Quantitative DNA hypomethylation of ligand Jagged1 and receptor Notch1 signifies occurrence and progression of breast carcinoma

Methylation alterations of Jagged1 and Notch1 genes have been reported in non-tumor lesions and a few cancers. However, methylation profiles of Jagged1 promoter and Notch1 exon25 in breast cancer and matched normal tissue and the association of methylation with clinicopathological characteristics still remain unclear. To explore the potential effects of aberrant DNA methylation of Jagged1 and Notch1 on occurrence and progression of breast cancer, we detected the quantitative DNA methylation of Jagged1 and Notch1 in 73 breast cancer (BC) and 20 adjacent normal breast tissues (ANBT) by using MassARRAY spectrometry. The methylation level of overall and majority individual CpG sites of the two genes were synergistically significantly lower in BC than in ANBT. The overall hypomethylation of the two genes, particularly of Jagged1 CpG_8.9.10 and Notch1 CpG_14.15.16 in primary tumors, were markedly associated with lymph node metastasis, advanced stage and high grade. The protein expressions of the both genes were examined by immunohistochemical staining in same cohorts. The expression was significantly inverse correlation with methylation. The two proteins in primary tumor were synergistically up-regulated and dramatically related to lymph node metastasis, advanced stage and high grade. Our findings suggest that the synergetic hypomethylation of Jagged1 and Notch1 genes, especially of Jagged1 CpG_8.9.10 and Notch1 CpG_14.15.16, may involve tumorigenesis and development of breast cancer. The negative relationship between methylation and expression indicates methylation role for expression regulation. The synergetic overexpression of the two proteins further indicates the effects on occurrence and progression of breast cancer.     

Jagged1基因甲基化与乳腺癌发生发展相关性探讨

目的 探讨Jagged1基因异常甲基化在乳腺癌发生发展过程中的意义.方法 采用MALDI-TOF MS法检测2004-01-16-2009-06-25石河子大学第一附属医院63例乳腺浸润性导管癌(invasive ductal carcinoma,IDC)、20例导管原位癌(ductal carcinoma in situ,DCIS)、20例非典型导管增生(atypical ductal hyperplasia,ADH)和20例普通型导管增生(usual ductal hyperplasia,UDH)组织中Jagged1基因甲基化水平,免疫组织化学方法检测4组乳腺组织中Jagged1蛋白表达状况,在IDC组中进一步分析Jagged1甲基化和表达与临床病理特征的相关性.结果 Jagged1基因总甲基化率在IDC(0.127 6±0.067 5)、DCIS(0.138 9±0.093 1)、ADH(0.168 0±0.014 6)和UDH(0.223 3±0.060 9)中逐渐升高,并且IDC组甲基化率分别与ADH(P=0.015)和UDH(P＜0.001)组比较,差异有统计学意义.CpG-2、CpG-6、CpG-13、CpG-20.21.22、CpG-23.24.25、CpG-26位点的甲基化率在IDC组最低,差异有统计学意义,P值均＜0.05.Jagged1蛋白在IDC(58.7％,37/63)、DCIS(45.0％,9/20)、ADH(40.0％,8/20)和UDH(25.0％,4/20)组中阳性表达率逐渐降低,其中IDC组的阳性率显著高于UDH组,P=0.003.Jagged1蛋白高表达与DNA低甲基化在IDC(P＜0.001)、DCIS(P=0.003)、ADH(P=0.004)和UDH组(P=0.007)均有相关性.Jagged1基因低甲基化和蛋白高表达与临床分期(P＜0.001;P=0.019)和组织学分级(P=0.003;P=0.025)均有相关性.结论 Jagged1基因低甲基化和CpG位点低甲基化,可能参与乳腺癌的发生,并且Jagged1基因低甲基化可能是调控蛋白高表达的方式之一,进而促进乳腺的癌变和进展.     

