAK1 (TGF-beta-activated kinase), HIPK2 (homeodomain-interacting protein kinase 2), and NLK (Nemo-like kinase). NLK and HIPK2 bind directly to c-Myb and phosphorylate c-Myb at multiple sites, inducing its ubiquitination and proteasome-dependent degradation. The mammalian myb gene family contains two members in addition to c-myb, A-myb, and B-myb. Here, we report that the Wnt-NLK pathway also inhibits A-Myb activity, but by a different mechanism. As in the case of c-Myb, both NLK and HIPK2 bound directly to A-Myb and inhibited its activity. NLK phosphorylated A-Myb, but did not induce A-Myb degradation. Overexpression of NLK inhibited the association between A-Myb and the coactivator CBP, thus, blocking A-Myb-induced trans-activation. The kinase activity of NLK is required for the efficient inhibition of the association between A-Myb and CBP, although the kinase-negative form of NLK also partly inhibits the interaction between A-Myb and CBP. Furthermore, NLK induced the methylation of histone H3 at lysine-9 at A-Myb-bound promoter regions. Thus, the Wnt-NLK pathway inhibits the activity of each Myb family member by different mechanisms.     

Association of Nuclear-Localized Nemo-Like Kinase with Heat-Shock Protein 27 Inhibits Apoptosis in Human Breast Cancer Cells

Nemo-like kinase (NLK), a proline-directed serine/threonine kinase regulated by phosphorylation, can be localized in the cytosol or in the nucleus. Whether the localization of NLK can affect cell survival or cell apoptosis is yet to be disclosed. In the present study we found that NLK was mainly localized in the nuclei of breast cancer cells, in contrast to a cytosolic localization in non-cancerous breast epithelial cells. The nuclear localization of NLK was mediated through direct interaction with Heat shock protein 27 (HSP27) which further protected cancer cells from apoptosis. The present study provides evidence of a novel mechanism by which HSP27 recognizes NLK in the breast cancer cells and prevents NLK-mediated cell apoptosis.     

Targeted disruption of Nemo‐like kinase inhibits tumor cell growth by simultaneous suppression of cyclin D1 and CDK2 in human hepatocellular carcinoma

The Wnt/β‐catenin signaling pathway regulates various aspects of development and plays important role in human carcinogenesis. Nemo‐like kinase (NLK), which is mediator of Wnt/β‐catenin signaling pathway, phosphorylates T‐cell factor/lymphoid enhancer factor (TCF/LEF) factor and inhibits interaction of β‐catenin/TCF complex. Although, NLK is known to be a tumor suppressor in Wnt/β‐catenin signaling pathway of colon cancer, the other events occurring downstream of NLK pathways in other types of cancer remain unclear. In the present study, we identified that expression of NLK was significantly up‐regulated in the HCCs compared to corresponding normal tissues in five selected tissue samples. Immunohistochemical analysis showed significant over‐expression of NLK in the HCCs. Targeted‐disruption of NLK suppressed cell growth and arrested cell cycle transition. Suppression of NLK elicited anti‐mitogenic properties of the Hep3B cells by simultaneous inhibition of cyclinD1 and CDK2. The results of this study suggest that NLK is aberrantly regulated in HCC, which might contribute to the mitogenic potential of tumor cells during the initiation and progression of hepatocellular carcinoma; this process appears to involve the induction of CDK2 and cyclin D1 and might provide a novel target for therapeutic intervention in patients with liver cancer. J. Cell. Biochem. 110: 687–696, 2010. © 2010 Wiley‐Liss, Inc.     

c-Myb截短突变体的构建及其对c-Myc蛋白表达的调节

目的:为了研究c-Myb蛋白各结构域的功能,构建c-Myb的6种截短突变体c-Myb-1~c-Myb-6的真核表达载体,测定不同突变体对c-Myc蛋白表达的影响。方法:以野生型c-Myb为模板,PCR扩增c-Myb-1~c-Myb-6片段,分别克隆到pcDNA3.0-FLAG载体,Western印迹检测克隆载体在293T细胞内的表达;将上述载体与野生型c-Myb转染乳腺癌细胞株MCF-7,检测其对c-Myb下游基因c-Myc表达的影响。结果:构建了c-Myb-1~c-Myb-6表达载体,与野生型c-Myb相比,c-Myb-1、c-Myb-4、c-Myb-5均能够促进MCF-7细胞中c-Myc的表达,而c-Myb-2、c-Myb-3、c-Myb-6则不能。结论:该研究为进一步探讨c-Myb在乳腺癌中的功能奠定了基础。     

c-Myb is an evolutionary conserved miR-150 target and miR-150/c-Myb interaction is important for embryonic development

