Prognostic impact of Notch ligands and receptors in nonsmall cell lung cancer

BACKGROUND:

Notch signaling plays a key role in embryonic vascular development and angiogenesis. The authors aimed to study the prognostic role of the angiogenesis‐related Notch ligands and receptors and investigate the prognostic impact of the coexpression of vascular endothelial growth factor‐A (VEGF‐A) and Notch signaling.

METHODS:

Tumor tissue samples from 335 resected patients with stage I to IIIA nonsmall cell lung cancer (NSCLC) were obtained, and tissue microarrays were constructed from duplicate cores of tumor cells and tumor‐related stroma from each specimen. Immunohistochemistry was used to evaluate the expression of the molecular markers Notch‐1, Notch‐4, Delta‐like ligand 4 (DLL4), and Jagged‐1.

RESULTS:

There were 191 squamous cell carcinomas (SCCs), 113 adenocarcinomas (ACs), and 31 large cell carcinomas. In AC, low tumor cell Delta‐like ligand 4 expression was an independent negative prognostic factor (hazard ratio [HR], 2.9; 95% confidence interval [CI], 1.4‐6.3 [P = .006]), whereas high tumor cell Notch‐1 expression was an independent negative prognostic factor (HR, 2.2; 95% CI, 1.2‐4.1 [P<.001]). In SCC, low stromal Delta‐like ligand 4 expression was an independent indicator of poor prognosis (HR, 3.3; 95% CI, 1.8‐6.1 [P<.001]). The coexpression of Notch‐1 and VEGF‐A had a significant prognostic impact (P<.001). For Notch‐1 and VEGF‐A, low/low (n = 142) versus high/high (n = 35) expression resulted in 5‐year survival rates of 69% and 32%, respectively.

CONCLUSIONS:

Delta‐like ligand 4 and Notch‐1 are independent prognostic factors in NSCLC, but have diverse impacts in SCC and AC. The coexpression of tumor cell Notch‐1/VEGF‐A has a major impact on survival. Cancer 2010. © 2010 American Cancer Society.     

Notch3和HES1蛋白在非小细胞肺癌中的表达及预后

目的:检测Notch3和HES1蛋白在非小细胞肺癌原发肿瘤组织中的表达,并分析其与预后的关系.方法:采用免疫组织化学法检测Notch3和HES1在2008年1月至2012年12月四川大学华西医院收集的199例肿瘤组织中的表达.数据分析采用SPSS 12.0.结果:Notch3和HES1在肺癌组织中的阳性表达率分别为39.7%,45.7%.Notch3阳性组总体生存率低于阴性组(P=0.0038),两组无进展生存率差异不具有统计学意义(P=0.07).HES1阳性组总体生存(P<0.001)及无进展生存率(P=0.001)均低于阴性组.Notch3-HES1共表达组总体生存(P<0.001)及无进展生存率(P=0.003)均低于非共表达组.多因素分析显示,HES1阳性组的总体生存率及无进展生存率更低(均P<0.001).结论:非小细胞肺癌中存在Notch3-HES1通路的过度活化,并且是不良预后的预测因子.     

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。     

Tanshinone IIA inhibits cell growth by suppressing SIX1-induced aerobic glycolysis in non-small cell lung cancer cells

Aerobic glycolysis plays a key role in cancer cell metabolism and contributes to tumorigenesis, including that of non-small cell lung cancer (NSCLC). Tanshinone IIA (Tan IIA), an active compound of Salvia miltiorrhiza, exhibits antitumor properties. Multiple mechanisms are involved in the antitumor action of Tan IIA in lung cancer, such as inhibiting cell growth, promoting cell apoptosis and influencing cellular metabolism. However, the effects of Tan IIA on NSCLC cells and its mechanisms of action remain unclear. The present study shows Tan IIA dose-dependently attenuated the growth of NSCLC cells and in vitro in a dose-dependent manner. Moreover, Tan IIA markedly decreased the ATP level, glucose uptake and lactate production in the NSCLC cells in vitro. Tan IIA also inhibited tumor growth in a xenograft model in vivo. Mechanically, Tan IIA treatment decreased sine oculis homeobox homolog 1 (SIX1) mRNA and protein levels, thus leading to the downregulation of pyruvate kinase isozyme M2, hexokinase 2 and lactate dehydrogenase A (LDHA) expression in A549 cells. SIX1 knockdown with small interfering-RNA inhibited glycolysis in NSCLC cells, suggesting that SIX1 plays a role in the antitumor effect of Tan IIA on NSCLC cells. More importantly, it was demonstrated that SIX1 expression was stimulated in patients with NSCLC and was positively correlated with the LDH serum level. Finally, SIX1 low expression levels predicted the poor prognosis of patients with NSCLC. In conclusion, the present study showed that Tan IIA functioned as an anti-glycolysis agent in NSCLC cells by downregulating SIX1 expression and inhibiting cell proliferation.     