Notch signalling in cancer progression and bone metastasis

Classically known for its indispensible role in embryonic development, the Notch signalling pathway is gaining recognition for its regulation of adult tissue homoeostasis and aberrant activation in disease pathogenesis. The pathway has been implicated in cancer initiation and development, as well as early stages of cancer progression by regulating conserved cellular programs such as the epithelial-to-mesenchymal transition. We recently extended the role of Notch signalling to late stages of tumour progression by elucidating a stroma-dependent mechanism for the pathway in osteolytic bone metastasis. Of clinical significance, disrupting the Notch pathway and associated molecular mediators of Notch-dependent bone metastasis may provide novel therapeutic strategies to combat aggressive bone metastatic disease.     

Jagged1/Notch3信号通路蛋白在人三阴乳腺癌中的表达及临床意义

目的：研究Jagged1/Notch3在TNBC患者中的表达水平及临床意义,阐明Jagged1和Notch3异常表达对TNBC患者的预后影响。方法：Ventana免疫组化法检测Jagged1、Notch3在石蜡标本的蛋白表达,分析其与临床病理及复发转移的关系。结果：共纳入患者70例,Jagged1和Notch3在TNBC组织中的阳性表达率分别为35.7%（25/70）和40.0%（28/70）。Jagged1在无淋巴结转移患者中表达高,Notch3则在4个以上淋巴结转移和Ⅲ期患者中的表达更高。结论：TNBC患者中Jagged1/Notch3蛋白的异常表达与淋巴结转移和分期相关。     

Jagged1促进乳腺癌发生发展及远处转移的研究进展

乳腺癌是一种严重危害女性身心健康的疾病。晚期乳腺癌患者的死亡在很大程度上归因于重要脏器的远处转移而非原发肿瘤。Jagged1(JAG1)作为Notch信号通路的重要配体之一,不仅表达于肿瘤细胞,亦可表达于肿瘤微环境中的间质细胞,通过激活Notch信号通路,促进乳腺癌的侵袭性进展与远处转移。本文就JAG1基因位置与JAG1蛋白的结构、JAG1促进乳腺癌远处转移相关机制、JAG1与乳腺癌脑转移、JAG1与乳腺癌骨转移及JAG1的靶向治疗等方面进行综合论述。     

不同人乳腺肿瘤细胞系中Jagged1基因和蛋白的表达水平

目的:研究Jagged1蛋白和mRNA在人乳腺癌细胞系中的表达。方法:分别使用蛋白印迹法及实时定量聚合酶链式反应检测Jagged1蛋白和基因在人乳腺癌细胞系中的表达情况。结果:Jagged1在不同的人乳腺肿瘤细胞系中蛋白和基因的表达水平不同。结论:Jagged1蛋白在人乳腺癌细胞系ZR-75-30和MDA-MB-231中高表达,Jagged1基因在人乳腺癌细胞系MDA-MB-231,HCC 1937中高表达,Jagged1可能是在人乳腺癌的发展进程中发挥着重要的作用。     

胃腺癌组织Notchl和Jaggedl蛋白表达临床意义分析

目的：探讨胃腺癌组织Notchl和Jaggedl蛋白的表达及其与临床病理学特征的关系和意义。方法：采用免疫组织化学SP法检测2009—01—01—2012—12—12泰山医学院附属莱芜医院60例胃腺癌组织和癌旁组织中Notchl和Jag—gedl蛋白表达水平。结果：Notchl在胃腺癌组织中的阳性表达率为41．7％（25／60）,癌旁组织为81．7％（49／60）,差异有统计学意义,x2=20．31,P〈0．001;Jaggedl在胃腺癌组织中的阳性表达率为71．7％（43／60）,癌旁组织为96．7％（58／60）,差异有统计学意义,x2=14．07,P〈0．001。Notchl表达与肿瘤分化程度（P=0．006）、淋巴结有无转移（P=0．005）和浸润深度（P=0．009）有关,Jaggedl的表达与肿瘤分化程度（P=0．004）、浸润深度（P=0．009）和淋巴结有无转移（P=0．006）有关。Spearman相关分析结果显示,胃腺癌组织中Notchl和Jaggedl蛋白表达呈正相关,r=0．460,P〈0．001。结论：胃腺癌组织中Notchl和Jaggedl蛋白表达下调,且两者呈正相关,可能与胃腺癌的发生、浸润及转移等多个环节有关;检测Notchl和Jaggedl的表达有助于判断胃腺癌的生物学行为。     

