LKB1对宫颈癌细胞转录谱的影响及抑癌机制研究与CIP2A调控宫颈腺癌多药耐药的研究
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摘要
第一部分LKB1对宫颈癌细胞转录谱的影响及抑癌机制研究
宫颈癌是全球范围内女性第二大常见恶性肿瘤,每年新发宫颈癌病例大约53万。尽管在发达国家随着筛查方法的改进,宫颈癌的发病率已经降低,但是在发展中国家,由于社会经济因素,宫颈癌的筛查方法不能广泛普及,因此宫颈癌仍然是威胁妇女的主要致死因素。
宫颈癌的发生有病毒方面的因素,高危型人乳头瘤病毒(High-risk type human papilloma virus, hr-HPV)感染与宫颈癌的发生密切相关。这一事实提供了预防宫颈癌的方法,即预防接种HPV疫苗阻止感染。尽管HPV疫苗已经上市,但是对于已经感染HPV或者患有免疫抑制的病人没有效果。
HPV是一类无包膜的小DNA病毒,可以在表皮和粘膜上皮细胞复制增殖。根据HPV的临床意义,可将其分为高危和低危型。持久的高危型HPV感染是宫颈癌发生的主要致病因子。全球大约70%的宫颈癌由HPV16,18型引起。高危型HPVs的两个癌蛋白E6和E7具有促进细胞增殖的作用,在宫颈癌的恶性转化过程中起重要作用。E6、E7分别与抑癌蛋白p53和pRb相互作用,促进它们的降解诱发肿瘤发生。尽管HPVs在宫颈癌的发生中发挥了重要的作用,但是只有一小部分感染了高危型HPVs的女性最终发展为宫颈癌,而且从最初感染HPVs到最终进展为宫颈癌,这一过程需要几十年的时间,提示高危型HPVs的感染对于宫颈的癌变是必要的但不是充分的条件。此外,有些宫颈癌检测不到HPVs。因此,在宫颈的癌变过程中,一定有一些其它协同因子或遗传事件参与其中。
肝激酶B1(liver kinaseB1,LKB1)又称丝氨酸-苏氨酸激酶(serine threonine kinase, STK11),最初在黑斑息肉综合征(Peutz-Jeghers syndrome, PJS)研究发现,LKB1基因突变是导致PJS发生的因素,PJS患者对胃肠道肿瘤,乳腺肿瘤,妇产肿瘤的易感性增加。继而在散发性肿瘤中,也发现了LKB1基因的体细胞突变,如非小细胞肺癌、结肠癌、乳腺癌和宫颈癌等,因而LKB1已作为一个比较公认的抑癌基因被广泛研究。LKB1抑癌的分子机制目前尚不十分清楚,现有的研究表明,LKB1是一种重要的上游蛋白激酶,LKB1与伪激酶STRAD和清道夫蛋白M025形成功能三聚体,这个复合体可以磷酸化至少14种在T环活化位点具有保守苏氨酸的丝氨酸-苏氨酸激酶,而使之激活。个研究非常多的LKB1底物是AMP-活化蛋白激酶(AMP-activated protein kinase,AMPK),AMPK是细胞、机体代谢的主要调节剂,LKB1可通过AMPK对外界各种生理病理刺激作出反应,例如：骨骼肌收缩,低氧,饥饿,H202,二甲双胍和苯乙双胍等。重要的是,LKB1通过激活AMPK调节细胞生长和细胞周期,活化的AMPK激活TSC2复合体和nTOR伴侣分子raptor,进而负向调控nTOR信号通路,抑制细胞生长。此外,LKB1参与调控细胞极性,细胞增殖,细胞迁移,细胞衰老,凋亡,DNA损伤反应,细胞分化和有氧糖酵解过程等,也与其抑制肿瘤发生的机制有关。
LKB1基因突变在宫颈癌中的研究最早始于1999年,根据不同的文献报道,宫颈癌中LKB1基因突变率在2-20%,但有关宫颈癌组织中LKB1蛋白表达状况的报道尚很少。
目前,宫颈癌细胞中LKB1抑制细胞增殖的分子机制研究主要集中于LKB1/AMPK通路。已有文献报道,在宫颈癌细胞HeLa、SiHa中过表达LKB1可以抑制其生长,且依赖于LKB1的激酶活性；LKB1可抑制HeLa细胞的锚定非依赖性生长,但对SiHa细胞没有此作用；应用AMPK激活剂(如AICAR、二甲双胍等)对于具有LKB1/AMPK/mTOR完整信号通路的宫颈癌细胞系有抑制作用。但是,LKB1抑癌分子机制的研究尚存在许多的未知,LKB1是否可调控更多的下游基因?是否有新的通路参与其抑癌作用机制?LKB1在宫颈正常组织及癌组织中的蛋白表达及功能?这些都是尚待解决的问题。深入研究LKB1的抑癌分子机制将有助于发现更多的关键分子,为治疗干预宫颈癌的发生发展提供新的思路和靶标。
基于LKB1在宫颈癌发生发展中的潜在重要作用,本研究在宫颈癌细胞中初步研究了LKB1的生物学活性,并对临床宫颈癌组织中LKB1蛋白的表达状况进行了检测。重要的是,我们首次利用基因芯片Microarray技术研究了LKB1对宫颈癌细胞基因转录谱的影响,通过生物信息学对差异表达基因进行了分析,继而在mRNA和蛋白水平给予验证。其中,在磷脂酰肌醇(PI3K)信号通路中,我们发现II型四磷酸肌醇磷酸酶(Inositol polyphosphate4-phosphatase type II, INPP4B)基因的表达在LKB1过表达后明显上调,进一步的细胞学实验进一步证明INPP4B是LKB1重要的下游靶分子。INPP4B也是一种潜在的肿瘤抑制因子,LKB1对INPP4B表达的调控作用尚未见报道。
一.LKB1表达对宫颈癌细胞增殖的影响
