新型药物组合抑制KRAS突变肿瘤的作用与机制
发布时间：2018-12-11 19:54
【摘要】：癌基因RAS (NRAS, KRAS 和 HRAS)在人类肿瘤中频发突变,约占人类所有恶性肿瘤突变的三分之一。KRAS作为RAS蛋白家族的主要亚型,其突变占所有RAS蛋白突变的86%,且多发于胰腺癌、结直肠癌和肺癌中,由此可见KRAS突变肿瘤极具危害性。然而,由于KRAS信号通路调节的复杂性以及KRAS突变肿瘤对临床药物的拮抗,迄今为止临床上仍无有效治疗KRAS突变肿瘤的药物和策略。近年来,协同致死筛选技术为癌基因突变肿瘤,如KRAS突变肿瘤的治疗开辟了新的方向,同时鉴于单一药物有限的治疗效果以及长期用药诱发的肿瘤细胞耐药,为此我们将协同致死化学筛选与药物组合筛选相结合,利用等基因细胞筛选体系,旨在发现有效的、安全的、可转化的临床靶向药物组合用于KRAS突变肿瘤的治疗。在本研究中,我们选取与KRAS信号通路相关的且已进入临床研究的小分子抑制剂进行药物组合筛选,包括KRAS上游信号通路(受体酪氨酸激酶)、KRAS的主要下游信号通路(RAF/MAPK信号通路和PI3K/AKT/mTOR信号通路)以及与癌基因KRAS具有协同致死关系的基因的特异性抑制剂。在KRAS等基因细胞株中对各备选药物进行细胞毒性分析,以检测单一药物对KRAS突变细胞的选择性并计算出各药物的半数抑制浓度(50% inhibitory concentration, IC50);随后以药物IC50的比值为参照,以此比值对两个药物进行固定浓度比的联合用药(0.125,0.25,0.5,1×ICso),利用Calcusyn 2.0软件计算联药指数(combination index, CI),当CI值小于1时,表示药物存在协同效应。筛选结果显示,在表现出协同效果的药物组合中,70%以上的组合中都含有KRAS协同致死基因抑制剂。在此种类型的组合中,PLK1的抑制剂BI-2536和ROCK的抑制剂Fasudil在KRAS突变细胞中表现出最佳的协同作用(CI=0.33)。为了验证此新型药物组合对KRAS突变肿瘤的协同抑制效果,本研究选用一系列以KRAS基因为主要突变类型的肿瘤,如肺癌、肠癌、胰腺癌细胞等进行体外细胞表型实验分析。我们发现这对新型的临床药物组合(BI-2536/Fasudil)在极低药物浓度(单一药物有效浓度的1/5)下就能显著地抑制不同组织器官来源的KRAS突变肿瘤细胞的生长,并诱导细胞周期阻滞和细胞凋亡,但对KRAS野生型细胞及正常细胞无明显毒性作用。为了探究药物组合的分子机理,本研究利用基因表达谱芯片对药物组合基因表达的差异进行分析,芯片分析的结果显示BI-2536/Fasudil对KRAS突变肿瘤的协同抑制作用与p53信号通路的激活相关；随后,通过蛋白免疫印迹、荧光实时定量等实验对芯片的结果进行深入分析和验证。我们发现这一新型药物组合可以特异的在KRAS突变的细胞中以p53非依赖的形式增加细胞周期蛋白依赖激酶抑制因子p21WAFI/CIP1的表达。为了进一步阐明p21WAFI/CIPI与KRAS的相关性,本研究利用cDNA转染以及药物作用在细胞中过表达p21,发现KRAS突变细胞的选择性抑制,这一结果提示了CDKN1A(p21)与突变KRAS之间存在协同致死关系,可以作为KRAS突变肿瘤治疗的潜在干预靶点。鉴于新型药物组合BI-2536/Fasudil在体外对KRAS突变肿瘤细胞显著的协同抑制作用,我们通过三种体内动物模型——KRAS突变细胞系的皮下荷瘤模型、肺癌原位模型以及人源肿瘤组织荷瘤模型,对此药物组合抑制体内肿瘤生长的作用进行评估。连续给药4-6周后,与对照组和单药组相比,BI-2536/Fasudil显著抑制恶性肿瘤的生长(P0.0001-0.05)。为了进一步验证新型药物组合协同抑制KRAS突变实体瘤生长的机制,提取肿瘤组织RNA和蛋白质进行Q-PCR分析及免疫印迹实验。实验结果显示,联合用药组上调肿瘤组织中p21的表达,上述结果说明联合抑制PLK1和ROCK信号通路可通过增加p21的表达诱导有丝分裂压力发挥强烈抑制KRAS突变肿瘤的作用,为KRAS突变肿瘤的治疗提供了新的策略。综上所述,本研究揭示了能扰乱KRAS对其协同致死基因的依赖性的新型药物组合,靶向肿瘤细胞基因型的药物组合研究具有相当的医学转化价值,可以指导KRAS突变肿瘤的临床治疗。
[Abstract]:Oncogene RAS (RAS, KRAS, and HRAS) frequently mutate in human tumors, accounting for approximately one-third of all human malignancies. KRAS is a major subtype of the RAS protein family, and its mutation accounts for 86% of all RAS protein mutations, and is multiple in pancreatic cancer, colorectal cancer and lung cancer. It can be seen that the KRAS mutant tumor is highly hazardous. However, because of the complexity of KRAS signal pathway regulation and the antagonism of KRAS mutant tumor to clinical medicine, the drug and strategy of KRAS mutant tumor have not been effectively treated to date. In recent years, the synergistic lethal screening technique opens a new direction for the treatment of cancer gene mutation tumors, such as the KRAS mutant tumor, and meanwhile, in view of the limited treatment effect of the single drug and the drug resistance of the tumor cells induced by the long-term administration, To this end, we combine the combination of lethal chemical screening with drug combination screening, and use the isogenic cell screening system to find effective, safe and convertible clinical targeting drug combination for the treatment of KRAS mutant tumors. in that present study, we select a small molecule inhibitor that is associate with the KRAS signal pathway and has entered the clinical study for drug combination screening, including the KRAS upstream signal pathway (receptor tyrosine kinase), KRAS's main downstream signal pathway (RAF/ MAPK signal pathway and PI3K/ AKT/ mTOR signaling pathway) and a specific inhibitor of a gene that has a synergistic lethal relationship with the oncogene KRAS. Each of the candidate drugs was subjected to a cytotoxicity assay in a KRAS isogenic cell line to detect the selectivity of a single drug to KRAS mutant cells and to calculate half of the inhibitory concentration of each drug (50% of the inhibition concentration, IC50); followed by a reference to the ratio of the drug IC50, In this ratio, the combined administration of the two drugs (0. 