【作者】 郝震锋；

【导师】 杨蓉娅；

【作者基本信息】 第三军医大学 ， 皮肤病与性病学， 2014， 博士

【摘要】 背景：皮肤癌是临床上最常见的恶性肿瘤之一，可笼统分为恶性黑色素瘤和非黑色素瘤性皮肤癌，后者主要包括鳞状细胞癌和基底细胞癌。皮肤癌严重者可危及生命，其发病率逐年上升，已经成为了一个全球性的公共健康问题。多种危险因素可导致皮肤癌的发生，包括内源性因素（遗传因素、基因突变等）和外源性因素（如紫外线暴露，化学致癌物质和其他环境压力）。早期诊断和治疗，可以减少因该病所造成的死亡和畸型。然而，皮肤癌发生的确切分子机制仍不清楚，这严重的影响了对于该病的治疗和预防。激活转录因子3（ATF3）是ATF/CREB转录因子家族成员之一，特征性的含有碱性亮氨酸拉链（bZIP）基序。正常细胞中ATF3的表达处于较低水平，但可由多种不同的胞外信号诱导而迅速上调表达，包括生长因子，细胞因子和某些基因毒性应激剂。做为转录因子，ATF3广泛参与人体的多种生理功能，包括维持身体内环境平衡，伤口愈合，细胞粘附，肿瘤细胞侵袭，细胞凋亡和信号通路等。新的证据表明，ATF3可能通过调整增殖和凋亡信号之间的微妙平衡关系，影响肿瘤的发生和发展。值得思考的是，ATF3在肿瘤发展中发挥的作用不尽相同，这可能取决于细胞的类型和所处的环境背景。有报道提示，免疫抑制宿主ATF3高表达可抑制p53依赖的细胞衰老，从而增加了皮肤鳞状细胞癌发生的可能性，但在皮肤癌背景下，ATF3与p53两者之间的相互作用关系，以及ATF3促进皮肤癌发生的分子机制仍不明确。信号传导蛋白和转录激活物3（Stat3）是生长因子受体（GFR）下游的信号转导蛋白，调节着参与细胞周期进程，细胞凋亡，血管生成，肿瘤细胞侵袭和转移相关靶基因的表达，其在肿瘤发生过程中发挥着重要的调控作用。有研究显示，Stat3在许多上皮癌，包括头和颈、乳腺、肺、皮肤、前列腺癌中存在组成型激活；表皮特异性Stat3缺陷小鼠皮肤癌发生相关实验结果表明，Stat3在上皮癌启动和促进阶段均发挥着重要作用。然而在ATF3促进皮肤癌发生的过程中，Stat3的角色作用尚不清楚。表遗传学相关改变是肿瘤发生的另一重要机制，这其中就包括组蛋白乙酰化模式的失调。能够阻断组蛋白脱乙酰酶（HDAC）活性的组蛋白脱乙酰酶抑制剂（HDACi）已被研发并用于多种肿瘤的靶向治疗。HDACi如曲古抑菌素A（TSA）和丁酸盐，可改变正常的染色质结构，从而导致相关基因表达和调控的改变。已有报道，TSA可引起肿瘤细胞的生长抑制并增加癌细胞的凋亡敏感性，同时有报道显示，ATF3可能与HDACs发生物理性的相互作用，通过对乙酰化修饰作用的调节，影响靶基因的表达，但ATF3在TSA诱导的肿瘤细胞生长抑制过程中的角色作用，特别是ATF3与TSA重要靶基因p21之间的相互作用关系，仍不明确。目的：在本实验中，我们拟利用qPCR技术和WB等技术分析ATF3在皮肤癌组织和皮肤癌细胞株中的表达情况，同时联合MTT法分析ATF3表达状态对细胞增殖活性的影响，从而进一步明确ATF3与皮肤癌发生之间的关系；基于SCC-13细胞和p53缺失的RTS3b细胞，利用细胞转染和荧光素酶报告基因检测等技术明确ATF3、p53和Stat3之间的相互作用关系，阐明ATF3对p53-Stat3级联信号的影响；从表遗传学角度，分析ATF3在TSA造成的表皮样癌细胞生长抑制过程中所发挥的作用，以及ATF3与TSA重要靶基因p21之间的相互作用关系。从而初步阐明ATF3促进皮肤癌细胞增殖的可能分子机制。方法：1、皮肤癌组织ATF3的表达，皮肤癌组织来源原代培养细胞、SCC-13细胞，和p53缺失的RTS3b细胞ATF3的转录表达及蛋白水平测定：临床来源的皮肤癌组织和正常皮肤组织各30例，利用qPCR技术，比较分析上述组织的ATF3转录表达水平，选取ATF3高表达的皮肤癌组织和ATF3低表达的正常皮肤组织各3例，建立原代培养细胞系，利用qPCR技术和WB技术分析皮肤癌组织来源细胞、正常组织来源细胞、SCC-13细胞，和p53缺失的RTS3b细胞ATF3的转录表达情况和蛋白表达水平。2、利用MTT法比较分析皮肤癌组织来源和正常皮肤组织来源的原代培养细胞、SCC-13细胞和P53缺失的RTS3b细胞的增殖活性。3、利用荧光素酶报告基因检测技术分析ATF3过表达对SCC-13细胞和P53缺失的RTS3b细胞Stat3转录活性的影响。4、利用细胞转染技术，分别使用ATF3shRNA质粒，pcDNA3载体，pcDNA3-p53，pcDNA3-Stat3C和/或pcDNA3-ATF3质粒转染SCC-13和P53缺失的RTS3b细胞，并联合使用Stat3抑制剂Stattic，后续利用MTT法对转染细胞的增殖活性变化情况进行比较分析，利用WB技术分析ATF3过表达对Stat3和p53蛋白水平的影响。5、利用细胞培养联合细胞转染、qPCR和WB等技术，分析ATF3在TSA造成的表皮样癌细胞生长抑制过程中所发挥的作用，以及ATF3与TSA重要靶基因p21之间的相互作用关系。结果：1、qPCR检测结果提示：与正常皮肤组织相比，皮肤癌组织中ATF3表达上调，而正常皮肤组织中ATF3表达处于较低水平。WB检测结果显示，ATF3高表达皮肤癌组织来源的原代培养细胞ATF3蛋白水平高于ATF3低表达的正常组织来源的原代培养细胞；蛋白表达水平与组间ATF3转录表达差异相一致。2、MTT法检测结果显示，皮肤癌组织来源的原代培养细胞增殖活性明显高于正常组织来源的原代培养细胞；利用细胞转染技术，敲低ATF3表达后，SCC-13细胞增殖活性明显降低，而ATF3增强表达则显著提高了该细胞的增殖活性。3、利用WB技术证实皮肤癌组织来源的原代培养细胞Stat3磷酸化水平明显高于正常皮肤组织来源的原代培养细胞。利用细胞转染技术证实ATF3过表达SCC-13细胞Stat3磷酸化水平明显高于ATF3敲低组，说明ATF3为Stat3活化所需要，ATF3过表达上调了Stat3磷酸化水平。荧光素酶报告基因检测实验结果提示ATF3正向调节Stat3的转录活性。实验同时发现，皮肤癌组织来源的ATF3高表达的原代培养细胞p53表达水平较低，ATF3敲低表达使SCC-13细胞p53表达上调，而ATF3过表达明显抑制了该细胞株p53的表达水平。4、P53缺失的RTS3b细胞Stat3磷酸化水平高于SCC-13细胞，利用细胞转染技术证实，ATF3过表达对P53缺失的RTS3b细胞的Stat3磷酸化水平和转录活性没有影响，提示ATF3可能通过下调p53的表达来阻遏后者对Stat3活性的抑制作用。5、组成型激活Stat3（Stat3C）显著促进了皮肤癌细胞生长，这与ATF3功能相一致。相反，Stat3抑制剂Stattic抑制了SCC-13细胞的增殖活性，提示ATF3对皮肤癌细胞的增殖促进作用被Stattic所阻遏，提示ATF3对皮肤癌细胞的增殖促进作用主要是通过调节Stat3的活性来实现的。此外，ATF3上调表达对SCC-13细胞增殖活性的促进作用可被p53过表达所抑制，而ATF3上调表达对P53缺失的RTS3b细胞的增殖活性无显著影响。提示，ATF3通过对p53-Stat3级联信号的调节实现了对皮肤癌细胞增殖的促进作用。6、我们还从表遗传学的角度对ATF3可能具有的促进癌细胞增殖的作用进行了初步研究，结果提示ATF3可能通过抑制p21基因表达干扰TSA引起的表皮样癌细胞的生长抑制。结论：在皮肤癌组织中存在ATF3上调表达，ATF3上调表达具有促进皮肤癌细胞增殖的作用。进一步的研究表明ATF3可抑制皮肤癌细胞P53的表达，进而削弱了p53对Stat3的抑制作用，致Stat3表达和活化水平上升，最终导致肿瘤细胞的增殖活性提高。ATF3通过调节p53-Stat3级联信号通路，实现了其对于肿瘤细胞的增殖促进作用，这可能是ATF3促进皮肤癌发生的主要机制之一。此外，ATF3促进肿瘤细胞增殖还可能存在着大量其他未知的机制，如在表遗传学方面，我们发现ATF3可能通过抑制p21基因表达干扰TSA引起的表皮样癌细胞生长抑制。上述结果有益于我们进一步认识皮肤癌发生的分子病理机制。ATF3过表达，增加了皮肤癌的发生风险，ATF3表达水平或可成为判断皮肤癌预后的指标，而ATF3-p53-Stat3级联信号通路可能成为预防皮肤癌发生和治疗该病的潜在靶点。更多还原

