新型藥物組合抑制KRAS突變腫瘤的作用與機制
發(fā)布時間：2018-12-11 19:54
【摘要】：癌基因RAS (NRAS, KRAS 和 HRAS)在人類腫瘤中頻發(fā)突變,約占人類所有惡性腫瘤突變的三分之一。KRAS作為RAS蛋白家族的主要亞型,其突變占所有RAS蛋白突變的86%,且多發(fā)于胰腺癌、結直腸癌和肺癌中,由此可見KRAS突變腫瘤極具危害性。然而,由于KRAS信號通路調節(jié)的復雜性以及KRAS突變腫瘤對臨床藥物的拮抗,迄今為止臨床上仍無有效治療KRAS突變腫瘤的藥物和策略。近年來,協(xié)同致死篩選技術為癌基因突變腫瘤,如KRAS突變腫瘤的治療開辟了新的方向,同時鑒于單一藥物有限的治療效果以及長期用藥誘發(fā)的腫瘤細胞耐藥,為此我們將協(xié)同致死化學篩選與藥物組合篩選相結合,利用等基因細胞篩選體系,旨在發(fā)現(xiàn)有效的、安全的、可轉化的臨床靶向藥物組合用于KRAS突變腫瘤的治療。在本研究中,我們選取與KRAS信號通路相關的且已進入臨床研究的小分子抑制劑進行藥物組合篩選,包括KRAS上游信號通路(受體酪氨酸激酶)、KRAS的主要下游信號通路(RAF/MAPK信號通路和PI3K/AKT/mTOR信號通路)以及與癌基因KRAS具有協(xié)同致死關系的基因的特異性抑制劑。在KRAS等基因細胞株中對各備選藥物進行細胞毒性分析,以檢測單一藥物對KRAS突變細胞的選擇性并計算出各藥物的半數(shù)抑制濃度(50% inhibitory concentration, IC50);隨后以藥物IC50的比值為參照,以此比值對兩個藥物進行固定濃度比的聯(lián)合用藥(0.125,0.25,0.5,1×ICso),利用Calcusyn 2.0軟件計算聯(lián)藥指數(shù)(combination index, CI),當CI值小于1時,表示藥物存在協(xié)同效應。篩選結果顯示,在表現(xiàn)出協(xié)同效果的藥物組合中,70%以上的組合中都含有KRAS協(xié)同致死基因抑制劑。在此種類型的組合中,PLK1的抑制劑BI-2536和ROCK的抑制劑Fasudil在KRAS突變細胞中表現(xiàn)出最佳的協(xié)同作用(CI=0.33)。為了驗證此新型藥物組合對KRAS突變腫瘤的協(xié)同抑制效果,本研究選用一系列以KRAS基因為主要突變類型的腫瘤,如肺癌、腸癌、胰腺癌細胞等進行體外細胞表型實驗分析。我們發(fā)現(xiàn)這對新型的臨床藥物組合(BI-2536/Fasudil)在極低藥物濃度(單一藥物有效濃度的1/5)下就能顯著地抑制不同組織器官來源的KRAS突變腫瘤細胞的生長,并誘導細胞周期阻滯和細胞凋亡,但對KRAS野生型細胞及正常細胞無明顯毒性作用。為了探究藥物組合的分子機理,本研究利用基因表達譜芯片對藥物組合基因表達的差異進行分析,芯片分析的結果顯示BI-2536/Fasudil對KRAS突變腫瘤的協(xié)同抑制作用與p53信號通路的激活相關；隨后,通過蛋白免疫印跡、熒光實時定量等實驗對芯片的結果進行深入分析和驗證。我們發(fā)現(xiàn)這一新型藥物組合可以特異的在KRAS突變的細胞中以p53非依賴的形式增加細胞周期蛋白依賴激酶抑制因子p21WAFI/CIP1的表達。為了進一步闡明p21WAFI/CIPI與KRAS的相關性,本研究利用cDNA轉染以及藥物作用在細胞中過表達p21,發(fā)現(xiàn)KRAS突變細胞的選擇性抑制,這一結果提示了CDKN1A(p21)與突變KRAS之間存在協(xié)同致死關系,可以作為KRAS突變腫瘤治療的潛在干預靶點。鑒于新型藥物組合BI-2536/Fasudil在體外對KRAS突變腫瘤細胞顯著的協(xié)同抑制作用,我們通過三種體內動物模型——KRAS突變細胞系的皮下荷瘤模型、肺癌原位模型以及人源腫瘤組織荷瘤模型,對此藥物組合抑制體內腫瘤生長的作用進行評估。連續(xù)給藥4-6周后,與對照組和單藥組相比,BI-2536/Fasudil顯著抑制惡性腫瘤的生長(P0.0001-0.05)。為了進一步驗證新型藥物組合協(xié)同抑制KRAS突變實體瘤生長的機制,提取腫瘤組織RNA和蛋白質進行Q-PCR分析及免疫印跡實驗。實驗結果顯示,聯(lián)合用藥組上調腫瘤組織中p21的表達,上述結果說明聯(lián)合抑制PLK1和ROCK信號通路可通過增加p21的表達誘導有絲分裂壓力發(fā)揮強烈抑制KRAS突變腫瘤的作用,為KRAS突變腫瘤的治療提供了新的策略。綜上所述,本研究揭示了能擾亂KRAS對其協(xié)同致死基因的依賴性的新型藥物組合,靶向腫瘤細胞基因型的藥物組合研究具有相當?shù)尼t(yī)學轉化價值,可以指導KRAS突變腫瘤的臨床治療。
[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.
【學位授予單位】：華東師范大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R73-36
，
本文編號：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.
【學位授予單位】：華東師范大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R73-36
，
本文編號：2373129
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