CN115105508B - 费德拉替尼在制备治疗krt18低表达的头颈鳞癌药物中的应用 - Google Patents
费德拉替尼在制备治疗krt18低表达的头颈鳞癌药物中的应用 Download PDFInfo
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Abstract
本发明公开了费德拉替尼在制备治疗KRT18低表达的头颈鳞癌药物中的应用。本发明首次发现JAK2抑制剂费德拉替尼具有抗头颈磷癌增殖活性，尤其是对于KRT18低表达头颈磷癌。本发明确定了抑制KRT18表达可以提高头颈磷癌对费德拉替尼的敏感性，KRT18抑制剂和费德拉替尼联合使用可以提升头颈磷癌对费德拉替尼的耐药性，可以有效指导临床的精准用药，本发明在头颈磷癌治疗领域具有广泛的应用前景。
Description
技术领域
本发明涉及生物医药领域，具体涉及费德拉替尼在制备治疗KRT18低表达的头颈磷癌药物中的应用。
背景技术
头颈癌是世界第六大常见癌症类型，包括起源于鼻窦、鼻腔、口腔、咽、喉等部位的上皮性恶性肿瘤，全球每年的发病人数超过55万，死亡人数超过30万，其中95％以上的头颈癌均为头颈鳞癌。目前，手术、放疗和化疗仍是头颈鳞癌的主要治疗手段，但在一定程度上也会造成头颈部器官及功能的损害，并导致患者的生活质量下降。近年来，随着测序技术及多组学分析技术的进展，靶向治疗特异性地作用于肿瘤发生、发展中起关键作用的靶分子及其调控的信号转导通路，显示出高效、低毒等特点，已成为多种癌症类型治疗的主流。然而头颈鳞癌的遗传学特征显示其以抑癌基因突变为主，正面临着靶向药物治疗策略极度匮乏的现状。
西妥昔单抗是唯一被批准用于头颈鳞癌的靶向药物，然而其临床应答率仅为13％，并且大多数研究未能发现EGFR表达或基因扩增与EGFR抑制剂的反应之间的关联，也没有前瞻性验证的生物标志物能够为西妥昔单抗靶向治疗选择合适的患者。头颈鳞癌出现这一治疗困境的根本原因与其特殊的遗传特征相关，由于头颈鳞癌高频变异的基因主要是抑癌基因，可供干预的致癌驱动基因较少，因此大部分针对头颈鳞癌的临床实验均以失败告终。
因此，亟需寻找新的药物用于头颈鳞癌的治疗，并且能够精准的根据生物标志物找到最佳的患者人群。
发明内容
本发明的目的是解决目前缺乏针对头颈鳞癌治疗疗效更好的药物，以及缺少指导临床治疗的耐药生物标志物的缺陷。
为了达到上述目的，本发明提供了费德拉替尼在制备治疗KRT18低表达的头颈鳞癌药物中的应用。
本发明提供了费德拉替尼在制备治疗KRT18低表达的头颈鳞癌药物中的应用。
较佳地，所述药物包含KRT18抑制剂。
较佳地，所述KRT18抑制剂包含与KRT18 mRNA结合的siRNA。
较佳地，所述siRNA选自si-KRT18-1、si-KRT18-2、si-KRT18-3中的任意一种，且所述si-KRT18-1、si-KRT18-2、si-KRT18-3的碱基序列如下：
si-KRT18-1：
正义链：5’-GCUCAGAUCUUCGCAAAUATT-3’；
反义链：5’-UAUUUGCGAAGAUCUGAGCTT-3’；
si-KRT18-2：
正义链：5’-GGUCAUUGAUGACACCAAUTT-3’；
反义链：5’-AUUGGUGUCAUCAAUGACCTT-3’；
si-KRT18-3：
正义链：5’-GGACUUUAAUCUUGGUGAUTT-3’；
反义链：5’-AUCACCAAGAUUAAAGUCCTT-3’。
较佳地，所述头颈磷癌至少包含口腔鳞癌、鼻咽癌、喉癌中的任意一种。
本发明还提供了一种用于制备治疗头颈鳞癌的药物组合物，所述药物组合物至少包含KRT18抑制剂和费德拉替尼。
较佳地，所述KRT18抑制剂包含与KRT18 mRNA结合的siRNA。
本发明还提供了一种生物标志物在制备用于提高头颈磷癌对费德拉替尼的敏感性的药物中的应用，所述生物标志物为KRT18。
本发明还提供了一种用于检测头颈鳞癌对费德拉替尼治疗敏感性的试剂盒，所述试剂盒至少包含与所述KRT18特异性结合的抗体。
较佳地，所述试剂盒选自qPCR试剂盒、免疫印迹检测试剂盒、流式细胞分析试剂盒、ELISA试剂盒中的任意一种。
