【作者】 孟辉；

【导师】 徐袛顺； 王晨光；

【作者基本信息】 山东大学 ， 泌尿外科， 2010， 博士

【摘要】 肿瘤是一种细胞周期性疾病,肿瘤的发生与细胞增殖失控、分化障碍及凋亡受阻密切相关,因此与细胞增殖有关的周期蛋白成为研究癌症发生的热点之一细胞周期的变化是一个复杂的过程,许多蛋白参与调控该过程。其中D型周期蛋白作用于G1期,分为3个亚型：Cyclin D1、Cyclin D2、Cyclin D3。但只有Cyclin D1被检测到在肿瘤中过度表达。Cyclin D1又存在Cyclin Dla和CyclinDlb两种亚型,通常Cyclin D1即指Cyclin D1a。正常的细胞周期有序的循环是通过激活和失活细胞周期蛋白依赖性激酶(CDK)来实现的。CyclinD1作为细胞周期进程的启动子,可以与CDK4结合形成复合体,通过磷酸化和失活视网膜母细胞瘤蛋白pRb,促进细胞周期从G1期到S期的进展,并通过这种方式介导DNA的合成、促进细胞的生长增殖。而Cyclin D1在不依赖CDK的情况下同样可以对细胞生长、分化和新陈代谢发挥作用。研究表明,Cyclin D1在人类多种肿瘤如乳腺癌、结肠癌、前列腺癌以及造血系统恶性肿瘤中存在过度表达。在侵袭性乳腺肿瘤中,50%病例存在Cyclin D1蛋白的过度表达。为了探讨Cyclin D1在肿瘤中的作用,我们做了大量研究。我们发现Cyclin D1缺失的小鼠会耐受致癌基因诱导的肿瘤发生；Cyclin D1基因的缺失也可以降低细胞存活和DNA合成,增加线粒体的大小和活性,抑制血管、骨髓巨噬细胞、成纤维细胞和乳腺上皮细胞的迁移能力。临床病例也显示CyclinD1过度表达与肿瘤转移存在联系。因此,疾病的恶性进展和转移可能与CyclinD1密切相关。细胞的迁移在细胞形态发生、组织修复和肿瘤转移中是必不可少的。肿瘤转移是从肿瘤原发病灶的肿瘤细胞的迁移开始的,经过进入血管的内渗和穿过血管壁的溢出过程,最后到达远处器官或组织从而产生新的肿瘤。抑制肿瘤转移是我们治疗肿瘤的重要途径。因此,了解细胞迁移或侵袭机制会帮助我们更好的理解肿瘤细胞的散布机制并为肿瘤治疗带来新的契机。探讨Cyclin D1在迁移或侵袭中的作用可以给我们的研究指明新的方向。大量研究发现Cyclin D1在调节雌激素受体α(ERα)、雄激素受体(AR)等的转录活性时,必须要结合辅助激活因子或转录因子。因此我们推测Cyclin D1在细胞生长、迁移及肿瘤中的作用是通过结合特殊的蛋白质形成复合物而产生的。为了研究Cyclin D1结合蛋白在调节Cyclin D1功能中的作用,我们纯化Cyclin D1结合蛋白复合物并发现了神经元蛋白激酶C酪蛋白激酶底物2(Protein kinase C and casein kinase substrate in neurons protein 2, PACSIN2)。PACSIN2是来源于鸡心脏的52KDa粘合蛋白(FAP52)的同源体,它的家族成员在功能上被看作是细胞黏着斑中的胞浆辅助蛋白,参与囊泡的形成与运输。本课题就Cyclin D与(?)PACSIN2在影响细胞迁移中的作用进行研究。一、发现并鉴定PACSIN是一种Cyclin D1结合蛋白,并探讨其结合机制目的：发现并鉴定出PACSIN2是一种Cyclin D1结合蛋白,探讨PACSIN2是否可以特异性的结合Cyclin D1而非其他家族成员及其结合机制。方法：1.通过免疫沉淀方法纯化Cyclin D1结合蛋白,并经过银染电泳凝胶、凝胶片切除及消化过程,最后进行质谱测定分析和序列分析。2.将带有Flag抗原标记的不同Cyclin D1质粒DNA,包括野生型和突变型DNA,分别和带有Myc抗原标记的(?)PACSIN2质粒DNA,使用磷酸钙转染的方法共转于293T细胞。通过免疫沉淀-免疫印迹的方法分析该细胞。3.将带有Flag抗原标记的不同Cyclin D质粒DNA,包括Cyclin D1,D2和D3及其突变型DNA,分别和带有Myc抗原标记的PACSIN2质粒DNA,使用磷酸钙转染的方法共转于293T细胞。通过免疫沉淀-免疫印迹的方法分析该细胞。4.将带有Myc抗原标记的不同(?)(?)ACSIN质粒DNA,包括PACSIN1,PACSIN2和(?)(?)ACSIN3 DNA分别和带有Flag抗原标记的Cyclin D1质粒DNA,使用磷酸钙转染的方法共转于293T细胞。通过免疫沉淀-免疫印迹的方法分析该细胞。5.将带有Myc抗原标记的不同PACSIN2质粒DNA,包括野生型和变异型DNA,分别和带有Flag抗原标记的Cyclin D1质粒DNA,使用磷酸钙转染的方法共转于293T细胞。通过免疫沉淀-免疫印迹的方法分析该细胞。结果：1.通过纯化Cyclin D1结合蛋白,得出CDK4(细胞周期蛋白依赖性激酶),70kD热休克同源蛋白(Hsc70)和一种额外的蛋白质。经过质谱测定分析和序列分析,鉴定出这种蛋白是PACSIN2。这种蛋白被看做是来源于鸡心脏的52KDa粘合蛋白(FAP52)的同源体。2.敲除细胞周期蛋白的酸性氨基酸序列后,Cyclin D1无法与PACSIN2结合。只有Cyclin D1和Cyclin D2可以与(?)(?)ACSIN2结合,但Cyclin D3、yclin D1b不能与其结合。3.免疫沉淀-免疫印迹实验发现PACSIN家族中只有PACSIN2可以和CyclinD1特异性的结合,PACSIN1和PACSIN3不能和Cyclin D1结合。4.通过敲除(?)pACSIN2蛋白序列上NPF结构域可以使得PACSIN2失去与Cyclin D1结合的能力。结论：1.发现并鉴定出PACSIN2是可以与Cyclin D1特异性结合的Cyclin D1结合蛋白。2. Cyclin D1通过其羟基末端的E-rich基序与PACSIN2结合。3. PACSIN2通过其NPF结构域与Cyclin D1结合。二. PACSIN2抑制正常细胞和肿瘤细胞的迁移能力,是通过与Cyclin D1a结合而非Cyclin D1b实现的；目的：观察Cyclin D1和(?)(?)ACSIN2在细胞中的蛋白结合共存位置,并确定作为CyclinDl结合蛋白的PACSIN2的生物学功能,以及其功能的发挥是否依赖于Cyclin D1。方法：1.在MRC5细胞(人胚胎肺成纤维细胞)中通过免疫组织化学染色方法观察Cyclin D1和(?)(?)ACSIN2蛋白结合共存位置2.通过Transwell migration assay分析PACSIN2基因敲除型和野生型小鼠皮肤成纤维细胞迁移能力的区别。3.通过特异性siRNA降低LNCaP细胞(人前列腺癌细胞)中PACSIN2蛋白表达水平后,Transwell migration assay分析其迁移能力的变化。4.通过特异性siRNA降低3T3细胞(小鼠胚胎成纤维细胞)中PACSIN2蛋白表达水平后,Cell spreading assay观察细胞伸展能力的变化。5.