PI3Kα选择性抑制剂抗乳腺癌作用及其疗效预测生物标志物研究

发布时间：2018-05-31 10:53

本文选题：乳腺癌 + PI3K　；参考：《中国科学院大学(中国科学院上海药物研究所)》2016年博士论文

【摘要】：PI3K信号通路参与细胞存活、生长、增殖和代谢等重要过程,在多种不同组织来源的肿瘤中异常活化。其中,约有70%的乳腺癌发生PI3K信号通路高度活化,编码PI3Kα催化亚基的PIK3CA基因突变率高达30%,散布于所有乳腺癌类型。大量分子生物学和遗传学研究证明PI3Kα异常激活能够促进乳腺癌发生发展,而抑制PI3K活性能够阻止乳腺癌形成和发展,因此PI3Kα是乳腺癌治疗的重要靶标。目前,国际上已有多个泛PI3K抑制剂和四个PI3Kα选择性抑制剂进入临床试验以单药或联合用药形式治疗不同类型乳腺癌,显示出良好的应用前景。因此,研究和开发新的具有我国自主知识产权的新型PI3Kα选择性抑制剂具有重要意义。已有的临床试验数据显示PI3Kα选择性抑制剂对乳腺癌的疗效显示出较大差异性,在部分患者中疗效显著。因此,发现和确证预测PI3Kα抑制剂疗效的生物标志物是促进PI3Kα选择性抑制剂研究和开发以及提高疗效的重要策略。本研究的目的是系统性地探索本实验室发现的新结构PI3Kα选择性抑制剂CYH33抗乳腺癌作用;探索能够预测PI3Kα抑制剂在乳腺癌中抗肿瘤疗效的生物标志物。CYH33是本实验室前期研究工作发现的全新PI3Kα选择性抑制剂,能够广泛抑制多种组织来源肿瘤细胞的增殖。通过检测CYH33对一系列乳腺癌细胞的增殖抑制作用,我们发现低浓度CYH33即能够显著抑制多种乳腺癌细胞增殖,其活性与泛PI3K抑制剂GDC0941相当,优于处于III期临床试验的同类型抑制剂BYL719。以对CYH33敏感的T47D和MCF-7细胞以及对CYH33耐受的MDA-MB-231细胞为例,我们初步探索了CYH33抑制乳腺癌细胞增殖的作用机制。CYH33能够浓度依赖性且同等程度地抑制T47D、MCF-7和MDA-MB-231细胞中PI3K下游蛋白激酶AKT磷酸化。CYH33能够下调敏感乳腺癌细胞中m TORC1下游p70S6K和S6磷酸化水平,而在耐受乳腺癌细胞中无此作用。在敏感乳腺癌细胞中,CYH33通过下调Rb磷酸化和cyclin D1含量以及p27累积,将细胞阻滞于G1期从而发挥细胞增殖抑制作用,而在耐受乳腺癌细胞中CYH33对细胞周期分布影响微弱。CYH33作用72小时不能诱导敏感或耐受乳腺癌细胞发生凋亡。因此,CYH33主要通过将细胞阻滞于G1期发挥细胞增殖抑制作用,而AKT磷酸化下调不能够预测乳腺癌细胞对CYH33的敏感性。与体外CYH33显著的乳腺癌细胞增殖抑制作用一致,CYH33能够有效地抑制T47D SCID小鼠移植瘤和PIK3CAH1047R基因工程小鼠自发瘤的生长,且活性优于BYL719。此外,CYH33和BYL719长期作用于乳腺癌模型动物对小鼠体重和血糖调节功能影响不大,均在安全范围之内。我们初步分析了不同分子分型乳腺癌细胞对CYH33的敏感性,发现管腔型和HER2扩增型乳腺癌细胞对CYH33更加敏感,而基底样乳腺癌细胞对CYH33耐受。分析PI3K信号通路内关键分子改变与乳腺癌细胞对CYH33敏感性的关系,我们发现PIK3CA突变和HER2分子扩增的乳腺癌细胞对CYH33更加敏感,而PTEN缺失的乳腺癌细胞对CYH33耐受。尽管乳腺癌细胞不同分子分型或PI3K信号通路内关键蛋白分子改变与乳腺癌细胞对CYH33的敏感性存在一定的相关性,部分基底样乳腺癌细胞或PI3K信号通路正常的乳腺癌细胞对CYH33敏感。因此,探索基于作用机制的PI3Kα选择性抑制剂疗效预测生物标志物是提高该类抑制剂疗效的重要策略。PI3K和MAPK信号通路是参与调节乳腺癌细胞增殖的两条关键信号通路,二者存在广泛的交互通话,抑制其中一条信号通路通常引起另一条信号通路的反馈性激活,提示同时靶向两条信号通路有希望达到更显著的抗肿瘤疗效。我们发现CYH33能够同时下调T47D细胞中AKT和ERK磷酸化,进一步研究CYH33这一作用机制有可能揭示其与CYH33抗肿瘤活性之间的关系。以敏感乳腺癌T47D和MCF-7细胞为研究对象,多个PI3K抑制剂能够同时抑制AKT和ERK磷酸化,而PDK1、AKT和m TORC抑制剂在发挥靶标抑制作用的同时对ERK磷酸化影响微弱甚至升高ERK磷酸化水平,表明PI3K调节ERK磷酸化不依赖其经典PDK1/AKT/m TOR下游信号通路。抑制RAF和MEK引起ERK磷酸化下调,而高表达RAF和MEK拮抗PI3K抑制剂对ERK磷酸化的抑制,表明PI3K调节ERK磷酸化依赖Raf/Mek。进一步研究发现RAS活性不受PI3K抑制剂影响,但是高表达RAS能够逆转PI3K抑制剂对ERK磷酸化的抑制作用,表明PI3K和RAS二者协同调节ERK磷酸化。激活细胞内EGFR活性能够升高胞内RAS-GTP含量继而阻止PI3K抑制剂对ERK磷酸化的抑制,进一步确证了PI3K和RAS在调节ERK磷酸化中发挥的协同作用,同时表明低活性的EGFR使ERK磷酸化依赖PI3K,高活性的EGFR使ERK磷酸化依赖RAS。基于EGFR的活性能够决定ERK磷酸化是否依赖于PI3K,我们研究了在乳腺癌组织中频繁高表达的受体酪氨酸激酶IGF-1R、HER3、FGFR和c-MET在PI3K调节ERK磷酸化中发挥的作用。激活EGFR、HER3、FGFR和/或c-MET活性能够拮抗BYL719对乳腺癌细胞T47D、MCF-7和HCC1954中ERK磷酸化的抑制,而且能够部分逆转BYL719对相应乳腺癌细胞的增殖抑制作用。然而,激活IGF-1R并未观察到上述现象。我们检测了一系列乳腺癌细胞中RTKs表达谱和PI3K抑制剂对其AKT和ERK磷酸化的影响,发现在PI3K抑制剂能够同时下调AKT和ERK磷酸化的乳腺癌细胞(该类细胞定义为A组)中,受体酪氨酸激酶IGF-1R和HER2表达水平较高,EGFR和c-MET表达水平较低;相反地,在PI3K抑制剂能够抑制AKT磷酸化但不抑制ERK磷酸化的乳腺癌细胞(该类细胞定义为B组)中,受体酪氨酸激酶EGFR和c-MET表达水平较高,而IGF-1R和HER2表达水平较低。统计分析A、B两组乳腺癌细胞对PI3Kα抑制剂CYH33和BYL719的敏感性差异,我们发现A组乳腺癌细胞对CYH33和BYL719更加敏感。在此基础之上,我们考察了乳腺癌细胞中RTKs表达量与PI3Kα抑制剂对其增殖抑制活性之间的相关性,发现EGFR和c-MET表达水平与PI3Kα抑制剂增殖抑制活性呈负相关性,即乳腺癌细胞内EGFR和c-MET表达水平越高,其对PI3Kα抑制剂更加耐受;而HER2和IGF-1R表达水平与其呈正相关性,即乳腺癌细胞内HER2和IGF-1R表达水平越高,其对PI3Kα抑制剂更敏感。BYL719对三例病人来源的乳腺癌移植瘤(PDX)的生长抑制活性与肿瘤组织RTKs表达量之间的关系进一步验证了RTKs表达谱合力决定乳腺癌对PI3Kα选择性抑制剂的敏感性。综上,我们发现CYH33具有显著的体内外抗肿瘤活性,且活性优于处于III期临床试验的同类抑制剂BYL719,有希望成为我国第一个具有自主知识产权的PI3Kα选择性抑制剂。我们发现乳腺癌细胞/组织中RTKs表达谱决定PI3K对ERK磷酸化和活性的调控,从而决定乳腺癌对PI3Kα选择性抑制剂的敏感性。其中IGF-1R和/或HER2高表达,EGFR,c-MET和/或FGFR1低表达预示PI3K调节ERK活性,乳腺癌对PI3K抑制剂敏感;相反地,IGF-1R和/或HER2低表达,EGFR,c-MET和/或FGFR1高表达预示ERK磷酸化不依赖PI3K,乳腺癌对PI3K抑制剂耐受。因此,RTKs表达谱有希望成为预测乳腺癌患者对PI3Kα抑制剂响应的生物标志物,为PI3Kα抑制剂临床患者的选择提供依据。
