Gli1与β-catenin相互作用促进二者在结肠癌细胞中的协同核输入

Wnt信号通路和Hedgehog（Hh）信号通路在胚胎和干细胞的发育中发挥重要作用.此外,这两条信号途径在结肠癌复发和浸润的过程也至关重要.然而,Wnt信号通路、Hedgehog信号通路二者之间具体的交互作用机制目前仍不清楚.本文发现,这两条途径的关键分子Gli1和β-联蛋白之间存在蛋白质相互作用.Gli1与β-联蛋白之间的分子相互作用有助于二者的核输入.同时发现,在肠癌细胞系中,Gli1与β-联蛋白协同上调表达. LiCl激活细胞Wnt信号通路使Gli1表达水平增加, RNA干扰抑制Wnt信号通路,Gli1的表达水平下降.同时,Gli1的过表达也提高了细胞内β-联蛋白的表达水平,并且用Hedgehog信号通路抑制剂GANT61处理细胞,降低Gli1的表达后细胞内β 联蛋白的表达相应下降.本研究揭示了Gli1 和 β-联蛋白的相互作用及二者协助核输入在Wnt、Hedgehog信号通路交互调节中发挥重要作用,Wnt、Hedgehog信号通路交互作用为大肠癌发生发展研究提供了细胞水平交互调控机制.     

β-catenin信号分子的生物学功能及其在肝癌中的研究进展

β-catenin(β-连环素)是一种由位于染色体3p21—22的CTNNB1基因编码的具有介导细胞间黏附及信号转导等多重功能的重要分子,其通过与多种蛋白质结合调控细胞的增殖和分化,在胚胎发育和肿瘤发生中发挥关键作用。近年来,越来越多的研究证实经典Wnt通路的异常激活在肝癌中具有重要作用,而作为Wnt信号转导通路中枢纽分子的β-catenin的异常表达与肝癌的发生、发展密切相关。就近几年β-catenin分子生物学功能及其在肝癌研究中的进展进行简要综述,为进一步阐明β-catenin在肝癌中的作用机理及以β-catenin为靶点的临床治疗提供理论依据。     

microRNA与Wnt/β-catenin通路在肿瘤发生及转移中的调控机制

microRNA(miR)和Wnt/β-catenin信号通路是肿瘤研究领域的两大传统模块,miR的表达失调与Wnt通路的激活共同驱动了肿瘤的发生、发展、转移及药物的耐药等性状.一方面,miR通过各种途径激活或抑制经典的Wnt信号通路;另一方面,Wnt与其启动子结合后激活转录,进而促进miR在肿瘤中的表达.本文对miR和经典Wnt信号通路之间在多种肿瘤中的相互关联和作用机制,以及其在多方面对肿瘤细胞的增殖、迁移、转移和药物反应等方面的作用进行了一一阐述.这将为miR和Wnt信号调控在肿瘤机制研究中提供更新、更全面的思路和途径,为肿瘤诊断、分子分型、药物筛选以及预后判断等方面提供新的指导思想.     

抗肿瘤干细胞药物盐霉素对Wnt/β-catenin信号通路的抑制

肿瘤干细胞(cancer stem cells, CSCs)是一小群具有自我更新和分化潜力,并能产生异质性细胞的肿瘤细胞亚群,该群细胞在肿瘤的发生发展、复发、转移和耐药中起着十分重要的作用,因此通过研发针对CSCs的靶向药物,将为抗肿瘤药物的研究提供新的方向,并为未来根治肿瘤带来新的希望.近年来,大量实验研究证实了盐霉素对多种类型的肿瘤干细胞具有明显的杀伤作用,这些肿瘤包括肝癌、乳腺癌、胃癌、结直肠癌、前列腺癌和骨肉瘤等.盐霉素可以抑制肿瘤干细胞的增殖、分化及转移,并诱导其凋亡. Wnt/β-catenin信号通路是一种在生物进化过程中高度保守的信号系统,与组织的发育和器官形成等生物学过程密切相关,该通路的异常活化在肿瘤及肿瘤干细胞的发生发展中起关键作用.通过靶向阻断异常活化的Wnt信号通路,可以抑制肿瘤细胞和肿瘤干细胞的增殖并诱导其凋亡,因此该通路已经成为抗肿瘤药物领域的研究热点. Wnt/β-catenin信号通路在盐霉素抗肿瘤干细胞过程中起重要作用,本文综述了盐霉素通过调节Wnt/β-catenin信号通路抑制肿瘤干细胞的研究进展.     

