ACC在肿瘤代谢中的功能及其机制研究

发布时间：2018-05-03 03:08

本文选题：肿瘤代谢 + 腺苷酸激活蛋白激酶　；参考：《天津医科大学》2016年博士论文

【摘要】：背景腺苷酸激活蛋白激酶(AMP-activated protein kinase,AMPK)与乙酰辅酶A羧化酶(Acetyl-Co A Carboxylase,ACC)作为其各自代谢调节过程中的关键环节,相互之间存在密切联系,形成细胞内的上游和下游的信号通路。AMPK是机体和细胞能量代谢的调节器,当细胞内ATP水平下降时,细胞处于能量应激状态下,AMPK激活,为细胞提供能量[1]。ACC是存在于胞浆中含有生物素的变构羧化酶,是脂肪酸代谢过程中的限速酶,其使乙酰辅酶A羧化成丙二酰辅酶A。在人类ACC有两种亚型,ACC1和ACC2。近年来发现肿瘤代谢在肿瘤的发生、侵袭、转移和耐药过程中起着重要的作用[2],而AMPK和ACC在肿瘤代谢中发挥着核心作用。在我们的前期研究中发现,一种表皮生长因子受体阻断抗体,西妥昔单抗可以抑制肿瘤代谢并且可以在肿瘤细胞中逆转Warburg效应(肿瘤有氧糖酵解)[3]。我们同样发现AMPK的短暂激活在应用西妥昔单抗治疗肿瘤细胞的早期是一项重要指标,但长期且持续激活的AMPK水平是肿瘤耐药的一种机制[4]。与西妥昔单抗治疗前的头颈鳞癌比较,治疗后组织及细胞高表达磷酸化AMPK、磷酸化ACC及ACC。我们设计本实验,进一步明确ACC在头颈鳞癌组织及细胞中的表达方式;从临床水平、细胞水平及动物实验水平探索ACC在肿瘤代谢过程中功能及其分子调控机制。方法1.应用分子克隆方法构建乏氧诱导因子-1α(Hypoxia inducible factor-1α,HIF-1α)与乙酰辅酶A羧化酶(Acetyl-Co A Carboxylase,ACC)过表达质粒,构建慢病毒稳定转染系统,建立稳定过表达HIF-1α的HEK293_HIF-1α细胞系;用Western blot,葡萄糖消耗实验(Glucose consumption assay)检测所构建稳系HEK293_HIF-1α的表达水平及其对于葡萄糖的消耗水平。2.应用分子克隆方法构建乙酰辅酶A羧化酶(Acetyl-Co A Carboxylase,ACC),ACC1_S79A及ACC2_S212A过表达质粒,构建慢病毒稳定转染系统,建立稳定过表达的ACC1_S79A和ACC2_S212A的HEK293_HIF-1α细胞系;用Western blot,细胞生存死亡实验(Live/dead cell viability assay)检测AMPK、ACC及其磷酸化蛋白的表达水平,检测在能量应激状况下(低糖)不同稳转细胞的存活率。3.应用Western blot、MTT检测不同头颈鳞癌细胞系代谢相关指标及对西妥昔单抗敏感性。应用RT-RCR检测不同细胞系ACC1和ACC2m RNA水平。应用放线菌酮(Cycloheximide,CHX)抑制蛋白合成,检测ACC降解水平。4.应用ACC1_S79A及ACC2_S212A过表达质粒及ACC1、ACC2小干扰RNA(si RNA)转染头颈鳞癌细胞系;用Western blot、细胞生存死亡实验(Live/dead cell viability assay)、细胞增殖实验(Cell proliferation assay)、凋亡实验(Apoptosis assay)检测肿瘤细胞经过西妥昔单抗治疗前后AMPK、ACC及其磷酸化蛋白的表达水平及细胞存活率,细胞凋亡状况。5.应用ACC及脂肪酸合成抑制剂5-十四烷氧基-2-呋喃甲酸(5-Tetradecyloxy-2-furoic acid,TOFA)抑制ACC,通过Western blot、流式细胞技术、细胞增殖实验(MTT)、凋亡实验(Apoptosis assay)检测TOFA对西妥昔单抗耐药头颈鳞癌细胞系的作用。6.构建裸鼠(Nude Mouse)皮下移植瘤模型,应用TOFA,检测肿瘤的增殖情况,并通过荧光成像(Luciferase image)和免疫组织化学的方法,探讨磷酸化AMPK、磷酸化ACC及ACC的表达与西妥昔单抗治疗的相关性。7.搜集头颈鳞癌组织石蜡标本资料进行回顾性研究,AMPK-T172p,ACC-S79p和ACC的表达表达水平与西妥昔单抗治疗的相关性。结果1.转染HIF-1α使HEK293细胞肿瘤化特征。予以正常胚胎肾细胞HEK293细胞系过表达HIF-1α_P402A/P564A和HIF-1α_(35)ODD后,发现转染HIF-1α的HEK293细胞对于葡萄糖的消耗明显增加(由于Warberg效应),同时伴随AMPK的激活(表现为AMPK T172磷酸化的增加)和ACC的抑制(表现为ACC S79磷酸化的增加)。转染HIF-1α的HEK293细胞对于低糖环境更敏感。2.能量应激(低糖)状况下ACC表现出对细胞重要的保护功能。过表达的ACC1_S79A和ACC2_S212A对于AMPK的激活没有作用,对于低糖所导致的内源性的ACC的抑制没有作用,这表明ACC可以保护低糖环境中的细胞存活是不由AMPK的激活所介导的。而且,敲除内源性ACC1和ACC2,特别是ACC1导致了在低糖环境下更多的HEK293和HEK293 HIF-1α_P402A/P564A细胞的死亡。在HEK293 HIF-1α_P402A/P564A细胞中敲除ACC1,16小时后约80%细胞死亡。3.磷酸化AMPK和磷酸化ACC的表达水平与头颈鳞癌细胞对西妥昔单抗的耐药相关。我们的前期研究发现西妥昔单抗可通过下调HIF-1α和抑制HIF-1α调节的糖酵解来激活AMPK并且抑制HNSCC细胞的增殖。进一步发现高表达的磷酸化AMPK T172和磷酸化ACC S79与头颈鳞癌细胞系对西妥昔单抗的敏感性呈负相关。这个结果同样在获得性耐药西妥昔单抗的两对等基因细胞中HN5/HN5-R和Fa Du/Fa Du-R中得到证实。进一步发现稳转HN5HIF-1α_P402A/P564A细胞激活AMPK抑制ACC,对西妥昔单抗耐药。4.ACC的表达水平与西妥昔单抗耐药相关。RT-PCR的结果显示增加的ACC不是由于转录水平的增加引起的,经过西妥昔单抗治疗后,HN5细胞中SREBP-1c下降明显,而HN5-R细胞中SREBP-1c无明显变化,相似的结果在另外两种耐药细胞UMSCC1和MDA1986中得到证实。ACC在HN5-R中明显比在HN5中更加稳定,代谢减慢,预示着一种转录后机制是ACC升高的原因。应用si RNA技术在HN5中敲除内源性ACC1和ACC2并没有影响西妥昔单抗治疗后凋亡的水平,而在两种耐药细胞HN5-R和UMSCC1中,可以看到明显的凋亡增加。5.西妥昔单抗耐药细胞对于TOFA更敏感,包括获得性耐药的HN5-R,天然耐药的UMSCC1和MDA1986。而且长期慢性暴露于TOFA治疗的UMSCC1-TOFA耐药细胞逆转为对西妥昔单抗敏感。TOFA与西妥昔单抗和用能明显的引起耐药细胞HN5-R、UMSCC1和MDA1986的凋亡,联合用药效果明显。6.体内动物实验证实联合用药对于西妥昔单抗耐药细胞有明显的效果。我们通过测量肿瘤的大小和IVIS肿瘤成像测量。应用TOFA治疗没有使裸鼠体重下降。免疫组化证实经西妥昔单抗治疗肿瘤组织磷酸化AMPK T172、磷酸化ACC S79和ACC与对照未治疗组增加,联合用药组免疫组化结果与体外细胞实验相符,应用UMSCC1细胞同样得到证实。7.我们收集了18例头颈鳞癌患者,其中6例经过化疗+西妥昔单抗治疗后手术,剩余18例为对照化疗后手术。其中第六例患者为化疗后手术,复发后给予化疗+西妥昔单抗治疗后再次手术。以第六例患者为例,可以明显发现经过西妥昔单抗治疗后,磷酸化AMPK T172、磷酸化ACC S79和ACC全部高表达。结论1.肿瘤处于应激状态下,ACC发挥着不可或缺的双重作用使肿瘤细胞得以存活、生长和增殖。2.ACC在肿瘤细胞由依赖于糖酵解转化为依赖于脂肪酸氧化的肿瘤代谢重新调整过程中发挥重要作用。3.对于西妥昔单抗耐药肿瘤,以抗ACC为靶点,我们可以发现合理的治疗手段,也可能增加其他以抗肿瘤Warberg效应为靶点的治疗效果。