乳腺癌WIF-1基因表达及其临床病理特征与该基因启动子区域甲基化的关联

  目的  研究WIF-1（Wnt inhibitory factor-1）mRNA在乳腺癌组织中的表达及其启动子区域甲基化情况,进一步探讨WIF-1基因甲基化与乳腺癌临床病理特征的关系。   方法  收集2009年9月1日至2009年12月30日青岛大学附属医院乳腺外科手术切除新鲜组织标本69例,其中良性病变组织9例,乳腺癌及癌旁组织各30例,应用RT-PCR及甲基化特异性PCR（methylation specific PCR,MSP）检测乳腺癌组织、相应癌旁组织和乳腺良性病变组织中WIF-1mRNA表达及其启动子甲基化情况。   结果   癌组织中WIF-1基因表达率明显低于相应癌旁组织及乳腺良性病变组织,具有显著性差异（χ2=41.786,P < 0.05）;与其他两组相比甲基化率在癌组织中明显升高（矫正χ2=16.484,P < 0.05）;WIF-1基因表达下降与其异常甲基化存在明显关联（P=0.023）;WIF-1异常甲基化与乳腺癌发病年龄、肿瘤分级、组织分型和淋巴结转移无相关性（P>0.05）。   结论  异常甲基化可能是乳腺癌WIF-1基因表达下降的重要原因,是乳腺癌发生、发展的重要机制。      

Endothelial Jagged1 promotes solid tumor growth through both pro-angiogenic and angiocrine functions

Angiogenesis is an essential process required for tumor growth and progression. The Notch signaling pathway has been identified as a key regulator of the neo-angiogenic process. Jagged-1 (Jag1) is a Notch ligand required for embryonic and retinal vascular development, which direct contribution to the regulation of tumor angiogenesis remains to be fully characterized.The current study addresses the role of endothelial Jagged1-mediated Notch signaling in the context of tumoral angiogenesis in two different mouse tumor models: subcutaneous Lewis Lung Carcinoma (LLC) tumor transplants and the autochthonous Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP).The role of endothelial Jagged1 in tumor growth and neo-angiogenesis was investigated with endothelial-specific Jag1 gain- and loss-of-function mouse mutants (eJag1OE and eJag1cKO). By modulating levels of endothelial Jag1, we observed that this ligand regulates tumor vessel density, branching, and perivascular maturation, thus affecting tumor vascular perfusion. The pro-angiogenic function is exerted by its ability to positively regulate levels of Vegfr-2 while negatively regulating Vegfr-1. Additionally, endothelial Jagged1 appears to exert an angiocrine function possibly by activating Notch3/Hey1 in tumor cells, promoting proliferation, survival and epithelial-to-mesenchymal transition (EMT), potentiating tumor development. These findings provide valuable mechanistic insights into the role of endothelial Jagged1 in promoting solid tumor development and support the notion that it may constitute a promising target for cancer therapy.     

Neuralized1 causes apoptosis and downregulates Notch target genes in medulloblastoma

Neuralized (Neurl) is a highly conserved E3 ubiquitin ligase, which in Drosophila acts upon Notch ligands to regulate Notch pathway signaling. Human Neuralized1 (NEURL1) was investigated as a potential tumor suppressor in medulloblastoma (MB). The gene is located at 10q25.1, a region demonstrating frequent loss of heterozygosity in tumors. In addition, prior publications have shown that the Notch pathway is functional in a proportion of MB tumors and that Neurl1 is only expressed in differentiated cells in the developing cerebellum. In this study, NEURL1 expression was downregulated in MB compared with normal cerebellar tissue, with the lowest levels of expression in hedgehog-activated tumors. Control of gene expression by histone modification was implicated mechanistically; loss of 10q, sequence mutation, and promoter hypermethylation did not play major roles. NEURL1-transfected MB cell lines demonstrated decreased population growth, colony-forming ability, tumor sphere formation, and xenograft growth compared with controls, and a significant increase in apoptosis was seen on cell cycle and cell death analysis. Notch pathway inhibition occurred on the exogenous expression of NEURL1, as shown by decreased expression of the Notch ligand, Jagged1, and the target genes, HES1 and HEY1. From these studies, we conclude that NEURL1 is a candidate tumor suppressor in MB, at least in part through its effects on the Notch pathway.     