BCL9与肿瘤及肿瘤靶点治疗研究

Wnt信号传导通路与肿瘤的关系是近年来肿瘤研究的热点,该通路包含许多信号成员,其中BCL9是近年来该通路上发现的新的癌基因,其在多种Wnt信号通路失调肿瘤中的异常表达表明其与肿瘤的发生、发展有密切关系.而针对Wnt信号通路不同基因靶点的高特异性基因药物开发以及肿瘤的分子诊断也相继出现,尤其是针对BCL9与β-catenin作用界面的小分子抑制剂基因药物的开发更是引起广泛的注意.现就BCL9及其与肿瘤的关系和BCL9基因为靶点的肿瘤基因治疗等三方面做一综述.     

EGCG通过抑制wnt/β-catenin信号通路调节猪骨骼肌卫星细胞的增殖和分化

为了阐明Wnt/β-catenin信号通路在猪骨骼肌卫星细胞增殖分化中的作用,利用Wnt/β-catenin信号通路抑制剂(-)-表没食子儿茶素没食子酸酯(EGCG)处理猪骨骼肌卫星细胞,采用MTT、流式细胞术、免疫荧光和Western印迹等方法检测了细胞增殖和分化情况.结果显示,与对照组相比,EGCG以时间、浓度依赖方式抑制猪骨骼肌卫星细胞的增殖.流式细胞术检测细胞周期结果表明,与对照组相比,经EGCG处理后,猪骨骼肌卫星细胞的G1期细胞比例上升,而G2和S期细胞比例下降,这说明细胞被阻滞在G1期,细胞的增殖受到抑制.免疫荧光检测分化过程中MyHC的表达,与对照组相比,EGCG促进猪骨骼肌卫星细胞的分化,并降低增殖标志基因MyoD以及细胞周期蛋白D的表达量,而提高了分化标志基因MyoG和MyHC的表达量.在猪骨骼肌卫星细胞增殖分化过程中,EGCG降低β-联蛋白的表达量,且核内的β-联蛋白明显减少.结果表明,EGCG通过抑制Wnt/β-catenin信号通路抑制猪骨骼肌卫星细胞的增殖,促进其分化.     

4-1BBL通过激活Wnt/β-catenin信号通路促进人胃癌细胞的增殖和迁移

4-1BB和4-1BB配体(4-1BBL),又被称为CD137和CD137配体,分别属于肿瘤坏死因子(TNF)受体和配体家族的成员。4-1BBL 与4-1BB相互作用可以激活T细胞免疫应答。因此,4-1BBL一直在抗肿瘤免疫应答中发挥经典的免疫共刺激分子作用。近期研究发现,4-1BBL在肿瘤细胞中另有其他的生物学功能,但4-1BBL在胃癌进展过程中的功能尚不明确。本文探讨了4-1BBL在人胃癌细胞中的生物学功能和分子作用机制。首先,通过检索TCGA和Kaplan Meier plotter数据库发现,4-1BBL在胃癌组织中的表达显著高于癌旁组织（P<0.001）,且4-1BBL的高表达与胃癌的不良预后正相关（P<0.05）。细胞生物学的结果显示,敲除4-1BBL明显抑制胃癌细胞的增殖（P<0.05）、侵袭和迁移（P<0.05）,促进胃癌细胞的凋亡（P<0.05）;另外,蛋白质免疫印迹结果表明,敲除4-1BBL可使β-联蛋白、c-Myc和细胞周期蛋白D1(cyclin D1)的蛋白质表达水平下降,抑制Wnt/β-catenin信号通路。相反,过表达4-1BBL则显著促进胃癌细胞增殖（P<0.05）、侵袭和迁移（P<0.05）,减少胃癌细胞的凋亡（P<0.05）;且过表达4-1BBL促进β-联蛋白(β-catenin)、c-Myc和细胞周期蛋白D1的蛋白质表达,激活Wnt/β-catenin信号通路。综上所述,4-1BBL可通过激活Wnt/β-catenin信号通路促进人胃癌细胞的增殖和迁移。     

