基于PDTX模型的胃癌分子靶向治疗研究

发布时间：2018-08-13 17:28

【摘要】：背景：胃癌作为全世界最常见的恶性肿瘤之一,也是全球癌症死亡的第二大原因。虽然在手术及化疗上取得了一定的进展,但是进展期胃癌的预后仍然较差,其五年生存率也只接近20%。在过去的十年里,靶向治疗极大的改善了许多恶性肿瘤的预后,包括乳腺癌、结直肠癌、肺癌等,然而在胃癌方面的进展却比较小。缺少与临床密切相关的动物模型是阻碍靶向药物发展的重要因素之一。小鼠体外细胞成瘤模型数十年来一直被用作临床前抗肿瘤药物研究的标准工具,但是药物进入临床试验后的高失败率让我们开始怀疑这个传统的成瘤模型对药物的预测能力。经过长期的体外培养及筛选的细胞移植成瘤后,通常已经丢失了来源肿瘤组织的分子特征和肿瘤异质性,这也是其对临床药物反应预测能力较差的原因。相比较体外细胞成瘤模型,病人组织来源肿瘤裸鼠移植瘤模型(patient derived tumor xenograft, PDTX)能很好的保持来源病人肿瘤组织在组织病理、遗传及表型方面的特征,因而能较好的预测药物的反应性。近年来,大量PDTX模型在各种肿瘤中被建立起来,包括肺癌、结直肠癌、乳腺癌、胰腺癌及胃癌等。PDTX模型目前逐渐成为研究肿瘤生物学及评价抗肿瘤药物的有效工具。曲妥珠单抗被批准用于治疗生长因子受体2(human epidermal growth factor receptor-2, HER2)阳性的胃癌开启了胃癌靶向治疗的时代。其后至今却只有雷莫芦单抗(ramucirumab)和阿帕替尼(apatinib)两个抗血管药物被批准用于晚期胃癌的二线治疗,但是目前胃癌靶向治疗对总生存率的提高仍然是有限的。因此研发新的药物,尤其是靶向药物,对胃癌治疗尤为重要。近年来,较多的研究集中在了受体络氨酸家族(RTK)成员上,这其中就包括了肝细胞生长因子受体(cMet)、成纤维细胞生长因子受体2(Gbroblast growth factor receptor2, FGFR2)、表皮生长因子受体(EGFR)等。目前许多研究正在探索相关通路抑制剂在胃癌中的抗肿瘤作用。胃癌PDTX模型可以作为筛选这些潜在靶向药物的理想平台,从而为进一步的临床试验提供可靠的数据支持。第一部分：背景：靶向治疗正在成为胃癌新的治疗选择之一。PDTX模型能很好的保持来源肿瘤组织的特征,为临床前药物疗效评估提供了一个有效的工具。本研究旨在建立胃癌PDTX模型,并探索针对IER2, cMet和FGFR2的靶向治疗,为临床进一步药效试验提供数据支持。方法：从32例胃癌病人获取胃癌组织移植入免疫缺陷小鼠中。利用免疫组织化学(IHC)和荧光原位杂交(FISH)评估一组胃癌病人HER2, cMet和FGFR2蛋白表达水平和基因扩增情况。最后,在PDTX模型中评价靶向药物的抗肿瘤作用。结果：9个能传代的胃癌PDTX模型被成功的建立起来。在检测的163例胃癌患者中分别有17(10.4%),32(19.6%)和6(3.7%)例出现HER2. cMet和FGFR2的分子改变,而在32例移植瘤供体中则分别为4(12.5%),8(25.0%)和1(3.1%)例。cMet的基因扩增或蛋白过表达(IHC3+)与胃癌的不良预后相关。Critozinib和AZD4547分别只在cMet扩增(G30, G31)或FGFR2扩增(G03)的PDTX模型中呈现明显的抗肿瘤作用。有趣的是,在G03(FGFR2扩增,cMet无扩增但IHC2+)模型中,联合Critozinib和AZD4547台疗能增加AZD4547的抗肿瘤作用。结论：我们建立了一系列胃癌PDTX模型为药物筛选和评价提供了一个理想的平台。cMet和FGFR2基因扩增的胃癌患者有可能从靶向或联合靶向cMet或FGFR2的治疗中获益。第二部分背景：cMet信号通路的异常激活参与肿瘤的生长、血管生成和转移。cMet过表达的胃癌因为肿瘤的高转移性及治疗选择上的局限性通常预后较差。木犀草素(Luteolin)是一种具有抗肿瘤作用的天然黄酮类化合物。但是目前Luteolin在cMet过表达胃癌中的作用还不清楚。方法：建立2株cMet过表达的胃癌PDTX模型,利用Luteolin来处理PDTX模型并评价其抗肿瘤作用。将肿瘤标本进行HE染色及免疫组化检测。利用不同浓度的Luteolin来处理两株高表达cMet的胃癌细胞系MKN45和SGC7901,通过Western blot、细胞活力检测、细胞凋亡检测、转移及侵袭试验等方法来评估Luteolin的抗肿瘤作用及对cMet信号通路的影响。结果：Luteolin在cMet过表达的胃癌PDTX模型中能抑制明显肿瘤的生长。免疫阻化提示cMet、MMP9和Ki-67蛋白的表达明显下调。在MKN45和SGC7901细胞系中,Luteolin能抑制细胞的增殖,促进细胞凋亡及降低细胞的侵袭能力。Western blot结果提示Luteolin能促进凋亡相关蛋白Caspase-3和PARP-1的活化,并下调侵袭相关蛋白MMP9。进一步研究表明Luteolin能下调cMet蛋白的表达及磷酸化水平,及下游AKT和ERK的磷酸化水平。此外,Luteolin也能在不依赖cMet的情况下抑制AKT的磷酸化。