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.     

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.     

PSMA specific single chain antibody-mediated targeted knockdown of Notch1 inhibits human prostate cancer cell proliferation and tumor growth

The down-regulation of Notch1 by small interfering RNA (siRNA) can significantly inhibit human prostate cancer cell growth. The delivery of siRNA into specific cells is a key requirement for its clinical application. Recent reports have indicated that antibody-mediated siRNA delivery is an effective approach for targeted knockdown of specific genes in appropriate cells. Prostate-specific membrane antigen (PSMA) is regarded as an ideal target for the delivery of therapeutic agents to prostate cancer cells. The purpose of the present study was to evaluate whether siRNA can be efficiently delivered into PSMA-positive prostate cancer cells using two fusion proteins, s-tP and sFH-tP. These fusion proteins are composed of an anti-PSMA single chain antibody (scFv, abbreviated as an “s”) and a truncated protamine (tP); and in sFH-tP a furin cleavage site and an HA2 fragment sequence (FH) were inserted between the scFv and tP domains. Our results showed that siRNA can be specifically delivered into PSMA-positive LNCaP cells by these two fusion proteins, with the sFH-tP fusion protein being more effective. Efficient knockdown of Notch1 by siNotch1 delivered by either fusion protein was observed in PSMA-positive LNCaP cells and in LNCaP xenografted nude mice. Further experiments confirmed that the fusion protein-delivered siNotch1 could efficiently inhibit PSMA-positive LNCaP cell proliferation and promote apoptosis both in vitro and in vivo. Our data describe a promising strategy for the targeted delivery of siRNA to PSMA-positive prostate cancer cells using anti-PSMA scFv fusion proteins.     

JAG1和Notch3在乳腺癌组织中的表达及预后价值

目的探讨JAG1和Notch3在乳腺癌组织中的表达水平及预后意义。方法通过在线Oncomine数据库和Kaplan-Meier Plotter数据库分析JAG1和Notch3在乳腺癌组织中的表达情况及对预后的影响。收集2008年1月至2013年12月乳腺浸润性导管癌组织及配对癌旁组织各80例,采用实时荧光定量PCR(QPCR)和免疫组织化学SP法分别检测JAG1和Notch3的mRNA和蛋白水平,分析JAG1和Notch3蛋白表达与乳腺癌临床病理特征和总生存时间(OS)的关系。多因素分析采用Cox比例风险回归模型。结果 Oncomine数据库分析显示,乳腺癌组织中JAG1和Notch3的mRNA水平均高于癌旁组织(P<0.05)。80例乳腺浸润性导管癌组织中JAG1 mRNA水平为1.58±0.56,高于癌旁组织的1.23±0.48(P=0.0039);乳腺浸润性导管癌组织中Notch3 mRNA水平为2.39±0.83,高于癌旁组织的1.95±0.64(P=0.0036)。JAG1和Notch3蛋白在乳腺癌组织中的高表达率为71.3%(57/80)和80.0%(64/80),分别高于癌旁组织的8.8%(7/80)和11.2%(9/80),差异均有统计学意义(P=0.005,P=0.003). JAG1和Notch3蛋白表达在乳腺癌组织中呈正相关(r=0.267,P=0.017)。JAG1和Notch3表达与乳腺癌淋巴结转移、TNM分期和组织学分级有关(P<0.05)。Kaplan-Meier Plotter在线分析显示, JAG1或Notch3高表达的乳腺癌患者的OS短于低表达者(P>0.05)。80例乳腺浸润性导管癌患者中,JAG1高表达者的中位OS为38.3个月,低于JAG1低表达者的80.4个月(P=0.0394);Notch3高表达者的中位OS为39.3个月,低于Notch3低表达者的80.2个月(P=0.0334),且JAG1和Notch3均高表达者的中位OS最短,为27.3个月。Cox多因素分析显示,淋巴结转移、JAG1表达和Notch3表达是影响乳腺癌患者OS的独立因素(P<0.05)。结论 JAG1和Notch3在乳腺癌组织中高表达,且与预后不良有关,有望成为临床评估乳腺癌预后的潜在生物标志物。     

