CN117849343A - 基于CHKα的非代谢功能作为癌症治疗、诊断和预后预测之靶标的应用 - Google Patents
基于CHKα的非代谢功能作为癌症治疗、诊断和预后预测之靶标的应用 Download PDFInfo
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Abstract
本发明属于肿瘤医学的技术领域,涉及基于CHKα的非代谢功能作为癌症治疗、诊断和预后预测之靶标的应用。本发明通过对胆碱激酶α(CHKα)在人癌症中肿瘤发生发展的机制进行分析,利用CHKα的非代谢功能在磷脂酰胆碱合成的调节、糖代谢(糖酵解)、谷胱甘肽合成、脂滴脂解、β‑氧化以及肿瘤生长中的作用,为癌症治疗、诊断和预后预测提供了新的作用靶标。
Description
本申请是分案申请,原申请的申请号是202110602766.7,申请日是2021年05月31日,发明名称为“基于CHKα的非代谢功能作为癌症治疗、诊断和预后预测之靶标的应用”。
技术领域
本发明属于肿瘤医学的技术领域,涉及基于CHKα的非代谢功能作为癌症治疗、诊断和预后预测之靶标的应用。
背景技术
胆碱激酶催化游离的胆碱磷酸化为磷酸胆碱,磷酸胆碱进一步被磷酸胆碱胞基转移酶转化为胞苷二磷酸胆碱,以及被胆碱磷酸转移酶转化为磷脂酰胆碱。胆碱激酶α促进肿瘤细胞增殖及生存,在40%–60%的人类肿瘤中高表达,与多个肿瘤的差预后正相关,在肿瘤的发生发展中发挥作用。但是胆碱激酶α(CHKα)增强肿瘤进展的具体机制尚不确切。
细胞代谢重新编程是肿瘤细胞的特征。肿瘤细胞中的诸多代谢酶的非代谢功能越来越受到人们的关注,并且在肿瘤的发生发展中发挥着重要作用。例如丙酮酸激酶(PKM)的蛋白激酶功能、磷酸甘油酸激酶(PGK)的蛋白激酶功能、磷酸烯醇丙酮酸羧激酶(PCK)的蛋白激酶功能等。
肿瘤细胞中活性氧及氧化应激增加,谷胱甘肽的合成可以抵消氧化应激并促进肿瘤进展,而胱硫醚β合成酶(CBS)的磷酸化可以促进谷胱甘肽的合成。
脂滴由极性的两亲性单层磷脂包围,其结构蛋白为脂滴包被蛋白(PLIN)。脂滴调节中性脂质的水解,例如甘油三酸酯、甾醇酯和视黄酯,它们被用于脂肪酸氧化的能量生产、膜的生物发生,以及蛋白质修饰等,从而对于肿瘤细胞的生长至关重要。
发明内容
本发明的目的在于对胆碱激酶α(CHKα)在人癌症中肿瘤发生发展的机制进行分析,利用CHKα的非代谢功能在磷脂酰胆碱合成的调节、糖代谢(糖酵解)、谷胱甘肽合成、脂滴脂解、β-氧化以及肿瘤生长中的作用,提出了基于CHKα调节的非代谢功能作为癌症治疗、诊断和预后预测之靶标的应用。
本发明是采用以下的技术方案实现的:
本发明提供了一种基于CHKα的非代谢功能作为癌症治疗、诊断之靶标的应用,所述应用包括:
1)确定检测患者癌细胞或者患者血液中包含:与参考水平相比升高的CBS Y484位点磷酸化水平、CHKαS279位点磷酸化水平、CHKαK247位点乙酰化水平、PLIN2 Y232位点磷酸化水平、PLIN3 Y251位点磷酸化水平、烯醇酶1(ENO1)Y44位点磷酸化水平、CHKα的C303和C307之间形成分子内二硫键;以及
(2)用抑制方法阻断CHKα蛋白激酶活性激活、CBS Y484位点磷酸化、CHKαS279位点磷酸化、CHKαK247位点乙酰化、PLIN2 Y232位点磷酸化、PLIN3 Y251位点磷酸化、烯醇酶1(ENO1)Y44位点磷酸化水平、CHKα的C303和C307之间形成分子内二硫键中的一种或两种以上状态;和/或
(3)预测患者对治疗方法的有利响应;
所述参考水平是来自非癌细胞或早期癌细胞或者患者血液的水平。
优选地,所述癌症是口腔癌、口咽癌、鼻咽癌、呼吸系统癌症、泌尿生殖系统癌症、胃肠癌、中枢或周围神经系统组织癌、内分泌或神经内分泌系统癌症或造血系统癌症、胶质瘤、肉瘤、上皮癌、淋巴瘤、黑素瘤、纤维瘤、脑脊膜瘤、脑癌、肾癌、胆道系统癌症、嗜铬细胞瘤、胰岛细胞癌、利-弗劳梅尼瘤、甲状腺癌、甲状旁腺癌、垂体瘤、肾上腺瘤、骨源性肉
瘤肿瘤、神经内分泌系统肿瘤、乳腺癌、肺癌、头颈癌、前列腺癌、食管癌、气管癌、肝癌、膀胱癌、胃癌、胰腺癌、卵巢癌、子宫癌、宫颈癌、睾丸癌、结肠癌、直肠癌或皮肤癌。
优选地,所述确定方法包括使用磷酸化特异性抗体,进行ELISA、免疫测定、放射免疫测定、免疫组织化学、免疫放射测定、荧光免疫测定、凝胶电泳、免疫印迹分析、原位杂交、流式细胞术或显微测定。
