RACK1对肝癌化疗耐药的调控及其相互作用蛋白CLEC-2的功能研究

【作者】 阮元元；

【导师】 顾建新； 谢建辉；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 第一部分RACK1对肝癌细胞化疗耐药的调控及其机制研究肝癌是目前全世界范围内最常见的并且具有高度致死性的肿瘤之一,在恶性肿瘤导致的死亡中居第三位。虽然常规的普查和监护可以使得肝癌在早期可以得到诊断和治疗,但由于肝癌细胞具有高度增殖的特性,绝大多数肝癌患者都是在进展期甚至在晚期发现；而此时,多数肿瘤已不能被切除而患者必须接受姑息治疗。化疗作为一种常用的姑息疗法,对于肝癌患者缺乏很好的效果。因为肝癌普遍对常用的化疗药物具有内在的抵抗性,全身性或者局部动脉内给药并不能达到抑制肿瘤生长和延长患者生存的效果。目前,肝癌细胞内在化疗耐药的机制尚不清楚。我们研究发现,构架蛋白RACK1 (receptor for activated C-kinase 1)在正常肝脏中高度表达,并且在肝癌患者中普遍表达增高。RACK1的表达增高可以促进肝癌细胞株对阿霉素的化疗抵抗,而且这一过程是依赖于其核糖体定位的,因为非核糖体定位的RACK1的突变体甚至可以促进阿霉素诱导的肝癌细胞株的凋亡。进一步研究表明,核糖体定位的RACK1可以促进肝癌细胞总体蛋白翻译水平的增加,其机制可能是通过作用于真核细胞翻译起始因子eIF4E来实现的。核糖体定位的RACK1可以与eIF4E相互作用,并且通过招募PKCβⅡ来促进eIF4E的209位丝氨酸的磷酸化并增强eIF4E的活性。随着eIF4E活性的增强,可以选择性的促进很多与细胞增殖和生存相关蛋白的翻译,包括cyclin D1、c-myc、surviving和Bcl-2。在肝癌患者的标本中,我们也发现RACK1的表达水平与cyclin D1、c-myc、surviving和Bcl-2的表达呈正相关。使用翻译的抑制剂CHX,或者抑制eIF4E的表达或者其活性,则可以抑制野生型RACK1介导的化疗抵抗。同时我们还发现,在化疗药物处理的情况下,过表达野生型RACK1可以介导应激颗粒的形成,而过表达其非核糖体定位的突变体则没有影响。抑制RACK1的表达或者过表达非核糖体定位的突变体可以抑制G3BP介导的应激颗粒的形成,提示RACK1及其核糖体定位与应激颗粒的形成密切相关。另外,RACK1的表达增高可以促进肝癌细胞中AKT和ERK的活化,并引起肝癌细胞的增殖。综上所述,我们第一次对RACK1在肝癌中的功能作了系统的研究,并为解释肝癌细胞内在的化疗抵抗的特性奠定了一定的基础。第二部分RACK1对C型凝集素样受体CLEC-2表达调控的研究CLEC-2是通过使用含有免疫学功能的C型凝集素结构域筛选基因组数据库时被鉴定的。人的CLEC-2 (hCLEC-2)是一个Ⅱ型跨膜的C型凝集素样受体,并且可以被N-糖基化修饰。其中胞外段含有单个的C型凝集素样结构域和一个颈部区,胞浆尾部含有非经典的免疫受体酪氨酸活化基序(D-x-Y-x-x-L)。最近研究表明,CLEC-2能够识别外源性配体蛇毒Rhodocytin和内源性配体Podoplanin,导致D-x-Y-x-x-L基序中的酪氨酸磷酸化,招募Syk酪氨酸激酶并激活血小板。另外,它还能够协同DC-SIGN促进血小板捕获HIV-1。鼠的CLEC-2 (mCLEC-2)与hCLEC-2具有高度的同源性,我们研究发现mCLEC-2有两个剪切异构体,同时mCLEC-2的全长形式可以形成同源二聚体并且其胞外段可以被剪切成可溶性形式。mCLEC-2在中性粒细胞表面也有表达,并且发挥着介导内化和活化中性粒细胞的功能。虽然目前对CLEC-2的功能已有一定的阐明,但是除Syk外与CLEC-2胞浆段相互作用的分子并没有报道。我们以hCLEC-2的胞浆段作为诱饵,利用Ga14酵母双杂交系统筛选人的白血病cDNA文库。经过三轮营养缺陷筛选,我们鉴定了构架蛋白RACK1作为与hCLEC-2的一个相互作用蛋白。通过体外结合实验我们证实了RACK1与hCLEC-2胞浆段之间直接的相互作用,体内实验中也通过免疫共沉淀证实了RACK1与hCLEC-2的相互作用,免疫荧光共聚焦分析也揭示了它们在细胞浆中存在共定位。进一步实验表明,RACK1可以通过降低hCLEC-2的蛋白稳定性来抑制hCLEC-2的表达,包括其未糖基化和糖基化两种修饰形式。体内泛素化实验表明,RACK1可以通过增强hCLEC-2的泛素化来促进其通过蛋白酶体途径降解；使用蛋白酶体抑制剂MG132和lactacystin可以逆转RACK1对hCLEC-2的表达抑制,而使用溶酶体抑制剂chloroquine则没有影响。虽然RACK1可以抑制细胞内总的糖基化CLEC-2的表达水平,但是却不影响细胞表面CLEC-2的表达及其所介导的细胞内总酪氨酸的磷酸化。这也提示了RACK1可能参与了糖蛋白CLEC-2的内质网折叠和蛋白质质量控制的过程,也为研究内源性CLEC-2的蛋白水平的调控奠定了基础。第三部分CLEC-2识别CD74在B-CLL白血病细胞中的功能研究人的CLEC-2 (hCLEC-2)是一种Ⅱ型跨膜的C型凝集素样受体,在肝脏、血小板、自然杀伤细胞和抗原提呈细胞中都有表达。然而,目前仅在血小板和巨核细胞表面检测到其分布。人血小板表面的CLEC-2有32kD和40kD两种修饰形式,其中32kD占主要形式。最近研究表明,CLEC-2能够识别外源性配体蛇毒Rhodocytin和内源性配体Podoplanin,导致D-x-Y-x-x-L基序中的酪氨酸磷酸化,招募Syk酪氨酸激酶从而激活血小板。另外,它还能够协同DC-SIGN促进血小板捕获HIV-1。为了进一步研究CLEC-2的生物学功能,我们以hCLEC-2的胞外段作为诱饵,利用Gal4酵母双杂交系统筛选人的白血病cDNA文库。经过三轮营养缺陷筛选,我们鉴定了CD74作为与hCLEC-2的一个相互作用蛋白。CD74又称为MHCⅡ类分子恒定链,在抗原提呈过程中发挥着重要功能。近些年研究表达,部分CD74分子可以定位到细胞表面,作为一个受体分子发挥信号传导功能。尤其是CD74在B淋巴细胞白血病(B-CLL)细胞表面高表达,可以促进白血病细胞的增殖。我们表达并纯化了CD74的胞外段与IgG1的Fc段的融合蛋白(Fc-CD74),体外结合实验证实Fc-CD74重组蛋白能够选择性地与40kD修饰形式的hCLEC-2相互作用,而不能与32kD修饰形式的hCLEC-2结合。通过生物素标记实验证实了32kD和40kD两种修饰的CLEC-2在细胞表面的分布,荧光共聚焦分析也揭示了Fc-CD74融合蛋白与hCLEC-2在细胞表面存在共定位。我们在CHO细胞中转染了hCLEC-2的真核表达载体,并且将其与人B细胞淋巴瘤细胞株Raji共孵育。结果显示,过表达hCLEC-2可以促进CHO细胞与Raji细胞的粘附,同时也可以促进Raji细胞中ERK信号的活化；使用CD74的拮抗性抗体LN2则可以抑制CHO/hCLEC-2所介导的Raji细胞中ERK磷酸化的增强。细胞周期和CFSE标记实验表明,CHO/hCLEC-2细胞也以CD74依赖的方式促进Raji细胞进入S期和增殖。同样,人巨核细胞株Dami也可以促进Raji细胞的增殖,而这一过程可以被hCLEC-2或者CD74单克隆抗体抑制。这些数据揭示了40kD修饰的hCLEC-2可以识别CD74并促进其下游信号传导,并且提示了血小板可能通过hCLEC-2与CD74之间相互作用来促进B-CLL细胞的增殖,从而为B-CLL病人的治疗提供新的依据和潜在的靶点。更多还原

