CN111450094A - 一种TGF-beta受体分子抑制剂在癌基因KrasG12D突变的胰腺癌治疗中的应用 - Google Patents
一种TGF-beta受体分子抑制剂在癌基因KrasG12D突变的胰腺癌治疗中的应用 Download PDFInfo
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Abstract
本发明涉及一种TGF‑beta受体分子抑制剂ly364947在癌基因KrasG12D突变的胰腺癌治疗中的应用。具体地，本发明提供了TGF‑beta受体‑I小分子抑制剂ly364947的新用途。该抑制剂可以抑制KrasG12D突变驱动的人胰腺癌细胞在体外和异种移植小鼠中表现的上皮‑间充质转化，及其胰腺癌的致瘤性和侵袭能力。本发明为TGF‑beta受体抑制剂在临床上治疗Kras突变导致的胰腺癌提供了一定的实验依据。
Description
技术领域
本发明属于生物技术领域，具体涉及一种TGF-beta受体-I分子抑制剂在癌基因KrasG12D突变的胰腺癌治疗中的应用。
背景技术
胰腺癌是一种恶性程度很高的消化道肿瘤，是所有实体肿瘤中患者生存率最低的癌症(Wang，J.，et al.，Gastrin regulates ABCG2 to promote the migration，invasionand side populations in pancreatic cancer cells via activation of NF-kappaBsignaling.Exp Cell Res，2016.346(1)：p.74-84.)。诊断后5年生存率小于3.5％，唯一可能治愈的治疗方法是手术切除。NAB-紫杉醇-吉西他滨的组合治疗，虽然没有明显延长生存期超过11个月，但也表现出显著的临床效益，已成为晚期和转移性胰腺癌的标准化疗方案(Schneider，G.，et al.，Pancreatic cancer：basic and clinicalaspects.Gastroenterology，2005.128(6)：p.1606-25.)。对于晚期且因为诸如年龄、转移、临床表现状态等因素不能进行手术切除的患者，只有标准和常见的临床或生物治疗方式被采用，目前尚没有分子药物可以有效治疗胰腺癌患者。
哺乳动物的ras基因家族有三个成员，分别是H-ras，K-ras，N-ras，这三种基因的突变在人类癌症中非常常见(Bos，J.L.，ras oncogenes in human cancer：areview.Cancer Res，1989.49(17)：p.4682-9.)。Ras蛋白是由ras基因编码的分子量约为21kDa的蛋白家族，是由190个氨基酸残基组成的小的单体GTP结合蛋白，具有GTPase活性，分布于质膜胞质一侧。Ras蛋白作为小分子GTPase的分子开关，对细胞的多种功能有着调控作用，如存活、增殖、分化以及细胞骨架的重排(Bourne，H.R.，D.A.Sanders，andF.MeCormick，The GTPase superfamily：conserved structure andmolecularmechanism.Nature，1991.349(6305)：p.117-27.)。
在ras基因家族中，K-Ras在癌症的发生中影响最大，行使着类似分子开关的作用。Ras蛋白是活化受体酪氨酸蛋白激酶受体下游的重要功能蛋白，当Kras基因被激活后，可以参与下游信号传导进而激活RAF-MAPK及PI3K/AKT等信号通路，从而调节细胞的增殖，凋亡，代谢及血管生成等生物学过程。COSMICS数据显示在70-95％的胰腺癌病例以及71％的胰腺 癌标本中均存在Kras突变(Forbes，S.A.，et al.，COSMIC：mining complete cancergenomes in the Catalogue of Somatic Mutations in Cancer.Nucleic Acids Res，2011.39(Database issue)：p.D945-50.)