优选地,所述抑制方法包括使用CHKα抑制剂、CBS Y484抑制剂、CHKαS279抑制剂、CHKαK247抑制剂、PLIN2 Y232抑制剂、PLIN3 Y251抑制剂,或其他任何抑制CHKα蛋白激酶活性的方法,或其他任何抑制CBS Y484位点磷酸化、CHKαS279位点磷酸化、CHKαK247位点乙酰化、PLIN2 Y232位点磷酸化、PLIN3 Y251位点磷酸化、烯醇酶1(ENO1)Y44位点磷酸化水平、CHKα的C303和C307之间形成分子内二硫键的方法。
优选地,所述CHKα抑制剂包括针对CHKα蛋白激酶活性的小分子抑制剂,CBS Y484、CHKαS279、CHKαK247、PLIN2 Y232、PLIN3 Y251抑制剂包括选择性针对CBS Y484位点磷酸化、CHKαS279位点磷酸化、CHKαK247位点乙酰化、PLIN2 Y232位点磷酸化、PLIN3Y251位点磷酸化、烯醇酶1(ENO1)Y44位点磷酸化水平、CHKα的C303和C307之间形成
分子内二硫键的多肽、小分子抑制剂或互补的抑制性多核苷酸。
优选地,所述有利响应包括肿瘤尺寸或负荷减小、肿瘤生长阻滞、肿瘤相关疼痛减轻、癌症相关病理状况减轻、癌症相关症状减轻、癌症无进展、无病间期延长、进展时间延长、诱导缓解、转移降低、患者存活延长或肿瘤对抗癌治疗的敏感性提高。
本发明还提供了基于CHKα的蛋白激酶活性作为癌症预后预测之靶标的应用,所述应用包括:
(1)确定检测患者癌细胞或者患者血液中是否包含:与参考水平相比升高的CBSY484位点磷酸化水平、CHKαS279位点磷酸化水平、CHKαK247位点乙酰化水平、PLIN2 Y232位点磷酸化水平、PLIN3 Y251位点磷酸化水平以及这五者的升高的各种联合表达水平;
(2)如果癌细胞或者患者血液包含(1)中任一项升高的水平,则预测患者具有侵袭性癌症;
(3)如果癌细胞或者患者血液包含(1)中任一项升高的水平,则预测患者具有的侵袭性癌症在进展期;
(4)如果癌细胞或者患者血液包含(1)中任一项升高的水平,则预测患者具有不良预后;
所述参考水平是来自非癌细胞或早期癌细胞或者患者血液的水平。
进一步地,如果所述测患者具有侵袭性癌症,则利用阻断CHKα蛋白激酶活性激活、CBS Y484位点磷酸化、CHKαS279位点磷酸化、CHKαK247位点乙酰化、PLIN2 Y232位点磷酸化、PLIN3 Y251位点磷酸化、烯醇酶1(ENO1)Y44位点磷酸化水平、CHKα的C303和C307之间形成分子内二硫键中的一种或两种以上的方式进行抑制剂抗癌治疗。
本发明的有益效果是:本发明利用,任何可使氧化应激信号激活的上游信号激活之后,导致CHKα的C303和C307之间形成分子内二硫键,进而磷酸化CBS Y484位点,增强CBS与磷酸吡哆醛结合,而促进肿瘤细胞中谷胱甘肽合成从头合成,细胞增殖及肿瘤生长。本发明利用,任何可使CHKα结合脂滴的上游信号(包括葡萄糖剥夺导致的CHKαS279位点磷酸化和CHKαK247位点乙酰化)激活之后,导致CHKα的催化结构域构象改变而具有蛋白激酶活性而磷酸化PLIN2 Y232位点和PLIN3 Y251位点。磷酸化的PLIN2/3从脂滴解离,被Hsc70介导的自噬所降解,进而促进脂滴脂解、β-氧化和肿瘤生长。强调了新鉴定的胆碱激酶CHKα的蛋白激酶活性在谷胱甘肽合成从头合成、脂滴脂解、β-氧化和肿瘤生长中的重要性。重要的是,CBS Y484位点磷酸化、CHKαS279位点磷酸化、CHKαK247位点乙酰化、PLIN2 Y232位点磷酸化、PLIN3 Y251位点磷酸化的表达水平在肿瘤组织中显著上调、表达水平彼此之间显著正相关、与肿瘤的进展相关、与肿瘤的不良预后相关。本发明利用,任何可使CHKα与ENO1结合以及磷酸化烯醇酶1(ENO1)Y44位点的信号(包括其结合位点突变CHKαF199N/P200N以及ENO1 Y44F突变)激活之后,阻断TRIM25结合并聚泛素化CHKα(K195位点)以及蛋白酶体降解CHKα,进而促进胆碱磷脂代谢及肿瘤生长。重要的是,ENO1 Y44位点磷酸化的表达水平在肿瘤组织中显著上调,与肿瘤患者的较长生存期负相关。
附图说明
附图用来提供对本发明的进一步理解,并且构成说明书的一部分,与本发明的实施例一起用于解释本发明,并不构成对本发明的限制。