【Abstract】 PartⅠStudy on the role of RACK1 in the innate chemotherapy resistance of hepatocellular carcinomaHepatocellular carcinoma (HCC) is among the most common and lethal cancers in the human population, ranked the third most common cause of cancer-related death worldwide. Though routine surveillance can lead to early diagnosis and treatment when the tumor might be resectable, most HCC patients are diagnosed at advanced or late stages and could only receive palliative treatments, possibly due to the rapid progression of HCC. However, chemotherapy, serving as a common choice of palliative therapy, showed little benefit in the treatment of HCC patients. HCC generally displays inherent high resistance to chemotherapeutic drugs, and systemic or selective intra-arterial administration of any chemotherapy agent, which has marginal anti-tumor activity and shows no benefit for survival, is not recommended in clinical practice. At present, the underlying mechanism of the inherent high chemotherapy resistance of HCC remains unclear.In our study, we demonstrate that RACK1, the receptor for activated C-kinase 1, is highly expressed in normal liver and frequently up-regulated in HCC. Aberrant expression of RACK1 contributes to the chemotherapy resistance of HCC relying on its ribosome localization in vitro and in vivo, and the non-ribosome-binding mutant of RACK1 even sensitizes HCC cells to chemotherapy-induced apoptosis. Further study reveals that ribosome-associated RACK1 promotes the global protein synthesis, probably by acting on the eukaryotic initiation factor 4E (eIF4E). Ribosomal RACK1 directly associates with eIF4E in vitro and in vivo, and modulates the activity of eIF4E by recruiting PKCβⅡand promoting the phosphorylation of eIF4E on Ser 209. With the elevation of eIF4E activity, ribosomal RACK1 preferentially enhances translation of select mRNAs, many of which encode potent growth and survival factors, such as cyclin D1, c-myc, surviving and Bcl-2. This effect is also observed in vivo that the protein level of RACK1 positively correlates with the expression of cyclin D1, c-myc, surviving and Bcl-2. Translation suppression by CHX, or inhibiting the expression or activity of eIF4E, abolishes the anti-apoptotic effect of RACK1. We also observe that wild-type RACK1, but not its non-ribosome-binding mutant, promotes the formation of stress granules (SGs) upon the chemotherapeutic stress. Depletion of RACK1, or overexpression of the non-ribosome-binding mutant, even suppress the G3BP-induced SGs formation, suggesting that RACK1 and its ribosome localization are required for the assembly of