【Abstract】 Background:Skin cancer or cutaneous carcinoma is the most common and life-risky type ofcarcinoma, categorized as melanoma and non-melanoma skin carcer (squamous cellcarcinoma (SCC) and basal cell carcinoma (BCC) are the two most common subtypes),affecting millions worldwide. The incidence of skin cancer is increasing yearly, making it apre-eminent public health threat, and identification of risk factors is needed to stop thisincreasing trend. Multiple risk factors exist, including endogenous factors (genetic factorsand gene mutations) and exogenous factors (sun exposure, chemical carcinogens and otherenvironmental stress). Early diagnosis and treatment are needed to decrease the number ofdeaths and disfigurations due to skin cancer. Whereas, the precise molecular mechanism ofskin cancer remains unclear, which make it difficult to treat and prevent of the disease.Activating transcription factor3(ATF3) is an ATF/CREB family member, whichcontains the basic region and leucine zipper (bZIP) motif. ATF3is expressed at low levels innormal cells but can be rapidly induced by multiple and diverse extracellular signalsincluding growth factors, cytokines and some genotoxic stress agents. The physiologicalfunction of ATF3has been addressed in several cell lines indicated that ATF3might beinvolved in homeostasis, wound healing, cell adhesion, cancer cell invasion, apoptosis andsignaling pathways. Emerging evidence suggests that ATF3may play an critical role in hostdefence by regulating the delicate balance between proliferative and apoptotic signals thatcontribute to the development of cancer. Interestingly, ATF3has been demonstrated to playdifferent roles in cancer development depending on the cell type and context. For example,over-expression of ATF3protein moderately suppresses cell growth through slowing downprogression from G1/S transition in Hela cells. Conversely, ATF3promotes growthfactor-independent proliferation in chick embryo and enhance serum-induced cellproliferation in rat fibroblasts. Although ATF3has been reported to promote skin tumor formation through suppression of p53-dependent senescence, the precise role of ATF3andthe underlying molecular mechanism in skin cancer remain unclear. Stat3is a member ofSTAT family which may be activated by many extracellular signals. In accordance with itscritical roles in the regulation of cellular processes associated with carcinogenesis, Stat3isconstitutively activated in many epithelial cancers including head and neck, breast, lung,skin, and prostate. Nonetheless there is little known about the relationship between ATF3,p53and Stat3signaling in the skin carcinogenesis.Epigenetic changes is another important mechanism of tumorigenesis, includingderegulated histone acetylation patterns. Histone deacetylase