【摘要】：第一章：在美國(guó)男性中，前列腺癌（Prostate cancer, PCa）是第二高發(fā)病率的腫瘤同時(shí)也是致死率第二高的腫瘤。以雄激素受體（Androgen receptor, AR）為靶標(biāo)的去勢(shì)療法會(huì)導(dǎo)致病人發(fā)展出去勢(shì)抵抗型前列腺癌（Castration-resistantPCa，CRPC），而治療去勢(shì)抵抗型前列腺癌的化學(xué)療法又會(huì)導(dǎo)致更惡性的化療抵抗型前列腺癌（Chemo-resistant prostate cancer），但是在這些復(fù)發(fā)型或難治型前列腺癌中，普遍存在配體結(jié)合結(jié)構(gòu)域（ligand binding domain, LBD）缺失的雄激素受體剪接變異體（Androgen receptor splicing variants, ARVs）的表達(dá)。在臨床數(shù)據(jù)中表明，AR-V7和ARV567es在ARVs中的表達(dá)量占絕大多數(shù)，所以研究AR-V7和ARV567es與前列腺癌之間的關(guān)系具有非常重大的意義。 我們?cè)谛奂に卮嬖诤托奂に睾谋M兩種條件下，對(duì)比了AR和ARVs功能的不同，例如轉(zhuǎn)錄活性，下游基因的表達(dá)，以及對(duì)前列腺癌細(xì)胞的生長(zhǎng)促進(jìn)等。在雄激素存在的條件下，我們通過(guò)熒光素酶實(shí)驗(yàn)（Luciferase assay）發(fā)現(xiàn)了AR-V7和ARV567es具有結(jié)合雄激素效應(yīng)元件（Androgen response element, ARE）的能力，利用qRT-PCR驗(yàn)證了AR-V7和ARV567ES可以替代AR的轉(zhuǎn)錄活性，激活了下游基因的轉(zhuǎn)錄，例如前列腺特異抗原（Prostate specific antigen，，PSA）等，用western blot證明了AR-V7和ARV567es促進(jìn)了AR下游基因PSA等的表達(dá)，而且細(xì)胞增殖實(shí)驗(yàn)的結(jié)果表明，AR-V7和ARV567es促進(jìn)了前列腺癌細(xì)胞的生長(zhǎng)，但是在雄激素耗盡的條件下，AR-V7和ARV567es的在蛋白表達(dá)、轉(zhuǎn)錄活性以及細(xì)胞增殖方面，比AR有更強(qiáng)的促進(jìn)作用。以上研究結(jié)果為無(wú)雄激素存在的情況下，AR信號(hào)通路為何還會(huì)被激活，病人為何會(huì)產(chǎn)生去勢(shì)抵抗，提供了潛在的解釋。 我們還比較了AR和ARVs對(duì)于化療藥物的不同反應(yīng)，我們利用紫杉醇（Paclitaxel，PTX）和多西紫杉醇（Docetaxel，DTX）這兩種常見化療藥物對(duì)表達(dá)AR和ARVs的前列腺癌細(xì)胞進(jìn)行處理，通過(guò)細(xì)胞核轉(zhuǎn)移實(shí)驗(yàn)（Translocationassay）、熒光霉素實(shí)驗(yàn)（Luciferase assay）、定量PCR和免疫印跡（Western blot）等實(shí)驗(yàn)手段，我們觀察到了無(wú)論是在有雄激素或無(wú)雄激素存在的情況下，PTX和DTX對(duì)AR的核轉(zhuǎn)移、表達(dá)水平、轉(zhuǎn)錄活性和對(duì)腫瘤細(xì)胞的生長(zhǎng)促進(jìn)方面都有很強(qiáng)的抑制效果，但是對(duì)于ARVs在以上這些方面則沒(méi)有明顯的抑制的效果。在無(wú)雄激素存在的情況下，當(dāng)AR與ARVs共表達(dá)時(shí)，ARVs可以協(xié)助AR完成核轉(zhuǎn)移，甚至ARVs可以協(xié)助AR逃脫紫杉醇對(duì)于AR核轉(zhuǎn)移的抑制。