[Abstract]:Background adenylate activated protein kinase (AMP-activated protein kinase, AMPK) and acetyl coenzyme A carboxylase (Acetyl-Co A Carboxylase, ACC) are the key links in their respective metabolic regulation processes. There is a close relationship between each other, and the formation of signal pathways in the upstream and downstream of the cell is the regulation of the body and cell energy metabolism. When the ATP level in the cell falls, the cell is in the state of energy stress, AMPK activates, and the energy [1].ACC is an allosteric carboxytransferase containing biotin in the cytoplasm. It is a speed limiting enzyme in the metabolic process of fatty acid. It makes acetyl coenzyme A carboxylation to propane two acyl coenzyme A. in two subtypes of human ACC, ACC1 and ACC2. in recent years. It is found that tumor metabolism plays an important role in the process of tumor occurrence, invasion, metastasis and drug resistance, and AMPK and ACC play a core role in tumor metabolism. In our previous study, a kind of epidermal growth factor receptor blocking antibody, cetuximab can inhibit tumor metabolism and can reverse W in tumor cells. The Arburg effect (tumor aerobic glycolysis) [3]. we also found that the transient activation of AMPK is an important indicator in the early use of cetuximab in the treatment of tumor cells, but the long-term and continuously activated AMPK level is a mechanism of tumor resistance, [4]. and cetuximab before the treatment of head and neck squamous cell carcinoma, after treatment, tissue and cell high surface AMPK, phosphorylated ACC and ACC., we designed this experiment to further clarify the expression of ACC in the tissues and cells of head and neck squamous cell carcinoma, and explore the function of ACC in the process of tumor metabolism and its molecular regulation mechanism from clinical level, cell level and animal experimental level. Method 1. the use of molecular cloning method to construct hypoxia inducible factor -1 alpha. Hypoxia inducible factor-1 alpha, HIF-1 alpha) and acetyl coenzyme A carboxylase (Acetyl-Co A Carboxylase, ACC) overexpressed plasmids, constructed the lentivirus stable transfection system, and established a HEK293_HIF-1 alpha cell line for stable overexpression of HIF-1 a. Expression level and glucose consumption level.2. application molecular cloning method to construct acetyl coenzyme A carboxylase (Acetyl-Co A Carboxylase, ACC), ACC1_S79A and ACC2_S212A overexpressed plasmids, construct lentivirus stable transfection system, establish a stable overexpressed ACC1_S79A and ACC2_S212A HEK293_HIF-1 alpha cell line. Cell survival and death test (Live/dead cell viability assay) test the expression level of AMPK, ACC and phosphorylated protein, detect the survival rate of different stable cells under energy stress (low sugar).3. application Western blot, MTT detection of different