【作者】 倪阳；

【导师】 刘奇；

【作者基本信息】 山东大学 ， 肿瘤学， 2014， 博士

【摘要】 恶性肿瘤是严重威胁人类健康的一类疾病,已成为我国城市居民死亡原因的首位,占全部死亡总数的25%。其中肺癌的发病在全球都呈现迅速上升趋势,死亡率在全球范围内居恶性肿瘤首位。过去30年间,我国肺癌死亡率上升了465%,成为上升速度最快的癌症,并已成为我国首位恶性肿瘤死亡原因。相关研究显示,目前我国肺癌发病率每年增长26.9%,肺癌的发病和死亡已成为一个严峻的问题。吸烟、空气污染被认为与肺癌的发病密切相关。肺癌分为两类：非小细胞肺癌(Non-small cell lung cancer, NSCLC)和小细胞肺癌(Small cell lung cancer,SCLC),其中非小细胞肺癌占所有肺癌的85%左右,是最常见、最致命的一种肺癌。尽管早期诊断技术不断发展,但仍有约七成的肺癌患者在确诊时已到晚期。目前临床上广泛采用的治疗手段如外科治疗、放疗、化疗等治疗效果均不理想。随着遗传学、分子生物学等医学科学的不断发展,基于致癌基因的分子靶向治疗正在逐步兴起。具有靶向性的表皮生长因子受体(EGFR)阻断剂,如吉非替尼、埃罗替尼等已被用于晚期的NSCLC二、三线治疗。但这些靶向药物又表现出对EGFR突变的人群有较好疗效,而对EGFR未突变患者效果不理想。所以,深入研究NSCLC中发挥关键作用的信号传导通路,对于揭示肺癌发生发展的分子机制及寻找NSCLC治疗新靶点具有重要的意义。蛋白激酶D (Protein kinase D, PKD)是一种新发现的钙离子／钙调蛋白依赖性的丝氨酸／苏氨酸蛋白激酶。研究发现PKD在受到G蛋白偶联受体(Guanosine-binding Protein Coupled Receptor, GPCR)激动剂、生长因子及佛波酯等刺激后活化,参与调控多条细胞内信号传导通路。PKD家族有三种同源结构的成员,分别是PKD1,PKD2和PKD3,其中PKD1是PKD家族中研究最为深入的成员。PKD1基因(又称为PRKD1)位于人类染色体14q11位置。PKD1在人体内脑、心脏、肺等多个器官中广泛表达,并在细胞增殖、凋亡,细胞侵袭,血管生成等一系列生物学行为中起重要的调控作用。同时,PKD1在维持心肌细胞功能和心血管系统健康及免疫调节方面还起到关键作用。因此,PKD1表达及功能的失衡与癌症、心血管疾病等多种疾病的发生发展密切相关。近年来的研究发现,PKD1在多种肿瘤中表达异常,如在前列腺癌、乳腺癌、胃癌和结直肠癌中PKD1显著下调,而过表达的PKD1在胰腺癌、皮肤癌的发展中起重要作用。由于PKD1作为一个关键激酶在介导细胞外刺激至胞内信号变化中发挥作用,使得通过调控PKD1表达水平和激酶活性来治疗肿瘤成为可能。肿瘤细胞中异常表达的PKD1通过调控多条信号传导通路影响肿瘤细胞的生物学行为。研究发现,细胞内PKD1可被GPCR激动剂激活并引起下游促分裂素原活化蛋白激酶(mitogen-activated protein kinases,MAPK), c-Jun N-terminal kinase (JNK)和NFκB等信号通路的活化。但PKD1在细胞分子信号通路网络中的作用和相关机制未完全阐明。同时,PKD1在NSCLC中的表达及功能尚无相关文献报道。在本课题中我们深入研究了PKD1对PI3K/Akt、Ⅱa型HDAC及InTOR-S6K1等信号通路的调控作用并探讨了PKD1在NSCLC中的表达、功能和调控机制。旨在进一步深入研究PKD1调控的分子信号通路及其对细胞生物学行为的作用,并阐明PKD1相关通路在NSCLC发生发展的作用和机制。为肺癌的防治提供新的治疗靶点和理论依据。目的1.探讨PKD1对P13K/Akt通路的调控作用和机制；2.明确PKD1调控组蛋白去乙酰酶(HDAC)4,5,7磷酸化及PKD1/HDAC通路对细胞周期和增殖的影响；3.确定PKD1在NSCLC中的表达及对NSCLC发生发展的作用；4.揭示PKD1在NSCLC细胞中作用的分子机制。第一部分PKDl对PI3K/Akt通路的调控作用和机制研究[研究目的]1.探讨特异性抑制或敲除PKD1对PI3K/Akt通路活性的作用；2.明确PKD1调控P13K/Akt通路的分子机制；3.在转基因小鼠模型中探讨验证PKD1对Akt的调控作用。[研究方法]1.应用不同浓度梯度的PKD1特异性抑制剂kb NB142-70抑制PKD1活性,观察在不同GPCR激动剂对Akt磷酸化(pAkt-Thr308,pAkt-Ser473)水平变化的作用。2.分别应用PKD1抑制剂CRT0066101和PKD1特异性siRNA处理或转染细胞,观察PKD1抑制或敲除对Akt磷酸化(pAkt-Thr308,pAkt-Ser473)水平的影响。3.向细胞内转染荧光蛋白(GFP)标记的AAKT-PH质粒,然后应用5μM kb NB142-70预处理细胞,荧光显微镜观察处理组和对照组在GPCR激动剂血管紧张素II(Angiotensin Ⅱ, Ang Ⅱ)刺激下AKT-PH的动态变化,以检测细胞膜磷脂酰肌醇三磷酸(phosphatidylinositol-3-phosphate,PIP3)的生成；免疫共沉淀检测选择性抑制PKD1对ANG II诱导的PI3K p85α亚基磷酸化及PI3K p85α--PTEN复合物形成的影响。4.构建PKD1过表达的转基因小鼠,取小鼠的肠上皮细胞,应用、；western blot免疫蛋白印迹法检测PKD1和pAkt-Thr308, pAkt-Ser473表达,从而在体内验证PKD1对pAkt的负性调控作用。[结果]1.特异性抑制PKD1可以增强GPCR诱导的Akt活性,主要表现在pAkt-Thr308, pAkt-Ser4乃的表达水平显著上调。Akt活性的增强与PKD1激酶活性的抑制程度呈负相关。2.通过转染两种PKD1特异性siRNA下调PKD1水平能显著增强ANG Ⅱ诱导的细胞内pAkt-Thr308, pAkt-Ser4刀的表达水平。3.抑制PKD1能显著增强PI3K介导的PIP3生成；Kb NB142-70能显著抑制PKD1诱导的PI3K p85α亚基磷酸化和PI3K p85α-PTEN复合物形成。4.转基因小鼠肠上皮中过表达的PKD1能抑制pAkt的表达。[结论]1.特异性PKD1能增强PI3K-Akt信号通路活性,PKD1负性调控PI3K-Akt信号通路。