雄激素受体剪接体与化学疗法抵抗之间的关系以及新型前列腺癌治疗药物的研究
发布时间：2018-08-21 07:07
【摘要】：第一章：在美国男性中，前列腺癌（Prostate cancer, PCa）是第二高发病率的肿瘤同时也是致死率第二高的肿瘤。以雄激素受体（Androgen receptor, AR）为靶标的去势疗法会导致病人发展出去势抵抗型前列腺癌（Castration-resistantPCa，CRPC），而治疗去势抵抗型前列腺癌的化学疗法又会导致更恶性的化疗抵抗型前列腺癌（Chemo-resistant prostate cancer），但是在这些复发型或难治型前列腺癌中，普遍存在配体结合结构域（ligand binding domain, LBD）缺失的雄激素受体剪接变异体（Androgen receptor splicing variants, ARVs）的表达。在临床数据中表明，AR-V7和ARV567es在ARVs中的表达量占绝大多数，所以研究AR-V7和ARV567es与前列腺癌之间的关系具有非常重大的意义。 我们在雄激素存在和雄激素耗尽两种条件下，对比了AR和ARVs功能的不同，例如转录活性，下游基因的表达，以及对前列腺癌细胞的生长促进等。在雄激素存在的条件下，我们通过荧光素酶实验（Luciferase assay）发现了AR-V7和ARV567es具有结合雄激素效应元件（Androgen response element, ARE）的能力，利用qRT-PCR验证了AR-V7和ARV567ES可以替代AR的转录活性，激活了下游基因的转录，例如前列腺特异抗原（Prostate specific antigen，，PSA）等，用western blot证明了AR-V7和ARV567es促进了AR下游基因PSA等的表达，而且细胞增殖实验的结果表明，AR-V7和ARV567es促进了前列腺癌细胞的生长，但是在雄激素耗尽的条件下，AR-V7和ARV567es的在蛋白表达、转录活性以及细胞增殖方面，比AR有更强的促进作用。以上研究结果为无雄激素存在的情况下，AR信号通路为何还会被激活，病人为何会产生去势抵抗，提供了潜在的解释。 我们还比较了AR和ARVs对于化疗药物的不同反应，我们利用紫杉醇（Paclitaxel，PTX）和多西紫杉醇（Docetaxel，DTX）这两种常见化疗药物对表达AR和ARVs的前列腺癌细胞进行处理，通过细胞核转移实验（Translocationassay）、荧光霉素实验（Luciferase assay）、定量PCR和免疫印迹（Western blot）等实验手段，我们观察到了无论是在有雄激素或无雄激素存在的情况下，PTX和DTX对AR的核转移、表达水平、转录活性和对肿瘤细胞的生长促进方面都有很强的抑制效果，但是对于ARVs在以上这些方面则没有明显的抑制的效果。在无雄激素存在的情况下，当AR与ARVs共表达时，ARVs可以协助AR完成核转移，甚至ARVs可以协助AR逃脱紫杉醇对于AR核转移的抑制。由于紫杉醇类化疗药物的作用靶标是微管（Microtubules，MTs），所以我们试图通过研究AR和ARVs与MTs的相互作用来解释ARVs对于紫杉醇的抗药机理，微管体内结合实验（Microtubule in vivo binding assay）的结果说明，AR可以和MTs结合，而ARVs则不能，我们进一步利用微管体外结合实验（Microtubule invitro binding assay）证明了在没有其他蛋白复合体参与的情况下，AR与微管可以直接结合。为了确定AR蛋白与微管结合的具体位置，我们构建了一系列AR功能区的缺失突变体，研究了他们与微管结合能力的差异和这些突变体在细胞内的定位差异，通过对比这些突变体与微管结合能力的差异并结合他们在细胞内分布的不同，结果发现了AR734-AR774片段对于AR和微管的结合有着至关重要的作用，而且这段序列不存在于ARVs，所以我们推断微管结合序列（MTAS）位于AR734-AR774。综上，ARVs中AR723-AR795的缺失，决定了ARVs不能与微管结合，进而失去了紫杉醇对微管结合蛋白的抑制，使得细胞对紫杉醇的敏感性降低，本论文结果为研究前列腺癌化疗抵抗提供了重要的理论依据，也为临床治疗化疗抵抗型前列腺癌具有重大的科学意义。第二章：有证据表明，Src作为一种原癌基因在前列腺癌中大量表达，尤其Src在雄激素非依赖的肿瘤生长和前列腺癌骨转移中起着非常重要的作用，一种新型的Src抑制剂KX-01（KX2-391）已经进入二期临床试验。我们通过研究核转移实验、荧光霉素实验、实时定量PCR和MTT等实验方法，发现了KX-01可以抑制AR的核转移、转录活性、下游基因的转录活性和对肿瘤生长的促进。同时发现了KX-01还可以抑制ARVs的转录活性，下游基因的表达以及肿瘤的生长促进，但是KX-01对AR和ARVs的蛋白稳定性没有显著的影响。我们利用双荧光素酶实验（Dual luciferase assay）证明了KX-01可以降低ARmRNA的合成，由于目前的研究证明了ARVs的mRNA合成依赖于AR的mRNA，所以KX-01可以同时抑制AR和ARVs的mRNA的合成进而抑制他们的表达。KX-01对AR和ARVs的表达抑制即KX-01对雄激素受体信号通路的抑制，揭示了KX-01抑制表达AR和ARVs前列腺癌细胞增殖的机理，结合第一章的结论ARVs可导致前列腺癌细胞的化疗抵抗，推断KX-01对于治疗ARVs表达的复发型和难治型前列腺癌具有重要作用。
[Abstract]:Chapter 1: Prostate cancer (PCa) is the second most common cancer and the second most lethal cancer among American men. Ovariectomy targeting androgen receptor (AR) leads to castration-resistant prostate cancer (CRPC) and castration-resistant prostate cancer (CRPC). Chemotherapy for resistant prostate cancer can lead to more malignant chemo-resistant prostate cancer, but ligand binding domain (LBD) deletion of androgen receptor splicin is common in these recurrent or refractory prostate cancer. The expression of G-variants and ARV567es is overwhelming in ARVs, so it is of great significance to study the relationship between AR-V7 and ARV567es and prostate cancer.