head and neck squamous cell carcinoma cell lines for Xie Xiang index and sensitivity to cetuximab. Use RT-RCR detection ACC1 and ACC2m RNA levels in different cell lines. Using Cycloheximide (CHX) inhibition protein synthesis, ACC degradation level.4. application ACC1_S79A and ACC2_S212A overexpressed plasmids and ACC1, ACC2 small interference RNA to transfect the head and neck squamous cell carcinoma cell lines. The cell proliferation test (Cell proliferation assay), apoptosis experiment (Apoptosis assay) was used to detect the expression level of AMPK, ACC and phosphorylated protein and cell survival rate before and after treatment with cetuximab, and the apoptosis status.5. applied ACC and fatty acid synthesis inhibitor 5- fourteen alkoxy -2- furanoic acid (5-Tetradecyloxy-2-furoic) (5-Tetradecyloxy-2-furoic) Acid, TOFA) inhibition of ACC, through Western blot, flow cytometry, cell proliferation test (MTT), apoptosis experiment (Apoptosis assay) detection of the effect of TOFA on cimiximab resistant head and neck squamous cell carcinoma cell line,.6. to construct nude mice (Nude Mouse) subcutaneous transplantation tumor model. Age) and immunohistochemical method, the relationship between the expression of phosphorylated AMPK, phosphorylated ACC and ACC and the treatment of cetuximab,.7. collection of paraffin specimens from the squamous cell carcinoma of the head and neck, the correlation between the expression level of AMPK-T172p, ACC-S79p and ACC and the correlativity of cetuximab treatment. Results 1. transfection of HIF-1 alpha to HEK293 The characteristics of cell oncology. After the expression of HIF-1 alpha _P402A/P564A and HIF-1 alpha (35) ODD in the normal embryonic renal cell line, it was found that the consumption of HEK293 cells transfected with HIF-1 a was significantly increased (due to Warberg effect), accompanied by the activation of AMPK (the increase of AMPK T172 phosphorylation) and the inhibition of ACC. The increase of 79 phosphorylation. HEK293 cells transfected with HIF-1 a are more sensitive to.2. energy stress (low sugar) in the low sugar environment. ACC shows an important protective function to the cells. The overexpressed ACC1_S79A and ACC2_S212A have no effect on the activation of AMPK, and the inhibition of the inhibition of endogenous ACC caused by low sugar shows that ACC can be found. The survival of cells in the protected low sugar environment was not mediated by the activation of AMPK. Moreover, the knockout of endogenous ACC1 and ACC2, especially ACC1, resulted in more HEK293 and HEK293 HIF-1 alpha _P402A/P564A cells in the low sugar environment. About 80% cell deaths in HEK293 HIF-1 alpha _P402A/P564A cells were phosphorylated. And the expression level of phosphorylated ACC is associated with the resistance of the head and neck squamous cell carcinoma cells to cetuximab. Our previous study found that cetuximab can activate AMPK and inhibit the proliferation of HNSCC cells by lowering the glycolysis of HIF-1 A and inhibiting HIF-1 a. The high expression of phosphorylated AMPK T172 and phosphorylated ACC S79 and head are also found. The sensitivity of cervical squamous cell