Hippo/YAP和Wnt/β-catenin通路的对话

Hippo/YAP通路和Wnt/β-catenin通路是在细胞的生长分化、组织器官形成以及成体干细胞的维持等方面都起着重要作用的两条信号通路。在哺乳动物细胞中,Wnt/β-catenin通路通过一系列胞质蛋白的相互作用,使β-catenin蛋白在胞质内累积,进而入核传递生长刺激信号。Hippo/YAP通路通过激酶级联反应磷酸化YAP/TAZ,使其滞留在细胞质中,抑制了YAP/TAZ的转录活性,从而限制细胞的生长增殖,诱导细胞凋亡。这两条通路的异常调控往往会导致肿瘤的发生。近年来越来越多的研究证实,Hippo/YAP和Wnt/β-catenin在很多方面相互影响,共同参与组织生长和胚胎发育的调控。研究这两个通路在肿瘤发生过程中的转导和调控以及它们相互作用的机制,有助于为肿瘤的防治提供新的思路与策略。文章对这两条通路的协同作用及其分子机制进行了综述。     

白藜芦醇对小鼠溃疡性结肠炎的影响*

目的:研究白藜芦醇通过调节Wnt/β-catenin信号通路抗溃疡性结肠炎的作用机制。方法:①葡聚糖硫酸钠盐(DSS)诱发溃疡性结肠炎实验:28只C57BL/6小鼠随机分为4组(n=7):control组、 DSS组、DSS+白藜芦醇(DSS+Res)组和Res组。实验周期为3周,小鼠饮用DSS水诱导溃疡性结肠炎并给予白藜芦醇灌胃。实验期间每天称小鼠体重并观察小鼠活动和粪便情况。处理结束后,安乐死小鼠,取小鼠脾脏称重,取小鼠结肠测量长度。苏木精-伊红染色法(H&E)染色观察小鼠结肠组织病理改变;实时荧光定量PCR(qPCR)检测小鼠结肠组织miRNA-31的表达;Western Blot检测小鼠结肠组织β-catenin、Cyclin D1蛋白的表达。②离体实验:以10 mg/ml浓度的白藜芦醇处理HCT 116细胞,检测HCT 116细胞β-catenin、低密度脂蛋白受体相关蛋白6(LRP-6)、卷曲蛋白3(FZD3)、c-Myc蛋白的表达;HCT 116细胞转染miRNA-31 mimic和inhibitor,检测β-catenin蛋白的表达。结果:①DSS组小鼠实验期间体重下降明显,精神萎靡,活动减少,出现血便;处理结束后小鼠的结肠长度缩短,脾脏增大。而给予白藜芦醇后小鼠的以上情况得到改善。②白藜芦醇抑制了溃疡性结肠炎小鼠结肠组织miRNA-31的表达及β-catenin、Cyclin D1蛋白的表达。③白藜芦醇下调HCT 116细胞β-catenin、LRP-6、FZD3、c-Myc蛋白的表达。转染miRNA-31 inhibitor后,HCT 116细胞中β-catenin蛋白表达减少。结论:白藜芦醇能够抑制DSS诱导的小鼠溃疡性结肠炎,这种作用与下调Wnt信号通路有关,其对Wnt 信号的下调作用与miRNA-31有关。     