利用PI3K抑制剂LY294002和／或ERK抑制剂PD98059能下调MMP9、cleaved caspase-3和cleaved PARP-1,与Luteolin的作用类似。结论：本课题首次提出了Luteolin在cMet过表达的胃癌PDTX模型具有显著的抗肿瘤作用,可能涉及到cMet/AKT/ERK信号通路。这些结果表明,Luteolin可以作为cMet过表达胃癌的潜在治疗选择。第三部分背景：甲胎蛋白分泌型胃癌(AFPGC)是一种具有高肝转移和不良预后的罕见胃癌类型。相比与常见胃癌,AFPGC在靶向治疗上的进展较小。本研究旨在探索几个已知的靶点HER2、cMet、EGFR和VEGF在AFPGC患者中的表达情况,并进一步在胃癌PDTX模型中评估潜在的靶向治疗方案。方法：采用免疫组织化学(IHC)和荧光原位杂交(FISH)方法,我们在35例AFPGC病人及70例分期匹配的非AFPGC病人中评估HER2、cMet、EGFR和VEGF的蛋白表达及HER2和cMet的基因扩增情况。同时我们建立了甲胎蛋白分泌型胃癌PDTX模型来评估靶向治疗的疗效。结果：我们发现在／AFPGC组中cMet过表达的比例为48.5%,而在非AFPGC组则为20%。在AFPGC组VEGF过表达的比例为71.4%,而在非AFPGC组则为47.1%。同时,我们发现在AFPGC组中cMet和VEGF共同过度表达的比例为42.8%,并且这部分AFPGC患者预后更差。我们观察到在cMet及VEGF共同过表达的胃癌PDTX模型中联合靶向cMet (Crizotinib)和V EGF (Bevacizumab)信号通路治疗能提高抗肿瘤作用。结论：AFPGC中高频率的出现cMet和VEGF共同过表达的情况,并且与不良预后相关。这一类特殊的AFPGC类型可能能够从联合靶向cMet和VEGF信号通路中受益。
[Abstract]:BACKGROUND: Gastric cancer is one of the most common malignant tumors in the world and the second leading cause of cancer death worldwide. Although some progress has been made in surgery and chemotherapy, the prognosis of advanced gastric cancer is still poor, and its five-year survival rate is only close to 20%. Targeted therapy has greatly improved many malignancies in the past decade. Prognosis of tumors, including breast, colorectal, and lung cancers, has made little progress in gastric cancer. Lack of clinical animal models is one of the important factors hindering the development of targeted drugs. The high failure rate of drugs in clinical trials has led us to question the predictive power of this traditional tumor-forming model for drugs. After long-term in vitro culture and screening of cells for tumor formation, the molecular characteristics and tumor heterogeneity of tumor origin have often been lost, which is also the ability to predict clinical drug response. Patient derived tumor xenograft (PDTX) can maintain the histopathological, genetic and phenotypic characteristics of the tumor tissues of the patients of origin, so it can better predict the drug responsiveness. In recent years, a large number of PDTX models have been used to predict the drug responsiveness. PDTX models have been established for cancer research, including lung, colorectal, breast, pancreatic and gastric cancer.