NACK的表达水平对非小细胞肺癌预后的影响

目的:探讨NACK在非小细胞肺癌(non-small cell lung cancer,NSCLC)中的表达及其与临床病理特征的关系.方法:采用RT-PCR及Western blot法对35例非小细胞肺癌患者的肺癌组织及癌旁组织的NACK表达水平进行定量分析,以及NACK的表达与非小细胞肺癌临床病理特征的关系,通过生存曲线分析随访病人的总体生存率(OS)和无病生存率(DFS).结果:非小细胞肺癌患者NACK相对表达水平高于正常对照组(P<0.05),其表达与临床分期、分化程度及淋巴结转移显著相关(P<0.05),而与患者的年龄、性别、肿瘤大小、抽烟、病理类型无关(P>0.05).随访3年NACK高表达的病人总体生存率(OS)和无病生存率(DFS)分别为30.7%和34.6%,而低表达者分别为OS 77.8%和DFS 81.8%,两组间差异有显著统计学意义(P<0.001).结论:我们认为NACK的高表达可以作为预测NSCLC预后不良的高危因素,为以后深入研究NACK-Notch调控机制和探索新的治疗靶点提供重要基础.     

siRNA下调Notch3表达对非小细胞肺癌骨转移能力的影响

目的:研究Notch3在肺癌骨转移中的作用.方法:通过RT-PCR方法检测Notch3在NSCLC及其伴骨转移患者的肺癌组织中的表达情况;利用慢病毒包装Notch3 siRNA载体抑制Notch3的表达后,通过体外迁移和侵袭试验观察Notch3 siRNA对肺癌细胞的迁移和侵袭能力的影响;通过体外ELISA法检测骨转移相关分子pTHrP和IL-6的表达,研究阻断Notch3的表达后是否能够逆转TGF-β诱导的肺癌骨转移.结果:Notch3在NSCLC发生骨转移的患者肺癌组织中过表达.体外试验发现,通过慢病毒包装Notch3 siRNA载体抑制Notch3的表达后,NSCLC细胞的转移能力下降,且Notch3-si可明显抑制NSCLC细胞中由TGF-β介导的pTHrP和IL-6的表达.结论:Notch3高表达在促进NSCLC骨转移中发挥重要作用.     

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.     

Aberrant Expression of Notch1 in Cervical Cancer

OBJECTIVE To investigate the putative role of the Notch1 receptor in cervical cancer carcinogenesis and progression. METHODS The expression of the Notch1 protein was analyzed by a Western-blotting approach in 40 cervical cancer and 30 normal cervical tissues. Some tissues were examined using RT-PCR to determine mRNA levels. Celluar localization of the Notch1 protein in the paraffin-embedded cervical tissues was also analyzed by immunohistochemistry. RESULTS The Notch1 protein was detected in all 30 normal cervical tissues. In contrast, only 6 samples of 40 cervical cancer tissues showed Notch1 expression. The level of the Notch1 protein expression was significantly lower in cervical cancer tissues than that in normal tissue samples. In agreement with these observations, levels of Notch1 mRNA were found to be substantially down-regulated in cervical cancer tissues. In the immunohistochemistry staining assay, the Notch1 protein was shown to localize predominantly in the cytoplasm and nucleoli of the normal cervical squamous epithelium of the cervix, but no staining was observed in the cervical cancer cells. Notch1 expression was observed to correlate with the clinical disease stage, but there were no correlations with age, tumor size, grade or lymph node metastasis （P〉0.05）. The levels of Notch1 protein expression were significantly higher in early stages （Ⅰ-Ⅱa, 66.7%） compared to those in the advanced stages （Ⅱb～Ⅳ,12.6%）（P=0.001）. CONCLUSION Notch1 may play a role as a tumor suppressor in cervical tumorigenesis. Determination of Notch1 expression may be helpful for preoperative diagnosis and accuracy of staging. But its clinical use for cervical cancer requires further investigation.     

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.     

白细胞介素-18抑制Lewis肺癌小鼠移植瘤生长与诱导移植瘤细胞凋亡的研究(英文)

Objective:The aim of our study was to investigate the effects of interleukin-18(IL-18) on implanted Lewis lung cancer in suppressing tumor growth and inducing tumor cells apoptosis.Methods:One week after hypodermic inoculation of Lewis cells, sixteen tumor-bearing syngeneic mice were randomly divided into two groups.The mice in the treatment group were intraperitoneal injected with the IL-18, in the control group were intraperitoneal injected with normal saline.The health conditions of the mice were measure...     