为检测LKB1蛋白表达对宫颈癌细胞增殖的影响,我们将编码LKB1的质粒成功转染了HeLa细胞(LKB1表达缺失)并经历短期G418筛选,Western blot检测此细胞中有明显的LKB1蛋白表达。进一步采用CCK-8方法检测细胞的增殖活性,结果显示,过表达LKB1的HeLa细胞增殖能力显著低于载体对照组。采用BrdU实验检测了细胞周期改变,结果发现HeLa细胞过表达LKB1后,位于S期的细胞数目显著降低,DNA复制合成减少,表明细胞生长增殖能力下降。以上结果说明LKB1蛋白抑制宫颈癌细胞HeLa的生长增殖。LKB1稳转细胞系HeLa-LKB1的研究结果表明：LKB1蛋白在稳转细胞系中有效表达,而且可检测至p-AMPK磷酸化水平的升高,说明在HeLa-LKB1细胞中LKB1可磷酸化其下游的MPK使其活化,LKB1具有活性,且LKB1-AMPK的信号通路是完整的。
二.LKB1蛋白在宫颈癌组织中的表达
为探究LKB1蛋白在临床宫颈癌组织中的表达状况,我们对78例宫颈癌(包括25例宫颈腺癌、53例宫颈鳞癌)和25例正常宫颈组织进行了免疫组化检测。结果表明,在44%(11/25)的宫颈腺癌、60.4%(32/53)的宫颈鳞癌组织中LKB1蛋白表达缺失。以上结果说明,宫颈癌组织中LKB1蛋白表达有较高的缺失率,这与公认的LKB1是一个抑癌基因一致。检测结果发现在正常宫颈组织中存在LKB1表达弱阳性或不表达,这与文献中报道的正常结直肠、胰腺组织LKB1的表达情况类似。
三.LKBl转录谱的研究以及LKB1的新靶标INPP4B的发现
尽管已知LKB1可以调控至少14种下游激酶,但是LKB1的作用机制仍然没有完全清楚,为探究宫颈癌细胞中受LKB1调控的基因,我们进行了Microarray基因芯片水平的转录谱研究。通过Microarray实验,在稳定表达LKB1蛋白的HeLa细胞系中,我们识别了222个LKB1调控的差异表达基因,其中下调的基因有117个,上调的基因有105个。qRT-PCR技术对七个差异表达基因LKB1、PLK2、ABCC2、GLP2R、KYNU、MAL2和AKAP12进行了mRNA水平的验证,结果证实Microarray数据真实反映了基因在转录水平的变化。
生物信息学方法进一步分析差异表达基因。基因本体方法(gene ontology,GO)对差异表达基因进行了分类,GO数据库分为生物学过程分类,分子功能分类以及细胞组分分类。分析结果显示,222个差异表达基因可归属在40个生物学过程分类,23个分子功能分类以及20个细胞组分分类中。LKB1调控的基因多参与信号转导,蛋白间相互作用以及定位于膜上。KEGG信号通路数据库对差异表达基因的分析发现了8个有统计学意义的生物学通路。其中两条通路是与代谢有关,分别是精氨酸和脯氨酸代谢通路、肌醇磷酸代谢通路,这与LKB1参与代谢的观点一致。另外一条重要的信号通路是磷脂酰肌醇(PI3K)信号通路,有文献报道LKB1-AMPK信号通路可与PI3K-Akt信号通路汇合在mTOR,参与调控细胞的生长与代谢。另外LKB1还参与了轴突发育通路和造血干细胞系分化通路。
在PI3K信号通路中的分析中我们发现一个重要的磷酸肌醇磷酸酶INPP4B基因的表达在LKB1过表达后明显上调。采用qRT-PCR进一步证实在过表达LKB1的HeLa中INPP4B mRNA水平显著增加。Western blot结果表明：LKB1稳转HeLa细胞中INPP4B蛋白水平升高1.4倍,同时在瞬转LKB1且经历G418短期筛选的HeLa细胞中INPP4B蛋白水平升高1.9倍。进一步采用特异性siRNA,在表达LKB1且LKB1-AMPK通路完整的宫颈癌CaSki细胞中敲低LKB1, Western blot检测INPP4B的表达变化,发现敲低LKB1后,INPP4B蛋白表达水平也随之降低。我们进一步检测了LKB1稳转HeLa细胞系中p-Akt水平改变,结果表明,与空载对照细胞HeLa-vetor相比,HeLa-LKB1细胞中Akt磷酸化水平明显下降。以上结果证明,抑癌蛋白LKB1可正向调控INPP4B的表达,INPP4B是LKB1下游的一个新靶蛋白。在过表达LKB1的HeLa细胞系中INPP4B表达上调,而磷酸化Akt水平下降,提示LKB1通过上调INPP4B参与了PI3K/Akt通路的负向调控,这可能是LKB1抑制细胞增殖的一个新通路。
综上所述,本研究探讨了抑癌蛋白LKB1在宫颈癌中的作用；并首次应用Microarray技术对表达LKB1的宫颈癌细胞进行了基因转录谱分析,进一步对发现的新靶标INPP4B进行了初步研究。研究证实,过表达LKB1对宫颈癌细胞生长增殖有抑制作用,在超过50%的宫颈癌组织中LKB1蛋白表达缺失,说明LKB1的抑癌活性在宫颈癌发生发展中具有重要作用。基于Microarray分析我们识别了222个受LKB1调控的基因,发现了8个有统计学意义的生物学通路。在PI3K信号通路中一个重要的抑癌基因INPP4B在LKB1过表达后明显上调,细胞学实验进一步证明INPP4B是LKB1下游的一个新靶标。在过表达LKB1的HeLa细胞系中INPP4B上调,而磷酸化Akt水平下降,提示LKB1通过上调INPP4B参与了P13K/Akt通路的负向调控,这可能是LKB1抑制细胞增殖的一个新通路。以上发现为深入研究LKB1调控的分子信号通路奠定了实验基础,为宫颈癌的治疗干预提供了新的分子靶标。第二部分CIP2A调控宫颈腺癌多药耐药的研究