。第2外显子的第12密码子发生单核苷酸突变使编码甘氨酸的GGT序列可以分别突变为编码天冬氨酸的GAT序列(G12D)、编码缬氨酸的GTT序列(G12V)、编码精氨酸的CGT序列(G12R)，或编码丙氨酸的GCT序列(G12A)。在第13和61密码子上也会发生点突变，但频率较低。在胰腺癌中约有75％的Kras突变发生在第12位密码子，使原来的甘氨酸突变为天冬氨酸(G12D)或者缬氨酸(G12V)。Kras基因发生G12D点突变后，该基因呈持续激活状态(constitutively active)，阻止Ras-GTP结合构象的水解，使细胞内信号传导紊乱，细胞增殖失控，恶性转化而癌变(Fatrai，S.，et al.，KRAS(G12V)enhances proliferation and initiates myelomonocytic differentiation in humanstem/progenitor cells via intrinsic and extrinsic pathways.J Biol Chem，2011.286(8)：p.6061-70.)。
上皮间充质转化(Epithelial-Mesenchymal Transition，EMT)是上皮细胞失去细胞极性和细胞间粘附的生物学过程，使上皮细胞转化为间充质干细胞从而具有迁移和侵袭的特性，这些多能基质细胞可分化为多种细胞类型。在上皮细胞中，E-cadherin(E-钙黏蛋白)表达水平较高，而在间质细胞中主要表达N-cadherin，纤连蛋白和波形蛋白。很多信号通路诸如TGF-β，FGF，EGF，HGF，Wnt/β-catenin和Notch等以及细胞低氧均会导致上皮间充质转化。EMT使癌细胞获得较强的侵袭能力，从而使癌症发生转移(Hanahan，D.andR.A.Weinberg，Hallmarks of cancer：the next generation.Cell，2011.144(5)：p.646-74.)。一些证据表明，细胞发生EMT后会获得干细胞的特性，从而成为肿瘤干细胞(CancerStem Cells，CSCs)。因此，EMT不仅能使癌细胞进入血液进行转移，并且也赋予了癌细胞干细胞特性，从而增加了肿瘤发生和癌细胞增殖的潜力(Singh，A.and J.Settleman，EMT，cancer stem cells and drug resistance：an emerging axis of evil in the war oncancer.Oncogene，2010.29(34)：p.4741-51.)。
转化生长因子(Tumor Growth Factor，TGF-β)是一种多功能的细胞因子，能够有效地抑制大多数类型正常细胞的增殖，包括上皮细胞、内皮细胞、造血细胞和淋巴细胞。同时，TGF-β通过各种细胞外基质蛋白的诱导能够导致组织纤维化。TGF-β通过激活Smad2/3/4等转录因子从而诱发EMT(Xu J，Lamouille S，Derynck R.TGF-beta-induced epithelialto mesenchymal transition.Cell Res.2009.19(2)：p.156-72.)。EMT是肿瘤侵袭和转移的重要步骤，而TGF-β通过EMT诱导肿瘤的进展。EMT的转录调控表现在上皮标记物的缺失以及间充质特征的获得，这一过程是通过转录程序而实现。转录程序涉及三个转录因子家族，包括Snail，ZEB和bHLH家族，它们因为TGF-β的刺激而进行表达。转录因子一方面可以通过Smad依赖机制来发挥作用，另一方面可以间接地通过其他转录因子的激活或解除其抑制状态而被活化。这些转录因子被激活后，抑制上皮标记基因的表达，并激活间充质基因的表达。