本发明的有益效果：
(1)本发明首次发现治疗骨髓纤维化的JAK2抑制剂费德拉替尼(Fedratinib)可以应用于头颈鳞癌的临床治疗，并且对KRT18低表达的头颈鳞癌患者具有更好的疗效；
(2)本发明首次发现生物标志物KRT18可作为头颈鳞癌耐药的生物标志物，用于制备提高头颈鳞癌对费德拉替尼敏感性的药物，也可以将KRT18用于制备检测头颈鳞癌对费德拉替尼治疗敏感性的产品，从而指导头颈鳞癌临床的精准用药，在头颈鳞癌治疗领域具有广泛的应用前景。
附图说明
图1为2248个化合物对13株肿瘤原代细胞的高通量药物筛选结果图。
图2为300个化合物对5株肿瘤原代细胞的高通量药物筛选结果图。
图3为Fedratinib对10株头颈鳞癌细胞的药效结果图。
图4为Fedratinib在敏感组与耐药组细胞间进行验证，排名前14的基因的表达量与药效敏感性关系图。
图5为利用siRNA敲减头颈磷癌患者来源的肿瘤原代细胞PDC_56和头颈磷癌商品化细胞系HN13中KRT18表达后，细胞中KRT18和GAPDH表达水平的Westernblot结果图。
图6为利用siRNA敲减PDC_56和HN13中KRT18后，各组细胞在1μM浓度的Fedratinib作用72小时后相对细胞存活率的对比图。
图7为在KRT18低表达的头颈鳞癌异种移植瘤PDX_8后，Fedratinib药物处理后肿瘤体积大小的变化图及统计图；其中，图7的a表示Fedratinib药物处理后肿瘤体积大小的变化图，图7的b表示Fedratinib药物处理后肿瘤体积大小的统计图。
图8为在KRT18高表达的头颈鳞癌异种移植瘤PDX_31后，Fedratinib药物处理后肿瘤体积大小的变化图及统计图；其中，图8的a表示Fedratinib药物处理后肿瘤体积大小的变化图，图8的b表示Fedratinib药物处理后肿瘤体积大小的统计图。
具体实施方式
以下结合附图和实施例对本发明的技术方案做进一步的说明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和仪器为本领域常规试剂、方法和仪器。
本发明中用以构建PDX模型的小鼠为雌性BALB/c-nu裸鼠，6-8周龄，购自上海市计划生育科学研究所实验动物经营部(动物许可证号：SCXK(沪)2018-0006)。小鼠饲养符合SPF级条件下分笼饲养。保持室温18-25℃，相对湿度40％-60％，专用鼠笼、垫料，饲料及饮水经121℃，30min高压灭菌，每周至少1次更换垫料。
实施例1构建头颈鳞癌患者来源的异种移植瘤模型
对于本发明构建PDX模型的对应患者，记录其临床信息，包括基本资料(性别、年龄、烟酒史等)、临床病理诊断(肿瘤大小及位置、TNM分期、HPV感染情况)、既往治疗史(手术、放化疗情况)、复发和转移等预后信息等。收集患者的肿瘤、癌旁及血液样本，对肿瘤样本进行病理组织形态学鉴定和遗传学信息鉴定。
肿瘤组织经手术切除后，观察组织颜色、形态、质地，切除坏死组织，选择病灶中央部分进行取材。因头颈鳞癌一般生长于口腔、鼻腔黏膜等污染部位，因此样本接种移植到小鼠前需要用0.05％次氯酸钠除菌，1％青霉素-链霉素双抗的PBS快速清洗30秒。轻轻刮除组织样本外周组织，将肿瘤切成1～2mm3大小的小块，在无菌条件下将患者组织移植到免疫缺陷小鼠血供、***较丰富的区域(如双侧腋窝)构建皮下异种移植瘤模型(Patient-Derived Xenograft,PDX)模型，或接种于动物双侧颌下间隙构建原位PDX模型。为提高接种成功率，可将Matrigel胶与患者肿瘤组织混合后接种，每个患者组织接种到3-5只小鼠。建模1-2周后开始追踪PDX模型生长轨迹，当肿瘤体积超过800mm3或肿瘤体积两周无明显增长时，对移植瘤进行序列传代，一般移植瘤传代至3代以上时认为该模型可以稳定传代。构建得到的PDX模型用于后续体内实验开展相关验证实验。
实施例2构建头颈鳞癌患者来源的肿瘤原代细胞