宫颈癌是世界范围内最常见的妇科肿瘤疾病之一,严重威胁女性的身体健康,死亡率位居发展中国家女性肿瘤的第二位。近年来,全球宫颈癌的发病率呈现上升和年轻化的趋势。流行病学研究结果表明,高危型人乳头瘤病毒(Human papillomavirus,HPV)感染是妇女宫颈癌和宫颈上皮内瘤变的高危因素。
虽然宫颈癌筛查方法的普及和HPV预防性疫苗的出现有效降低了全球宫颈癌的发病率。然而,在发展中国家宫颈癌仍然是影响和威胁女性健康的重要因素。手术和放疗是宫颈癌的主要治疗方法,早期以手术治疗为主,中晚期多采用放射治疗。过去,一直认为宫颈癌属于化疗不敏感的肿瘤,仅在晚期及复发的患者中将化疗作为综合治疗的一部分。近年来,国内外学者对化疗在宫颈癌中的应用进行了大量的基础和临床研究,发现经化疗后患者5年生存率明显提高,因此确立了化疗在宫颈癌治疗中的重要地位。化疗的优势在于可以治疗肿瘤周围肉眼看不见的微小转移灶以及可能存在的全身亚临床转移和复发的病人。近10年来对宫颈癌的综合治疗日益受到关注,综合治疗已成为现代治疗宫颈癌的一个重要策略。
然而,尽管人们已经认识到化疗在宫颈癌中的作用,并且已经将化疗作为宫颈癌治疗的重要手段,但是宫颈癌多药耐药的产生却大大限制了化疗药物的疗效和应用,导致治疗失败,肿瘤复发。宫颈癌细胞产生多药耐药的机制分为两大类：①对天然化疗药物的耐药机制,主要包括P糖蛋白的过度表达及DNA拓扑异构酶酶含量与活性的改变；②对铂类化合物和烷化剂耐药的机制,主要包括药物在细胞内积聚减少,巯基化合物(GSH、GST、MT)对药物的解毒力增加,DNA损伤修复能力增加等。
P糖蛋白(P-glycoprotein, P-gp)是一种跨膜糖蛋白,由多药耐药基因(MDR1)编码。P糖蛋白是ABC转运蛋白家族的一员,通过与抗癌药物结合后再与ATP结合,经ATP供能将细胞内药物逆浓度梯度运出胞外,使细胞内药物浓度不断下降从而使之达不到有效杀伤浓度,最终导致肿瘤细胞耐药性的产生。在使用天然化疗药物后,几乎50%的肿瘤都有P-gp蛋白的表达增加。宫颈癌的耐药问题也与P-gp的表达增加密切相关。
Cancerous inhibitor of protein phosphatase2A (CIP2A)是一种2007年发现的癌蛋白,CIP2A可抑制PP2A对c-Myc62位丝氨酸(S62)的去磷酸化,从而增加细胞中c-Myc的蛋白水平。研究发现,CIP2A在很多人类恶性肿瘤中过表达,例如胃癌、乳腺癌、非小细胞肺癌、急性白血病、前列腺癌、宫颈癌等。CIP2A还可以促进细胞的恶性生长增殖,并与化疗药物导致肝细胞癌、乳腺癌、白血病细胞的凋亡有关。那么,CIP2A是否也和宫颈癌的耐药性相关?进而是否参与了宫颈癌多药耐药机制的产生?CIP2A在宫颈癌中与P-gp蛋白的表达有无关联?这些问题是我们此项研究的重点。
一.敲低CIP2A提高了宫颈癌细胞对化疗药物的敏感性
我们在宫颈腺癌细胞系HeLa中特异性敲除CIP2A后,使用临床常用的三种肿瘤化疗药物多柔比星(Dox)、顺铂(Cis)和紫杉醇(Pac)处理细胞,通过MTT实验观察药物对HeLa细胞的杀伤作用。
实验结果显示,多柔比星(Dox)、顺铂(Cis)和紫杉醇(Pac)三种化疗药物抑制HeLa细胞增殖,在特异性敲低CIP2A后,三种化疗药物对HeLa细胞增殖的抑制能力显著增加。这表明,在HeLa细胞中CIP2A与化疗药物疗效有关,敲低CIP2A能够提高HeLa细胞对化疗药物的敏感性,因此本部分实验提示CIP2A可能参与肿瘤的多药耐药(MDR)。二. CIP2A和P-gp蛋白的表达在宫颈癌组织中密切相关
我们在山东大学齐鲁医院收集了103例石蜡包埋组织块,其中包括15例正常宫颈组织、16例CIN I、17例CIN II、12例CIN III、43例宫颈腺癌,利用免疫组化技术检测CIP2A和P-gp的蛋白表达情况。实验结果显示CIP2A和P-gp在15例正常组织、16例CIN I、17例CIN II组织中均不表达；12例CIN III组织中有1例(8.3%)CIP2A阳性,在43例宫颈腺癌组织中有16例(37.2%)有CIP2A的表达；P-gp在12例CIN III组织中表达阴性,在43例宫颈腺癌组织中有13例(30.2%)有P-gp的表达。CIP2A主要表达于胞浆,而P-gp主要位于细胞膜上。免疫组化结果显示,CIP2A和P-gp的表达在同一个宫颈癌标本上强度一致。并且统计学分析显示在宫颈腺癌组织中,CIP2A和P-gp表达正相关((r2=0.617,p<0.001)),P-gp表达和病人年龄、肿瘤大小、临床分期、肿瘤淋巴结转移、淋巴转移无相关性,但与肿瘤分化成度有关(p=0.029)。以上结果显示,在宫颈腺癌组织标本中,CIP2A和P-gp表达成正相关。三. CIP2A通过调控P-gp蛋白影响宫颈癌的多药耐药
为了进一步验证CIP2A与P-gp蛋白之间的关系,我们进行了如下实验：多柔比星(Dox)、顺铂(Cis)和紫杉醇(Pac)三种化疗药物处理HeLa细胞,检测化疗药物作用后CIP2A与P-gp蛋白的表达水平；同时检测抗Dox HeLa细胞系HeLa/Dox中CIP2A与P-gp蛋白的表达水平,同时在该细胞系中特异敲低CIP2A后,检测P-gp蛋白的水平变化；在HeLa、HeLa/Dox细胞系中特异性敲低CIP2A后,罗丹明外排实验检测P-gp蛋白的功能。