Wnt/β-catenin信号通路相关蛋白在胃癌组织中的表达和肿瘤转移的关系

目的:探讨了Wnt信号通路相关蛋白在胃癌组织中的表达及与肿瘤转移的关系。方法:选取2011年6月到2012年6月我院胃癌术后47例肿瘤标本作为研究对象,并选取同一患者的正常胃组织作为对照研究。采用实时荧光定量PCR和Western blot对胃癌组织和正常胃组织Wnt信号通路相关蛋白进行分析,并分析了肿瘤转移和非转移患者Wnt信号通路相关蛋白的变化。结果:与正常胃组织比较,胃癌组织中Wnt1、Wnt3、Wnt3a、β-catenin、CyclinD1和c-Myc等分子的mRNA水平明显上调,差异有显著统计学意义(P0.05)。胃癌组织中总β-catenin和核内β-catenin蛋白较正常胃组织明显增加,而磷酸化β-catenin较正常组明显下降、差异有显著统计学意义(P0.05)。与非转移组比较,转移组患者胃癌组织中Wnt1、Wnt3、Wnt3a等分子mRNA水平显著上调,差异有统计学意义(P0.05)。结论:Wnt信号通路异常激活在胃癌发生和癌细胞转移中发挥着重要的作用,为临床治疗提供了一定靶点。     

Wnt3a regulates proliferation,apoptosis and function of pancreatic NIT‐1 beta cells via activation of IRS2/PI3K signaling

Wnt‐signaling pathway is implicated in pancreatic development and functional regulation of mature beta‐cells. Wnt3a/Wnt pathway activation expands islet cell mass in vitro by increasing proliferation and decreasing apoptosis of beta‐cells, thereby enhancing its function. However, the signaling pathways that mediate these effects remain unknown. By using a clonal beta‐cell line (NIT‐1), we examined the role of IRS2/PI3K in the mediation of Wnt3a‐stimulated beta‐cell growth. Real‐time PCR and Western blot were employed to investigate the activity of Wnt/β‐catenin and IRS2/PI3K signaling. Proliferation of NIT‐1 cells was assessed by BrdU incorporation, and apoptosis was quantitatively determined by TUNEL and flow cytometry (FCM). Dkk1, an inhibitor of Wnt signaling, and wortmannin, an inhibitor of PI3K, were also used. Results showed that Wnt3a rapidly activated Wnt/β‐catenin signaling, promoted IRS2 expression and Akt phosphorylation in NIT‐1 cells. These effects were completely abrogated by Dkk1 or partially eliminated by wortmannin. Wnt3a also promoted NIT‐1 cell proliferation, inhibited cytokine‐induced beta‐cell apoptosis, and increased insulin secretion. Both of these effects were also eliminated by Dkk1 or wortmannin. Our results demonstrated that Wnt3a regulates proliferation, apoptosis and enhances function of pancreatic NIT‐1 beta cells via activation of Wnt/β‐catenin signaling, involving crosstalk with IRS2/PI3K signaling, with the effect of Wnt signaling on beta‐cells also being IRS2/PI3K/AKT dependent. J. Cell. Biochem. 114: 1488–1497, 2013. © 2013 Wiley Periodicals, Inc.     

The role of Wnt/β‐catenin signaling in proliferation and regeneration of the developing basilar papilla and lateral line

Canonical Wnt/β‐catenin signaling has been implicated in multiple developmental events including the regulation of proliferation, cell fate, and differentiation. In the inner ear, Wnt/β‐catenin signaling is required from the earliest stages of otic placode specification through the formation of the mature cochlea. Within the avian inner ear, the basilar papilla (BP), many Wnt pathway components are expressed throughout development. Here, using reporter constructs for Wnt/β‐catenin signaling, we show that this pathway is active throughout the BP (E6‐E14) in both hair cells (HCs) and supporting cells. To characterize the role of Wnt/β‐catenin activity in developing HCs, we performed gain‐ and loss‐of‐function experiments in vitro and in vivo in the chick BP and zebrafish lateral line systems, respectively. Pharmacological inhibition of Wnt signaling in the BP and lateral line neuromasts during the periods of proliferation and HC differentiation resulted in reduced proliferation and decreased HC formation. Conversely, pharmacological activation of this pathway significantly increased the number of HCs in the lateral line and BP. Results demonstrated that this increase was the result of up‐regulated cell proliferation within the Sox2‐positive cells of the prosensory domains. Furthermore, Wnt/β‐catenin activation resulted in enhanced HC regeneration in the zebrafish lateral line following aminoglycoside‐induced HC loss. Combined, our data suggest that Wnt/β‐catenin signaling specifies the number of cells within the prosensory domain and subsequently the number of HCs. This ability to induce proliferation suggests that the modulation of Wnt/β‐catenin signaling could play an important role in therapeutic HC regeneration. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 438–456, 2014     