miR-124通过调节Jagged1/Notch信号通路影响肾细胞癌OS-RC-2细胞的恶性生物学行为

目的：探讨miR-124通过调节Jagged1(JAG1)/Notch信号通路对肾细胞癌(RCC)细胞增殖、凋亡、迁移和侵袭的影响。方法：收集2018年6月至2021年10月在武汉市第三医院治疗的38例RCC患者的RCC组织和癌旁组织标本,并体外培养RCC细胞(Caki-2、A498、ACHN、786-O、OS-RC-2)和人正常肾细胞(293T),采用免疫组织化学法、qPCR和WB法检测miR-124和JAG1蛋白在RCC组织和细胞中的表达水平。选择miR-124表达与293T细胞差异最大的OS-RC-2细胞进行转染,按转染物不同分为Control组、NC mimic组、miR-124 mimic组、miR-124 mimic+pcDNA组和miR-124 mimic+pc-JAG1组。采用双荧光素酶报告基因实验验证miR-124与JAG1的关系;q PCR法检测miR-124、JAG1 mRNA表达;免疫组化法分析JAG1蛋白表达;WB法检测JAG1、凋亡相关蛋白(cleaved caspase-3、BAX和Bcl2)和Notch信号通路相关蛋白(NICD、HES1和HES5)的表...     

乳腺癌组织Notch1和JAG1蛋白表达与分子分型相关性研究

目的 Notch信号通路在乳腺癌中存在异常表达,但在不同分子分型乳腺癌中的表达情况鲜见报道.本研究将探讨不同分子分型乳腺浸润性导管癌组织中Notch1和JAG1蛋白表达及其与临床病理特征之间的关系.方法 收集滨州医学院附属医院病理科2012-01-06-2014-12-30存档的乳腺浸润性导管癌病例240例,根据ER、PR、HER2、Ki-67免疫组化结果分为管腔A型、管腔B型、HER2过表达型及三阴性型4组,每组60例,采用免疫组化En-Vision法检测不同分子分型乳腺癌中Notch1、JAG1蛋白的表达情况,对各分型乳腺癌组织的阳性表达率进行统计学分析.结果 各分型乳腺癌组织中Notch1的阳性表达差异有统计学意义(P=0.010,P＜0.05),其中三阴性乳腺癌(86.67％)与管腔A型(56.67％)比较(P＜0.001),HER2过表达型(83.33％)与管腔A型(56.67％)比较,差异均有统计学意义,P＜0.001;Notch1的阳性表达与淋巴结转移相关(P=0.017,P＜0.05),与临床分期(P=0.005,P＜0.01)、组织学分级(P=0.009,P＜0.01)显著相关,与ER表达(rs=-0.206,P=0.001,P＜0.01)、PR表达(rs=-0.187,P=0.005,P＜0.01)显著负相关,而与患者年龄、肿块大小无关.各分型乳腺癌组织中JAG1的阳性表达差异有统计学意义(P=0.035,P＜0.05),以三阴性乳腺癌阳性表达率最高,但各组间两两比较差异无统计学意义;JAG1阳性表达与ER表达(rs=-0.142,P=0.015,P＜0.05)、PR表达(rs=-0.127,P=0.035,P＜0.05)呈负相关;Notch1和JAG1之间无明显相关性.结论 Notch1阳性表达与ER、PR表达显著负相关,与乳腺癌的组织学分级、淋巴结转移及临床分期密切相关,并在HER2过表达型,尤其是三阴性乳腺癌组织中存在高表达,有可能成为三阴性乳腺癌新的治疗靶点.     