在附图中:
图1为实施例1的H2O2处理LN229细胞后做生物素下拉实验,之后做免疫印迹分析,使用微管蛋白作为标志物;其中,WLC,全细胞裂解液;Cyto,胞浆提取物;Nuc,核提取物。
图2为实施例1的人类CHKα蛋白结三维构图。
图3为实施例1的生物素下拉实验;其中WLC,全细胞裂解液。
图4为实施例2的Ni-NTA下拉实验,H2O2处理后,Flag、His的免疫印迹分析图,使用微管蛋白作为标志物。
图5为实施例2的体外激酶测定实验,H2O2处理后,CHKα、CBS、32P-CBS、CBS pY484的免疫印迹分析图。
图6为实施例3的体外激酶测定实验(左侧)以及CBS活性测定实验(右侧)换柱状图填充用纹样。
图7为实施例4的ROS水平检测实验(左侧)和GSH/GSSG比例检测实验(右侧)换柱状图填充用纹样。
图8为实施例5的肿瘤大小检测实验,***P<0.001(双尾学生t检验)。C1,克隆1;C2,克隆2。换柱状图填充用纹样
图9为实施例6的脑胶质瘤患者的CBS Y484位点磷酸化水平的生存曲线。
图10为实施例7的缺糖处理肿瘤细胞后,免疫印迹分析检测其CHKα和CHKβ的表达,使用微管蛋白作为标志物。
图11为实施例8的缺糖处理肿瘤细胞后,免疫荧光分析CHKα、ATGL、Beclin1与脂滴的共定位。
图12为实施例9的免疫印迹分析CHKα与脂滴的结合情况,使用微管蛋白作为标志物。
图13为实施例10的免疫印迹分析CHKα与脂滴的结合情况,使用微管蛋白作为标志物。
图14为实施例11的免疫沉淀及免疫印迹分析CHKα与PLIN2和PLIN3的结合情况,使用微管蛋白作为标志物。
图15为实施例12的免疫沉淀及免疫印迹分析CHKα结合PLIN2/3的情况,使用微管蛋白作为标志物。
图16为实施例13的Ni-NTA琼脂糖珠做下拉分析及免疫印迹分析CHKα磷酸化PLIN2Y232和PLIN3 Y251位点。使用微管蛋白作为标志物。
图17为实施例14的分子动力学模拟分析CHKα。
图18为实施例15的免疫沉淀及免疫印迹分析PLIN2/3与Hsc70的相互作用,使用微管蛋白作为标志物。
图19为实施例16的免疫荧光分析PLIN2/3与脂滴的共定位情况。
图20为实施例16的免疫荧光分析ATGL、Beclin1、LC3B与脂滴的共定位情况。
图21为实施例17的在2-DG处理的条件下检测肿瘤细胞增殖统计图。
图22为实施例18的小鼠脑胶质瘤大小检测结果。
图23为实施例18的小鼠脑胶质瘤组织样本中脂滴累积检测结果。
图24为实施例19的ACC S79磷酸化抗体、CHKαS279磷酸化抗体、CHKαK247乙酰化抗体、PLIN2 Y232磷酸化抗体和PLIN3 Y251磷酸化抗体在100例人胶质瘤样本中的免疫组化试验。
图25为实施例19的所示肿瘤标志物在60例人胶质瘤样本中表达水平的相关性检测。
图26为实施例19的脑胶质瘤患者的ACC S79磷酸化水平、CHKαS279磷酸化水平、CHKαK247乙酰化水平、PLIN2 Y232磷酸化水平和PLIN3 Y251磷酸化水平的生存曲线。
图27为实施例20的GST下拉实验及免疫印迹分析ENO1与CHKα的结合情况。
图28为实施例21的免疫沉淀及免疫印迹分析CHKα的泛素化情况。
图29为实施例22的免疫沉淀及免疫印迹分析TRIM25与CHKα的结合情况。
图30为实施例22的免疫沉淀及免疫印迹分析CHKα的泛素化位点。
图31为实施例23的免疫沉淀及免疫印迹分析TRIM25与CHKα的结合情况。
图32为实施例24的细胞内磷脂酰胆碱的产量检测。
图33为实施例25的体外激酶实验。
图34为实施例26的乳酸产量检测。
具体实施方式
为了使本发明目的、技术方案更加清楚明白,下面结合附图,对本发明作进一步详细说明。下述实施例中所述实验方法,如无特殊说明,均为常规方法;实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行;所述试剂和材料,如无特殊说明,均可从商业途径获得。
一、检测方法:
1、磷酸化蛋白质的水平检测:
通过使样品与特异性结合磷酸化多肽的抗体接触并确定结合的抗体的量,例如通过检测或测量抗体与多肽之间复合物的行程。抗体可被标记(放射性、荧光等)以便于检测复合物。