专利号US8298825公开了一个多靶点蛋白激酶抑制剂的小分子化合物ly364947，它对TGF-beta受体-I(TGF-beta RI)具有很强的抑制作用，IC50值为59nM。如下式(I)所示，其化学名为4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline，(4-[3-(2-吡啶基)-1H-吡唑-4-基]-喹啉)。研究表明，ly364947可用于体细胞重编程的制备，并应用在慢性腹膜炎小鼠模型，CT26直肠癌和BxPC3胰腺癌的血管生成(Kano M，Komuta Y，et al.Comparisonof the effects of the kinase inhibitors imatinib，sorafenib，and transforminggrowth factor-b receptor inhibitor on extravasation of nanoparticles fromneovasculature.Cancer Sci.2009.100(1)：p.173-180.)，过表达或经VEGF-C处理的胰腺癌细胞的淋巴血管生成的体外体内实验(Oka M，Iwata C，et al.Inhibition ofendogenous TGF-β signaling enhanceslymphangiogenesis.Blood.2008.111：p.4571-4579.)，BCR-ABL活化的慢性髓系白血病(Naka，K.，et al.，TGF-b-FOXO signallingmaintains leukaemia-initiating cells in chronic myeloid leukaemia.Nature，2010.463：p.676-680)，以及近期小鼠模型来源的胰腺癌细胞ly364947和PD-L1联合免疫治疗中(Sow H，Ren J，et al.Combined inhibition of TGF-β signaling and the PD-L1immune checkpoint is differentially effective in tumor models.Cells.2019.8：320.)。以上研究暗示ly364947可能用于治疗上述疾病，但未涉及ly364947在癌基因KrasG12D突变导致人胰腺癌细胞上皮-间充质转化(EMT)及其在异种移植小鼠的胰腺癌致瘤性和侵袭的研究。
胰腺癌恶性程度高，诊断和治疗困难，因为其具有很强的侵袭和迁移能力。分子生物学分析超过90％的胰腺癌具有Kras基因点突变(Hashimoto D，Arima K，Yokoyama N，etal.Heterogeneity of KRAS mutations in pancreatic ductaladenocarcinoma.Pancreas，2016.45(8)：p.1111-1114.)。因此，本研究选用人胰腺导管腺癌细胞系PANC-1和MIAPaCa-2。PANC-1细胞具有内源性Kras基因编码子12(p.G12D；GGT＞GAT)杂合的错义突变，MIAPaCa-2细胞具有内源性Kras基因编码子12(p.G12C；GGT＞TGT)纯合的错义突变(Gradiz R，Silva H，et al.MIA PaCa-2 and PANC-1-pancreas ductaladenocarcinoma cell lines with neuroendocrine differentiation andsomatostatin receptors.Sci Rep.2016.17(6)：p.21648.)。为了模拟胰腺癌细胞中KrasG12D突变，研究KrasG12D突变在胰腺癌中的作用机制及药物治疗，我们在本研究设计中，在PANC-1和MIAPaCa-2细胞系中过表达KrasG12D点突变基因来探讨胰腺癌的药物治疗和分子机制。我们在前期的基因表达谱芯片数据中发现KrasG12D持续性激活导致TGF-beta信号通路激活。