Visfatin promotes cell and tumor growth by upregulating Notch1 in breast cancer

Overexpression of Notch1 has been associated with breast cancer. We recently showed that visfatin stimulates breast cancer cell proliferation and invasion. The present study was undertaken to determine whether Notch1 signaling is affected by visfatin and to characterize the functional role of the visfatin-Notch1 axis in breast cancer. Visfatin and Notch1 were expressed at higher levels in breast tumors than in matched control tissues. Visfatin induced Notch1 expression in MDA-MB-231 breast cancer cell line and in nontransformed MCF10A mammary epithelial cells, whereas visfatin depletion reduced Notch1 mRNA and protein levels. Depletion of Notch1 in MDA-MB-231 cells attenuated cell growth in vitro and in vivo; visfatin depletion produced similar effects, but was less potent. Additionally, Notch1 depletion inhibited cell proliferation induced by visfatin. Analysis of the signaling pathways underlying visfatin-mediated Notch1 upregulation revealed that visfatin activated NF-κB p65. Blockade of NF-κB signaling suppressed the effects of visfatin on Notch1 upregulation and breast cancer cell proliferation. Breast tumors expressing high levels of NF-κB p65 exhibited increased expression of Notch1. Our results demonstrate that the visfatin-Notch1 axis contributes to breast tumor growth through the activation of the NF-κB pathway. Study of the visfatin-Notch1 axis may offer new therapeutic directions for breast cancer.     

VEGF与Notch1在乳腺癌浸润和转移中的作用及其相关性

目的探讨血管内皮生长因子(VEGF)和Notch1在乳腺癌组织中的表达及与肿瘤浸润和转移之间的关系。方法采用免疫组化S-P法检测VEGF和Notch1蛋白在60例乳腺癌组织及20例癌旁正常乳腺组织中的表达情况,并分析两者与乳腺癌患者临床病理学特征的关系。结果 VEGF和Notch1蛋白在乳腺癌组织中的表达高于癌旁正常组织(P均<0.05)。VEGF的表达与乳腺癌的淋巴结转移、组织学分级、临床分期有关(P均<0.05),Notch1蛋白的表达与乳腺癌的淋巴结转移、组织学分级有关(P均<0.05),两者在乳腺癌组织中的表达呈正相关关系(r=0.414,P=0.001)。结论 VEGF和Notch1可能在乳腺癌的发生、发展中起重要作用,两者的高表达有可能作为预测乳腺癌浸润、转移的指标。     

Stromal expression of ALDH1 in human breast carcinomas indicates reduced tumor progression

Interactions between cancer cells and microenvironment are emerging issue in tumor progression. Aldehyde dehydrogenase 1 (ALDH1) is a recognized cancer stem cell marker but little is known about its role in intratumoral stroma. Therefore, we focused on ALDH1 expression in tumor-associated stroma of breast carcinomas (BrCa).Stromal and tumoral ALDH1 expression was evaluated immunohistochemically in BrCa and their lymph node metastases (LNMs), and related to clinico-pathological characteristics, patients’ outcome, presence of CD68, HLADR, retinoic acid (RA) in stroma, and selected proteins in tumor cells.ALDH1(+) stromal cells were detected in 53% of 374 BrCa and 61% of 102 LNMs. ALDH1(+) stroma in primary tumor correlated to longer disease-free (p = 0.030), metastasis-free (p = 0.024), and overall survival (p = 0.043) having an independent prognostic impact on DFS (multivariate analysis, p = 0.047). It was associated with concomitant presence of HLA-DR(+) stromal cells and RA in tumor cells (both p < 0.001), and inversely associated with vimentin expression in tumor cells (p = 0.036). ALDH1(+) stroma in LNMs correlated inversely to presence of disseminated tumor cells in patients’ bone marrow (p = 0.014) and was independent prognosticator of shorter DFS and MFS (multivariate analysis, p = 0.004 and p = 0.002, respectively).In conclusion, ALDH1 expression in tumor-associated stromal cells indicates reduced BrCa progression, possibly via RA secretion.     