研究结果显示,化疗药物处理HeLa细胞后,CIP2A与P-gp蛋白的表达水平均明显上升；耐药细胞系HeLa/Dox中CIP2A与P-gp蛋白的表达水平较HeLa细胞显著增高,在干扰CIP2A后,P-gp蛋白的表达水平随之降低；在HeLa、 HeLa/Dox细胞系干扰CIP2A后,罗丹明外排实验结果显示细胞内荧光信号显著增强,表明P-gp蛋白的药物外排功能受到抑制。上述结果提示,CIP2A通过P-gp蛋白实现对宫颈腺癌耐药的调控。
综上,本研究利用体内外实验首次验证了CIP2A参与宫颈腺癌多药耐药的形成,这种作用主要是CIP2A通过上调P-gp蛋白的表达来实现的。在宫颈腺癌组织中CIP2A和P-gp蛋白的表达呈正相关；敲低CIP2A可提高宫颈腺癌细胞对多种化疗药物的敏感性,P-gp蛋白的表达水平也随之降低。因此,我们的研究结果提示,CIP2A作为一个新的基因治疗靶标,在宫颈腺癌的综合治疗中有着潜在的应用前景。
Part One:Transcription profiling and molecular mechanism of the LKB1tumor suppressor in cervical cancer
Cervical cancer is the second most common cancer in women worldwide with an annual incidence of530,000cases. Although the incidence of cervical cancer in developed countries has decreased due to improved screening methods, it is still a main mortality factor for women in developing countries.
Cervical cancer has a viral infectious etiology. Infection with high-risk types of human papillomavirus (HPVs) is strongly-associated with the development of cervical cancer. This fact provides a great opportunity for primary prevention of cervical cancer by prophylactic vaccination to prevent cervical HPV infection. Although HPV vaccine has become available, they are less likely to be effective for those who have already been infected by HPV and those who are immunosuppressed.
Papillomaviruses are small DNA nonenvelopedviruses that replicate in epidermal or mucosal epithelial cells. HPVs can be classified into high-or low-risk types depending on their clinical associations. Persisting high-risk HPV infections are the most significant risk factor for development of cervical cancer. HPV types16and18are responsible for about70%of cervical cancers worldwide. The two oncogenes E6and E7of high-risk HPVs possess proliferation-stimulating activity and play a critical role during malignant transformation of cervical cancer. E6and E7interact with p53and pRB respectively and promote their degradation. Though HPVs play significant roles in the progression to cervical neoplasia, only a small percentage of high-risk HPV-infected women develop cancer eventually and it often takes decades after the initial infection, suggesting that infection by high-risk HPVs is not sufficient for cervical carcinogenesis. In addition, some cervical cancers are negative for HPVs. Therefore, there must be other cofactors or genetic events involved in cervical carcinogenesis.