用于本发明的多肽水平的检测系统包括:放射自显影免疫测定RIA、免疫荧光、凝胶电泳、Western免疫印迹测定。
所用抗体(添加):tubulin(sc-8035)购自Santa Cruz Biotechnology(SantaCruz,CA);
Anti-CHKα、ENO1、LC3B、ATGL、Beclin 1、ACC pS79、CHK antibodies购自CellSignaling Technology(Danvers,MA);Rabbit antibodies that recognize CHK、CBS(pY484)、CBS、CHKαpS279、CHKαAcK247、PLIN2 pY232、PLIN3 pY251购自SignalwayBiotechnology(Pearland,TX);Mouse monoclonal anti-Flag(F1804)、rabbit anti-Flag(F7425)、anti-His(SAB1305538)antibodies购自Sigma-Aldrich(St.Louis,MO)。
2、小鼠肿瘤样品获取与分析:
Huh7细胞(1×106)颅内注射到无胸腺裸鼠中(n=7/组)。注射28天后使小鼠安乐死并检查HCC肿瘤生长。
3、细胞中蛋白质的免疫组织化学IHC染色检测(组织微阵列构建):
通过手术切除获得福尔马林固定的石蜡包埋的组织,并用Mayer的苏木精和伊红(H&E;Biogenex实验室,加利福尼亚州,圣拉蒙)进行染色。
对癌症病例肿瘤样本和正常组织样本进行组织微阵列(TMA)处理。使用自动组织阵列仪器(Alphelys,Plaisir,法国),从每个标本中提取癌组织(直径为2mm,由病理学家选择),并固定在石蜡块中。质量控制后,将TMA块切成切片进行免疫组织化学分析。
二抗anti-Rabbit IgG heavy chain(HRP)(ab99702)antibodies购自Abcam(Cambridge,MA)。
4、皮尔逊相关检验
本发明使用皮尔逊相关检验来进行人脑胶质瘤标本中蛋白表达之间的相关性。
免疫组织化学分析是根据以前的出版物进行的(参见Nucleus-TranslocatedACSS2 Promotes Gene Transcription for Lysosomal Biogenesis andAutophagy.Molecular cell.2017;66(5):684-97e9)。去石蜡,再水化和抗原修复后,将TMA玻片与一抗兔抗人AKT pS473(稀释度1:200),一抗兔抗人磷酸化PCK1 pS90(稀释度1:200),一抗兔抗人孵育INSIG1 pS207和INSIG2 pS151(稀释度1:500),一抗兔抗人SREBP1(稀释度1:100)或非特异性IgG(作为阴性对照)在4℃过夜。然后将玻片与抗兔二抗(即用型溶液;Cell Signaling Technology;#8114)孵育,然后进行二原色二氨基联苯胺(DAB)染色(Cell Signaling Technology)和苏木精染色,并在二甲苯上固定。根据阳性细胞的百分比和染色强度在显微镜下对组织玻片进行定量评分。本发明分配了以下比例得分:0,0%的细胞为阳性;1,0%至1%;2,2%至10%;3、11%至30%;4、31%至70%;和5,从71%到100%。还以0到3:0(负)的等级对染色强度进行了评级。1,弱;2,适中;3,强壮。如前所述文献记载,然后将比例和强度得分相加以获得总得分(范围0-8)。两位不了解临床信息的病理学家独立地验证了评分系统的可重复性。
5、患者的总生存率Kaplan-Meier绘图
使用SPSS20.0版软件(SPSS Inc.,美国伊利诺伊州,芝加哥)进行数据分析。使用独立样本t检验比较了生物标志物在肿瘤和正常组织中的表达水平。使用单因素方差分析(ANOVA,post hoc Bonferroni检验)对生物标志物的表达水平与患者的临床病理特征之间的相关性进行多重比较和最低显著性差异检验。使用Pearson相关系数分析生物标志物表达水平之间的相关性。总生存期(OS)定义为从诊断之日至死亡之日或最后一次随访的持续时间。使用K-means聚类分析对相关标志物的表达水平进行分类、Kaplan-Meier方法绘制生存曲线、对数秩检验以比较生存率以及带有双向Wald检验的Cox回归模型计算危险比(HR)和95%置信区间(CIs)进行生存分析。审查的数据用于上次随访中活着或因随访而遗失的患者。P值小于0.05的单变量分析变量纳入多变量分析。P<0.05被认为具有统计学意义。所有统计检验都是双面的。