Trastuzumab has been approved for the treatment of human epidermal growth factor receptor-2 (HER2)-positive gastric cancer, opening the era of targeted therapy for gastric cancer. Nowadays, the total survival rate of gastric cancer is still limited by targeted therapy. Therefore, it is very important to develop new drugs, especially targeted drugs, for gastric cancer treatment. Receptor 2 (Gbroblast growth factor receptor 2, FGFR2), epidermal growth factor receptor (EGFR) and so on. At present, many studies are exploring the anti-tumor effects of inhibitors of related pathways in gastric cancer. Hold.
Part one:
BACKGROUND: Targeted therapy is becoming one of the new therapeutic options for gastric cancer. PDTX model can maintain the characteristics of tumor tissue and provide an effective tool for pre-clinical evaluation of drug efficacy. Provide data support.
METHODS: Gastric cancer tissues were obtained from 32 patients with gastric cancer and transplanted into immunodeficient mice. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) were used to evaluate the expression and gene amplification of HER2, cMet and FGFR2 in a group of patients with gastric cancer. Finally, the anti-tumor effect of targeted drugs was evaluated in PDTX model.
Results: Nine PDTX models of gastric cancer were successfully established. Among 163 gastric cancer patients examined, 17 (10.4%), 32 (19.6%) and 6 (3.7%) had molecular changes in HER2.cMet and FGFR2, respectively, while in 32 transplanted tumor donors, 4 (12.5%), 8 (25.0%) and 1 (3.1%) had gene amplification or protein overexpression (IHC3+) and Critozinib and AZD4547 showed significant antitumor effects in PDTX models with cMet amplification (G30, G31) or FGFR2 amplification (G03), respectively.
Conclusion: We have established a series of PDTX models for gastric cancer to provide an ideal platform for drug screening and evaluation. Patients with gastric cancer amplified by cMet and FGFR2 genes may benefit from targeted or combined targeted therapy with cMet or FGFR2.
The second part
BACKGROUND: Abnormal activation of cMet signaling pathway is involved in tumor growth, angiogenesis and metastasis. Overexpression of cMet in gastric cancer usually leads to poor prognosis because of its high metastasis and limited treatment options. Luteolin is a natural flavonoid compound with antitumor activity. However, Luteolin is overexpressed in cMet at present. The role of gastric cancer is unclear.
Methods: Two gastric cancer cell lines with overexpression of cMet, MKN45 and SGC7901, were treated with Luteolin, and their antitumor effects were evaluated. Apoptosis detection, metastasis and invasion assay were used to evaluate the antitumor effect of Luteolin and its effect on cMet signaling pathway.
Results: Luteolin could inhibit the growth of gastric cancer in PDTX model with overexpression of cMet. Immunosuppression indicated that the expressions of cMet, MMP9 and Ki-67 were down-regulated. Luteolin could inhibit the proliferation, promote apoptosis and decrease the invasiveness of cells in MKN45 and SGC7901 cell lines. Further studies have shown that Luteolin can down-regulate the expression and phosphorylation of cMet protein and the phosphorylation levels of downstream AKT and ERK. In addition, Luteolin can also inhibit the phosphorylation of AKT without cMet dependence. / or ERK inhibitor PD98059 could down-regulate MMP 9, cleaved caspase-3 and cleaved PARP-1, similar to Luteolin.
CONCLUSION: Luteolin PDTX model of gastric cancer overexpressing cMet has been proposed for the first time in this study. It may be involved in the cMet/AKT/ERK signaling pathway. These results suggest that Luteolin may be a potential therapeutic option for gastric cancer overexpressing cMet.
The third part
BACKGROUND: Alpha-fetoprotein-secreting gastric cancer (AFPGC) is a rare type of gastric cancer with high liver metastasis and poor prognosis. Compared with common gastric cancer, AFPGC has made less progress in targeted therapy. Evaluation of potential targeted therapies.
METHODS: Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) were used to evaluate the expression of HER2, cMet, EGFR and VEGF in 35 AFPGC patients and 70 stage-matched non-AFPGC patients. Curative effect.
Results: We found that the overexpression of cMet was 48.5% in the / AFPGC group and 20% in the non-AFPGC group, 71.4% in the AFPGC group and 47.1% in the non-AFPGC group. The combination of targeted cMet (Crizotinib) and Bevacizumab (Bevacizumab) signaling pathways in a PDTX model of gastric cancer with co-expression of cMet and VEGF can enhance the anti-tumor effect.
CONCLUSION: High frequency of co-expression of cMet and VEGF in AFPGC may be associated with poor prognosis. This special type of AFPGC may benefit from co-targeting cMet and VEGF signaling pathways.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.2

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