分别在Cyclin D1敲除的小鼠上皮成纤维细胞Cyclin D1-/-细胞,以及该细胞经过Cyclin D1a、Cyclin D1b质粒DNA转导后的两种细胞Cyclin D1-/-+D1a细胞和Cyclin D1-/-+D1b细胞中,使用特异性siRNA降低PACSIN2蛋白表达水平后,通过Transwell migration assay观察细胞迁移能力的变化,Cell spreading assay分析其伸展能力的变化。结果：1.MRC5细胞中,Cyclin D1和(?)(?)ACSIN2结合共存于细胞膜皱褶中。2. PACSIN2基因敲除后,小鼠皮肤成纤维细胞的迁移能力增强；(?)PACSIN2 siRNA可以增强人前列腺癌细胞LNCaP的迁移能力,降低小鼠胚胎成纤维细胞3T3细胞的伸展能力。3. PACSIN2 siRNA可以增强Cyclin D1-/-+D1a细胞的迁移能力,降低细胞的伸展能力；但是在Cyclin D1-/-细胞和Cyclin D1-/-+D1b两组细胞中没有变化。结论：1.内源性PACSIN2可以抑制人前列腺癌细胞和小鼠成纤维细胞的迁移能力,增强小鼠成纤维细胞的伸展能力。2.在Cyclin Dla存在的前提下,PACSIN2才能发挥其抑制细胞迁移、增强细胞伸展的能力。总之,作为Cyclin D1的结合蛋白,(?)ACSIN2通过其结构中NPF区域与CyclinD1a的羟基末端特异性结合而发挥其抑制细胞迁移、增强细胞伸展的能力。因此,研究Cyclin D1及其结合蛋白PACSIN2对细胞迁移的影响,为进一步探讨CyclinD1在肿瘤中的作用提供了一个新的方向。更多还原

【Abstract】 Tumor is regarded as a cellular periodic disease; Tumorigenesis is associated with deregulation of proliferation, dysdifferentiation and the inhibition of apoptosis. Cyclin D, as a member of the Cyclin family of cell cycle regulators, may play an important role in cancer. While there are three D-type Cyclins, Cyclin D1, Cyclin D2 Cyclin D3, only Cyclin Dl is observed to be overexpressed frequently in cancer. Cyclin D1 is best known as the regulatory subunit of a dimeric holoenzyme including the cell cycle-dependent kinase CDK4/6, which phosphorylates and inactivates the retinoblastoma protein Rb to promote progression through the G1-S phase of the cell cycle. And in this manner promote cellular proliferation.The orderly transition through the cell cycle is orchestrated by the sequential activation and inactivation of Cyclin-dependent kinases. The regulatory subunits of the CDK4/6 kinase are encoded by the D-type Cyclins. Cyclin D1 is induced early during G1 phase progression by mitogenic stimuli, which activate both the transcription of Cyclin D1, through specific DNA sequences in the Cyclin D1 promoter, and through the assembly of complexes that require sustained mitogenic stimulation. The Cyclin D1/CDK complexes phosphorylate and inactivate the retinoblastoma (pRb) protein, and the NRF-1 (Nuclear respiratory factor 1) protein to induce nuclear DNA synthesis and inhibit mitochondrial metabolism respectively.In addition to its CDK-binding function, a lot of evidence now show that D-type Cyclins have CDK-independent properties. As previously proposed, the role of these properties in cellular growth, metabolism, and cellular differentiation are substantial. Cyclin Dl forms physical associations with more than 30 transcription factors or transcriptional coregulators. Cyclin D1 directly interacts with and represses or induces several different nuclear receptors and transcription factors, including the estrogen receptor (ER), androgen receptor (AR), peroxisome proliferator activated receptor gamma (PPARy), CCAAT enhance binding proteinβ(C/EBPβ), and signal transducer and activator of transcription 3 (STAT3).Given its role in promoting cellular proliferation and modulating transcription, it is not surprising that Cyclin D1 is deregulated in