[Abstract]:PI3K signaling pathway plays an important role in cell survival, growth, proliferation and metabolism, and is abnormally activated in a variety of different tissue sources. About 70% of breast cancers are highly activated by PI3K signaling pathway, and the PIK3CA gene mutation rate of the encoded PI3K a catalytic subunit is up to 30%, scattered in all types of breast cancer. A large number of molecular biology And genetic studies have shown that abnormal activation of PI3K a can promote the development of breast cancer, and the inhibition of PI3K activity can prevent the formation and development of breast cancer. Therefore, PI3K alpha is an important target for the treatment of breast cancer. Currently, there are many universal PI3K inhibitors and four PI3K alpha selective suppressor agents entering clinical trials with single or combined drug use. It is of great significance to study and develop new PI3K alpha selective inhibitors with independent intellectual property rights in China. The clinical trial data show that PI3K alpha selective inhibitors have a greater difference in the efficacy of breast cancer and are treated in some patients. Therefore, the discovery and confirmation of the biomarkers for predicting the efficacy of PI3K alpha inhibitors is an important strategy to promote the study and development of PI3K alpha selective inhibitors and to improve the efficacy of this study. The purpose of this study is to systematically explore the effect of the new structure of the new PI3K a selective inhibitor, CYH33, on the anti breast cancer effect found in our laboratory, and to explore the prediction of PI3K The biomarker of the antitumor effect of alpha inhibitor in breast cancer,.CYH33, is a new PI3K alpha selective inhibitor found in the previous research work in our laboratory. It can widely inhibit the proliferation of tumor cells from a variety of tissue sources. By detecting the proliferation inhibition effect of CYH33 on a series of breast cancer cells, we found that low concentration of CYH33 is capable of being able to inhibit the proliferation of a series of breast cancer cells. The proliferation of multiple breast cancer cells was significantly inhibited, and its activity was equivalent to that of the pan PI3K inhibitor GDC0941. It was superior to the same type of inhibitor BYL719. in III clinical trials, as an example of CYH33 sensitive T47D and MCF-7 cells and MDA-MB-231 cells tolerant to CYH33. We initially explored the mechanism.CYH33 of CYH33 to inhibit the proliferation of breast cancer cells. In a concentration dependent and equal degree of inhibition of T47D, the downstream protein kinase AKT phosphorylated.CYH33 in MCF-7 and MDA-MB-231 cells can downregulate the downstream p70S6K and S6 phosphorylation levels of M TORC1 in sensitive breast cancer cells, but not in breast cancer cells. In sensitive breast cancer cells, CYH33 by down-regulation of Rb phosphorylation and depletion. The content of in D1 and the accumulation of p27, inhibit the cell proliferation and inhibit the proliferation of cells in G1 stage, but in the tolerance of breast cancer cells, CYH33 can not induce sensitivity or tolerance to the apoptosis of breast cancer cells for 72 hours. Therefore, CYH33 mainly inhibits cell proliferation and inhibits cell proliferation by blocking the cells in G1 phase. The inhibition of AKT phosphorylation can not predict the sensitivity of breast cancer cells to CYH33. It is consistent with the inhibitory effect of CYH33 on the proliferation of breast cancer cells in vitro. CYH33 can effectively inhibit the