二、下列实施例采用的材料如下:
1、细胞种类:
Huh7细胞(人肝癌细胞株Huh7细胞)、LN229细胞(人胶质瘤细胞),来自ATCC;
2、无胸腺裸鼠是BALB/c无胸腺裸鼠;
3、病人样本:
从浙江大学转化研究院(中国杭州)的生物库中回顾性收集了经手术切除、福尔马林固定、石蜡包埋的NSCLC组织样品。选择100例经病理诊断为脑胶质瘤的未接受过手术治疗的患者的组织样本作为独立队列。通过回顾患者的病史获得了临床数据。病理分期由美国癌症联合委员会/国际癌症控制TNM分类系统联合会第8版评估。
4、基因敲降所用shRNA序列如下:
CHKα:TGATACTAAAGACGGTATTAA
PLIN2:CAGAAGCTAGAGCCGCAAATT
PLIN3:CTGGACCACATGGTGGAATAT
ATGL:CCTGCCACTCTATGAGCTTAA
Beclin 1:GCTTGGGTGTCCTCACAATTT
ENO1:CGCATTGGAGCAGAGGTTTAC
TRIM25:CCGGAACAGTTAGTGGATTTA
实施例1、CHKα是直接的氧化应激传感器
1、CHKα分子内二硫键形成是细胞应对氧化应激的直接事件
用50μM H2O2处理LN229细胞。用生物素-马来酰亚胺标记氧化的蛋白,并用链霉亲和素-琼脂糖珠纯化。如图1所示,H2O2处理2分钟的时候CHKα分子内二硫键形成。
2、CHKα分子内二硫键在C303和C307之间形成
(1)如图2所示,人类的CHKα结构(PDB:2CKO)显示了C303和C307的空间位置。C303和C307的周围区域被框出并放大。C303和C307的硫原子之间形成分子内二硫键。
(2)在LN229细胞中表达HA标记的野生型CHKα,CHKαC303A,或者CHKαC307A。细胞用还原试剂N-乙酰半胱氨酸(NAC)处理30分钟,然后用50μM H2O2处理10分钟。用生物素-马来酰亚胺标记氧化的蛋白,并用链霉亲和素-琼脂糖珠纯化。WLC,全细胞裂解液。如图3所示,在LN229细胞中CHKα的C303或C307(CHKα结构中仅相距)的突变消除了二硫键的形成和CHKα更快的迁移。
实施例2、氧化应激导致CHKα结合并磷酸化CBS
1.H2O2处理之后CHKα与CBS结合
表达Flag标签的CHKα和His标签的CBS的LN229细胞用NAC处理10分钟,然后用50μMH2O2处理10分钟。Ni-NTA下来实验结果显示,H2O2处理导致CHKα与CBS结合,而这种结合被NAC处理所阻断。(图4)
2.氧化应激使得CHKα磷酸化CBS的Y484位点
纯化的野生型Flag-CHKα固定在磁珠上,加入H2O2处理10分钟,PBS漂洗,之后在32P-ATP存在下与纯化的野生型His-CBS或者His-CBS Y484F孵育,进行体外激酶测定。结果如图5,H2O2处理使得CBS磷酸化,而CBS的Y484F突变阻断了这种磷酸化。
实施例3、CHKα介导的CBS磷酸化激活了CBS
纯化的野生型Flag-CHKα或者其突变蛋白固定在磁珠上,加入50μM H2O2处理10分钟,10mM DTT处理30分钟,PBS漂洗,之后在ATP存在下与纯化的野生型His-CBS或者His-CBSY484F孵育,进行体外激酶测定(左侧)以及CBS活性测定(右侧)。结果如图6,CBS的Y484磷酸化增强了CBS活性。
实施例4、CHKα介导的CBS磷酸化增强了谷胱甘肽的从头合成
LN229野生型细胞以及CHKαC307A、CHKαS298A/P299A、或者CBS Y484F突变的LN229细胞用0.25mM的百草枯处理6小时之后,检测细胞内的ROS水平(左侧)、GSH/GSSG比例(右侧)。如图7所示,CHKα介导的CBS Y484位点磷酸化激活CBS,增加谷胱甘肽的从头合成(肿瘤细胞抵消氧化应激的直接反应)。
实施例5、CHKα调节的谷胱甘肽的合成促进了肿瘤生长
