靶向肿瘤干细胞“Akt-Oct4”调控通路的小分子药物初步研究

发布时间：2018-04-24 15:20

本文选题：肿瘤干细胞 + 干细胞样肿瘤细胞　；参考：《浙江大学》2016年博士论文

【摘要】：肿瘤在疾病死亡率及死亡原因构成中占第一位。肿瘤的发生是多因素、多阶段、多基因相互作用的结果,其治疗后发生的复发转移、药物耐受和放疗抵抗等导致迄今仍无法根治肿瘤。近年来,肿瘤的发生与胚胎发育间的相似性一直受到关注。随着干细胞研究的迅猛发展,肿瘤干细胞(Cancer stem cell)假说及相关研究逐渐成为热点。该假说认为,肿瘤组织或肿瘤细胞系中存在着数量极少的细胞亚群,具有类似干细胞的自我更新和分化能力,在肿瘤的发生、发展、复发和转移过程中起决定性作用,被称为肿瘤干细胞。肿瘤干细胞通过自我更新,使其致瘤性得以维持；通过分化形成肿瘤组织中不同表型的非干性肿瘤细胞,是肿瘤异质性的基础。Oct4是由POU5F1基因编码的转录因子,对于维持多能干细胞的多能性和自我更新具有无可替代的作用。长期以来,Oct4基因的表达被认为是多能干细胞所特有的,但近年来"Oct4是多能干细胞标志物”的结论却受到了挑战。因为陆续有报道在一些分化的肿瘤细胞系和肿瘤组织中检测到Oct4 mRNA或蛋白,但Oct4在分化的正常体细胞和肿瘤细胞中究竟发挥何种功能,尚有待进一步研究。课题组前期工作表明,Oct4蛋白的第235位苏氨酸可被蛋白激酶Akt磷酸化(Oct4-pT235),而Oct4又能以正反馈方式激活AKT基因转录,形成‘'Akt-Oct4调控通路”,从而在干细胞自我更新和凋亡之间建立起全新的联系。为探究在分化的肿瘤细胞和肿瘤干细胞中是否存在被过分激活的"Akt-Oct4调控通路”、以及该通路是否可作为特异性的药物靶标,本论文开展了一系列研究,在多种人肿瘤细胞系中检测到Oct4和Oct4-pT235的表达,且发现Oct4-pT235主要定位于脑胶质瘤细胞的细胞核中。Oct4-pT235表达水平升高可使干细胞样胶质瘤细胞中Oct4蛋白稳定性增加。在干细胞样胶质瘤细胞中,PI3K-Akt和干细胞信号通路被激活,且在细胞的自我更新中发挥重要作用。通过抑制Akt的活性,可降低Oct4-pT235水平,抑制细胞的增殖。有研究表明,小分子药物曲尼司特可通过作用于芳香烃受体(AhR)而降低Oct4的表达水平,但具体作用机制不清楚。本论文研究发现,U87胶质瘤细胞中可检测到AhR的内源配体ITE,缺氧条件下内源ITE含量下降会伴随Oct4转录的上调。外源ITE可抑制干细胞样胶质瘤细胞中的Oct4转录,降低Oct4-pT235和Oct4总蛋白水平。外源ITE抑制干细胞样胶质瘤细胞的增殖和分化,并可抑制人源胶质瘤细胞在移植裸鼠体内的增殖。此外,有研究发现用于二型糖尿病治疗的二甲双胍也能在转录水平抑制Oct4的表达。基于在转录水平和翻译后水平双重抑制Oct4的构想,本论文尝试将二甲双胍和Akti-1/2进行联合使用。结果发现,双药联用后,可共同作用于"Akt-Oct4调控通路”,降低Oct4-pT235和Oct4总蛋白、pAkt-S473和pAkt-T308的表达水平。同时,AMPK和AKT之间存在着双向抑制作用,Akti-1/2可去除AKT对AMPK的抑制作用,同时二甲双胍可激活AMPK,因此双药联用后可通过"Akt-Oct4 调控通路”协同抑制干细胞样胶质瘤细胞的增殖,并抑制裸鼠体内移植人胶质瘤细胞的增殖。综上所述,本论文工作证实了干细胞样胶质瘤细胞中存在可作为潜在药物靶标的"Akt-Oct4调控通路”,Akt抑制剂可通过在翻译后水平下调Oct4-pT235而有效抑制干细胞样胶质瘤细胞的增殖。ITE可在转录水平上抑制Oct4,并抑制干细胞样胶质瘤细胞的增殖,促进其分化。因此,ITE可作为靶向肿瘤干细胞的候选药物。Akt抑制剂Akti-1/2与二甲双胍联用可通过阻抑"Akt-Oct4调控通路”等机制有效抑制干细胞样胶质瘤细胞增殖,双药联用或可成为治疗人脑胶质瘤的一种新策略。
[Abstract]:Tumor is the first in the mortality and cause of death. The occurrence of tumor is multifactor, multistage, multi gene interaction, recurrence and metastasis after treatment, drug tolerance and radiation resistance, which are still unable to cure the tumor so far. In recent years, the similarity between the occurrence of tumor and the development of embryo has been closely related to the recent years. Note. With the rapid development of stem cell research, the Cancer stem cell hypothesis and related research have gradually become a hot spot. This hypothesis suggests that there are very few cell subgroups in the tumor tissue or tumor cell lines, which have the ability to renew and differentiate like stem cells, and in the occurrence, development, recurrence and metastasis of tumor. The tumor stem cells are known as tumor stem cells. The tumor stem cells maintain their tumorigenicity by self renewal; by differentiating into non dry tumor cells of different phenotypes in the tumor tissue, the base.Oct4 of tumor heterogeneity is the transcription factor encoded by the POU5F1 gene, and the pluripotent and self-determination of the pluripotent stem cells. My update has an irreplaceable role. For a long time, the expression of the Oct4 gene is considered to be specific to the pluripotent stem cells. However, in recent years, the conclusion of "Oct4 is a pluripotent cell marker" has been challenged. Since there have been reports of the detection of Oct4 mRNA or protein in some differentiated tumor cell lines and tumor tissues, Oct4 is in a fraction. What function in normal somatic cells and tumor cells remains to be further studied. Earlier work in the group showed that the 235th threonine of Oct4 protein could be phosphorylated by protein kinase Akt (Oct4-pT235), and Oct4 can