cancer. Overexpression of the Cyclin D1 gene has been reported in a variety of human cancers including breast, colon, prostate, and hematopoietic malignancies. In invasive breast cancers, Cyclin D1 is overexpressed in up to 50% of cases. And in keeping with a role for Cyclin D1 in diverse human cancers, mice deficient in Cyclin D1 are resistant to oncogene-induced tumorigenesis. Also, Gastrointestinal tumors induced by mutation of the Apc gene are reduced in number by Cyclin D1 deficiency. Consistent with findings that Cyclin D1 antisense abrogates mammary epithelial cell growth induced by ErbB2, Cyclin D1-deficient mice are resistant to mammary gland tumorigenesis induced by Ras or ErbB2.Cyclin D1-deficient cells demonstrate reduced cellular survival and DNA synthesis increased mitochondrial size and activity and reduced cellular migration of diverse cell types including blood vessels, bone marrow macrophages, fibroblasts and mammary epithelial cells.Cell migration is essential for developmental morphogenesis, tissue repair, and tumor metastasis. Cancer cells spread from the initial site of tumor growth by first invading the surrounding tissue (migration), then entering the blood or lymph vessels (intravasation), and finally crossing the vessel wall to exit the vasculature (extravasation) in distal organs. The cancer cells then colonize the new site and proliferate to form a second tumor mass.Preventing metastasis presents an important therapeutic approach to cancer treatment. So as the first step of metastasis, learning more about the cellular different migration/invasion programmes will help us to understand how cancer cells disseminate and lead to new treatment strategies. By frequent overexpression of Cyclin D1 in cancer cells, new finding suggested that Cyclin D1 may have a central role in mediating invasion and metastasis of cancer cells by controlling Rho/ROCK signaling and matrix deposition of TSP-1. But how Cyclin D1 promotes cellular migration, we still do not know.A common polymorphism of the human Cyclin D1 gene has been associated with increased rate of cancer development. The polymorphism (A870G) is located at the splice donor region at the exon4-intron4 boundary and modulates the efficiency of alternate splicing. Alternate splicing results in distinct carboxyl terminal amino acid sequences. Characterization of the functional properties of the canonical Cyclin D1a and the alternate Cyclin D1b isoform has revealed each encodes subunits capable of phosphorylating pRb, but distinguishable abilities to regulate cellular migration. Cyclin D1a promotes migration of fibroblasts and mammary epithelial cells; however the Cyclin D1b is defective in promoting migration.It has been predicted the diverse functions regulated by Cyclin D1 in cellular differentiation, proliferation and migration may be governed by subpartitioning of Cyclin D1 into distinct subcellular compartments, or through physical association with distinct