inhibitors (HDACi) which canblock the activity of histone deacetylase (HDAC) have been developed and used for avariety of targeted tumor therapy. HDACi such as trichostatin A (TSA) and butyrate canalter the normal chromatin structure, leading to changes in gene expression and regulation.It has been reported that TSA can cause tumor cell growth inhibition and increasedapoptotic sensitivity of cancer cells, meanwhile as reported, ATF3may interact physicallywith HDACs, but the role of ATF3in TSA-induced inhibition of tumor cell growth,especially the interaction between ATF3and p21, the key target gene of TSA remainsunclear.Aims:First, to confirm the role of ATF3in skin carcinogenesis by detecting its expression inskin cancer tissue, meanwhile find out the relationship between the ATF3protein levels andthe proliferation activity of the cells drived from skin cancer tissue and the controlrespectively; then clarify the interactions between ATF3and p53-Stat3cascade in SCC-13and RTS3b (p53null) cells; determine the role of ATF3in TSA-induced inhibition of tumorcell growth, especially the interaction between ATF3and p21, the key target gene of TSA,finally find out the potential mechanism of ATF3promoting skin cancer development.Methods:1.The expression of ATF3in skin cancer tissue and the protein level of ATF3, Stat3andp53in different cell lines.A total of30surgically resected examples of skin cancer were enrolled, thirty skinspecimens from healthy donors were collected as normal control. The ATF3expression ofall the samples were detected by quantitative real-time PCR (qPCR). Then the3pieces of skin cancer tissues with highest ATF3(C1, C2, C3) and the3pieces of normal skin tissueswith lowest ATF3(N1, N2, N3) were primarily cultured and established as cell lines. Thenthe transcriptional expression of the ATF3and the protein level of ATF3, Stat3and p53inthe different cell lines, as well as in the human squamous carcinoma SCC-13cells andp53-null RTS3b cells, were analyzed by qPCR and Western blot analysis.2. Transcriptional activity of Stat3in SCC-13and p53-null RTS3b cells.The luciferase reporter construct was co-transfected with pRL-TK and over expressingplasmids or vectors into SCC-13or RTS3b cells. After transfection for48h, cells wereharvested for the luciferase reporter assay.3. Influence of ATF3, Stat3and p53, as well as their interaction on the proliferation ofthe diffenent Cell lines.SCC-13or RTS3b cells were transfected with control shRNA plasmids, ATF3shRNAplasmids, pcDNA3vector, pcDNA3-p53, pcDNA3-Stat3C and/or pcDNA3-ATF3plasmidsby using Lipofectamine2000. Otherwise the cell lines were treated with Stat3inhibitorStattic. Then the cell proliferation activity of SCC-13cells and RTS3b cells was determinedby MTT at indicated time points after transfection.4. ATF3interferes activation of p21in HDACi-induced growth inhibition ofepidermoid carcinoma cells.Based on epidermoid carcinoma cell A431, joint use of cell culture，cell transformation,qPCR and WB techniques, the relationship between ATF3that can promote tumor cellgrowth and TSA with the opposite effect was analyzed, meanwhile the interaction betweenATF3and the