SGs. Moreover, overexpression of RACK1 promotes the activation of AKT and ERK, and induces the proliferation of HCC cells. Our research first gain insight into the role of RACK1 in HCC, and provide clues to understanding the underlying mechanism of inherent chemotherapy resistance in HCC. PartⅡStudy on the role of RACK1 in the regulation of CLEC-2 expressionCLEC-2 was first identified as one member of non-classical C-type lectins by sequence similarity to C-type lectin-like molecules with immune functions. Human CLEC-2 is a typeⅡtransmembrane receptor with N-glycosylation, displaying a single extracellular C-type lectin-like domain (CTLD) connected to transmembrane region by a stalk and a non-classic immunoreceptor tyrosine-based activation motif (D-x-Y-x-x-L motif, ITAM) in its cytoplasmic tail. Recently, CLEC-2 has been demonstrated as a novel activating receptor that is likely to underlie platelet activation by the snake toxin Rhodocytin and endogenous ligand Podoplanin through the phosphorylation of ITAM and recruitment of Syk to initiate downstream signaling pathway. Additionally, CLEC-2 also co-operates with DC-SIGN to facilitate the capture of HIV-1 by platelets. Mouse CLEC-2 (mCLEC-2) shares high homology with human counterpart. We have reported two new alternatively spliced transcripts of mCLEC-2, the homologous dimerization of full-length mCLEC-2 and its cleavage into a soluble form. MCLEC-2 is also expressed on the surface of murine peripheral blood neutrophils, mediating internalization as well as the activation of neutrophils. So far, there is no report about the interacting partners with the cytoplasmic region of CLEC-2 except for Syk.In this study, by using the cytoplasmic region of human CLEC-2 (hCLEC-2) as bait, we perform a yeast two-hybrid screening in human leukemia cDNA library and identify the scaffold protein RACK1 as a potential interacting partner with hCLEC-2 in yeast. The direct interaction between RACK1 and hCLEC-2 is further identified by GST pull-down assay in vitro and co-immunoprecipation in vivo. Confocal analysis also reveals that RACK1 and hCLEC-2 co-localize in the cytoplasm of cells. Further research demonstrate that RACK1 decreases the stability of hCLEC-2 and inhibits the expression of both the unglycosylated and glycosylated forms of hCLEC-2. In vivo ubiquitination assay indicates that RACK1 promotes the proteosome-mediated degradation of hCLEC-2 by enhancing its ubiquitination. Proteosome inhibitors MG132 and lactacystin attenuate the degradation of CLEC-2 mediated by RACK1, while the treatment of choloquine, a lysosome inhibitor, shows little effect. Though RACK1 decreases the expression of glycosylated CLEC-2 in whole cell lysates, it does not impair the surface expression and signaling of CLEC-2. Taken together, these results suggest that RACK1 might be involved in the folding and protein quality control of CLEC-2, and provide clues to the understanding