因而我们使用TGF-beta受体RI抑制剂ly364947进行KrasG12D点突变导致人 胰腺癌细胞侵袭迁移和致瘤性的分子靶向药物治疗。本研究没有使用内源性Kras基因为野生型的人胰腺导管腺癌细胞系BxPC-3，其具有TGF-beta通路的18q21位点关键转录因子Smad4基因纯合缺失(Nicolas F，Hill C.Attenuation of the TGF-b-Smad signalingpathway in pancreatic tumor cells confers resistance to TGF-b-induced growtharrest.Oncogene 2003.22：p.3698-3711.)，因此此细胞系无法用于TGF-beta信号通路的研究。
发明内容
本发明的目的是提供一种TGF-beta受体-I分子抑制剂ly364947在治疗癌基因KrasG12D突变的人胰腺导管腺癌的药物应用。本发明提供了一种TGF-beta受体-I抑制剂ly364947的用途，用于制备(i)抑制KrasG12D突变的人胰腺癌细胞上皮-间充质细胞转化、和/或(ii)治疗其引发的胰腺癌致瘤性和侵袭的药物组合物。
本发明发现，TGF-beta受体-I分子抑制剂对于癌基因KrasG12D突变的人胰腺导管腺癌体外和体内实验研究具有良好的治疗效果。在本发明优选的实施方案中，所述的TGF-beta受体-I小分子抑制剂为如下的式(I)所示化合物或其可药用盐。本发明中，所述的TGF-beta受体-I抑制剂的用量可以在1-20mg/kg，优选10mg/kg，所述的用量以化合物(I)的形式计量。
式(I)化合物或其药学上可接受的盐也可以与药学上可接受的载体一起制成本领域熟知的形式，如片剂、胶囊、颗粒剂、注射剂等。本发明涉及含有选自式(I)化合物或者其药学上可接受的盐的化合物在制备治疗前述胰腺癌的药物中的用途。
附图说明
图1显示构建稳定过表达KrasWT和KrasG12D胰腺癌细胞系，进行细胞增殖活性的检测。(A)RT-PCR以及(B)Westernblot Blot验证所得细胞系能分别稳定过表达KrasWT和KrasG12D突变。使用CCK-8(C)和EdU渗入实验(D)进行细胞增殖活力检测。KrasG12D细胞系的增殖活力明显强于KrasWT细胞系，并均高于对照组。Overlay为暗点的细胞核Hoechst33342染色和亮点的EdU增殖细胞核的照片融合图片。
图2显示KrasG12D突变上调TGF-beta下游蛋白，EMT标记物以及EMT转录因子的表达。(A)TGF-betaR1和Foxd4的QPCR结果；(B)pSmad2/3和Foxd4的Westernblot结果；(C)EMT标记物以及EMT转录因子的QPCR结果；(D)EMT标记物以及EMT转录因子的Westernblot结果。
图3显示化合物(I)对胰腺癌细胞的EMT细胞表型的影响。(A，B)加药处理后EdU检测细胞增殖活性变化的结果；暗点为细胞核Hoechst33342染色，亮点为EdU渗入的增殖细胞核，照片为两种染色的融合图片。(C)光镜下观察药物处理对细胞上皮-间质细胞转化形态的影响；(D)加药处理后Transwell细胞迁移实验结果。
图4显示化合物(I)处理胰腺癌细胞系对EMT表型的分子水平的影响。
A.加药处理对胰腺癌细胞mRNA水平的影响，为QPCR结果；B.加药处理对胰腺癌细胞蛋白水平的影响，为Western Blot结果。
图5显示化合物(I)对KrasG12突变胰腺癌异种移植体内肿瘤生长，侵袭和EMT水平的影响。(A)肿瘤生长曲线；(B)第40天肿瘤拍照结果；(C)第40天肿瘤重量；(D)肿瘤组织EMT相关蛋白Western Blot结果；(E)肿瘤组织HE染色结果显示胰腺癌细胞的侵袭生长。KrasG12D和对照组胰腺癌细胞的体内生长和侵袭表型能明显被化合物(I)抑制。
具体实施方式
