Clinical Value of Differential Expression of Pim1 and Notch1 During Carcinogenesis of Breast Cancer
-
摘要:目的
探讨Pim1和Notch1在乳腺组织中差异性表达的临床意义及与预后的关系。
方法qRT-PCR、免疫组织化学法和数据库检测评估Pim1、Notch1 mRNA和蛋白在乳腺癌旁正常组织、普通型导管增生、导管原位癌和浸润性导管癌中的表达水平,分析Pim1和Notch1表达与乳腺癌患者临床病理参数的关系,Kaplan-Meier Plotter数据库发掘Pim1和Notch1在乳腺癌预后中的价值。
结果乳腺癌组织中Pim1 mRNA水平升高,Pim1蛋白表达明显降低;Pim1 mRNA与淋巴结转移和TNM晚期密切相关,Pim1蛋白在淋巴结转移及高分级患者中表达显著减少,其低表达的患者无复发生存期更短(P=0.000)。Notch1 mRNA和蛋白在乳腺癌中显著高表达,Notch1 mRNA与淋巴结转移、高分级和晚分期明显相关,Notch1蛋白在淋巴结转移组中表达显著升高,其高表达患者总生存时间减少(P=0.025)。Pim1和Notch1蛋白表达在乳腺癌中负相关(r=-0.385,P=0.001)。
结论Pim1低表达和Notch1高表达可能是促进乳腺癌发生发展及预后不良的危险因素,可能成为评估乳腺癌潜在生物标志物。
Abstract:ObjectiveTo investigate the differential expression levels of Pim1 and Notch1 in breast tissues and their clinical and prognostic significance.
MethodsqRT-PCR, immunohistochemistry and databases were used to detect the mRNA and protein levels of Pim1 and Notch1 genes in adjacent normal breast tissues (ANBT), usual ductal hyperplasia (UDH), ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). The relation between Pim1/Notch1 expression and the clinicopathological parameters of breast cancer patients was analyzed. The prognostic value of Pim1 and Notch1 protein in breast cancer was evaluated by Kaplan-Meier Plotter databases.
ResultsThe expression of Pim1 mRNA in breast cancer tissues was increased. The expression of Pim1 protein in the breast cancer tissues was significantly decreased. The expression of Pim1 mRNA was markedly associated with lymph node metastasis and advanced TNM stage. The expression of Pim1 protein was significantly decreased in the patients with lymph node metastasis or histological grade 3. The patients with low expression of Pim1 had a shorter relapse-free survival (P=0.000). The expression of Notch1 mRNA and protein in breast cancer were higher than those in normal breast tissues. Notch1 mRNA was closely related to the lymph node metastasis, high grade and advanced stage. Notch1 protein expression in lymph node metastasis group had markedly increased. The overall survival time of patients with high level of Notch1 was shorter (P=0.025). The protein levels of Pim1 and Notch1 were negatively correlated in breast cancer tissues (r=-0.385, P=0.001).
ConclusionLow Pim1 expression and high Notch1 expression may promote the carcinogenesis and progression of breast cancer and may be risk factors for poor prognosis. Pim1 and Notch1 may be used as potential biomarkers for clinical evaluation of breast cancer patients.