growth of spontaneous tumor in T47D SCID mice and PIK3CAH1047R gene engineering mice, and the activity is better than BYL719. in addition to CYH33 and BYL719 for a long time. The model animals used in breast cancer had little influence on the weight and blood glucose regulation of mice and were within the safety range. We preliminarily analyzed the sensitivity of different molecular types of breast cancer cells to CYH33, and found that the lumen and HER2 expanded breast cancer cells were more sensitive to CYH33, and the base like breast cancer cells were tolerant to CYH33. The PI3K letter was analyzed. The relationship between the changes of key molecules in the pathway and the sensitivity of breast cancer cells to CYH33 sensitivity, we have found that the PIK3CA mutation and HER2 amplification of breast cancer cells are more sensitive to CYH33, while the PTEN deficient breast cancer cells are tolerant to CYH33. Although the key protein molecules in the different molecular types of the breast cancer cells or the key protein molecules in the PI3K signaling pathway are altered with the mammary gland There is a certain correlation between the sensitivity of cancer cells to CYH33 and the sensitivity of some basal like breast cancer cells or normal breast cancer cells in the PI3K signaling pathway to CYH33. Therefore, the exploration of biomarkers based on the mechanism of PI3K alpha selective inhibitors is an important strategy to improve the efficacy of these inhibitors,.PI3K and MAPK signaling pathways. There are two key signaling pathways involved in the regulation of breast cancer cell proliferation. There are extensive interaction between the two and one of the signaling pathways that usually cause feedback activation of another signal pathway, suggesting that the target two signal pathways are expected to achieve a more significant antitumor effect. We found that CYH33 can reduce the T47D finer at the same time. AKT and ERK phosphorylation in the cell, further research on the mechanism of CYH33 may reveal its relationship with the anti-tumor activity of CYH33. T47D and MCF-7 cells in sensitive breast cancer are the research object, and multiple PI3K inhibitors can inhibit AKT and ERK phosphorylation simultaneously, while PDK1, AKT and M inhibitors are at the same time playing the target inhibition effect. Phosphorylation weakly affects or even increases the phosphorylation level of ERK, indicating that PI3K regulated ERK phosphorylation is not dependent on its classical PDK1/AKT/m TOR downstream signal pathway. Inhibition of RAF and MEK causes ERK phosphorylation down down, while high expression RAF and MEK antagonize the inhibition of PI3K inhibitor to ERK phosphorylation. RAS activity is not affected by PI3K inhibitors, but high expression of RAS can reverse the inhibitory effect of PI3K inhibitors on ERK phosphorylation, which indicates that PI3K and RAS two regulate ERK phosphorylation. Activation of EGFR activity in cells can increase the intracellular RAS-GTP content and prevent the inhibition of ERK phosphorylation by PI3K inhibitors. The synergistic effect of ERK phosphorylation indicates that the low activity of EGFR makes ERK phosphorylation dependent on PI3K, and the highly active EGFR makes ERK phosphorylation dependent on RAS. based on EGFR activity to determine whether ERK phosphorylation is dependent on PI3K. 3K regulates the role of ERK phosphorylation. Activation of EGFR, HER3, FGFR and / or