Inhibition of Notch1 reverses EMT and chemoresistance to cisplatin via direct downregulation of MCAM in triple-negative breast cancer cells

Resistance to chemotherapy continues to be a critical issue in the clinical therapy of triple-negative breast cancer (TNBC). Epithelial–mesenchymal transition (EMT) is thought to contribute to chemoresistance in several cancer types, including breast cancer. Identification of the key signaling pathway that regulates the EMT program and contributes to chemoresistance in TNBC will provide a novel strategy to overcome chemoresistance in this subtype of cancer. Herein, we demonstrate that Notch1 positively associates with melanoma cell adhesion molecule (MCAM), a unique EMT activator, in TNBC tissue samples both at mRNA and protein levels. High expression of Notch1 and MCAM both predicts a poor survival in basal-like/TNBC patients, particularly in those treated with chemotherapy. The expression of Notch1 and MCAM in MDA-MB-231 cells gradually increases in a time-dependent manner when exposing to low dose cisplatin. Moreover, the expressions of Notch1 and MCAM in cisplatin-resistant MDA-MB-231 cells are significantly higher than wild-type counterparts. Notch1 promotes EMT and chemoresistance, as well as invasion and proliferation of TNBC cells via direct activating MCAM promoter. Inhibition of Notch1 significantly downregulates MCAM expression, resulting in the reversion of EMT and chemoresistance to cisplatin in TNBC cells. Our study reveals the regulatory mechanism of the Notch1 pathway and MCAM in TNBC and suggesting that targeting the Notch1/MCAM axis, in conjunction with conventional chemotherapies, might be a potential avenue to enhance the therapeutic efficacy for patients with TNBC.     

肝癌组织DLK1和Jagged1表达临床意义探讨

目的探讨DLK1和Jagged1在原发性肝细胞性肝癌组织中的表达情况,深入分析两者与肝癌临床病理资料的关系及其临床意义。方法收集2001-01-01-2009-10-30顺德第一人民医院病理科采用免疫组化SP法检查10例正常肝组织、50例肝炎、50例肝硬化和150例肝细胞性肝癌组织中DLK1和Jagged1的表达情况。结果 DLK1阳性表达率在正常肝组织为0(0/10)、肝炎组为6.00%(3/50)、肝硬化组为28.00%(14/50)、肝癌组为46.67%(70/150),四组间阳性率比较差异有统计学意义,χ2=34.38,P<0.05。正常肝组织和肝炎组,正常肝组织和肝硬化组,正常肝组织和肝癌组,肝硬化和肝癌组两两比较差异均无统计学意义,P值均>0.05。肝炎和肝硬化组(χ2=8.575,P=0.003),肝炎和肝癌组(χ2=26.757,P<0.001)两两比较差异均有统计学意义。Jagged1阳性表达率在正常肝组织中为40.00%(4/10)、肝炎组为40.00%(20/50)、肝硬化组为62.00%(31/50)、肝癌组为71.33%(107/150),四组间阳性率比较差异有统计学意义,χ2=17.92,P<0.05。正常肝组织和肝炎组,正常肝组织和肝硬化组,正常肝组织和肝癌组,肝炎组和肝硬化组,肝硬化组和肝癌组,两两比较差异均无统计学意义,P值均>0.05。肝炎组和肝癌组比较差异有统计学意义,χ2=15.885,P<0.001。DLK1表达与年龄有关(χ2=15.01,P<0.05),Jagged1表达与年龄(χ2=7.94,P<0.05)、分化程度(χ2=14.79,P<0.05)、HBsAg(χ2=4.53,P<0.05)和预后(χ2=3.92,P<0.05)有关。DLK1和Jagged1在肝细胞癌组织中表达密切相关,χ2=12.79,P<0.05。结论 DLK1和Jagged1的异常表达与肝癌的发生、发展和分化密切相关。