本发明人首次报道了一种TGF-beta受体-I分子抑制剂ly364947在癌基因KrasG12D突变的人胰腺癌细胞的体外和异种移植体内实验进行有效治疗的应用。实验结果表明ly364947即化合物(I)抑制KrasG12D突变胰腺癌细胞的增殖，迁移和侵袭，以及EMT分子过程。体内实验进一步表明化合物(I)治疗明显抑制胰腺癌的生长和侵袭，以及EMT水平。这为使用TGF-beta受体-I分子抑制剂在临床上治疗KrasG12D突变的胰腺癌提供了一定的实验依据。
以下结合实施例用于进一步描述本发明，但这些实施例并非限制本发明的范围。
受试药物
受试化合物(I)配置方法：以2mg/mL在DMSO中溶解，并在100μL生理盐水中稀释。
人胰腺癌细胞系
PANC-1和MIAPaCa-2，购自中科院上海细胞库。
实验动物
BALB/C裸鼠，5周大雄性，购自北京华阜康生物科技有限公司。实验小鼠在天津医科大学动物中心饲养。饲养环境：SPF级。
实验步骤
建立稳定过表达KrasWT和KrasG12D胰腺癌细胞系
将pMXs-KrasWT和pBabe-KrasG12D质粒中的KrasWT和KrasG12D基因片段分别构建到pCDH-CMV-MCS-EF1-copGFP-T2A-Puro慢病毒载体中。慢病毒载体与pSpax2和pMD2G共转染293T细胞，形成病毒包装。收集病毒上清液，感染胰腺癌细胞，使用Puromycin进行药杀和筛选。经RT-PCR和Westernblot验证目的细胞系，用于后续实验。
细胞迁移实验
24孔板中每孔加入含10％胎牛血清的完全培养基。将Transwell小室加入2％胎牛血清培养基后，加入1x105个细胞，放入CO2培养箱培养24h。取出24孔板中的小室，放入24孔板干净的孔中，加入无水甲醇固定30min，PBS清洗后加入结晶紫染色30min，PBS清洗后风干，在200x显微镜下观察拍照。
构建小鼠胰腺癌模型
5周大BALB/c雄性裸鼠，预培养一周，随机分为四组，每组5只，分别为对照组，对照加药组，实验组，实验加药组。用15cm培养盘大量培养对照组和实验组胰腺癌细胞系，调整细胞浓度，保证两种细胞系在200μl PBS中含有2x107个细胞。用1ml注射器吸取细胞悬液，在小鼠胸部脂肪垫位置进行皮下注射，每只小鼠注射200μl细胞悬液。荷瘤7天后，开始用游标卡尺测量肿瘤的长度、宽度及深度，利用公式体积＝0.52.(长度.宽度.深度)计算肿瘤体积，每3天测量一次。荷瘤10天后，对对照加药组小鼠和实验加药组小鼠进行给药，，每两天给药一次，给药周期为30天。给药周期结束后处死小鼠，剥离皮下瘤组织，拍照并称重，取部分瘤组织浸泡于4％多聚甲醛，或液氮速冻后存放于-80℃冰箱以备后期使用。
动物研究由南开大学和天津医科大学动物护理和使用委员会批准。所有动物实验均按照美国国立卫生研究院实验动物护理和使用指南(NIH，第8版，2011)进行。经南开大学和天津医科大学伦理委员会批准。
数据分析
用GraphPad Prism 6分析数据，柱状图结果以mean SEM显示，两组数据之间用student t test检验进行差异显著性分析，三组或多组数据之间用单因素方差分析进行差异显著性分析，P 0.05具有统计学意义。
实施例1
构建稳定过表达KrasWT和KrasG12D胰腺癌细胞系，进行细胞增殖活性的检测。
通过分子克隆技术将KrasWT和KrasG12D两个基因片段分别连接到慢病毒质粒载体中。包装病毒后，用病毒上清分别感染PANC-1和MIAPaca-2胰腺导管腺癌细胞系。药物筛选得到目的细胞系。通过(图1A)RT-PCR(图1B)Westernblot Blot验证所得细胞系能分别稳定过表达KrasWT和KrasG12D突变。我们使用CCK-8(图1C)和EdU渗入实验(图1D)进行细胞增殖活力检测。KrasG12D细胞系的增殖活力明显强于KrasWT细胞系，并均高于对照组。
实施例2
KrasG12D突变上调TGF-beta下游蛋白，EMT标记物以及EMT转录因子的表达。