125, 0.25, 0.5, 1, ICso) was used to calculate the combination index (CI). When the CI value was less than 1, it was indicated that the drug had a synergistic effect. The results showed that in the combination of drug with synergistic effect, the KRAS co-lethal gene inhibitor was contained in more than 70% of the combination. In this type of combination, the inhibitor BI-2536 of PLK1 and inhibitor of ROCK showed the best synergistic effect in KRAS mutant cells (CI = 0.33). In order to verify the synergistic effect of the new drug combination on the KRAS mutant tumor, a series of tumor with KRAS gene as the main mutation type, such as lung cancer, intestinal cancer, pancreatic cancer cells and the like, were selected for the in vitro cell phenotypic analysis. We found that this new clinical drug combination (BI-2536/ Dialodil) can significantly inhibit the growth of KRAS mutant tumor cells from different tissue organ sources at very low drug concentrations (1/ 5 of a single drug effective concentration), and induce cell cycle arrest and cell apoptosis, but no obvious toxic effect on KRAS wild-type cells and normal cells. In order to explore the molecular mechanism of the drug combination, the difference of the expression of the drug combination gene was analyzed by using the gene expression profile chip. The results of the chip analysis showed that the synergistic inhibition of the BI-2536/ budil on the KRAS mutant tumor was related to the activation of the p53 signal pathway; and then, by the protein immunoblotting, The results of the chip are analyzed and verified by real-time fluorescence quantitative and other experiments. We have found that this novel combination of drugs can specifically increase the expression of the cyclin-dependent kinase inhibitor p21WAI/ CIP1 in a non-dependent form of p53 in the KRAS mutant cells. In order to further clarify the correlation between p21WAI/ CIPI and KRAS, this study uses cDNA transfection as well as drug action to overexpress p21 in the cells, and the selective inhibition of KRAS mutant cells is found, which results in a synergistic lethal relationship between the CDKN1A (p21) and the mutant KRAS. and can be used as a potential intervention target for the treatment of the KRAS mutant tumor. In view of the remarkable synergistic inhibition of the novel drug combination BI-2536/ Prodil in vitro on the KRAS mutant tumor cells, we adopted the subcutaneous tumor-bearing model of the three in vivo animal model _ KRAS mutant cell line, the in-situ model of the lung cancer and the tumor-bearing model of the human source tumor, The effect of the combination of the drug on the growth of the tumor in the body is evaluated. After 4-6 weeks of continuous administration, the growth of the malignant tumor was significantly inhibited by BI-2536/ Dialo1 compared to the control group and the single-drug group (P. 0001-0.05). In order to further verify the mechanism of the new drug combination to inhibit the growth of the KRAS mutant solid tumor, the Q-PCR and the immunoblotting test of the RNA and the protein of the tumor tissue were extracted. The results showed that the combined inhibition of PLK1 and ROCK signaling pathway could significantly inhibit the KRAS mutant tumor by increasing the expression of p21 and provide a new strategy for the treatment of KRAS mutant tumors. To sum up, this study has disclosed a new type of drug combination which can disturb the dependence of KRAS on its synergistic lethal gene, and the drug combination study targeting the tumor cell genotype has considerable medical conversion value and can guide the clinical treatment of the KRAS mutant tumor.