Direct interaction of β‐catenin with nuclear ESM1 supports stemness of metastatic prostate cancer

Wnt/β‐catenin signaling is frequently activated in advanced prostate cancer and contributes to therapy resistance and metastasis. However, activating mutations in the Wnt/β‐catenin pathway are not common in prostate cancer, suggesting alternative regulations may exist. Here, we report that the expression of endothelial cell‐specific molecule 1 (ESM1), a secretory proteoglycan, is positively associated with prostate cancer stemness and progression by promoting Wnt/β‐catenin signaling. Elevated ESM1 expression correlates with poor overall survival and metastasis. Accumulation of nuclear ESM1, instead of cytosolic or secretory ESM1, supports prostate cancer stemness by interacting with the ARM domain of β‐catenin to stabilize β‐catenin–TCF4 complex and facilitate the transactivation of Wnt/β‐catenin signaling targets. Accordingly, activated β‐catenin in turn mediates the nuclear entry of ESM1. Our results establish the significance of mislocalized ESM1 in driving metastasis in prostate cancer by coordinating the Wnt/β‐catenin pathway, with implications for its potential use as a diagnostic or prognostic biomarker and as a candidate therapeutic target in prostate cancer.     

Sporadic Early-Onset Colorectal Cancer Is a Specific Sub-Type of Cancer: A Morphological,Molecular and Genetics Study

Sporadic early onset colorectal carcinoma (EOCRC) which has by definition no identified hereditary predisposition is a growing problem that remains poorly understood. Molecular analysis could improve identification of distinct sub-types of colorectal cancers (CRC) with therapeutic implications and thus can help establish that sporadic EOCRC is a distinct entity. From 954 patients resected for CRC at our institution, 98 patients were selected. Patients aged 45–60 years were excluded to help define “young” and “old” groups. Thirty-nine cases of sporadic EOCRC (patients≤45 years with microsatellite stable tumors) were compared to both microsatellite stable tumors from older patients (36 cases, patients>60 years) and to groups of patients with microsatellite instability. Each group was tested for TP53, KRAS, BRAF, PIK3CA mutations and the presence of a methylator phenotype. Gene expression profiles were also used for pathway analysis. Compared to microsatellite stable CRC from old patients, sporadic EOCRC were characterized by distal location, frequent synchronous metastases and infrequent synchronous adenomas but did not have specific morphological characteristics. A familial history of CRC was more common in sporadic EOCRC patients despite a lack of identified hereditary conditions (p = 0.013). Genetic studies also showed the absence of BRAF mutations (p = 0.022) and the methylator phenotype (p = 0.005) in sporadic EOCRC compared to older patients. Gene expression analysis implicated key pathways such as Wnt/beta catenin, MAP Kinase, growth factor signaling (EGFR, HGF, PDGF) and the TNFR1 pathway in sporadic EOCRC. Wnt/beta catenin signaling activation was confirmed by aberrant nuclear beta catenin immunostaining (p = 0.01). This study strongly suggests that sporadic EOCRC is a distinct clinico-molecular entity presenting as a distal and aggressive disease associated with chromosome instability. Furthermore, several signaling pathways including the TNFR1 pathway have been identified as potential biomarkers for both the diagnosis and treatment of this disease.     