-
Key words:
- Breast cancer /
- Pim1 /
- Notch1 /
- Development /
- Prognosis
-
0 引言
膀胱癌(bladder cancer, BC)是中国泌尿生殖系统常见的恶性肿瘤之一[1],约75%新诊断的BC为非肌层浸润性膀胱癌(non-muscle invasive bladder cancer, NMIBC),局限于膀胱壁黏膜或黏膜下层,主要治疗手段为经尿道膀胱肿瘤切除术(transurethral resection of the bladder tumor, TURBT)联合膀胱内灌注化疗药物。但TURBT术后1年肿瘤复发率为40%~80%,25%的患者进展为肌层浸润性膀胱癌(muscle invasive bladder cancer, MIBC)[2]。目前普通白光膀胱镜检查是BC诊断和术后随访的标准技术手段,其对微小肿瘤、原位癌、术中残留肿瘤的识别能力有限,导致肿瘤的漏诊,而常规膀胱内灌注药物的不良反应严重影响患者术后生活质量[3]。因此,如何精准地发现病灶及界定肿瘤的边界、既能彻底切除肿瘤又能减少正常组织细胞的损伤成为了BC临床诊疗亟需解决的问题。
随着BC靶向分子载体、靶向特异性作用机制、靶向光学分子影像和靶向光免疫疗法(photoimmunotherapy, PIT)研究的不断深入,BC的靶向诊疗有望取得新的进展。为此,本文结合国内外最新文献及本研究组的前期工作,将已发现的BC靶向分子载体的研究现状综述如下。
1 靶向基团的种类及特性
靶向分子载体是指能够特异识别抗原蛋白并与之紧密结合的靶向基团,主要包括抗体、抗体片段、支架蛋白、肽和小分子等,见表 1[4]。抗体与抗原特异性结合以及成熟的抗体制备技术,使其成为临床应用最多的一类靶向基团,但单克隆抗体分子量较大(约150 kDa)、肾脏不能排泄、体内主要通过肝脏分解代谢,导致抗体具有血液内循环时间长、非特异性组织蓄积和组织穿透能力差等缺点[5-6]。而采用化学技术制备的抗体片段(微抗体、抗原结合片段、双抗体、单链可变片段等),分子量约为15~80 kDa,既保留了完整抗体特异性结合抗原的生物学特性,又具有组织渗透性强和血液滞留时间短的优点[7]。
另外,最新关注的靶向基团还有非免疫球蛋白类支架蛋白(scaffolds protein),如亲和体、结蛋白和Centyrin等[8-10],这些蛋白支架既有类似抗体的靶向功能,又具有相对分子量小、高亲和力、折叠速率快、理化性能稳定、能接受化学修饰等优点。
此外,肽类和小分子的分子量最小,能与肿瘤特异性结合,而不与或很少与正常组织、细胞结合的小分子肽被称为肿瘤导向肽(tumor homing peptide, THP)。THP为基础的载体系统能够有效穿透组织且几乎无免疫原性,其半衰期短、肾清除率快、组织蓄积少以及生产工艺便捷、成本更低等优势[11],具有良好的应用前景。
2 膀胱癌靶向分子载体
2.1 抗体及其衍生物
CD47是一种普遍存在于人类细胞膜表面的跨膜糖蛋白,与巨噬细胞表面的信号调节蛋白α结合后能抑制其吞噬功能使肿瘤产生免疫逃逸[12]。CD47在80%以上的BC细胞膜上高表达,包括乳头状尿路上皮癌、鳞状细胞癌、微乳头状瘤及腺癌[13]。本项目组使用Alexa Fluor 790标记的抗-CD47孵育26例新鲜离体人BC组织进行近红外荧光成像,肿瘤组织的平均荧光强度明显高于临近正常组织(分别为132.31±6.67和52.27±12.09, P<0.001),见图 1[14]。Pan等[13]评价了抗-CD47-Qdot625介导的内镜分子成像检测BC的诊断准确性,21例新鲜完整膀胱标本经抗-CD47-Qdot625孵育后,在蓝光下检测到119个可疑区域,通过病理学验证,其诊断敏感度和特异性分别为82.9%和90.5%。
图 1 膀胱癌组织图像Figure 1 Images of bladder cancer tissuesA: The image of tumor specimen under white light; B: After anti-CD47-Alexa Fluor 790 incubating, the image of tumor specimen under NIR light; C: After anti-CD47-Alexa Fluor 790 incubating, the pseudo-color image of fresh integrated tumor specimen under NIR light; D: The HE staining image of fresh integrated tumor specimen; E: The HE staining representative image of tumor area; NIR: near-infrared.CAⅨ是碳酸酐酶家族中的一员,在缺氧条件下调节细胞内pH值,进而改变肿瘤细胞的黏附、增殖和进展的生物学特性[15]。免疫组织化学染色显示CAⅨ在BC组织中的阳性率高达67.1%,在正常的尿路上皮和膀胱慢性炎性病变组织中均呈阴性(P<0.01)[16]。Wang等[17]在8例新鲜完整膀胱标本内灌注抗-CAⅨ-Qdot625,白光膀胱镜诊断BC的敏感度为76.0%,特异性为90.5%,而抗-CAⅨ介导的蓝光内镜分子成像下的肿瘤检测获得了较高的诊断准确率,总体敏感度和特异性分别为88.00%和93.75%。
PIT是一种新型的分子靶向光动力治疗模式,亲水性酞菁染料IR700与单克隆抗体结合进行的靶向性PIT,有效减少了单纯IR700光动力疗法的不良反应[18]。Kiss等[19]研究发现,抗-CD47-IR700介导的PIT治疗,IR700使用剂量低,但显著增加了人膀胱肿瘤细胞系和原代膀胱肿瘤细胞的细胞毒作用。通过尾静脉注射抗-CD47-IR700对BC异种移植小鼠模型进行近红外光免疫治疗(NIR-PIT),单次治疗发现NIR-PIT组肿瘤生长明显减慢,在连续5周的治疗后,NIR-PIT能够有效抑制肿瘤生长(P=0.0104),明显延长治疗组小鼠的生存期(P=0.009)[20]。