binding proteins. In this regard, Cyclin D1 associates with p160 coactivator SRC-1 to regulate estrogen receptor a activity. In association with transcription factors, Cyclin D1 regulates CDK-independent transcriptional activities of the androgen receptor, CEBPβand PPARγ.In view of the diverse functions of Cyclin D1 in DNA synthesis, oncogenesis, and migration, we hypothesized that Cyclin D1-associated proteins may mediate these functions by forming a complex with Cyclin D1.Thus, In order to determine adapter proteins regulating Cyclin D1 function, we immunopurified Cyclin D1-associated proteins by Mass spectrometry and sequence analysis and identified PACSIN2, original named chicken FAP52, which family members have been shown to function as cytoplasmic adapter proteins in focal adhesions, as Cyclin D1-associated proteins.In our study we will conduct the physical association between Cyclin D1 and PACSIN2, and the role of Cyclin Dl and PACSIN2 in the cellular migration.Part I PACSIN2 is Identified as a Cyclin D1-binding Protein and PACSIN2 can specifically Bind Cyclin D1 Through its DPF DomainObjective:To identify PACSIN2 as a Cyclin D1-binding protein, and investigate whether Cyclin D1 can specifically binding PACSIN2, rather than other family members, and the mechanism for this specific binding.Methods:1. Purification of Cyclin D1-associated proteins were analyzed by silver-stained gel, the associated proteins excised and in gel tryptic digest in analyzed by mass spectrometry2. Using IP-Westernblot to conduct the cell co-transfected with expression vector for Myc epitope-tagged PACSIN2 and Flag-tagged Cyclin Dl wild type or mutant expression vector.3. Using IP-Westernblot to conduct the cell co-transfected with PACSIN2 association with D-type Cyclin D1, including D1 Wild type or Mutant4. Using IP-Westernblot to conduct the cell co-transfected with Cyclin D1 association with PACSIN family, including PACSIN1,2,35. Identify the domain of PACSIN2 required for physical interaction with Cyclin D1, by using IP-Westernblot to conduct the cell co-transfected with C-terminus truncated mutant constructs of PACSIN2 together with Vector encoding flag-Cyclin D1.Results:1. Co-purifing proteins, include CDK4, Hsc70 and an additional protein was identified by mass spectrometry with sequence identified to the homolog of chicken52, now identified as PACSIN22. Delection of the acidic rich stretch of Cyclin D1 abrogated the binding to PACSIN2. Cyclin Dl and Cyclin D2, but not Cyclin D3 and Cyclin Dlb, co-precipitated with PACSIN2. 