Human c-Myb proto-oncogene is highly expressed in hematopoietic progenitors as well as leukemia and certain solid tumor. However, the regulatory mechanisms of its expression and biological functions remain largely unclear. Recently, c-Myb has been shown to be targeted by microRNA-150 (miR-150) which thereby controls B cell differentiation in mice. In this study, we demonstrated that c-Myb is an evolutionary conserved target of miR-150 in human and zebrafish, using reporter assays. Ectopic expression of miR-150 in breast cancer and leukemic cells repressed endogenous c-Myb at both messenger RNA (mRNA) and protein levels. Among several leukemia cell lines, primary leukemia cells, and normal lymphocytes, expression levels of miR-150 inversely correlated with c-Myb. The miR-150 overexpression or c-Myb silencing in zebrafish zygotes led to similar and serious phenotypic defects in zebrafish, and the phenotypic aberrations induced by miR-150 could be reversed by coinjection of c-Myb mRNA. Our findings suggest that c-Myb is an evolutionally conserved target of miR-150 and miR-150/c-Myb interaction is important for embryonic development and possibly oncogenesis.     

NLK is a key regulator of proliferation and migration in gallbladder carcinoma cells

Gallbladder cancer (GBC) is one of the most lethal neoplasm and is the fifth most common malignancy of gastrointestinal tract. The prognosis of gallbladder cancer is extremely terrible partially due to metastasis. Thus, understanding the molecular pathways controlling metastasis of this lethal disease may provide new targets for targeted therapeutic approach. In this study, we investigated the function of nemo-like kinase (NLK) in GBC growth and migration. Lentivirus-mediated siRNA was employed to alleviate the expression level of NLK in GBC cell lines (GBC-SD and SGC-996). Real-time PCR and western-blot analysis demonstrated that both mRNA and protein levels of NLK in GBC-SD and SGC-996 cells were decreased after infection with NLK-siRNA-expressing lentivirus (Lv-shNLK). The proliferation and in vitro tumorigenesis (colony formation) ability as well as migration of GBC-SD and SGC-996 cells with low NLK expression decreased significantly. Our results suggested that NLK is a key regulator involved in proliferation and migration of GBC, and it could be used as a potential therapeutic target for GBC.     

乳腺癌中CELF6的表达水平及其与预后的关系

目的:研究CELF6在乳腺癌组织与正常组织中的表达差异以及其在乳腺癌中的预后意义。方法:采用GEPIA分析乳腺癌组织与正常乳腺组织中CELF6的表达差异,免疫组化检测乳腺癌组织CELF6蛋白的表达。KM-plotter在线分析TCGA数据库中CELF6的表达差异与乳腺癌患者生存预后的关系,CCK8实验分析不同CELF6表达水平对乳腺癌细胞生长增殖的影响。结果:GEPIA在线软件分析结果显示乳腺癌CELF6表达较与正常组织显著降低(P0.001)。免疫组化检测结果显示乳腺癌患者的乳腺癌组织CELF6的阳性表达显著低于癌旁的正常组织。KM-plotter在线分析的生存曲线显示CELF6高表达的乳腺癌患者生存预后显著优于低表达者(P0.001)。CCK8实验显示敲低乳腺癌细胞MDA-MB-231中CELF6的表达,细胞增殖速度显著变快,而过表达CELF6的MDA-MB-231细胞的增殖速度减慢。结论:CELF6可能作为潜在的抑癌基因,在乳腺癌组织中低表达,与乳腺癌患者预后不良有关。     

The active component of ginseng,ginsenoside Rb1, improves erythropoiesis in models of Diamond–Blackfan anemia by targeting Nemo-like kinase