The liver kinase LKB1(also known as serine threonine kinase, STK11) was originally identified as the causative gene of Peutz-Jeghers syndrome (PJS), an autosomal dominant disease. Patients with PJS have increased cancer predisposition in GI tract, breast, and gynecological organs. Importantly, somatic LKB1mutations are frequently found in significant number of sporadic non-small cell lung carcinomas (approximately30%)、colon cancer、breast cancer and cervical carcinomas (approximately20%). In recent years, LKB1has become more established as a tumor suppressor. However, the molecular mechanism of LKB1has not been fully understood. LKB1is a master upstream kinase that forms a complex with a pseudokinase STRAD and the scaffold protein MO25. This complex can phosphorylate at least14serine-threonine kinases with conserved threonine in the "T-loop" activation site. A well-known substrate of LKB1is AMP-Activated Protein Kinase (AMPK), which is the master regulator of cellular and organismal metabolism. AMPK subsequently activates the TSC2complex or mTOR-binding partner raptor, which negatively regulates mTOR signaling. LKB1is involved in response to diverse signals under physiological and pathological conditions via AMPK, e.g.:skeletal muscle contraction, hypoxia, starvation, H2O2, metformin and phenformin. In addition, LKB1exerts its effects on cell polarity, proliferation, migration, senescence, apoptosis, DNA damage response, differentiation and aerobic glycolysis.
Mutation of LKB1in cervical cancer was originally studied in1999. The frequency of LKB1mutation in cervical cancer is between2%to20%according to different reports. But the expression level of LKB1protein in cervical cancer is not known.
At present, the molecular mechanism of LKB1as a tumor suppressor in cervical cancer is mainly focused on LKB1/AMPK pathway. It was reported that overexpression of LKB1in HeLa and SiHa could inhibit the growth of cells depending on its kinase activity. Moreover, LKB1can inhibit the anchorage-independent growth of HeLa not SiHa. AMPK activators, such as:AICAR, Metformin etc., are able to inhibit the growth of cervical cancer cell lines which have intact LKB1/AMPK/mTOR pathway. However, there are still a lot of questions about LKB1as a tumor suppressor. Are there more downstream targets of LKB1? Any new pathways involved into tumor-suppressing activity of LKB1? The expression of LKB1protein in normal cervix and cervical cancer?Detailed study of the molecular mechanisms of LKB1will help find more key molecules, provide new insights and targets for the treatment of cervical cancer.
Given the importance of LKB1in tumor suppression and the potential role in development of cervical cancer, we characterized the biological activities of LKB1, performed the immunohistochemical (IHC) study for LKB1in cervical cancers and initiated the first microarray experiment to identify LKB1-regulated genes. We used the bioinformatics to analyze the differentially expressed genes and verified the data at the mRNA and protein levels. More importantly, we identified inositol polyphosphate4-phosphatase type Ⅱ, INPP4B from PI3K pathway was upregulated after LKB1overexpression. And it was further confirmed that INPP4B is an important downstream target of LKB1. INPP4B is also a potential tumor suppressor and the regulation of LKB1on INPP4B is not reported untill now.
1. Characterization of LKB1expressing cervical cancer cells
To examine the effects of LKB1expression on cervical cancer cells, we transfected a plasmid encoding LKB1into HeLa cells, which contains a deletion in the gene for LKB1, followed with short period of G418selection. Upon transfection of the plasmid encoding LKB1, it becomes readily detectable by western blotting. We then examined the proliferation of LKB1expressing HeLa cells using the CCK8assay. The data showed that,HeLa cells expressing LKB1proliferate significantly slower than the vector control cells. We also examined cell cycle profiles in HeLa cells transfected with LKB1by BrdU assay. We found that overexpression of LKB1significantly reduced the number of cells in S-phase. These results demonstrate that, after transient transfection of LKB1in HeLa, proliferation and percentage in S phase significantly decreased, so expression of LKB1inhibits cervical cancer cell proliferation. Next we also examined p-AMPK of HeLa cells stably expressing LKB1. We can detect the expression of LKB1and increase of p-AMPK in HeLa-GFP-LKB1cell. The data indicated that LKB1can activate AMPK and the pathway was intact.
2. LKB1expression in cervical cancer
To characterize the expression of LKB1protein in cervical cancer, we performed immunohistochemical analysis on a series of cervical carcinoma tissues as well as normal controls. Paraffin-embedded tissue blocks were obtained (78cervical cancer:25cervical adenocarcinoma and53cervical squamous carcinoma samples, and25normal cervical tissues) from Qilu Hospital of Shandong University. For cervical adenocarcinoma tissues,56%showed positive LKB1stain while the other44%stained negative. For squamous cell carcinoma of the cervix,60.4%(32/53) of the samples were negative for LKB1, only39.6%(21/53) showed positive stain. Among the cervical cancer tissues, more than50%of samples did not express LKBl. The results were consistent with the notion for LKB1as a tumor suppressor. The observation of weak or no LKB1protein expression in normal cervix was consistent with the reports of LKB1expression in normal epithelia of colorectum and pancreas.
3. Transcriptional profiling of LKB1and INPP4B as a LKB1-regulated target
Although it's well known that LKB1can activate at least14downstream kinases, molecular mechanism of LKB1was still not fully understood. Therefore we performed Microarray analysis to study transcriptional profiling of LKB1. We took advantage of the HeLa cell line stably expressing LKB1, to advoid the alteration of a large number of genes by transfection. We identified222LKB1-regulated genes by microarray,117of them were down-regulated while105were up-regulated after LKBl overexpression. We then performed real-time PCR (qRT-PCR) experiment to confirm the results obtained by microarray. LKB1and the other six randomly selected genes PLK2, ABCC2, GLP2R, KYNU, MAL2and AKAP12were verified at mRNA level. Thus the qRT-PCR data suggest that probe sets used in the microarray experiments are likely to accurately measure the levels of transcripts.