2.PKD1通过抑制P13K介导的PIP3生成、PI3K p85α亚基的磷酸化以及增加PI3K p85α-PTEN复合物形成参与调控P13K-Akt信号通路。第二部分PKI)1依赖的Ⅱa型HDAC磷酸化在调控细胞增殖中的作用和机制研究[研究目的]1.探讨PKD1介导的II a型HDAC (HDAC4, HDAC5, HDAC7)磷酸化；2.明确PKD1在诱导HDAC5由细胞核向细胞质迁移中的作用；3.探讨PKD1/Ⅱa型HDAC调控轴在细胞周期、细胞DNA合成和细胞增殖中的作用；4.在PKD1转基因小鼠模型中,研究并验证PKD1对IIa型HDAC磷酸化的调控作用。[研究方法]1.分别应用PKD1抑制剂(5μM kb NB142-70和CRT0066101)及PKD1特异性siRNA处理或转染细胞,western blot免疫蛋白印迹检测在GPCR或佛波酯等PKD1激动剂刺激下,细胞内HDAC4Ser246, HDAC5Ser259, HDAC7Ser155及HDAC4Ser632, HDAC5Ser498, HDAC7Ser486磷酸化蛋白表达水平。2.预先应用3.5μM kb NB142-70处理细胞2小时,随后使用50nMAngII处理细胞1小时,然后将细胞清洗、固定。应用细胞染色技术观察HDAC5从胞核向胞质的定向迁移。3.向细胞内分别转染FLAG标记的突变型HDAC5(HDAC5Ser259, Ser498突变为Ala),然后使用3.5μM kb NB142-70处理细胞2小时,随后应用50nM Ang Ⅱ刺激细胞1小时,刺激结束后将细胞固定、染色,观察FLAG标记的HDAC5在细胞内的定位。4.应用HDAC抑制剂MC1568(5μM)和TMP269(3μM)处理细胞,[3H]胸腺嘧啶检测细胞DNA合成,流式细胞术检测细胞周期,细胞计数法检测细胞增殖。5.构建PKD1转基因小鼠模型,在体内观察PKD1对Ⅱa型HDAC磷酸化的作用。[研究结果]1.PKD1抑制剂及特异性siRNA能抑制GPCR激动剂或佛波酯诱导的IIa型HDAC磷酸化。2.特异性抑制PKD1能诱导HDAC5从胞核向胞质移动。3.HDAC5磷酸化位点突变为非磷酸化位点时,HDAC5胞质的移动受到抑制。4.tHDAC抑制剂能显著降低细胞DNA合成和细胞增殖,抑制细胞从G0/G1期转入G2/M期。5.在PKD1高表达小鼠模型中磷酸化HDAC水平较未处理小鼠组显著升高。[结论]1.PKD1是诱导Ⅱa型HDAC磷酸化及HDAC5出核的关键上游调控因子。2.PKD1-HDAC信号通路能调控细胞增殖和DNA合成,抑制细胞向G2/M期转化。第三部分PKD1在NSCLC中表达、功能及作用机制研究[研究目的]1.明确PKD1在NSCLC中瘤组织中表达水平及其与临床病理因素的关系；2.探讨PKD1对mTOR-S6K1信号通路的调控作用,明确PKD1调控mTOR-S6K1信号的机制；3.探讨PKD1下调对NSCLC细胞增殖的作用。[研究方法]1.收集34例NSCLC手术病人肿瘤组织及癌旁正常组织,并于-80℃保存。提取组织RNA,荧光定量PCR (qRT-PCR)分析肿瘤组织和正常组织中PKD1表达量,并分析PKD1与病人临床病理因素间的关系。2.分别应用不同浓度PKD1抑制剂Kb NB142-70和PKD1特异性siRNA处理或转染NSCLC A549和H520细胞,western blot免疫蛋白印迹检测细胞在佛波酯刺激后pS6K1,pS6,pERK,pAkt的表达量,分析PKD1抑制对pS6K1表达量的作用。3.分别应用P13K抑制剂-LY294002,BKM120和MEK抑制剂-U0126,PD0325901,观察上述抑制剂对Kb NB142-70激活的mTOR-S6K1信号通路的影响。4.转染PKD1特异性siRNA,比较转染细胞和对照组A549和H520细胞在佛波酯刺激下细胞增殖情况的差异。[结果]1.在34例NSCLC患者中有26例患者肿瘤组织中PKD1表达量显著低于癌旁正常组织(P<0.05),其中,低表达的PKD1与NSCLC血管侵袭和淋巴结转移呈显著相关(P<0.05)。2.PKD1抑制剂或特异性siRNA能激活S6K1(表现为pS6Kl Ser235/236表达升高),同时伴随细胞内pERK和pAkt表达量升高。3.PI3K和MEK/ERK抑制剂能有效阻断Kb NB142-70激活的S6K1和S6。4.PKD1siRNA转染组的NSCLC细胞增殖较对照组明显增加。[结论]1.PKD1在NSCLC肿瘤组织中显著低表达,且与肺癌的疾病进展相关。2.在NSCLC细胞受到佛波酯PMA刺激时,PKD1能负向调控mTOR-S6K1信号通路的活性。3.PKD1调控mTOR-S6K1的机制是PKD1通过抑制pAkt和pEK的表达近进而影响其下游mTOR信号通路。4.特异性敲除PKD1增强细胞增殖能力,PKD1具有抑制细胞增殖的作用。PKD1表达的缺失可能在NSCLC的发生发展中发挥重要作用。PKD1有望成为NSCLC治疗的新靶点。更多还原

【Abstract】 Malignant tumor is a serious threat to human health and disease. As the Number one cause of all deaths in China, it accounts for25%of all deaths. Globally, the morbidity of lung cancer is increasing rapidly and remains the leading cause of cancer death. In the past30years, the mortality for lung cancer increased for465%. It was reported that the morbidity of lung cancer is increasing26.9%every year. Smoking, air pollution is strongly associated with lung cancer. Lung cancer comprises non-small cell lung cancer(NSCLC) and