【学位授予单位】：华东师范大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R73-36
，
本文编号：2373129
[Abstract]:Oncogene RAS (RAS, KRAS, and HRAS) frequently mutate in human tumors, accounting for approximately one-third of all human malignancies. KRAS is a major subtype of the RAS protein family, and its mutation accounts for 86% of all RAS protein mutations, and is multiple in pancreatic cancer, colorectal cancer and lung cancer. It can be seen that the KRAS mutant tumor is highly hazardous. However, because of the complexity of KRAS signal pathway regulation and the antagonism of KRAS mutant tumor to clinical medicine, the drug and strategy of KRAS mutant tumor have not been effectively treated to date. In recent years, the synergistic lethal screening technique opens a new direction for the treatment of cancer gene mutation tumors, such as the KRAS mutant tumor, and meanwhile, in view of the limited treatment effect of the single drug and the drug resistance of the tumor cells induced by the long-term administration, To this end, we combine the combination of lethal chemical screening with drug combination screening, and use the isogenic cell screening system to find effective, safe and convertible clinical targeting drug combination for the treatment of KRAS mutant tumors. in that present study, we select a small molecule inhibitor that is associate with the KRAS signal pathway and has entered the clinical study for drug combination screening, including the KRAS upstream signal pathway (receptor tyrosine kinase), KRAS's main downstream signal pathway (RAF/ MAPK signal pathway and PI3K/ AKT/ mTOR signaling pathway) and a specific inhibitor of a gene that has a synergistic lethal relationship with the oncogene KRAS. Each of the candidate drugs was subjected to a cytotoxicity assay in a KRAS isogenic cell line to detect the selectivity of a single drug to KRAS mutant cells and to calculate half of the inhibitory concentration of each drug (50% of the inhibition concentration, IC50); followed by a reference to the ratio of the drug IC50, In this ratio, the combined administration of the two drugs (0. 125, 0.25, 0.5, 1, ICso) was used to calculate the combination index (CI). When the CI value was less than 1, it was indicated that the drug had a synergistic effect. The results showed that in the combination of drug with synergistic effect, the KRAS co-lethal gene inhibitor was contained in more than 70% of the combination. In this type of combination, the inhibitor BI-2536 of PLK1 and inhibitor of ROCK showed the best synergistic effect in KRAS mutant cells (CI = 0.33). In order to verify the synergistic effect of the new drug combination on the KRAS mutant tumor, a series of tumor with KRAS gene as the main mutation type, such as lung cancer, intestinal cancer, pancreatic cancer cells and the like, were selected for the in vitro cell phenotypic analysis. We found that this new clinical drug combination (BI-2536/ Dialodil) can significantly inhibit the growth of KRAS mutant tumor cells from different tissue organ sources at very low drug concentrations (1/ 5 of a single drug effective concentration), and induce cell cycle arrest and cell apoptosis, but no obvious toxic effect on KRAS wild-type cells and normal cells. In order to explore the molecular mechanism of the drug combination, the difference of the expression of the drug combination gene was analyzed by using the gene expression profile chip. The results of the chip analysis showed that the synergistic inhibition of the BI-2536/ budil on the KRAS mutant tumor was related to the activation of the p53 signal pathway; and then, by the protein immunoblotting, The results of the chip are analyzed and verified by real-time fluorescence quantitative and other experiments. We have found that this novel combination of drugs can specifically increase the expression of the cyclin-dependent kinase inhibitor p21WAI/ CIP1 in a non-dependent form of p53 in the KRAS mutant cells. In order to further clarify the correlation between p21WAI/ CIPI and KRAS, this study uses cDNA transfection as well as drug action to overexpress p21 in the cells, and the selective inhibition of KRAS mutant cells is found, which results in a synergistic lethal relationship between the CDKN1A (p21) and the mutant KRAS. and can be used as a potential intervention target for the treatment of the KRAS mutant tumor. In view of the remarkable synergistic inhibition of the novel drug combination BI-2536/ Prodil in vitro on the KRAS mutant tumor cells, we adopted the subcutaneous tumor-bearing model of the three in vivo animal model _ KRAS mutant cell line, the in-situ model of the lung cancer and the tumor-bearing model of the human source tumor, The effect of the combination of the drug on the growth of the tumor in the body is evaluated. After 4-6 weeks of continuous administration, the growth of the malignant tumor was significantly inhibited by BI-2536/ Dialo1 compared to the control group and the single-drug group (P. 0001-0.05). In order to further verify the mechanism of the new drug combination to inhibit the growth of the KRAS mutant solid tumor, the Q-PCR and the immunoblotting test of the RNA and the protein of the tumor tissue were extracted. The results showed that the combined inhibition of PLK1 and ROCK signaling pathway could significantly inhibit the KRAS mutant tumor by increasing the expression of p21 and provide a new strategy for the treatment of KRAS mutant tumors. To sum up, this study has disclosed a new type of drug combination which can disturb the dependence of KRAS on its synergistic lethal gene, and the drug combination study targeting the tumor cell genotype has considerable medical conversion value and can guide the clinical treatment of the KRAS mutant tumor.
【学位授予单位】：华东师范大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R73-36
，
本文编号：2373129
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