LN229野生型细胞以及CHKαC307A、CHKαS298A/P299A、或者CBS Y484F突变的LN229细胞颅内注射到无胸腺的裸鼠(每组14只),一个星期之后每三天一次(共四次)颅内注射脂质体阿霉素(20mg/kg)(每组7只)。一个月后检测肿瘤大小。如图8所示,阿霉素抑制肿瘤生长,而CHKαC307A、CHKαS298A/P299A、或者CBS Y484F突变表达减少了肿瘤生长,并增强了阿霉素抑制肿瘤生长的效果。
实施例6、CBS Y484位点磷酸化水平与脑胶质瘤患者的生存期负相关
CBS Y484位点磷酸化水平在脑胶质瘤样本中的表达分为高表达和低表达,绘制患者总体生存Kaplan-Meier图。如图9所示,CBS Y484位点磷酸化水平与脑胶质瘤患者的生存期负相关。
实施例7、CHKα结合脂滴并为脂滴脂解所需
Huh7、U87、GP06、GP08细胞缺糖处理1小时,检测全细胞裂解液、细胞质、脂滴中的CHKα表达。如图10所示,缺糖处理使得一小部分胞质CHKα,而不是CHKβ,结合在脂滴上。
实施例8、CHKα为脂滴脂解所需
缺糖处理有或者无敲降CHKα或者敲降CHKβ的Huh7细胞1小时,细胞用BODIPY或者DAPI或者识别Beclin 1或者ATGL的抗体做免疫荧光。如图11所示,只有CHKα敲降抑制缺糖处理诱导的ATGL和自噬蛋白Beclin1与脂滴的共定位。这个结果证明在缺糖的情况下CHKα为脂滴脂解所需。
实施例9、缺糖条件下AMPK介导的CHKαS279位点磷酸化为CHKα结合脂滴所需
5μM Compound C(AMPK抑制剂)处理Huh7细胞30分钟,缺糖处理1小时,或者0.5mMA769662(AMPK激活剂)处理30分钟。纯化脂滴,做免疫印迹分析。如图12所示,Compound C处理阻断了缺糖诱导的CHKα与脂滴结合,而即使在无缺糖的条件下A769662处理依然促进了这种结合。
实施例10、CHKαS279位点磷酸化导致KAT5介导的CHKαK247位点乙酰化以及CHKα被招募到脂滴
表达野生型Flag-CHKα或者Flag-CHKαK247R突变的Huh7细胞做缺糖处理1小时,纯化脂滴,用Flag抗体做免疫沉淀分析。如图13所示,K247R突变的CHKα(CHKαS279位点磷酸化不受影响)不会转位到脂滴。
实施例11、CHKα结合PLIN2/3
通过免疫沉淀及免疫印迹分析,如图14所示,缺糖处理1小时Huh7细胞,CHKα与PLIN2和PLIN3结合。
实施例12、单体CHKα结合PLIN2/3
表达Flag-PLIN2、Flag-PLIN3、野生型His-CHKα或者CHKα突变的Huh7细胞,缺糖处理1小时,获得全细胞裂解液。用Flag抗体琼脂糖珠和Ni-NTA琼脂糖珠做下拉分析。如图15所示,CHKα从二聚体转为单体使得其可以结合PLIN2和PLIN3。
实施例13、CHKα磷酸化PLIN2 Y232和PLIN3 Y251位点
表达野生型His-PLIN2、His-PLIN2 Y232F突变、野生型His-PLIN3、His-PLIN3Y251F突变的Huh7细胞,缺氧处理1小时,用Ni-NTA琼脂糖珠做下拉分析。如图16所示,缺糖处理导致PLIN2 Y232和PLIN3 Y251位点的磷酸化,这种磷酸化被PLIN2 Y232F突变和PLIN3Y251F突变阻断。
实施例14、CHKαK247位点乙酰化介导的CHKα单体化改变了其催化结构域的结构并磷酸化PLIN2 Y232和PLIN3 Y251位点
分子动力学模拟分析CHKα二聚体和单体,结果如图17所示,上方显示单体CHKα对接PLIN2 Y232(LHSRAYQQALS)肽或者PLIN3 Y251(LRQHAYEHSLG)肽的整个概貌。下方为放大图像。测量ATP的γ-磷酸根与PLIN2 Y232或PLIN3 Y251的OH根之间的距离。计算机对接分析结果显示只有单体的、扩张的胆碱结合口袋的CHKα,使得PLIN2 Y232或PLIN3 Y251肽与CHKα的催化结构域互作;并且PLIN2 Y232或者PLIN3 Y251/>的OH根与ATP的γ-磷酸根之间的距离足够接近以使得磷酸基团转移。
实施例15、在缺糖条件下CHKα磷酸化的PLIN2/3促进PLIN2/3与Hsc70结合
表达HA-Hsc70、野生型Flag-PLIN2、野生型Flag-PLIN3、PLIN2 Y232F突变或者PLIN3Y251F突变的Huh7细胞,用结合BSA的油酸处理细胞12小时,以及缺糖处理1小时,接着用Flag琼脂糖珠做免疫沉淀分析。结果如图18所示,PLIN2 Y232F突变或者PLIN3 Y251F突变阻断了PLIN2/3与Hsc70的相互作用。