单链可变片段(single-chain variable fragment, scFv)是抗体内部结合抗原的最小功能结构域,Rezaei等[20]开发了新型抗-CD47-scFv磁性纳米粒子(magnetic nanoparticles, MNP),体外研究表明抗-CD47-scFv-MNP对BC细胞系EJ138和5637具有高亲和力,经外磁场作用的靶向热疗后能显著降低肿瘤细胞存活率。
2.2 肿瘤导向肽(THP)
目前,利用噬菌体展示肽库技术和组合化学方法,先后筛选出四种与BC结合的靶向配体,分别是九肽序列Bld-1[21]、环九肽序列PLZ4[22]、环七肽序列NYZL1[23]和PLSWT7[24]。其中前两种筛选自MIBC细胞系,后两种则来自NMIBC细胞系。研究显示,四种THP均能在离体细胞、组织和小鼠体内与BC特异性结合,但它们结合肿瘤细胞的分子位点目前还不清楚[21-24]。
PLSWT7是目前唯一运用于内镜下分子成像的多肽载体。Peng等[24]将PLSWT7-IRDye800CW分子探针灌注入8例离体人膀胱腔内,分析了40个膀胱内感兴趣区域,进行了近红外分子成像诊断与组织病理学比较研究,发现PLSWT7-IRDye800CW分子影像诊断BC的敏感度和特异性分别为84.00%和86.70%。
常规尿液脱落细胞检查是一种简单易行的非侵入性诊断方法,但检测的敏感度较低。Jia等[25]使用Bld-1-FITC探针结合80例血尿患者尿液中的肿瘤细胞,其诊断BC的敏感度和特异性分别为79.31%和100%,优于尿脱落细胞学检查和荧光原位杂交技术检查(敏感度分别为20.69%和72.41%)。另外一项包含66例BC的研究发现,NYZL1-FITC探针与尿液中肿瘤细胞的结合与肿瘤恶性程度成正比,平均阳性结合百分比在Ta、T1、T2和T3~T4期分别为30%、57%、73%和85%[26]。
在靶向化疗方面,Bld-1能够与凋亡肽KLA和阿霉素(DOX)构建靶向制剂,并内化进入人膀胱癌HT1376细胞[27-28]。Jung等[27]评价了Bld-1-KLA在小鼠体内的治疗效能,静脉注射给药4周后,Bld-1-KLA组小鼠的肿瘤体积明显小于对照组(P<0.001)。Wei等[28]使用Bld-1-DOX治疗荷瘤小鼠16天后,Bld-1-DOX组的肿瘤体积明显缩小,且小鼠无明显不良反应,而单用DOX治疗组出现明显的心脏和肝脏损害。Lin等[29]开发了PLZ4靶向纳米胶束,在靶向递送方面的效率是非靶向胶束的1.5倍(P<0.05)且是游离染料的14.3倍(P<0.01),靶向胶束不仅能黏附在肿瘤细胞表面,还能被靶细胞摄取。使用PLZ4纳米胶束介导的紫杉醇(PTX)进行靶向化疗,成功将荷瘤小鼠的中位存活期从单纯紫杉醇治疗组的55天提高到了靶向治疗组的69.5天(P=0.03)[30]。
术中光学分子影像引导的外科手术被认为是继开放手术、微创外科手术之后的第三代外科手术新模式。传统的依据白光内窥镜图像和操作者的经验确定肿瘤浸润深度和手术切缘的方式具有主观性。一项涉及8 490例经TURBT治疗BC患者的系统回顾显示,17%~67%的初发Ta期患者和20%~71%的初发T1期患者在复发时发现了残留肿瘤,36%~86%残留肿瘤位于初次切除部位[3]。Peng[24]等采用PLSWT7-IRDye800CW进行了分子成像引导BC手术治疗的临床前研究,将小鼠异种移植模型随机分为两组:对照组(n=20)和实验组(n=20)分别在自然光和光学分子成像下进行肿瘤切除术。1周后,对照组和实验组肿瘤复发率分别为95%和5%;术后30天两组的总存活率分别为0和90%。
3 膀胱癌的多载体靶向和多模态诊疗
BC具有较强的异质性,单纯的一种靶向分子载体很难使全部患者受益,使用抗-CD47、抗-CAⅨ和无靶向性IgG4三种单抗对离体BC组织进行多路复合分子成像发现,诊断图像较单靶点成像噪声更低,分辨率更加精准(ROC AUC为0.93(0.73, 1.0))[31]。多模态分子探针能集成多种成像和(或)治疗模式,Lin等[32]开发的PLZ4-纳米卟啉平台,在进行BC光动力学诊断的同时实现BC的靶向光动力学治疗、靶向光热治疗和靶向化疗结合的三模态治疗,显著提升BC的临床诊断水平和治疗水平。
4 展望与挑战
综上所述,靶向分子载体介导的靶向性定位功能在膀胱癌早期诊断、完整切除、靶向治疗等方面具有巨大的应用潜力,但仍有许多科学问题和技术难点需要解决,比如靶向载体、连接体、荧光染料等的安全性验证,临床有效性的标准化评价体系的建立等,需要临床医生、化学家、物理学家、药理学家和工程师之间的多学科交叉协作。
Competing interests: The authors declare that they have no competing interests.作者贡献熊世双:实验操作与数据统计分析、构思与撰写文章陈晓文:参与文章整体设计穆亚男、张德瑞:参加部分实验操作蒋金芳:实验技术操作指导李军:文章撰写思路指导曹玉文:文章整体构思、内容修改 -
表 1 Pim1和Notch1蛋白表达相关性
Table 1 Association between Pim1 and Notch1 of protein expression
表 2 Pim1和Notch1中蛋白和mRNA与乳腺癌患者临床病理参数的相关性
Table 2 Correlation of Pim1/Notch1 protein and mRNA with clinicopathological features of breast cancer patients
-
[1] Li Q, Jiang C, Wang Y, et al. Resibufogenin suppresses tumor growth and inhibits glycolysis in ovarian cancer by modulating PIM1[J]. Naunyn Schmiedebergs Arch Pharmacol, 2019, 392(12): 1477-1489. doi: 10.1007/s00210-019-01687-2