3. Immmunoprecipitation of Cyclin Dl was found to co-precipitated PACS1N2, but not PACSIN1 and PACSIN3.4. The deletion the amino acid residues from aa363 to aa382 abrogates the association of Cyclin D1 with PACSIN2Conclusion:1. Identification and confirmation of PACSIN2 as a Cyclin D1-binding protein2. Cyclin D1 binds to PACSIN2 through its C-terminal E-rich motif3. NPF domain of PACSIN2 is required for Cyclin D1 bindingPartⅡ.PACSIN2 Represses Cellular Migration through Direct Association Cyclin Dla, but not the Cyclin DlbObjective:To find out the co-localization of Cyclin Dl and PACSIN2, Identify the biological function of PACSIN2 as a Cyclin D1-binding protein, and whether PACSIN2 acts it role depend on Cyclin D1 or not.Methods:1. Identify the co-localization of Cyclin D1 and PACSIN2 by Immunohistochemical straing of MRC5 cell2. Transwell migration assay in PACSIN2+/+ and PACSIN2-/- mouse skin fibroblast cell lines3. Transwell migration assay by Knocking down PACSIN2 with siRNA (h) in Prostate Cancer cell line LNCaP4. Cell spreading Assay by Knocking down PACSIN2 with siRNA (m) in Mouse embryonic fibroblast cell line NIH 3T3.5. Transwell migration assay by Knocking down PACSIN2 in Cyclin D1-/- cells, and Cyclin D1-/- cell transduced with either Cyclin D1a or Cyclin Dlb retrovirus.6. Cell spreading Assay by Knocking down PACSIN2 in Cyclin D1-/- cell transduced with either Cyclin Dla or Cyclin D1b retrovirus.Results:1. Cyclin D1 and PACSIN2 were co-localized in cell ruffle. 2. PACSIN2-/- mouse skin fibroblast cell migration ability increases, PACSIN2 siRNA (h) increases cellular migration in Prostate Cancer cell line LNCaP. 3. PACSIN2 siRNA (m) reduced cell spreading in Mouse embryonic fibroblast cell line NIH3T34. PACSIN2 siRNA increases cell migration in Cyclin-/-+Dla cell, but has no effection on Cyclin D1-/- and Cyclin-/-+Dlb cells5. PACSIN2 siRNA represses cell spreading in Cyclin-/-+Dla cell, but has no effection on Cyclin-/-+Dlb cell.Conclusion:1. PACSIN2 represses cellular migration both in normal cell and prostate cancer cell lines.2. Endogenous PACSIN2 enhances cell spreading via Cyclin D1a, and Cyclin Dla is required for PACSIN2 repression of cellular migration.So, we hypothesize Cyclin D1 protein binding partners that contribute to the diverse functions of Cyclin D1 such as governing DNA synthesis, contact-independent growth, angiogenesis, and cellular migration, are critical for understanding Cyclin D1 function. Through identifying polypeptides associated with Cyclin D1, by tandem mass spectrometry, the current studies have identified PACSIN2 as a new Cyclin D1-associated protein, which contributes to the induction of cellular migratory directionality. In the current study, Cyclin D1 colocalized with PACSIN2 at the leading edge of MRC5 cells, Endogenous PACSIN2 enhances cell spreading via Cyclin Dla, and PACSIN2 siRNA can increase cellular migration in prostate cancer cell, indicating a role for PACSIN2 in repressing cellular migration. Together with the requirement of Cyclin Dla for PACSIN2 to repress cellular migration and PACSIN2 association with cytoskeletal proteins, we conclude that PACSIN2 provides a likely candidate protein coordinating a subset of the cellular migratory functions of Cyclin D1, which leads to a better understanding of the complexes contributing to Cyclin D1-mediated cellular migration.更多还原