实施例16、缺糖条件下CHKα介导的PLIN2/3磷酸化导致ATGL和自噬体募集到脂滴以发生脂滴脂解
U87野生型及CHKαS279A突变、CHKαK247R突变、PLIN2 Y232F突变或者PLIN3 Y251F突变的U87细胞,用结合BSA的油酸处理细胞12小时。接着缺糖处理2小时。细胞用BODIPY、DAPI、PLIN2抗体、PLIN3抗体染色,做免疫荧光分析。结果如图19和图20,CHKαS279A突变、CHKαK247R突变、PLIN2 Y232F突变或者PLIN3 Y251F突变阻断了缺糖诱导的PLIN2/3从脂滴解离(图19),以及ATGL、Beclin1、LC3B从脂滴解离(图20)。
实施例17、CHKα介导的脂解促进肿瘤细胞生存
表达CHKαshRNA、PLIN2 shRNA、PLIN3 shRNA、重构的野生型Flag-rCHKα、重构的野生型Flag-rPLIN2、重构的野生型HA-PLIN3或者如图所示突变的Huh7细胞,用25mM 2-DG处理48小时,之后做细胞计数统计。如图21所示,在2-DG处理的条件下,表达CHKα或者PLIN2/3的突变进一步减少了细胞增殖。
实施例18、CHKα介导的脂解促进脑肿瘤生长
U87细胞以及表达CHKαS279A突变、CHKαK247R突变、PLIN2 Y232F/PLIN3 Y251F突变(PLIN2/3Mut)、ATGL shRNA或者Beclin1 shRNA的U87细胞,颅内注射无胸腺裸鼠,两周之后腹腔每天注射0.2ml 2-DG(500mg/kg),持续注射14天,之后检测肿瘤大小(如图22),左侧HE染色结果展示了代表性的肿瘤异种移植,右侧结果为肿瘤体积检测。肿瘤组织做冰冻切片,之后用红油O染色,统计红油O染色区域百分比(如图23)。结果显示,表达这些突变或者ATGL shRNA或者Beclin1 shRNA抑制了肿瘤生长,增加了脂滴累积。
实施例19、CHKα介导的脂解在脑胶质瘤的恶性临床表现中起关键作用
用如图24所示抗体在100例脑胶质瘤样本中做免疫组化,结果显示ACC S79磷酸化水平、CHKαS279磷酸化水平、CHKαK247乙酰化水平、PLIN2 Y232磷酸化水平和PLIN3 Y251磷酸化水平之间彼此正向相关(如图25)。在其中的60例患者的样本中,分别把ACC S79磷酸化水平、CHKαS279磷酸化水平、CHKαK247乙酰化水平、PLIN2 Y232磷酸化水平和PLIN3Y251磷酸化水平分为高表达和低表达,绘制总体生存时间Kaplan-Meier图,结果如图26所示,CHKαS279磷酸化水平、CHKαK247乙酰化水平、PLIN2 Y232磷酸化水平和PLIN3 Y251磷酸化水平与脑胶质瘤患者的差预后正相关。
实施例20、ENO1与CHKα结合
把纯化的GST-CHKα与纯化的ENO1共同孵育之后,通过GST下拉试验,结果如图27,NO1与CHKα之间存在结合。
实施例21、ENO1抑制CHKα的泛素化
在U87细胞中表达Flag-CHKα、His-Ub和ENO1 shRNA,通过免疫沉淀及免疫印迹分析,如图28所示,敲降ENO1增强了CHKα的泛素化。
实施例22、TRIM25结合并泛素化CHKα的K195位点
在U87细胞中表达Flag-CHKα、HA-TRIM25、CHKαK195R、CHKαK273R、CHKαK276R、CHKαK325R突变,通过免疫沉淀及免疫印迹分析,如图29,TRIM25结合CHKα;如图30,CHKαK195R突变降低了其泛素化水平。
实施例23、ENO1与CHKα的结合减少了TRIM25与CHKα的结合
在U87细胞中表达HA-TRIM25、Flag-CHKα、Flag-CHKαF199N、Flag-CHKαP200N,通过免疫沉淀及免疫印迹分析,如图31所示,ENO1与CHKα的结合位点突变增加了TRIM25与CHKα的结合。
实施例24、ENO1上调的CHKα促进了磷脂酰胆碱生产
在U87细胞中表达ENO1 shRNA、TRIM25 shRNA,检测细胞中的磷脂酰胆碱的含量。如图32所示,敲低ENO1降低了磷脂酰胆碱的产量,而敲低TRIM25增强了磷脂酰胆碱的产量;在敲低TRIM25的基础上,敲低ENO1,削弱了磷脂酰胆碱的产量。