[2] Cheng H, Huang C, Xu X, et al. PIM-1 mRNA expression is a potential prognostic biomarker in acute myeloid leukemia[J]. J Transl Med, 2017, 15(1): 179. doi: 10.1186/s12967-017-1287-4
[3] Xie Y, Bayakhmetov S. PIM1 kinase as a promise of targeted therapy in prostate cancer stem cells[J]. Mol Clin Oncol, 2016, 4(1): 13-17. http://search.ebscohost.com/login.aspx?direct=true&db=aph&AN=111447791&site=ehost-live
[4] Ouhtit A, Muzumdar S, Gupta I, et al. Understanding the functional discrepancy of Pim-1 in cancer[J]. Front Biosci (Elite Ed), 2015, 7: 208-214. http://europepmc.org/abstract/MED/25553374?source=rss
[5] 刘雪嘉, 肖区龙, 伍希, 等. Pim1蛋白在乳腺不同病变组织中的表达差异及临床特征[J].海南医学院学报, 2019, 25(1): 37-40. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=hainanyxyxb201901010 Liu XJ, Xiao OL, Wu X, et al. Differential expression and clinical characteristics of Pim1 protein in different lesions of breast[J]. Hainan Yi Xue Yuan Xue Bao, 2019, 25(1): 37-40. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=hainanyxyxb201901010
[6] Zhang X, Song M, Kundu JK, et al. PIM Kinase as an Executional Target in Cancer[J]. J Cancer Prev, 2018, 23(3): 109-116. doi: 10.15430/JCP.2018.23.3.109
[7] Santio NM, Landor SK, Vahtera L, et al. Phosphorylation of Notch1 by Pim kinases promotes oncogenic signaling in breast and prostate cancer cells[J]. Oncotarget, 2016, 7(28): 43220-43238. doi: 10.18632/oncotarget.9215
[8] Rhodes DR, Yu J, Shanker K, et al. ONCOMINE: a cancer microarray database and integrated data-mining platform[J]. Neoplasia, 2004, 6(1): 1-6. doi: 10.1016/S1476-5586(04)80047-2
[9] Chandrashekar DS, Bashel B, Balasubramanya SAH, et al. (2017) UALCAN: a portal for facilitating tumor subgroup gene expression and survival analyses[J]. Neoplasia, 2017, 19(8): 649-658. doi: 10.1016/j.neo.2017.05.002
[10] Györffy B, Lanczky A, Eklund AC, et al. An online survival analysis tool to rapidly assess the effect of 22, 277 genes on breast cancer prognosis using microarray data of 1, 809 patients[J]. Breast Cancer Res Treat, 2010, 123(3): 725-731. doi: 10.1007/s10549-009-0674-9
[11] Gao X, Liu X, Lu Y, et al. PIM1 is responsible for IL-6-induced breast cancer cell EMT and stemness via c-myc activation[J]. Breast Cancer, 2019, 26(5): 663-671. doi: 10.1007/s12282-019-00966-3
[12] Deng D, Wang L, Chen Y, et al. MicroRNA-124-3p regulates cell proliferation, invasion, apoptosis, and bioenergetics by targeting PIM1 in astrocytoma[J]. Cancer Sci, 2016, 107(7): 899-907. doi: 10.1111/cas.12946
[13] Pei J, Wang B. Notch-1 promotes breast cancer cells proliferation by regulating LncRNA GAS5[J]. Int J Clin Exp Med, 2015, 8(8): 14464-14471. http://www.ncbi.nlm.nih.gov/pubmed/26550436?dopt=Abstract