small cell lung cancer (SCLC), in which NSCLC accounts for85%of all cases and is identified as the most commom and fatal lung cancer. Despite the development of early diagnosis menthods, around70%of patients therefore present with advanced stage IIIB or IV disease. Surgery, radical radiotherapy and chemotherapy is the major treatment method but5-year cure rates have only barely improved. With these problems in mind, it is timely that a new approach to treatment is emerging with targeted therapy. Mutations in the epidermal growth factor receptor (EGFR) have been identified in NSCLC, and overexpression of EGFR and its ligands has made it an attractive target for various antitumor strategies. EGFR inhibitors such as gefitinib and erotinib are gradually applied in advanced NSCLC patients. However, EGFR-TKI inhibitors represent better efficacy for specific population, and the tolerance of these drugs urgently need to be resolved. Therefore, further understanding of the related cancer cell signaling pathway and exploring novel therapy target is necessary.Protein kinase D (PKD) is a novel serine-threonine protein kinase family within the CAMK group. PKD is activated by a number of different agents, including GPCR agonists, growth factors and phorbol esters. The PKD family contains3members that are homologous in structure and function, namely, PKD1, PKD2,and PKD3. PKD1is the founding and the most studied member of the family. The PKD1gene, located on human chromosome14q11, is broadly expressed in many organs, including the thyroid, brain, heart, and lungs. PKD1has been shown to play important roles in a variety of cellular functions that regulate intracellular signal transduction pathways, cell survival, proliferation, motility, invasion, angiogenesis, and apoptosis. PKD1also plays a critical role in cardiac cell functioning and maintenance of cardiovascular health. Thus, the deregulation of PKD1has been connected with the development of cancers, cardiovascular hypertrophy and other diseases. Recently, PKD1has been shown to be downregulated in prostate cancer, breast cancer, gastric cancer and colon cancer. However, the overexpression of PKD1has been shown to play a role in the development of pancreatic cancer and skin cancers. Because PKD1functions as a critical kinase that integrates extracellular signals into intracellular processes by modulating a multitude of signaling pathways, the regulation of PKD1levels and/or activity through pharmacological or genetic intervention might aid in cancer treatment.PKD1modulates a variaty of cancer-related signaling pathways and therefore regulates multiple biological functions which are critical for the functions