Next we performed bioinformatic analysis of genes differentially expressed in LKB1expressing HeLa cells. We first analyzed the differentially expressed genes using gene ontology (GO) system. In the GO database, the genes are classified into three ontologies, i.e., biological process, molecular function and cellular component. Based on the GO annotation, the222genes whose expression was significantly altered in LKB1expressing HeLa cells can be classified into40,23,20functional groups in the biological process ontology, molecular function ontology and cellular component ontology, respectively. Most LKB1-regulated genes were involved in signal transduction, protein binding and relocated on the membrane. Then we searched the differentially expressed genes against KEGG pathway maps. Eight biological pathways were identified to be significantly regulated. Two pathways involved in metabolism, i.e., arginine and proline metabolism and inositol phosphate metabolism were represented, which was consistent with a role of LKB1in metabolism. Notably, the phosphatidylinositol signaling system is also identified. It is known that the PI3K-AKT pathway and LKB1-AMPK pathway converge at mTOR. In addition, LKB1involved in axon guidance pathway and the hematopoietic cell lineage pathway.
We found inositol polyphosphate4-phosphatase type II (INPP4B) in PI3K pathway was up-regulated after LKB1overexpression. We confirmed the increase of INPP4B at mRNA level and protein level by qRT-PCR and Western blot. Western blot results demonstrated that INPP4B increased1.4fold in HeLa stably-expressing LKBl and1.9fold in HeLa cells transfected with LKB1and followed with short period of G418selection. Moreover, knock-down of LKB1in the cervical carcinoma CaSki cells, which have an intact LKB1-AMPK pathway, reduced the steady state level of INPP4B. We further examined the level of p-Akt in HeLa stably-expressing LKB1and found the significant decrease in the level of p-Akt compared to the control cell of HeLa-vector. These results provide further evidence for INPP4B upregulation by LKB1and being a target for LKB1. INPP4B upregulation and decrease in level of p-Aktin HeLa stably-expressing LKB1suggest that LKB1regulates PI3K/Akt pathway negatively by upregulation of INPP4B and maybe this is a new signaling pathway for inhibition of cell growth by LKB1.
In summary, we characterized the biological activities of LKB1in cervical cancer, performed transcriptional profiling of LKB1and found INPP4B as a new target in this study. The results showed that, transiently transfected LKB1inhibits the growth of cervical cancer cell, loss of LKB1expression was found in more than50%cervical cancer samples and identified LKB1-regulated222genes and8pathways. We performed bioinformatics analysis and therefore identified a target of LKB1in PI3K signaling pathway, INPP4B. INPP4B upregulation and decrease in level of p-Aktin HeLa stably-expressing LKB1suggest that LKB1regulates PI3K/Akt pathway negatively by upregulation of INPP4B and maybe this is a new signaling pathway for inhibition of cell growth by LKB1. This study sheds light on the novel signaling pathways regulated by LKB1and provides new therapeutic targets in cervical cancer in the future.
Part Two:Regulation of multidrug resistance of cervical adenocarcinoma by CIP2A
Cervical cancer is one of the most common cancer in women worldwide. It is a major problem for women's health and is the second mortality factor among female tumors in the developing countries. In recent years, the incidence of cervical cancer worldwide rises and patients become younger. Epidemiological studies suggest that infection of high-risk human papillomavirus (hr-HPV) is a risk factor for cervical cancer and cervical intraepithelial neoplasia.
The incidence of cervical cancer worldwide has decreased due to improved screening methods and HPV prophylactic vaccines. However, prevention and early screening for cervical cancer has not been popularized in developing countries, due to economic factors and other medical conditions. Cervical cancer is still a mortality factor to women's health. It's well known that surgery and radiation therapy are the main treatments for cervical cancer, surgical treatment for early disease, radiation therapy for advanced. In the past, cervical cancer was considered to be not sensitive to chemotherapy and used in patients with advanced and recurrent tumors as part of a multimodality therapy.However, people performed a lot of basic and clinical research about chemotherapy in cervical cancer, and found that5-year survival rate was significantly improved in patients, thus establishing chemotherapy as an important part in cervical cancer treatment. Chemotherapy can be used in patients with tumor micrometastases, subclinical metastasis and recurrence. During the past10years, multimodality therapy has been used including surgery, radiation, and adjuvant and neoadjuvant chemotherapy for management of cervical cancer.
Although the importance of chemotherapy in cervical cancer has been established, the crucial factor that influences the chemotherapeutic response is the limited sensitivity of carcinoma of the cervix to cytotoxic agents and acquired drug resistance frequently following chemotherapeutic regimens. During the formation of the drug resistance, the worst is the development of multidrug resistance which can severely impede the efficacy of chemotherapy treatment. There are two categories of mechanisms of multidrug resistance for cervical cancer cells:①natural resistance, including the overexpression of P-glycoprotein and change in DNA topoisomerase enzymes activity;②cquired resistance, including the reduction in the intracellular accumulation of the drug, increase of the drug detoxification capacity of mercapto compounds (GSH, GST, MT) and increased DNA repair capacity.