之前我们对急性髓系白血病KrasG12D转基因小鼠体内的白血病干细胞进行基因表达谱芯片测定，发现KrasG12D突变导致TGF-beta信号通路失控。因此，我们在稳定过表达KrasG12D突变的胰腺癌细胞检测TGF-beta信号通路的下游蛋白磷酸化和转录因子的表达。(图2A)TGF-betaR1和Foxd4的QPCR结果；(图2B)p-Smad2/3和Foxd4的Westernblot结果说明KrasG12D＞KrasWT＞对照细胞系中TGF-beta信号通路的激活。
在癌症中TGF-β信号通路对于调节肿瘤细胞发生上皮-间充质转化(EMT)发挥着重大作用。由此我们猜想KrasG12D突变是否也是通过调控TGF-β信号通路，从而实现细胞的上皮间充质转化，进而癌症的侵袭和转移。对此，我们通过QPCR和Western Blot对胰腺癌细胞的mRNA和蛋白水平进行检测。结果发现Fibronectin(FN1)、Vimentin(VIM)、E-cadherin(CDH1)、N-cadherin(CDH2)、ZEB1的mRNA和蛋白水平均发生明显变化，其中，KrasG12D组和Kraswt组细胞中FN1、VIM、CDH2、ZEB1的mRNA和蛋白水平明显上调，CDH1的mRNA和蛋白水平明显下调，且KrasG12D组结果较KrasWT组明显(图2C和D)，并均强于对照组。说明KrasG12D过表达在分子水平显著增强EMT表型。
实施例3
评价化合物(I)对胰腺癌细胞的EMT细胞表型的影响。
为明确在KrasG12D突变的胰腺癌中EMT水平与TGF-β信号通路的作用关系，我们靶向对TGF-βR1的活性进行了调节，通过分析细胞表型发生的变化以及对EMT markers和EMT-TFs的表达水平的影响，以初步确定KrasG12D导致胰腺癌细胞发生上皮间充质转化的作用机制。化合物(I)LY364947是一种ATP竞争性TGF-β受体抑制剂，能够使TGF-βR1受体活性降低，进而影响下游效应蛋白及转录因子的表达。TGF-β1细胞因子与化合物(I)作用相反。
我们用10μM的化合物(I)和5ng/ml的TGF-β1分别处理各组胰腺癌细胞系，处理5天后，用EdU试剂盒检测两种药物对于肿瘤细胞增殖活性的影响，结果发现化合物(I)有效抑制了各组细胞的增殖，而TGF-β1细胞因子对各组细胞的增殖起到了促进作用(图3A，B)。显微镜下观察细胞形态并拍照，发现KrasG12D突变组细胞和KrasWT组细胞均呈现出一定程度的间质表型，且KrasG12D突变组的间质表型比KrasWT组更加明显；化合物(I)有效抑制了肿瘤细胞的间质表型，细胞呈现上皮瓦楞状；而TGF-β1细胞因子使肿瘤细胞呈现出较多的间质表型，细胞形态如梭形(图3C)。细胞迁移实验分析药物处理对细胞迁移能力的影响，结果显示化合物(I)明显抑制了各组细胞的迁移，而TGF-β1使各组细胞迁移能力增强(图3D，E)。
实施例4
评价化合物(I)处理胰腺癌细胞系对EMT表型的分子水平的影响。
我们从细胞水平直观分析了LY364947和TGF-β1对细胞形态、迁移和增殖的影响，为了更深入探究药物作用，我们又从分子水平进行了实验研究。首先，通过QPCR实验分别检测对照组、KrasWT组及KrasG12D组胰腺癌细胞经药物处理后FN1、VIM、CDH1、CDH2、ZEB1mRNA水平的变化，结果发现肿瘤细胞经LY364947处理后，CDH1的mRNA水平升高，FN1、VIM、CDH2、ZEB1的mRNA水平均下降；经TGF-β1处理后，CDH1的mRNA水平下降，FN1、VIM、CDH2、ZEB1的mRNA水平均升高(图4A)。通过Western Blot实验从蛋白水平进一步验证上述结果，结果显示肿瘤细胞经LY364947处理后，CDH1的蛋白水平升高，FN1、VIM、CDH2、ZEB1的蛋白水平均下降；经TGF-β1处理后，CDH1的蛋白水平下降，FN1、VIM、CDH2、ZEB1的蛋白水平均升高(图4B)。以上结果，药物处理后KrasG12D细胞的变化明显强于KrasWT细胞，并均强于对照组细胞。