of the cancer cells. PKD1mediates the activation of MAPK, JNK and NFκB signaling pathways in response to GPCR agonists. However, the exact functions of PKD1in the signaling pathways regulation is still unknown. In the present study, we aim to explore the regulation of PI3K/Akt and HDAC mediated by PKD1. Also, we examine the expression of PKD1in NSCLC tissues and explore the function and mechanism of PKD1in NSCLC. These results raise the possibility that tumor-specific delivery of the PKD1gene or PKD1activators may have potential therapeutic value in NSCLC patients. Objectives:1. To identify the activity of PI3K/Akt regualted by PKD1;2. To explore the phosphorylations of HDAC4,5,7mediated by PKD1and its impacts for cell cycle and cell proliferation;3. To examine the expression patterns and functions of PKD1in NSCLC;4. To explore the possible mechanism of PKD1functions in NSCLC.Section Ⅰ PKD1Mediates Negative Feedback of PI3K/Akt Activation in Response to G Protein-Coupled ReceptorsObjectives:1. To examine the impacts of PKD1inhibitors and siRNAs to PI3K/Akt;2. To explore the mechanism of PKD1regulating PI3K/Akt;3. To validate the regulation pathway in transgenic animal models.Methods:1. Stimulation of intestinal epithelial IEC-18cells with angiotensin Ⅱ (ANG Ⅱ), a mitogenic agonist that activates Gq-coupled receptors endogenously expressed by these cells. Cultures of IEC-18cells were treated with increasing concentrations of the selective PKD family inhibitor kb NB142-70for1h and then challenged with50nM ANG Ⅱ. Akt phosphorylation at Thr308and Ser473was examined by western blot.2. IEC-18cells were treated with another PKD family inhibitor CRT0066101or transfected with siRNAs. Akt phosphorylation at Thr308and Ser473was examined by western blot.3. Cultures of IEC-18cells were treated without or with kb NB142-70or CRT0066101, stimulated with ANG Ⅱ and lysed. The p85aregulatory subunit of PI3K was immunoprecipitated from the lysates and the resulting immunoprecipitates were analyzed by immunoblotting with a motif-specific antibody that detects Ser/Thr phosphorylated by PKD family members. We then examined whether PKD1stimulates binding of p85ato PTEN in intestinal epithelial cells. 4. we used transgenic mice that express elevated PKD1protein in the small intestine epithelium. Total PKD1and pAkt was examined by western blot.Results:1. Exposure of IEC-18cells to the selective PKD family inhibitor kb NB142-70potentiates GPCR-induced Akt activation. The selective PKD family inhibitor CRT0066101and knockdown of PKD1potentiate GPCR-induced Akt phosphorylation at Thr308and Ser473.2. Inhibition of PKD1increases Akt translocation to the plasma membrane in