Nemo-like kinase (NLK) is a member of the mitogen-activated protein kinase family of kinases and shares a highly conserved kinase domain with other mitogen-activated protein kinase family members. The activation of NLK contributes to the pathogenesis of Diamond–Blackfan anemia (DBA), reducing c-myb expression and mechanistic target of rapamycin activity, and is therefore a potential therapeutic target. Unlike other anemias, the hematopoietic effects of DBA are largely restricted to the erythroid lineage. Mutations in ribosomal genes induce ribosomal insufficiency and reduced protein translation, dramatically impacting early erythropoiesis in the bone marrow of patients with DBA. We sought to identify compounds that suppress NLK and increases erythropoiesis in ribosomal insufficiency. We report that the active component of ginseng, ginsenoside Rb1, suppresses NLK expression and improves erythropoiesis in in vitro models of DBA. Ginsenoside Rb1–mediated suppression of NLK occurs through the upregulation of miR-208, which binds to the 3′-UTR of NLK mRNA and targets it for degradation. We also compare ginsenoside Rb1–mediated upregulation of miR-208 with metformin-mediated upregulation of miR-26. We conclude that targeting NLK expression through miRNA binding of the unique 3′-UTR is a viable alternative to the challenges of developing small-molecule inhibitors to target the highly conserved kinase domain of this specific kinase.     

Modulation of Myb-induced NF-kB -STAT3 signaling and resulting cisplatin resistance in ovarian cancer by dietary factors

c-Myb regulates tumorigenesis in multiple cancers. Here we show, for the first time, the mechanism of c-Myb-mediated proliferation, invasion, and drug resistance in ovarian cancer (OC), the most lethal gynecological cancer, and a comparative analyses of dietary agents, curcumin, epigallocatechin-3-gallate (EGCG), and sulforaphane in inhibiting c-Myb activity. We evaluated myb expression in patients with OC and found its increased expression in patients with cancer, compared with normal controls and in higher grade tumors, compared with low-grade tumors. Using ES2 and OVCAR3 cell line models, along with the silencing or overexpression of c-Myb, we establish a role of c-Myb in determining resistance to cisplatin. c-Myb overexpression activated NF-κB and STAT3 signaling leading to enhanced proliferation, invasion, and cisplatin resistance. Contrary to this, silencing of c-Myb inhibited proliferation, invasion, and sensitized OC cells to cisplatin. Further, among the dietary agents tested, EGCG almost completely inhibited the c-Myb-induced proliferation and invasion whereas sulforaphane also had significant inhibitory effect. Both compounds significantly sensitized OC cells to cisplatin, reversing the c-Myb effects. Higher c-Myb levels in patients with ovarian cancer lead to poor survival and our results indicate a possible effect of dietary factors EGCG and sulforaphane against c-Myb-mediated ovarian cancer progression and chemoresistance.     

Integrated analysis of pivotal biomarker of LSM1, immune cell infiltration and therapeutic drugs in breast cancer

The discovery of early diagnosis and prognostic markers for breast cancer can significantly improve survival and reduce mortality. LSM1 is known to be involved in the general process of mRNA degradation in complexes containing LSm subunits, but the molecular and biological functions in breast cancer remain unclear. Here, the expression of LSM1 mRNA in breast cancer was estimated using The Cancer Genome Atlas (TCGA), Oncomine, TIMER and bc‐GenExMiner databases. We found that functional LSM1 inactivation caused by mutations and profound deletions predicted poor prognosis in breast cancer (BRCA) patients. LSM1 was highly expressed in both BRCA tissues and cells compared to normal breast tissues/cells. High LSM1 expression is associated with poorer overall survival and disease‐free survival. The association between LSM1 and immune infiltration of breast cancer was assessed by TIMER and CIBERSORT algorithms. LSM1 showed a strong correlation with various immune marker sets. Most importantly, pharmacogenetic analysis of BRCA cell lines revealed that LSM1 inactivation was associated with increased sensitivity to refametinib and trametinib. However, both drugs could mimic the effects of LSM1 inhibition and their drug sensitivity was associated with MEK molecules. Therefore, we investigated the clinical application of LSM1 to provide a basis for sensitive diagnosis, prognosis and targeted treatment of breast cancer.