头颈鳞癌肿瘤组织在4℃环境下运输回实验室后，消毒外包装，通过无菌传递仓进入细胞房，利用无菌显微剪、显微镊进行组织分离剪碎。配制组织消化液(1mg/ml四型胶原酶，200U/ml透明质酸酶，200U/ml DNase I型酶，1×无酚红胰蛋白酶)，组织消化液体积约为肿瘤体积的5-10倍。
将剪碎的组织进行1500rpm，离心3min后，用组织消化液重悬后，加入组织解离管，放置于美天旎组织处理器，按照组织韧度选择不同分离模式进行分离。碾碎组织后，放入37℃环境内摇床上消化30-60min，其间每10min将组织解离管进行上下摇晃，混匀组织，避免组织成团，影响消化。待消化液浑浊，无明显组织剩余时，即可终止消化。利用含10％胎牛血清的培养基中和消化，并分别通过100μm和40μm的滤网后，收集未消化团块，收集细胞悬液。1500rpm，离心3min后，若发现较多红细胞污染，可以加入1ml红细胞裂解液，静置3min后，加入10ml PBS稀释后，1500rpm，3min离心。
弃去上清，添加5ml完全培养基重悬，完全培养基配方为高糖DMEM培养基：DMEM/F12培养基＝3:1、胰岛素5μg/ml、两性霉素B 250ng/ml、庆大霉素10μg/ml、霍乱毒素0.1nM、EGF0.125 ng/ml、氢化可的松25ng/ml、ROCK抑制剂Y-2763210μM，利用0.22μm无菌过滤器对溶液进行消毒，并在4℃下保存2个月。细胞悬液置于37℃、5％CO2加湿培养箱中持续培养。成功传代5次以上仍然保持较高细胞增殖活性的肿瘤原代细胞(Patient-Derived Cell,PDC)即为构建成功。构建得到的PDC用于后续体外实验开展相关验证实验。
实施例3利用头颈鳞癌肿瘤原代细胞及细胞系进行高通量药物筛选
选取了2248个小分子化合物，以美国食品与药品监督管理局批准的药物为主(1800/2248)，还纳入了319个临床试验评估药物以及129个临床前化合物。非抗肿瘤药物占了50％以上(1419/2248)，剩余的肿瘤相关的化合物针对了常见的一些作用机制，靶向药物有718个，化疗药物111个。包含针对常见的癌基因靶点的抑制剂，包括PI3K抑制剂，mTOR抑制剂，CDK抑制剂，以及HDAC抑制剂等，总计覆盖了286个不同的药物靶点。
随后本研究先选取13株(分别为PDC_10、PDC_22、PDC_6、PDC_13、PDC_11、PDC_14、PDC_19、PDC_4、PDC_5、PDC_20、PDC-16、PDC_12、PDC_15)患者肿瘤原代细胞，以单剂量(1μM)对2248个化合物进行三次重复筛选，测定药物作用72小时后细胞的增殖活力。
实验结果：如图1、图2所示，根据初筛结果，选取能够在一个以上的细胞模型中引起50％的细胞杀伤效果的300个化合物，进行5株肿瘤原代细胞的3浓度药物筛选(5μM、1μM、0.2μM)。其中，总共有171种化合物被排除，原因是它们没有浓度梯度依赖性，或者在一个以上的细胞模型中不能减少至少50％的活细胞数量。最后，筛选出129个化合物具有较好的抗头颈鳞癌活性的药物，这些药物形成了本发明的药物筛选库。
实施例4对药物筛选库进行药物基因组学研究，筛选药效敏感生物标志物
对129个药物组成的药物筛选库开展了54株头颈鳞癌细胞模型(包括40株肿瘤原代细胞和14株商品化细胞系)进行10浓度梯度的响应曲线分析。药物处理72小时后，利用Rpackage GRmetrics计算药物响应指标IC50、Emax和AUC，以及对应的GR50、GRmax和GRAOC。
为了进行药物基因组学分析，所有肿瘤原代细胞和商品化细胞系均进行了全外显子组测序和转录组测序。为了寻找药物反应相关特征并建立预测模型，将药效数据，结合使用基因组改变、拷贝数变异和基因表达特征来评估它们对药物反应的贡献。描绘各个指标作为生物标志物指导药物使用的潜力。通过Spearman相关性及P值评价预测效果。
实施例5药物基因组学分析验证Fedratinib对KRT18低表达头颈鳞癌具有高敏感性