c-MET activity can antagonize the inhibition of BYL719 on T47D, MCF-7 and HCC1954 ERK phosphorylation in breast cancer cells, and can partly reverse the inhibitory effect of BYL719 on the proliferation of corresponding breast cancer cells. The expression of RTKs in a series of breast cancer cells and the effect of PI3K inhibitor on its phosphorylation of AKT and ERK found that the expression level of AKT and ERK phosphorylated breast cancer cells (defined as A group) at the same time the PI3K inhibitor was defined as the A group, the expression level of receptor tyrosine kinase IGF-1R and HER2 was higher, and the expression level of EGFR and c-MET was lower; on the contrary, it was suppressed. The preparation can inhibit AKT phosphorylation but not inhibit ERK phosphorylation of breast cancer cells (the cell defined as B), receptor tyrosine kinase EGFR and c-MET expression level is higher, and IGF-1R and HER2 expression level is lower. Statistical analysis A, B two group breast cancer cells for PI3K alpha inhibitors CYH33 and BYL719 sensitivity difference, we found breast breast Cancer cells were more sensitive to CYH33 and BYL719. On this basis, we examined the correlation between the expression of RTKs in breast cancer cells and the inhibitory activity of PI3K a inhibitor on its proliferation. It was found that the expression level of EGFR and c-MET was negatively correlated with the inhibitory activity of PI3K a inhibitor, that is, the higher the level of EGFR and c-MET expression in breast cancer cells. It is more tolerant to PI3K alpha inhibitors, and the expression level of HER2 and IGF-1R is positively correlated with the higher expression level of HER2 and IGF-1R in breast cancer cells, which is more sensitive to the relationship between the inhibitory activity of.BYL719 and the growth inhibitory activity of the breast cancer transplantation tumor (PDX) in three patients with the PI3K alpha inhibitor, and the relationship between the expression of the tumor tissue and the expression of the tumor tissue is further examined. It is demonstrated that the RTKs expression profile determines the sensitivity of breast cancer to the PI3K alpha selective inhibitors. To sum up, we have found that CYH33 has significant antitumor activity in the body and in vivo, and the activity is better than that of the same kind of inhibitor, BYL719, which is in the clinical trial of III. We hope to be the first selective inhibitor of PI3K alpha with self principal intellectual property rights. The expression profiles of RTKs in breast cancer cells / tissues determine the regulation of the phosphorylation and activity of ERK by PI3K, which determines the sensitivity of breast cancer to PI3K alpha selective inhibitors. IGF-1R and / or HER2 are highly expressed, EGFR, c-MET and / or FGFR1 low expression indicates PI3K regulation ERK activity, breast cancer is sensitive to PI3K inhibitors; conversely, and / or low High expression of EGFR, c-MET and / or FGFR1 indicates that ERK phosphorylation is not dependent on PI3K, and breast cancer is tolerated to PI3K inhibitors. Therefore, the RTKs expression profile is expected to be a biomarker for predicting the response of breast cancer to PI3K a inhibitors, and the basis for the selection of clinical patients with the PI3K alpha inhibitors.
【学位授予单位】：中国科学院大学(中国科学院上海药物研究所)
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R96

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