实施例5
评价化合物(I)对KrasG12突变胰腺癌异种移植体内肿瘤生长，侵袭和EMT水平的影响。
我们进一步用异种移植小鼠体内研究，来验证KrasG12D突变对于肿瘤生长及EMT水平的影响，以及使用化合物(I)靶向抑制TGF-β信号通路活性对于胰腺癌治疗的作用。我们将20只免疫缺陷型小鼠随机分为4组：对照组、对照加药组、KrasG12D组、KrasG12D加药组。荷瘤7天后测量肿瘤大小，每3天测量一次，荷瘤10天后开始对加药组进行给药处理，给药30天后处死小鼠剥离瘤组织拍照称重。分析结果显示，4组小鼠中KrasG12D组小鼠肿瘤生长速度比对照组小鼠快，LY364947有效抑制了肿瘤的生长(图5A)。拍照称重结果说明KrasG12D组小鼠肿瘤的体积和重量均大于对照组小鼠肿瘤，LY364947使肿瘤体积变小，重量变轻(图5B和C)。4组小鼠之间体重无明显差异。
我们提取了肿瘤组织的蛋白，通过Western Blot实验发现与对照组比较，KrasG12D突变使肿瘤组织的EMT水平上升，VIM、CDH2和ZEB1的表达量升高，化合物(I)有效抑制了肿瘤组织的EMT相关蛋白的表达水平(图5D)。我们将组织石蜡包埋切片，经HE染色发现，KrasG12D突变使肿瘤的侵袭能力增强，化合物(I)有效抑制了肿瘤的侵袭(图5E)。实线表示胰腺癌细胞侵袭的组织边缘。因而，TGF-beta受体-I抑制剂化合物(I)有效防止KrasG12D突变胰腺癌的成瘤和侵袭，提示KrasG12D胰腺癌细胞在TGF-beta缺陷的微环境中减弱其恶性癌变能力。
结论
目前尚且没有分子药物是可以有效治疗胰腺癌患者。70-95％的胰腺癌病例存在Kras突变，而在胰腺癌中约有75％的Kras突变发生在第12位密码子，使原来的甘氨酸突变为天冬氨酸或者缬氨酸。KrasG12D突变使Kras基因处于一种被持续激活的状态，从而导致Kras持续激活性过表达。我们在本项研究中，分别从细胞、分子及个体三个水平进行了探索和验证，发现KrasG12D突变在胰腺癌中促进癌细胞的增殖、迁移和侵袭，是因为其通过TGF-β信号通路导致胰腺癌细胞发生上皮间充质转化(EMT)。我们针对TGF-β受体对胰腺癌细胞进行化合物(I)药物处理，发现TGF-β细胞通路被抑制后细胞上皮间充质转化减弱，被TGF-β细胞因子过度激活后细胞上皮间充质转化增强，这说明KrasG12D突变细胞系发生EMT是由TGF-β-Smad信号通路高表达介导。并且，小鼠胰腺癌移植实验证明，KrasG12D突变促进了肿瘤的生长，侵袭和EMT；与之对比，化合物(I)对肿瘤生长有明显抑制作用，并有效抑制了胰腺癌的侵袭和EMT。以上结果提示化合物(I)能有效辅助胰腺癌的治疗，为后期临床药物治疗评估提供实验基础。
在本发明提及的文献在本申请中引用作为参考。此外应理解，在阅读了本发明的上述内容之后，本领域研发和技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
Claims (3)
1.TGF-beta受体分子抑制剂在治疗癌基因KrasG12D突变的胰腺癌的药物应用，选择所述TGF-beta受体分子抑制剂是TGF-beta受体-I小分子抑制剂ly364947。其特征在于，用于制备(i)抑制KrasG12D突变的人胰腺癌细胞上皮一间充质细胞转化、和/或(ii)治疗其引发的胰腺癌致瘤性和侵袭的药物组合物。
2.根据权利要求1所述的用途，其中所述的TGF-beta受体-I小分子抑制剂ly364947是式(I)所示化合物或其可药用盐，
3.根据权利要求2所述的用途，其中所述的TGF-beta受体-I小分子抑制剂ly364947的胰腺癌体内注射用量为1-20mg/kg，优选10mg/kg。
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