activate AKT gene transcription in a positive feedback manner, forming a ''Akt-Oct4 regulation pathway' and thus in stem cells. In order to explore the existence of an excessively activated "Akt-Oct4 regulation pathway" in differentiated tumor cells and tumor stem cells, and whether the pathway can be used as a specific drug target, a series of studies have been carried out to detect Oct4 and Oct4-p in a variety of human tumor cell lines. The expression of T235 and the increase of the expression level of Oct4-pT235 mainly in the nucleus of brain glioma cells increase the stability of Oct4 protein in the stem like glioma cells. In the stem like glioma cells, the PI3K-Akt and stem cell signaling pathways are activated and play an important role in the self renewal of the cells. By inhibiting the activity of Akt, the level of Oct4-pT235 can be reduced and the proliferation of cells can be inhibited. Some studies have shown that the small molecular drug, a drug, can reduce the expression level of Oct4 by acting on the aromatic hydrocarbon receptor (AhR), but the specific mechanism of action is not clear. In this paper, we found that the endogenous ligand of AhR in the U87 glioma cells can be detected in ITE and hypoxia. The decrease of endogenous ITE content was associated with up regulation of Oct4 transcription. Exogenous ITE inhibited Oct4 transcription in stem like glioma cells and reduced the total protein level of Oct4-pT235 and Oct4. Exogenous ITE inhibited the proliferation and differentiation of stem like glioma cells and inhibited the proliferation of human glioma cells in nude mice. It is found that metformin used in the treatment of type two diabetes can also inhibit the expression of Oct4 at transcriptional level. Based on the idea of double inhibition of Oct4 at transcriptional level and post translation level, this paper attempts to combine metformin and Akti-1/2. The results show that the combination of double drugs can work together on the "Akt-Oct4 regulation pathway" and reduce Oct4. -pT235 and Oct4 total protein, pAkt-S473 and pAkt-T308 expression level. Meanwhile, there is a bidirectional inhibition between AMPK and AKT, Akti-1/2 can remove the inhibitory effect of AKT on AMPK, and the metformin activates AMPK. Therefore, the double drug combination can inhibit the proliferation of stem like glioma cells through the "Akt-Oct4 regulation pathway" and inhibit the proliferation and inhibit the proliferation of the stem cell like glioma cells. The proliferation of human glioma cells in nude mice. In summary, the work demonstrated that there is a "Akt-Oct4 regulation pathway" in the stem like glioma cells that can be used as a potential drug target. The Akt inhibitor can effectively inhibit the proliferation of stem like glioma cells by reducing the level of Oct4-pT235 after translation, and the.ITE can be transcribed in the transcriptional water. Oct4 can inhibit the proliferation of stem like glioma cells and inhibit the proliferation of stem cell like glioma cells. Therefore, ITE can be used as a candidate drug for targeting tumor stem cells, the combination of.Akt inhibitor Akti-1/2 and metformin can effectively inhibit the proliferation of stem like glioma cells by blocking the "Akt-Oct4 regulation pathway". Double drug combination or treatment can be used as a cure. A new strategy for the treatment of human glioma.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.5

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