Notch3 is activated by chronic hypoxia and contributes to the progression of human prostate cancer

Prostate cancer (PC) is still the second cause of cancer‐related death among men. Although patients with metastatic presentation have an ominous outcome, the vast majority of PCs are diagnosed at an early stage. Nonetheless, even among patients with clinically localized disease the outcome may vary considerably. Other than androgen sensitivity, little is known about which other signaling pathways are deranged in aggressive, localized cancers. The elucidation of such pathways may help to develop innovative therapies aimed at specific molecular targets. We report that in a hormone‐sensitive PC cell line, LNCaP, Notch3 was activated by hypoxia and sustained cell proliferation and colony formation in soft agar. Hypoxia also modulated cellular cholesterol content and the number and size of lipid rafts, causing a coalescence of small rafts into bigger clusters; under this experimental condition, Notch3 migrated from the non‐raft into the raft compartment where it colocalized with the γ‐secretase complex. We also looked at human PC biopsies and found that expression of Notch3 positively correlated with Gleason score and with expression of carbonic anhydrase IX, a marker of hypoxia. In conclusion, hypoxia triggers the activation of Notch3, which, in turn, sustains proliferation of PC cells. Notch3 pathway represents a promising target for adjuvant therapy in patients with PC.     

Downregulation of CPA4 inhibits non small–cell lung cancer growth by suppressing the AKT/c‐MYC pathway

Carboxypeptidase A4 (CPA4) is a member of the metallocarboxypeptidase family. A previous study indicated that CPA4 may participate in the modulation of peptide hormone activity and hormone‐regulated tissue growth and differentiation. However, the role of CPA4 in lung tumorigenesis remains unclear. Our study revealed that CPA4 expression was higher in both lung cancer cells and tumor tissues. We performed 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assays, colony‐formation assays, and Cellomics ArrayScan Infinity analysis to demonstrate that CPA4 knockdown inhibited non small–cell lung cancer (NSCLC) cell proliferation. Conversely, ectopic expression of CPA4 enhanced lung cancer cell proliferation. Consistent with these observations, we generated xenograft tumor models to confirm that CPA4 downregulation suppressed NSCLC cell growth. Mechanistically, we revealed that CPA4 downregulation may induce apoptosis and G1‐S arrest by suppressing the protein kinase B/c‐MYC pathway. These results suggest that CPA4 has an oncogenic effect on lung cancer growth. Taken together, we identified a novel gene in lung cancer that might provide a basis for new therapeutic targets.     

miR-139-5p通过靶向Notch1抑制上皮性卵巢癌细胞的增殖与侵袭

目的：探讨miR-139-5p靶向Notch1抑制上皮性卵巢癌（epithelial ovarian cancer,EOC）细胞增殖和侵袭的作用机制。方法：选取2018年1月至2018年12月在河南省南阳市中心医院妇科手术切除的24例EOC患者的癌和相应的癌旁组织标本,以及人卵巢癌细胞系SKOV3、ES2、HEY-T30和人卵巢上皮细胞株IOSE80,用qPCR检测EOC组织和细胞系中miR-139-5p和Notch1 mRNA的表达。将过表达miR-139-5p载体、重组质粒pLV-Notch1转染至SKOV3细胞,并设置空白对照组（Ctrl组）和阴性对照组（NC组）,用双荧光素酶报告基因实验验证miR-139-5p与Notch1 3’-UTR靶向关系,用CCK-8、Transwell、划痕愈合实验分别检测细胞的增殖、侵袭和迁移能力,用Western blotting检测细胞中增殖和迁移相关蛋白的表达。结果：与癌旁组织和IOSE80细胞比较,EOC组织和细胞系中miR-139-5p表达显著降低、Notch1 mRNA表达显著升高（均P<0.01）。双荧光素酶报告基因实验结果证实,Notch1是miR-139-5p的靶基因。与NC组比较,miR-139-5p mimic组3 d时SKOV3细胞的增殖、侵袭、迁移能力和Notch1、NICD、Cyclin D1、Cyclin A1、Snail1、β-catenin及N-cadherin表达水平均明显降低（均P<0.01）,E-cadherin表达水平明显升高（P<0.01）;同时过表达Notch1可逆转miR-139-5p抑制SKOV3细胞增殖、侵袭与迁移的作用。结论：miR-139-5p可靶向Notch1抑制EOC细胞的增殖、侵袭和迁移能力,可能与其下调NICD、Cyclin D1、Cyclin A1、Snail1、β-catenin、N-cadherin而上调E-cadherin的表达水平有关。