In the presence of androgen and androgen depletion, we compared the different functions of AR and ARVs, such as transcriptional activity, downstream gene expression, and growth promotion of prostate cancer cells. The ability of hormone response element (ARE) was verified by qRT-PCR that AR-V7 and ARV567ES could replace AR and activate the transcription of downstream genes such as prostate specific antigen (PSA). Western blot showed that AR-V7 and ARV567es promoted the expression of PSA and other downstream genes of AR. Furthermore, the results of cell proliferation experiments showed that AR-V7 and ARV567es promoted the growth of prostate cancer cells, but AR-V7 and ARV567es had stronger effects on protein expression, transcriptional activity and cell proliferation than AR under androgen depletion. Why is it activated? Why do patients develop castration resistance? This provides a potential explanation.
We also compared the different reactions of AR and ARVs to chemotherapeutic agents. Paclitaxel (PTX) and Docetaxel (DTX) were used to treat prostate cancer cells expressing AR and ARVs. Translocation assay, Luciferase assay were used to determine the effects of these two chemotherapeutic agents. By means of quantitative PCR and Western blot, we observed that PTX and DTX had strong inhibitory effects on AR nuclear metastasis, expression level, transcriptional activity and tumor cell growth in the presence or absence of androgens, but not on ARVs. In the absence of androgen, when AR and ARVs co-express, ARVs can help AR complete nuclear metastasis, and even ARVs can help AR escape the inhibition of paclitaxel on AR nuclear metastasis. Microtubule in vivo binding assay showed that AR could bind to MT, whereas ARVs could not. We further used microtubule in vitro binding assay to prove that no other protein complexes were involved. In order to determine the specific location of AR protein binding to microtubules, we constructed a series of mutants with deletion of AR functional region, studied their binding ability to microtubules and the location of these mutants in cells, and compared the differences between these mutants and microtubule binding ability. Combined with their different intracellular distribution, we found that AR734-AR774 fragment plays an important role in the binding of AR to microtubules, and this sequence does not exist in ARVs. Therefore, we infer that the microtubule binding sequence (MTAS) is located in AR734-AR774. In conclusion, the deletion of AR723-AR795 in ARVs determines that ARVs can not bind to microtubules, and consequently, ARVs are absent. The results of this study provide an important theoretical basis for the study of chemotherapy resistance of prostate cancer, and also have great scientific significance for the clinical treatment of chemotherapy-resistant prostate cancer. Chapter 2: Evidence shows that Src is a proto-oncogene. Prostate cancer is highly expressed, especially Src plays an important role in androgen-independent tumor growth and bone metastasis of prostate cancer. A novel inhibitor of Src, KX-01 (KX2-391), has entered phase II clinical trials. KX-01 can inhibit AR nuclear metastasis, transcriptional activity, transcriptional activity of downstream genes and promote tumor growth. KX-01 can also inhibit the transcriptional activity of ARVs, the expression of downstream genes and tumor growth promotion, but KX-01 has no significant effect on the stability of AR and ARVs proteins. The Dual luciferase assay has shown that KX-01 can reduce the synthesis of AR mRNA. Since current studies have shown that the synthesis of ARVs mRNA depends on AR mRNA, KX-01 can inhibit both AR and ARVs mRNA synthesis and their expression. KX-01 inhibits AR and ARVs expression, i.e. KX-01 inhibits the androgen receptor signaling pathway. The results indicate that KX-01 may inhibit the proliferation of prostate cancer cells expressing AR and ARVs. Combining with the conclusion of Chapter 1, ARVs can induce chemotherapy resistance of prostate cancer cells, it is concluded that KX-01 may play an important role in the treatment of recurrent and refractory prostate cancer expressing ARVs.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.25
本文编号：2194926
[Abstract]:Chapter 1: Prostate cancer (PCa) is the second most common cancer and the second most lethal cancer among American men. Ovariectomy targeting androgen receptor (AR) leads to castration-resistant prostate cancer (CRPC) and castration-resistant prostate cancer (CRPC). Chemotherapy for resistant prostate cancer can lead to more malignant chemo-resistant prostate cancer, but ligand binding domain (LBD) deletion of androgen receptor splicin is common in these recurrent or refractory prostate cancer. The expression of G-variants and ARV567es is overwhelming in ARVs, so it is of great significance to study the relationship between AR-V7 and ARV567es and prostate cancer.