response to GPCR agonists. Inhibitors of class Ⅰ A PI3K and EGFR prevent the potentiation of Akt induced by suppression of PKD1activity.3. ANG Ⅱ markedly increased the phosphorylation of p85adetected by a PKD motif-specific antibody and enhanced the association of p85awith PTEN.4. Transgenic mice overexpressing PKD1showed a reduced phosphorylation of Akt at Ser473in intestinal epithelial cells compared to wild type littermates.Conclusions:1. PKD1activation mediates feedback inhibition of PI3K/Akt signaling.2. PKD1-mediated phosphorylation of p85α mediates negative feedback of PIP3accumulation and Akt phosphorylation in GPCR-stimulated cells, at least in part, by enhancing the stimulatory association of p85a with PTEN. Section Ⅱ Protein Kinase D1mediates Class Ⅱa Histone Deacetylase Phosphorylation and Nuclear Extrusion:Role in Mitogenic SignalingObjectives:To examine whether class Ⅱa histone deacetylases (HDACs) play a role in mitogenic signaling mediated by protein kinase D1(PKD1) Methods:1. Cell lysates were analyzed by Western blotting using antibodies thatrecognize class Ⅱa HDACs4,57and9. We then used an antibody that detects the phosphorylated state of HDAC4at Ser246, HDAC5at Ser259and HDAC7at Ser155and a second antibody that recognizes the phosphorylated state HDAC4at Ser632, HDAC5at Ser498and HDAC7at Ser486in cells stimulated with GPCR agonists(ANGII).2. We used the recently identified preferential PKD family inhibitors kb NB142-70and CRT0066101which act as potent PKD1inhibitors in intact IEC-18cells. Cultures of IEC-18cells were treated with increasing concentrations of kb NB142-70or CRT0066101for1h and then stimulated with ANGⅡ.3. The effect of ANG Ⅱ on endogenous HDAC5nucleocytoplasmic shuttling was examined by immunofluorescence analysis. Cultures of IEC-18cells were transfected with epitope (FLAG)-tagged HDAC5or an identical construct in which Ser259and Ser498were mutated to non-phosphorylatable Ala.4. Cultures of IEC-18cells in serum-free medium were stimulated with ANG Ⅱ in the absence or presence ofincreasing concentrations of the specific class Ⅱa HDAC inhibitor MC1568and DNA synthesis was assessed by measuring [3H]thymidine incorporation into acid-precipitable material.5. We then used transgenic mice that express elevated PKD1protein in the small intestine epithelium and display a marked increase in DNAsynthesizing cells in their intestinal crypts and a significant increase in the length and total number of cells per crypt.Results:1. Class Ⅱa HDAC4,5and7are prominently expressed in these cells. Simulation with angiotensin Ⅱ (ANG Ⅱ), a potent mitogen for IEC-18cells, induced a striking increase in the phosphorylation of HDAC4at