important target gene p21of TSA in the process of growth inhibition inducedby TSA in epidermoid carcinoma was investigated.Results:1. ATF3was significantly upregulated in skin cancer tissue, while it was maintainedat low level in normal epitheliums；the primary cultured skin cancer cells possessed higherATF3protein level, which was consistent with transcriptional expression differencebetween skin cancer and normal tissues.2. ATF3promotes skin cancer cell proliferation. Among the6lines of primarycultured skin cells, the cells from skin cancer (C1-C3) proliferated much faster than thatfrom the normal skin. The proliferation rate of SCC-13cells was suppressed by ATF3 knockdown (KD); conversely, forced expression of ATF3in SCC-13cells significantlypromoted cell growth; these results demonstrated that ATF3is a positive regulator of skincancer cell proliferation.3. Phosphorylated Stat3level was much higher in skin cancer cells with higher ATF3expression. The results of Luciferase reporter assay combined with cell transfectionrevealed that ATF3was required for Stat3phosphorylation, ATF3overexpressionupregulated Stat3phosphorylation level, furthermore, ATF3positively regulated Stat3transcriptional activity. Meanwhile p53showed lower mRNA level in established skincancer cell lines with higher ATF3expression. Correspondingly, ATF3knockdownupregulated p53expression and ATF3overexpression strongly inhibited p53transcription.4. The constitutive active Stat3form, Stat3C overexpression significantly promotedskin cancer cell growth. Conversely, the Stat3inhibitor Stattic inhibited cell proliferation ofSCC-13cells and the promotion effect of ATF3was prevented by Stattic. Moreover, theproliferation rate of skin cancer cells was suppressed by p53overexpression in SCC-13cells. Whereas, ATF3had no significant effects on the cell proliferation of p53-null RTS3bcells. Collectively, these data demonstrate that ATF3might act trough modulating p53-Stat3signaling cascade to enhance skin cancer cell proliferation.5. We also investigate the potential function of ATF3in promoting cancer cell growthfrom the perspective of epigenetic, the results suggest that ATF3may interfere withTSA-induced epidermoid cancer cell growth inhibition by inhibiting p21expression.Conclusion:In summary, ATF3was upregulated in skin cancer tissues and promoted theproliferation of skin cancer cells. The molecular mechanism of ATF3in skin carcinogenesiswas investigated and we found that ATF3enhanced skin cancer cell growth throughmodulating p53-Stat3signaling activity. This study discovered the functions of ATF3-p53-Stat3signal cascade in skin cancer development and will assist to understand the pathologicmechanism of skin carcinogenesis. ATF3might increase the risk of skin cancer and serve asan important prognostic indicator for the disease，and the ATF3-p53-Stat3signaling maybe apotential target for skin cancer prevention. Furthermore, ATF3promote skin cancer cellproliferation may also have a large number of other unknown mechanisms, as shown inepigenetics, ATF3may interfere with TSA-induced epidermoid cancer cell growthinhibition by inhibiting p21expression.更多还原