对129个化合物组成的头颈鳞癌药物筛选库进行分析，筛选得到IC50排名前十五的药物：Bortezomib、Ceritinib、Foretinib、CUDC-101、Pelitinib、Osimertinib、dactolisib、OTSSP167、LY2874455、Dacomitinib、Neratinib、Brigatinib、Fedratinib、Poziotinib、Ponatinib。本申请发现Fedratinib作为IC50排名前十五药物中唯一未在头颈鳞癌中开展过临床试验的靶向药物，具有极高的抗头颈鳞癌潜力。鉴于此，本发明对其进行药物基因组学分析，结果鉴定了2862个特征与Fedratinib药效相关。
本申请筛选得到的Fedratinib购自Selleck，其CAS号：936091-26-8，分子式：C27H36N6O3S。
随后选取未进行过药物筛选的独立的6株肿瘤原代细胞和4株商品化细胞系组成验证细胞集合进行Fedratinib药效测试，将细胞分成Fedratinib敏感组与耐药组细胞。
实验结果：如图3所示，经过Fedratinib药效测试后，敏感组细胞为PDC_53、HSC3、SCC25、PDC_55、PDC_51，耐药组细胞为TU686、HN13、PDC_54、PDC_52、PDC_56。如图4所示，随后检测在上述10株细胞中排名前14的药效生物标志物表达情况，结果显示KRT18的低表达与Fedratinib的敏感性存在显著相关性(P＝0.001)。
KRT18编码I型中间丝链角蛋白18，根据相关报道，KRT18高表达与包括结直肠癌、胃癌、非小细胞肺癌、肝癌在内的多种癌症类型的不良预后相关。KRT18是上皮间质转换过程中关键的参与蛋白，在上皮间质转换发生过程中，上皮细胞极性丧失，与周围细胞和基质细胞的接触减少，细胞间的相互作用减少，细胞迁移和运动能力增强，同时细胞表型发生改变，丧失上皮表型，表现为角蛋白如KRT18表达水平下降，将导致细胞的粘附力降低，使细胞获得易于侵袭和转移的特性。
实施例6体外实验验证Fedratinib对KRT18低表达头颈鳞癌细胞具有更高敏感性
siRNA的设计和合成：siRNA由吉玛基因生物公司合成出目的序列，具体siRNA序列如表1所示。
表1 siRNA序列
| sense(5'-3') | antisense(5'-3') | |
| si-KRT18-1 | GCUCAGAUCUUCGCAAAUATT | UAUUUGCGAAGAUCUGAGCTT |
| si-KRT18-2 | GGUCAUUGAUGACACCAAUTT | AUUGGUGUCAUCAAUGACCTT |
| si-KRT18-3 | GGACUUUAAUCUUGGUGAUTT | AUCACCAAGAUUAAAGUCCTT |
| si-NC | UUCUCCGAACGUGUCACGUTT | ACGUGACACGUUCGGAGAATT |
siRNA转染：转染前一天，在孔板中每个孔内加入目的细胞，每孔放2.5mL不含抗生素的生长培养基。从细胞中去除生长培养基，加入1.5mL新鲜的不含血清的生长培养基。在250μL的无血清生长培养基中加入100pmolsiRNA，轻柔混匀。在250μL生长培养基中加入5μL的转染试剂进行稀释，室温下温育5min。混合前两步稀释好的siRNA和转染试剂，室温下温育20min。将混合物加入前一天加有细胞的孔板中。在37℃培养箱中培养细胞5-6h。更换含有血清的培养基并培养细胞24-48h，进行转染后的其它检测步骤。培养3-4天后检测蛋白表达量。
蛋白免疫印迹：在裂解细胞后，进行BCA蛋白定量，随后加入SDS loading buffer，95℃加热10分钟变性，然后将10-50μg蛋白样品进行SDS-PAGE电泳。电泳后将PAGE胶上的蛋白通过转膜***转移到PVDF膜上，分别进行一抗和二抗孵育。使用的抗体包括：Anti-KRT18和anti-GAPDH，进而检测在头颈鳞癌中敲减KRT18的敲减效率。
实验结果：如图5所示，相比对照组，siRNA处理组的目的基因表达均显著降低。
细胞增殖实验：将头颈鳞癌细胞以3000细胞/孔的密度接种到96孔板中，培养箱培养过夜。随后进行siRNA转染，再用Fedratinib进行药物处理，72小时后用Cell CountingKit-8检测细胞增殖情况。