P-glycoprotein (P-gp) is a transmembrane glycoprotein and is encoded by multidrug resistance gene (MDR1). P-gp is a member of sub-family B of the ATP-binding cassette (ABC) transporter superfamily, The protein displays basal ATPase activity, which is often (but not always) stimulated by substrates, and it hydrolyzes ATP to power active transport, generating a drug concentration gradient across the membrane and extrudes the drug out of the cells leading to the multidrug resistance (MDR). Almost50%of cancers have increased P-gp expression after treatment of natural chemotherapeutic regimens. Overexpression of P-gp is related with MDR in cervical cancer.
CIP2A is a newly identified oncoprotein that stabilizes c-Myc protein level by inhibiting the activity of protein phosphatase2A (PP2A) dephosphorylating c-Myc S62in cancer cells. We and other researchers have reported that CIP2A is overexpressed in many carcinomas such as gastric cancer, breast cancer, non small-cell lung cancer, acute myeloid leukemia, prostate cancer and cervical cancer. Overexpression of CIP2A promotes the malignant growth of cancer cells. Furthermore, CIP2A has been demonstrated as a major determinant mediating chemotherapeutic drug-induced apoptosis in hepatocellular carcinoma, breast cancer andleukemia cells. It is still unknown whether CIP2A is related with P-gp and involved in the multi-drug resistance of cervical cancer cells in vitro and in vivo.
1. CIP2A knockdown increased the sensitivity of HeLa cells to chemotherapeutics
After efficient depletion of CIP2A protein expression in HeLa cell,3chemotherapeutic agents, Dox, Cis and Pac, were used to observe the cytotoxic effect by MTT assay.
Dox, Cis and Pac, dose-dependently inhibited cell proliferation of HeLa cells. Such inhibition was facilitated after CIP2A siRNA knockdown. The data indicates that CIP2A might be associated with MDR of HeLa cells.
2. Expression of CIP2A and P-gp in cervical caner
We obtained103paraffin-embedded tissue blocks (43cervical adenocarcinoma,16CIN I,17CIN II,12CIN III and15normal cervical tissues) from Qilu Hospital for immunohistochemistry staining of CIP2A and P-gp. CIP2A and P-gp expression were absent in15normal cervical tissue samples and16CIN I and17CIN II samples. CIP2A was present in1of12CIN III samples (8.3%) and in16of43adenocarcinoma samples (37.2%). Meanwhile, P-gp expression was not detected in any CIN III samples but in13of43adenocarcinoma samples (30.2%). CIP2A was positively stained in cytoplasm and P-gp in the cell membrane. P-gp and CIP2A immunoreactivity were uniformly both in location and intensity on the same cancer specimens. Additionally, P-gp and CIP2A expression levels were positively correlated in cervical adenocarcinoma (r2=0.617, p<0.001). P-gp expression was associated with differentiation grade (p=0.029) of tumors but not age, tumor size, grade, tumor-node-metastasis stage or lymph-node metastasis. The data showed that CIP2A and P-gp expression might be linked in cervical adenocarcinoma.
3. CIP2A contributes to MDR of HeLa cells via P-gp
To further confirm that upregulation of P-gp by CIP2A, we performed the following experiments:examined P-gp, CIP2A protein levels in HeLa cells treated with Dox, Cis and Pac; examined P-gp, CIP2A protein levels in HeLa/Dox cells and P-gp protein levels after CIP2A knockdown by specific siRNA in HeLa/Dox cells; conducted Rh123efflux assay after CIP2A knockdown by specific siRNA in HeLa, HeLa/Dox cells.
The results demonstrated that protein levels of P-gp and CIP2A increase in drug-treated HeLa cells; the P-gp and CIP2A levels in the drug-resistant HeLa/Dox cells were remarkably elevated compared with the HeLa cell line, P-gp protein level was significantly decreased after CIP2A knockdown by specific siRNA in HeLa/Dox cells; Flow cytometry results revealed that when the CIP2A expression was knocked down by specific siRNA, the Rh123fluorescence signal in HeLa, HeLa/Doxcells was significantly increased and indicated that the function of P-gp was inhibited.
CIP2A might enhance the P-gp function via up-regulating the P-gp expression, and thus induce the drug resistance of cervical adenocarcinoma cells.
In summary, our study first demonstrated CIP2A is involved in multidrug resistance of cervical adenocarcinoma via upregulation of P-gp and CIP2A.CIP2Aand P-gp are positively related in cervical adenocarcinoma.CIP2A knockdown maybe increase the sensitivity of cervical adenocarcinoma cells to chemotherapeutics. Our study suggests, CIP2A is a good target for gene therapy for clinical application.