实施例25、CHKα磷酸化ENO1 Y44位点
通过体外激酶实验、免疫沉淀和免疫印迹实验,如图33所示,CHKα磷酸化ENO1 Y44位点。
实施例26、CHKα介导的ENO1磷酸化增强了肿瘤细胞糖酵解
如图34所示,敲除CHKα或这敲降ENO1均使得肿瘤细胞LN229的乳酸产量降低,而降低糖酵解。
当然,以上仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种检测CHKαS279位点磷酸化水平、CHKαK247位点乙酰化水平和/或CHKα的C303和C307之间形成分子内二硫键水平的试剂在制备癌症诊断产品中的应用。
2.根据权利要求1所述的应用,其特征在于,当CHKαS279位点磷酸化水平、CHKαK247位点乙酰化水平和/或CHKα的C303和C307之间形成分子内二硫键水平与参考水平相比升高时,代表患者具有侵袭性癌症或侵袭性癌症在进展期;
当参考水平为非癌细胞的水平,代表患者具有侵袭性癌症;
当参考水平为早期癌细胞的水平,代表患者具有侵袭性癌症在进展期。
3.根据权利要求1或2所述的应用,其特征在于,所述癌症是口腔癌、口咽癌、鼻咽癌、呼吸系统癌症、泌尿生殖系统癌症、胃肠癌、中枢或周围神经系统组织癌、内分泌或神经内分泌系统癌症或造血系统癌症、胶质瘤、肉瘤、上皮癌、淋巴瘤、黑素瘤、纤维瘤、脑脊膜瘤、脑癌、肾癌、胆道系统癌症、嗜铬细胞瘤、胰岛细胞癌、利-弗劳梅尼瘤、甲状腺癌、甲状旁腺癌、垂体瘤、肾上腺瘤、骨源性肉瘤肿瘤、神经内分泌系统肿瘤、乳腺癌、肺癌、头颈癌、前列腺癌、食管癌、气管癌、肝癌、膀胱癌、胃癌、胰腺癌、卵巢癌、子宫癌、宫颈癌、睾丸癌、结肠癌、直肠癌或皮肤癌。
4.一种抑制CHKα蛋白激酶活性激活、CHKαS279位点磷酸化、CHKαK247位点乙酰化和/或CHKα的C303和C307之间形成分子内二硫键的试剂在制备癌症治疗产品中的应用。
5.根据权利要求4所述的应用,其特征在于,所述癌症治疗产品包括CHKα抑制剂、CHKαS279抑制剂、CHKαK247抑制剂、CHKα的C303和C307之间形成分子内二硫键抑制剂。
6.根据权利要求5所述的应用,其特征在于,所述CHKα抑制剂包括针对CHKα蛋白激酶活性的小分子抑制剂,所述CHKαS279、CHKαK247抑制剂包括选择性针对CHKαS279位点磷酸化、CHKαK247位点乙酰化、CHKα的C303和C307之间形成分子内二硫键的多肽、小分子抑制剂或互补的抑制性多核苷酸。
7.根据权利要求4-6任一项所述的应用,其特征在于,所述癌症是口腔癌、口咽癌、鼻咽癌、呼吸系统癌症、泌尿生殖系统癌症、胃肠癌、中枢或周围神经系统组织癌、内分泌或神经内分泌系统癌症或造血系统癌症、胶质瘤、肉瘤、上皮癌、淋巴瘤、黑素瘤、纤维瘤、脑脊膜瘤、脑癌、肾癌、胆道系统癌症、嗜铬细胞瘤、胰岛细胞癌、利-弗劳梅尼瘤、甲状腺癌、甲状旁腺癌、垂体瘤、肾上腺瘤、骨源性肉瘤肿瘤、神经内分泌系统肿瘤、乳腺癌、肺癌、头颈癌、前列腺癌、食管癌、气管癌、肝癌、膀胱癌、胃癌、胰腺癌、卵巢癌、子宫癌、宫颈癌、睾丸癌、结肠癌、直肠癌或皮肤癌。
8.一种检测CHKαS279位点磷酸化水平和/或CHKαK247位点乙酰化水平的试剂在制备癌症预后预测产品中的应用。
9.根据权利要求8所述的应用,其特征在于,当CHKαS279位点磷酸化水平和/或CHKαK247位点乙酰化水平与参考水平相比升高时,代表患者具有不良预后;
所述参考水平是来自非癌细胞或早期癌细胞的水平。
10.根据权利要求8-9任一项所述的应用,其特征在于,所述癌症是口腔癌、口咽癌、鼻咽癌、呼吸系统癌症、泌尿生殖系统癌症、胃肠癌、中枢或周围神经系统组织癌、内分泌或神经内分泌系统癌症或造血系统癌症、胶质瘤、肉瘤、上皮癌、淋巴瘤、黑素瘤、纤维瘤、脑脊膜瘤、脑癌、肾癌、胆道系统癌症、嗜铬细胞瘤、胰岛细胞癌、利-弗劳梅尼瘤、甲状腺癌、甲状旁腺癌、垂体瘤、肾上腺瘤、骨源性肉瘤肿瘤、神经内分泌系统肿瘤、乳腺癌、肺癌、头颈癌、前列腺癌、食管癌、气管癌、肝癌、膀胱癌、胃癌、胰腺癌、卵巢癌、子宫癌、宫颈癌、睾丸癌、结肠癌、直肠癌或皮肤癌。
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