实验结果：如图6所示，相比于对照组细胞和空白组细胞，在PDC_56和HN13中Fedratinib显著抑制了KRT18敲减细胞增殖，表明KRT18低表达头颈鳞癌细胞对Fedratinib更敏感。
实施例7体内移植瘤实验验证Fedratinib对KRT18低表达的头颈鳞癌具有更高敏感性
选择两例头颈鳞癌PDX模型(PDX_8,PDX_31)用于评价Fedratinib对不同KRT18表达水平头颈鳞癌的体内治疗效果。每个PDX模型构建15只子代模型，待肿瘤体积达到100-200mm3后选择10-12只小鼠进行分组，分别分到Fedratinib药物处理组和溶剂对照组。
①Fedratinib药物处理组，口服，Fedratinib 120mg/kg，溶剂为2％DMSO+30％PEG300+5％Tween-80+63％水，每天。
②溶剂对照组：对应药物溶剂2％DMSO+30％PEG300+5％Tween-80+63％水，每天。
持续测量模型肿瘤体积及体重直到肿瘤体积达到1000-1500mm3。
实验结果：如图7所示，PDX_8为KRT18低表达模型，表达水平为KRT18RPKM＝33.61，在PDX_8模型中Fedratinib药物处理组相比于对照组显著抑制了肿瘤生长。如图8所示，PDX_31为KRT18高表达模型，表达水平为KRT18 RPKM＝185.53。在PDX_31模型中Fedratinib药物处理组相比于对照组对肿瘤生长无明显影响。可以说明费德拉替尼在治疗KRT18低表达头颈磷癌更具有敏感性。在PDX模型中，KRT18 RPKM低于33.61定义为KRT18低表达。
一些实施例中，本发明还提供了一种用于检测头颈磷癌患者对费德拉替尼的敏感性的试剂盒，该试剂盒至少包含与KRT18特异性结合的抗体，可以通过检测头颈磷癌组织中KRT18的表达水平，来判断头颈磷癌对费德拉替尼的敏感性。所述试剂盒选自qPCR试剂盒、免疫印迹检测试剂盒、流式细胞分析试剂盒、ELISA试剂盒中的任意一种。
本发明首次发现了费德拉替尼具有抗头颈磷癌增殖的活性，尤其对于KRT18低表达的头颈磷癌患者。KRT18作为生物标志物可以预测头颈磷癌对费德拉替尼治疗的敏感性，KRT18抑制剂与费德拉替尼的联合用药可以提升费德拉替尼治疗头颈磷癌的药效，在临床用药上具有较佳的指导效果，具有极大的应用前景。
尽管本发明的内容已经通过上述优选实施例作了详细介绍，但应当认识到上述的描述不应被认为是对本发明的限制。在本领域技术人员阅读了上述内容后，对于本发明的多种修改和替代都将是显而易见的。因此，本发明的保护范围应由所附的权利要求来限定。
Claims (4)
1.费德拉替尼在制备治疗KRT18低表达的头颈鳞癌药物中的应用，其特征在于，所述药物包含KRT18抑制剂，所述KRT18抑制剂包含与KRT18mRNA结合的siRNA，所述siRNA选自si-KRT18-1、si-KRT18-2、si-KRT18-3中的任意一种，且所述si-KRT18-1、si-KRT18-2、si-KRT18-3的碱基序列如下：si-KRT18-1：
正义链：5’-GCUCAGAUCUUCGCAAAUATT 3’；
反义链：5’-UAUUUGCGAAGAUCUGAGCTT 3’；
si-KRT18-2：
正义链：5’-GGUCAUUGAUGACACCAAUTT 3’；
反义链：5’-AUUGGUGUCAUCAAUGACCTT 3’；
si-KRT18-3：
正义链：5’-GGACUUUAAUCUUGGUGAUTT 3’；
反义链：5’-AUCACCAAGAUUAAAGUCCTT 3’。
2.如权利要求1所述的应用，其特征在于，所述头颈磷癌至少包含口腔鳞癌、鼻咽癌、喉癌中的任意一种。
3.一种用于制备治疗头颈鳞癌的药物组合物，其特征在于，所述药物组合物包含如权利要求1中所述KRT18抑制剂和费德拉替尼。
4.一种生物标志物抑制剂在制备用于提高头颈磷癌对费德拉替尼的敏感性的药物中的应用，其特征在于，所述生物标志物抑制剂为权利要求1中所述的KRT18抑制剂。
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