[14] Zhu HH, Wang XT, Sun YH, et al. Pim1 Overexpression Prevents Apoptosis in Cardiomyocytes After Exposure to Hypoxia and Oxidative Stress via Upregulating Cell Autophagy[J]. Cell Physiol Biochem, 2018, 49(6): 2138-2150. doi: 10.1159/000493817
[15] Zhang G, Liu Z, Cui G, et al. MicroRNA-486-5p targeting PIM-1 suppresses cell proliferation in breast cancer cells[J]. Tumour Biol, 2014, 35(11): 11137-11145. doi: 10.1007/s13277-014-2412-0
[16] 李秀芹, 韩玉贞, 曾令瑞, 等.乳腺癌组织Notch1和JAG1蛋白表达与分子分型相关性研究[J].中华肿瘤防治杂志, 2017, 24(5): 297-301. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=qlzlzz201705003 Li XQ, Han YZ, Zeng LR, et al. Expression and significance of Notch1 and JAG1 proteins in different subtypes of breast cancer[J]. Zhonghua Zhong Liu Fang Zhi Za Zhi, 2017, 24(5): 297-301. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=qlzlzz201705003
[17] Zhao W, Qiu RY, Li P, et al. PIM1: a promising target in patients with triple-negative breast cancer[J]. Med Oncol, 2017, 34(8): 142. doi: 10.1007/s12032-017-0998-y
[18] Giuli MV, Giuliani E, Screpanti I, et al. Notch Signaling Activation as a Hallmark for Triple-Negative Breast Cancer Subtype[J]. J Oncol, 2019, 2019: 8707053. http://www.researchgate.net/publication/334407398_Notch_Signaling_Activation_as_a_Hallmark_for_Triple-Negative_Breast_Cancer_Subtype
[19] 陈悦, 刘淑真, 张盼.乳腺癌组织中SPARC和Notch-1的表达及其临床意义[J].湖北民族学院学报(医学版), 2018, 35(4): 14-18. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=hbmzxyxb-yxb201804004 Chen Y, Liu SZ, Zhang P. The expression of SPARC and Notch-1 in breast cancer and their clinical significances[J]. Hubei Min Zu Xue Yuan Xue Bao(Yi Xue Ban), 2018, 35(4): 14-18. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=hbmzxyxb-yxb201804004
[20] Wan G, Tian L, Yu Y, et al. Overexpression of Pofut1 and activated Notch1 may be associatedwith poor prognosis in breast cancer[J]. Biochem Biophy Res Commun, 2017, 491(1): 104-111. doi: 10.1016/j.bbrc.2017.07.053
[21] Cao YW, Li WQ, Wan GX, et al. Correlation and prognostic value of SIRT1 and Notch1 signaling in breast cancer[J]. J Exp Clin Cancer Res, 2014, 33(1): 97. doi: 10.1186/s13046-014-0097-2
[22] Yuan X, Zhang M, Wu H, et al. Expression of Notch1 Correlates with Breast Cancer Progression and Prognosis[J]. PLoS One, 2015, 10(6): e0131689. doi: 10.1371/journal.pone.0131689
[23] Xu J, Song F, Jin T, et al. Prognostic values of Notch receptors in breast cancer[J]. Tumor Biol, 2016, 37(2): 1871-1877. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=7510b24cb806d6e1c274950fda51d9c8