肝细胞癌中转录因子E2F7对Wnt/β-catenin信号通路活性的影响

探讨肝细胞癌(HCC)中非典型性E2F家族成员E2F7在肝癌细胞生长、分化中的作用及可能涉及的分子机制.本研究运用实时荧光定量PCR检测38例原发性肝细胞癌及对应的癌旁组织中E2F7基因mRNA的表达情况;分别通过基因过表达和RNA干扰技术上调或下调E2F7基因表达,并运用实时荧光定量PCR和Western印迹检测肝癌细胞株MHCC-H中β-catenin及其靶基因cmyc的表达情况;双荧光素酶报告基因系统检测E2F7对Wnt/β-catenin信号通路活性的影响;核浆分离实验检测过表达E2F7基因对β-catenin入核的影响;免疫共沉淀实验检测异位表达E2F7与内源β-catenin的相互作用.结果显示,肝细胞癌组织中E2F7基因的表达量显著高于相应的癌旁组织(P0.001);转录因子E2F7可与β-catenin相互作用并促进β-catenin进入细胞核.转录因子E2F7可以促进Wnt/β-catenin信号通路的活性.     

Rspo2/Int7 regulates invasiveness and tumorigenic properties of mammary epithelial cells

Rspo2 was identified as a novel common integration site (CIS) for the mouse mammary tumor virus (MMTV) in viral induced mouse mammary tumors. Here we show that Rspo2 modulates Wnt signaling in mouse mammary epithelial cells. Co‐expression of both genes resulted in an intermediate growth phenotype on plastic and had minor effects on the growth‐promoting properties of Wnt1 in soft agar. However, individual Rspo2 and Wnt1 HC11 transfectants as well as the double transfectant were tumorigenic in athymic nude mice, with tumors from each line having distinctive histological characteristics. Rspo2 and Rspo2/Wnt1 tumors contained many spindle cells, consistent with an epithelial–mesenchymal transformation (EMT) phenotype. When Rspo2 and Rspo2/Wnt1 tumor cells were transferred into naïve mice, they exhibited greater metastatic activity than cells derived from Wnt1 tumors. For comparison, C57MG/Wnt1/Rspo2 co‐transfectants exhibited invasive properties in three‐dimensional (3D) Matrigel cultures that were not seen with cells transfected only with Wnt1 or Rspo2. Use of Dickkopf‐1, a specific antagonist of the Wnt/β‐catenin pathway, or short hairpin RNA targeting β‐catenin expression demonstrated that the invasive activity was not mediated by β‐catenin. Our results indicate that Rspo2 and Wnt1 have mutually distinct effects on mammary epithelial cell growth and these effects are context‐dependent. While Rspo2 and Wnt1 act synergistically in the β‐catenin pathway, other mechanisms are responsible for the invasive properties of stable double transfectants observed in 3D Matrigel cultures. J. Cell. Physiol. 227: 1960–1971, 2012. © 2011 Wiley Periodicals, Inc.     

The Tumor Suppressor SCRIB is a Negative Modulator of the Wnt/β‐Catenin Signaling Pathway

SCRIB is a scaffold protein containing leucine‐rich repeats (LRR) and PSD‐95/Dlg‐A/ZO‐1 domains (PDZ) that localizes at the basolateral membranes of polarized epithelial cells. Deregulation of its expression or localization leads to epithelial defects and tumorigenesis in part as a consequence of its repressive role on several signaling pathways including AKT, ERK, and HIPPO. In the present work, a proteomic approach is used to characterize the protein complexes associated to SCRIB and its paralogue LANO. Common and specific sets of proteins associated to SCRIB and LANO by MS are identified and an extensive landscape of their associated networks and the first comparative analysis of their respective interactomes are provided. Under proteasome inhibition, it is further found that SCRIB is associated to the β‐catenin destruction complex that is central in Wnt/β‐catenin signaling, a conserved pathway regulating embryonic development and cancer progression. It is shown that the SCRIB/β‐catenin interaction is potentiated upon Wnt3a stimulation and that SCRIB plays a repressing role on Wnt signaling. The data thus provide evidence for the importance of SCRIB in the regulation of the Wnt/β‐catenin pathway.     

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.