In the presence of androgen and androgen depletion, we compared the different functions of AR and ARVs, such as transcriptional activity, downstream gene expression, and growth promotion of prostate cancer cells. The ability of hormone response element (ARE) was verified by qRT-PCR that AR-V7 and ARV567ES could replace AR and activate the transcription of downstream genes such as prostate specific antigen (PSA). Western blot showed that AR-V7 and ARV567es promoted the expression of PSA and other downstream genes of AR. Furthermore, the results of cell proliferation experiments showed that AR-V7 and ARV567es promoted the growth of prostate cancer cells, but AR-V7 and ARV567es had stronger effects on protein expression, transcriptional activity and cell proliferation than AR under androgen depletion. Why is it activated? Why do patients develop castration resistance? This provides a potential explanation.
We also compared the different reactions of AR and ARVs to chemotherapeutic agents. Paclitaxel (PTX) and Docetaxel (DTX) were used to treat prostate cancer cells expressing AR and ARVs. Translocation assay, Luciferase assay were used to determine the effects of these two chemotherapeutic agents. By means of quantitative PCR and Western blot, we observed that PTX and DTX had strong inhibitory effects on AR nuclear metastasis, expression level, transcriptional activity and tumor cell growth in the presence or absence of androgens, but not on ARVs. In the absence of androgen, when AR and ARVs co-express, ARVs can help AR complete nuclear metastasis, and even ARVs can help AR escape the inhibition of paclitaxel on AR nuclear metastasis. Microtubule in vivo binding assay showed that AR could bind to MT, whereas ARVs could not. We further used microtubule in vitro binding assay to prove that no other protein complexes were involved. In order to determine the specific location of AR protein binding to microtubules, we constructed a series of mutants with deletion of AR functional region, studied their binding ability to microtubules and the location of these mutants in cells, and compared the differences between these mutants and microtubule binding ability. Combined with their different intracellular distribution, we found that AR734-AR774 fragment plays an important role in the binding of AR to microtubules, and this sequence does not exist in ARVs. Therefore, we infer that the microtubule binding sequence (MTAS) is located in AR734-AR774. In conclusion, the deletion of AR723-AR795 in ARVs determines that ARVs can not bind to microtubules, and consequently, ARVs are absent. The results of this study provide an important theoretical basis for the study of chemotherapy resistance of prostate cancer, and also have great scientific significance for the clinical treatment of chemotherapy-resistant prostate cancer. Chapter 2: Evidence shows that Src is a proto-oncogene. Prostate cancer is highly expressed, especially Src plays an important role in androgen-independent tumor growth and bone metastasis of prostate cancer. A novel inhibitor of Src, KX-01 (KX2-391), has entered phase II clinical trials. KX-01 can inhibit AR nuclear metastasis, transcriptional activity, transcriptional activity of downstream genes and promote tumor growth. KX-01 can also inhibit the transcriptional activity of ARVs, the expression of downstream genes and tumor growth promotion, but KX-01 has no significant effect on the stability of AR and ARVs proteins. The Dual luciferase assay has shown that KX-01 can reduce the synthesis of AR mRNA. Since current studies have shown that the synthesis of ARVs mRNA depends on AR mRNA, KX-01 can inhibit both AR and ARVs mRNA synthesis and their expression. KX-01 inhibits AR and ARVs expression, i.e. KX-01 inhibits the androgen receptor signaling pathway. The results indicate that KX-01 may inhibit the proliferation of prostate cancer cells expressing AR and ARVs. Combining with the conclusion of Chapter 1, ARVs can induce chemotherapy resistance of prostate cancer cells, it is concluded that KX-01 may play an important role in the treatment of recurrent and refractory prostate cancer expressing ARVs.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.25
【参考文献】
相关期刊论文 前1条
1 沈棋;胡帅;李峻;王静华;何群;;膀胱前列腺切除术中前列腺偶发癌发生率及临床病理特点分析[J];北京大学学报(医学版);2014年04期
本文编号：2194926
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