Ser246and Ser632, HDAC5at Ser259and Ser498and HDAC7at Ser155.2. Treatment with the PKD family inhibitors kb NB142-70and CRT0066101or siRNA-mediated knockdown of PKD1prevented ANG Ⅱ-induced phosphorylation of HDAC4,5and7.3. PKD1-mediated phosphorylation of HDAC5induces its nuclear extrusion into the cytoplasm. In contrast, HDAC5with Ser259and Ser498mutated to Ala was localized to the nucleus in both unstimulated and stimulated cells.4. Treatment of IEC-18cells with specific inhibitors of class ⅡaHDACs, including MC1568and TMP269, prevented cell cycle progression, DNA synthesis and proliferation induced in response to GPCR/PKD1activation.5. The PKD1/class Ⅱa HDAC axis also functions in intestinal epithelial cell in vivo.Conclusions:Our results reveal a PKD1/classIIa HDAC axis in intestinal epithelial cells leading to mitogenic signaling. Section Ⅲ PKD1is downregulated in non-small cell lung cancer and mediates the feedback inhibition of mTORCl-S6K1axis in responseto phorbol esterObjectives:1. To determine the expression patterns and the role of PKD1in NSCLC;2. To elucidate the regulation of the mTORC1activity by PKD1.Methods:1. Thirty-four pairs of human NSCLC and matched normal bronchiolar epitheliums were enrolled and evaluated for PKD1expression by quantitative real-time PCR.2. Exposure of NSCLC A549and H520cells to the PKD family inhibitor kb NB 142-70(Kb), S6K1phosphorylation at Thr389and S6phosphorylation at Ser235/236was determined by western blot.3. We then used the PI3K inhibitors LY294002, BKM120and MEK inhibitors U0126, PD0325901to block the enhanced S6K1activity induced by the PKDl inhibition by Kb.Results:1. PKD1was downregulated in26of34cancer tissues in comparison with matched normal epitheliums. Moreover, patients with venous invasion or lymph node metastasis showed significant lower expression of PKD1.2. Exposure of NSCLC A549and H520cells to the PKD family inhibitor kb NB142-70(Kb), at concentrations that inhibited PKD1activation, strikingly potentiated S6K1phosphorylation at Thr389and S6phosphorylation at Ser235/236in response to phorbol ester (PMA).3. Knockdown of PKD1with siRNA also strikingly enhanced S6K1phosphorylation in response to PMA stimulation.4. exposure of cells to either Kb/PI3K inhibitor or Kb/MEK inhibitor strikingly inhibits S6K1and S6phosphorylation in response to PMA.Conclusions:Our results identify decreased expression of the PKD1as a marker for NSCLC and the loss of PKD1expression increases the malignant potential of NSCLC cells. This may be due to the function of PKD1as a negative regulator of mTORCl-S6K1. Re-expression or activation of PKDl might serve as a potential therapeutic target for NSCLC treatment.更多还原