由于紫杉醇類化療藥物的作用靶標(biāo)是微管（Microtubules，MTs），所以我們?cè)噲D通過(guò)研究AR和ARVs與MTs的相互作用來(lái)解釋ARVs對(duì)于紫杉醇的抗藥機(jī)理，微管體內(nèi)結(jié)合實(shí)驗(yàn)（Microtubule in vivo binding assay）的結(jié)果說(shuō)明，AR可以和MTs結(jié)合，而ARVs則不能，我們進(jìn)一步利用微管體外結(jié)合實(shí)驗(yàn)（Microtubule invitro binding assay）證明了在沒(méi)有其他蛋白復(fù)合體參與的情況下，AR與微管可以直接結(jié)合。為了確定AR蛋白與微管結(jié)合的具體位置，我們構(gòu)建了一系列AR功能區(qū)的缺失突變體，研究了他們與微管結(jié)合能力的差異和這些突變體在細(xì)胞內(nèi)的定位差異，通過(guò)對(duì)比這些突變體與微管結(jié)合能力的差異并結(jié)合他們?cè)诩?xì)胞內(nèi)分布的不同，結(jié)果發(fā)現(xiàn)了AR734-AR774片段對(duì)于AR和微管的結(jié)合有著至關(guān)重要的作用，而且這段序列不存在于ARVs，所以我們推斷微管結(jié)合序列（MTAS）位于AR734-AR774。綜上，ARVs中AR723-AR795的缺失，決定了ARVs不能與微管結(jié)合，進(jìn)而失去了紫杉醇對(duì)微管結(jié)合蛋白的抑制，使得細(xì)胞對(duì)紫杉醇的敏感性降低，本論文結(jié)果為研究前列腺癌化療抵抗提供了重要的理論依據(jù)，也為臨床治療化療抵抗型前列腺癌具有重大的科學(xué)意義。第二章：有證據(jù)表明，Src作為一種原癌基因在前列腺癌中大量表達(dá)，尤其Src在雄激素非依賴的腫瘤生長(zhǎng)和前列腺癌骨轉(zhuǎn)移中起著非常重要的作用，一種新型的Src抑制劑KX-01（KX2-391）已經(jīng)進(jìn)入二期臨床試驗(yàn)。我們通過(guò)研究核轉(zhuǎn)移實(shí)驗(yàn)、熒光霉素實(shí)驗(yàn)、實(shí)時(shí)定量PCR和MTT等實(shí)驗(yàn)方法，發(fā)現(xiàn)了KX-01可以抑制AR的核轉(zhuǎn)移、轉(zhuǎn)錄活性、下游基因的轉(zhuǎn)錄活性和對(duì)腫瘤生長(zhǎng)的促進(jìn)。同時(shí)發(fā)現(xiàn)了KX-01還可以抑制ARVs的轉(zhuǎn)錄活性，下游基因的表達(dá)以及腫瘤的生長(zhǎng)促進(jìn)，但是KX-01對(duì)AR和ARVs的蛋白穩(wěn)定性沒(méi)有顯著的影響。我們利用雙熒光素酶實(shí)驗(yàn)（Dual luciferase assay）證明了KX-01可以降低ARmRNA的合成，由于目前的研究證明了ARVs的mRNA合成依賴于AR的mRNA，所以KX-01可以同時(shí)抑制AR和ARVs的mRNA的合成進(jìn)而抑制他們的表達(dá)。KX-01對(duì)AR和ARVs的表達(dá)抑制即KX-01對(duì)雄激素受體信號(hào)通路的抑制，揭示了KX-01抑制表達(dá)AR和ARVs前列腺癌細(xì)胞增殖的機(jī)理，結(jié)合第一章的結(jié)論ARVs可導(dǎo)致前列腺癌細(xì)胞的化療抵抗，推斷KX-01對(duì)于治療ARVs表達(dá)的復(fù)發(fā)型和難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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R737.25

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 沈棋;胡帥;李峻;王靜華;何群;;膀胱前列腺切除術(shù)中前列腺偶發(fā)癌發(fā)生率及臨床病理特點(diǎn)分析[J];北京大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2014年04期

本文編號(hào)：2194926