of the regulation of endogenous CLEC-2 expression. Part III Study on the recognition of CD74 by CLEC-2 and its functional effect on B-CLL leukemia cellsCLEC-2 is a type II transmembrane C-type lectin-like receptor, mainly distributed in liver, platelet, megakaryocyte, natural killer cells (NK cells) and antigen presentation cells (APC cells). However, surface expression CLEC-2 is only readily detected on platelet and megakaryocyte. In human platelet, CLEC-2 is detected as a doublet by western blot, with a major band migrating at 32kD and a minor band at 40kD. Recent studies demonstrate that CLEC-2 recognizes snake venom Rhodocytin as an exogenous ligand and Podoplanin as the endogenous ligand. In addition, CLEC-2 also co-operates with DC-SIGN to facilitate the capture of HIV-1 by platelets.In this study, to explore the potential endogenous ligand of CLEC-2 and gain insight into its biological function, we perform a yeast two-hybrid screening in human leukemia cDNA library by using the extracellular region of human CLEC-2 (hCLEC-2) as bait. We identify CD74 as a potential ligand for CLEC-2 in yeast. CD74, which is originally known as the MHC class II invariant chain, plays a critical role in the process of antigen presentation. Recent studies reveal that CD74 is also expressed on cell surface and functions as receptor to initiate downstream signaling. Importantly, CD74 is highly expressed on the surface of and mediated the proliferation of B chronic lymphocyte leukemia (B-CLL) cells. By expressing and purifying the recombinant protein of the extracellular part of CD74 fused to the Fc region of IgG1 (Fc-CD74), we identify in vitro that CD74 selectively binds to the 40kD form of hCLEC-2, but not its 32kD form. Confocal analysis also reveales that Fc-CD74 fusion protein co-localizes well with CLEC-2 on cell surface. We also transfect the hCLEC-2 construct into CHO cells (CHO/hCLEC-2), and co-culture them with Raji (B lymphoma cell line) cells. Results indicate that overexpression of CLEC-2 in CHO cells promotes the Raji adhesion, and enhances the activation of ERK in Raji cells. Blocking CD74 by using its antagonist antibody LN2 attenuates the ERK activation induced by CHO/hCLEC-2 cells. Cell cycle analysis and CFSE labeling assay demonstrate that co-culture with CHO/hCLEC-2 facilitates the S phase progression and proliferation of Raji cells in CD74-dependent manner. A megakaryocytic leukemia cell line, Dami, also promotes the proliferation of Raji cells through direct contact, while this effect could be blocked by using the CLEC-2 or CD74 monoclonal antibody. These results indicate that the 40kD form of CLEC-2 recognizes CD74 and promotes the initiation of its downstream signaling, and suggest that platelet may be involved in the progression and development of B-CLL by CLEC-2-CD74 interaction, thus providing CLEC-2 as a potential target for the treatment of B-CLL patients.更多还原