carcinoma cell line to cetuximab is negatively correlated. This result is also confirmed in HN5/HN5-R and Fa Du/Fa Du-R in the two peer-to-peer gene cells of acquired resistant cetuximab. It is further found that stable HN5HIF-1 alpha _P402A/P564A cells activate AMPK to inhibit ACC and the expression level of resistant.4.ACC to cetuximab. The results of cetuximab resistance related.RT-PCR showed that the increase of ACC was not caused by the increase of transcriptional level. After cetuximab treatment, SREBP-1c decreased significantly in HN5 cells, while SREBP-1c in HN5-R cells did not change significantly. The similar results were confirmed in the other two resistant cells, UMSCC1 and MDA1986, in HN5-R. It is obviously more stable and slower than in HN5, indicating that a post transcriptional mechanism is the cause of the increase of ACC. The use of Si RNA technique to knock out endogenous ACC1 and ACC2 in HN5 does not affect the level of apoptosis after cetuximab, and a significant increase in apoptosis in two resistant cells, HN5-R and UMSCC1, can be seen in.5. cetuximab. Drug-resistant cells are more sensitive to TOFA, including acquired resistance to HN5-R, naturally resistant UMSCC1 and MDA1986., and the long-term chronic exposure to TOFA for UMSCC1-TOFA resistant cells reverses the apoptosis of the sensitive.TOFA and cetuximab against cetuximab and the drug resistant cells HN5-R, UMSCC1 and MDA1986, combined with the effect of drug use. .6. in vivo animal experiments confirmed that the combined use of combined drugs has a significant effect on cetuximab resistant cells. We measured tumor size and IVIS tumor imaging. TOFA treatment did not reduce the weight loss of nude mice. Immunohistochemistry proved that cetuximab was used to treat tumor tissue phosphorylated AMPK T172, phosphorylated ACC S79 and ACC. Compared with the untreated group, the results of the immunization of the combined drug group were consistent with the in vitro cell test. The UMSCC1 cells were also confirmed by the UMSCC1 cells. We collected 6 cases of head and neck squamous cell carcinoma, of which 6 cases were treated after chemotherapy plus cetuximab, and the remaining 18 cases were treated after chemotherapy. Of these, sixth patients underwent postoperative chemotherapy surgery. After treatment with chemotherapy plus cetuximab, reoperation was performed after treatment. In sixth patients, it was found that after cetuximab treatment, phosphorylated AMPK T172, phosphorylated ACC S79 and ACC were all highly expressed. Conclusion the 1. tumor is in stress state, and ACC plays an indispensable double role in the survival, growth and growth of the tumor cells. Proliferating.2.ACC plays an important role in the process of tumor metabolism dependent on glycolysis to tumor metabolism dependent on fatty acid oxidation..3. is a target for resistant to cetuximab resistant tumor, with anti ACC as a target. We can find a reasonable therapeutic method and may also increase the therapeutic effect of his target on the anti tumor Warberg effect. Fruit.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.2

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