宫颈癌是全球范围内女性第二大常见恶性肿瘤,每年新发宫颈癌病例大约53万。尽管在发达国家随着筛查方法的改进,宫颈癌的发病率已经降低,但是在发展中国家,由于社会经济因素,宫颈癌的筛查方法不能广泛普及,因此宫颈癌仍然是威胁妇女的主要致死因素。
宫颈癌的发生有病毒方面的因素,高危型人乳头瘤病毒(High-risk type human papilloma virus, hr-HPV)感染与宫颈癌的发生密切相关。这一事实提供了预防宫颈癌的方法,即预防接种HPV疫苗阻止感染。尽管HPV疫苗已经上市,但是对于已经感染HPV或者患有免疫抑制的病人没有效果。
HPV是一类无包膜的小DNA病毒,可以在表皮和粘膜上皮细胞复制增殖。根据HPV的临床意义,可将其分为高危和低危型。持久的高危型HPV感染是宫颈癌发生的主要致病因子。全球大约70%的宫颈癌由HPV16,18型引起。高危型HPVs的两个癌蛋白E6和E7具有促进细胞增殖的作用,在宫颈癌的恶性转化过程中起重要作用。E6、E7分别与抑癌蛋白p53和pRb相互作用,促进它们的降解诱发肿瘤发生。尽管HPVs在宫颈癌的发生中发挥了重要的作用,但是只有一小部分感染了高危型HPVs的女性最终发展为宫颈癌,而且从最初感染HPVs到最终进展为宫颈癌,这一过程需要几十年的时间,提示高危型HPVs的感染对于宫颈的癌变是必要的但不是充分的条件。此外,有些宫颈癌检测不到HPVs。因此,在宫颈的癌变过程中,一定有一些其它协同因子或遗传事件参与其中。
肝激酶B1(liver kinaseB1,LKB1)又称丝氨酸-苏氨酸激酶(serine threonine kinase, STK11),最初在黑斑息肉综合征(Peutz-Jeghers syndrome, PJS)研究发现,LKB1基因突变是导致PJS发生的因素,PJS患者对胃肠道肿瘤,乳腺肿瘤,妇产肿瘤的易感性增加。继而在散发性肿瘤中,也发现了LKB1基因的体细胞突变,如非小细胞肺癌、结肠癌、乳腺癌和宫颈癌等,因而LKB1已作为一个比较公认的抑癌基因被广泛研究。LKB1抑癌的分子机制目前尚不十分清楚,现有的研究表明,LKB1是一种重要的上游蛋白激酶,LKB1与伪激酶STRAD和清道夫蛋白M025形成功能三聚体,这个复合体可以磷酸化至少14种在T环活化位点具有保守苏氨酸的丝氨酸-苏氨酸激酶,而使之激活。个研究非常多的LKB1底物是AMP-活化蛋白激酶(AMP-activated protein kinase,AMPK),AMPK是细胞、机体代谢的主要调节剂,LKB1可通过AMPK对外界各种生理病理刺激作出反应,例如：骨骼肌收缩,低氧,饥饿,H202,二甲双胍和苯乙双胍等。重要的是,LKB1通过激活AMPK调节细胞生长和细胞周期,活化的AMPK激活TSC2复合体和nTOR伴侣分子raptor,进而负向调控nTOR信号通路,抑制细胞生长。此外,LKB1参与调控细胞极性,细胞增殖,细胞迁移,细胞衰老,凋亡,DNA损伤反应,细胞分化和有氧糖酵解过程等,也与其抑制肿瘤发生的机制有关。
LKB1基因突变在宫颈癌中的研究最早始于1999年,根据不同的文献报道,宫颈癌中LKB1基因突变率在2-20%,但有关宫颈癌组织中LKB1蛋白表达状况的报道尚很少。
目前,宫颈癌细胞中LKB1抑制细胞增殖的分子机制研究主要集中于LKB1/AMPK通路。已有文献报道,在宫颈癌细胞HeLa、SiHa中过表达LKB1可以抑制其生长,且依赖于LKB1的激酶活性；LKB1可抑制HeLa细胞的锚定非依赖性生长,但对SiHa细胞没有此作用；应用AMPK激活剂(如AICAR、二甲双胍等)对于具有LKB1/AMPK/mTOR完整信号通路的宫颈癌细胞系有抑制作用。但是,LKB1抑癌分子机制的研究尚存在许多的未知,LKB1是否可调控更多的下游基因?是否有新的通路参与其抑癌作用机制?LKB1在宫颈正常组织及癌组织中的蛋白表达及功能?这些都是尚待解决的问题。深入研究LKB1的抑癌分子机制将有助于发现更多的关键分子,为治疗干预宫颈癌的发生发展提供新的思路和靶标。
基于LKB1在宫颈癌发生发展中的潜在重要作用,本研究在宫颈癌细胞中初步研究了LKB1的生物学活性,并对临床宫颈癌组织中LKB1蛋白的表达状况进行了检测。重要的是,我们首次利用基因芯片Microarray技术研究了LKB1对宫颈癌细胞基因转录谱的影响,通过生物信息学对差异表达基因进行了分析,继而在mRNA和蛋白水平给予验证。其中,在磷脂酰肌醇(PI3K)信号通路中,我们发现II型四磷酸肌醇磷酸酶(Inositol polyphosphate4-phosphatase type II, INPP4B)基因的表达在LKB1过表达后明显上调,进一步的细胞学实验进一步证明INPP4B是LKB1重要的下游靶分子。INPP4B也是一种潜在的肿瘤抑制因子,LKB1对INPP4B表达的调控作用尚未见报道。
一.LKB1表达对宫颈癌细胞增殖的影响
为检测LKB1蛋白表达对宫颈癌细胞增殖的影响,我们将编码LKB1的质粒成功转染了HeLa细胞(LKB1表达缺失)并经历短期G418筛选,Western blot检测此细胞中有明显的LKB1蛋白表达。进一步采用CCK-8方法检测细胞的增殖活性,结果显示,过表达LKB1的HeLa细胞增殖能力显著低于载体对照组。采用BrdU实验检测了细胞周期改变,结果发现HeLa细胞过表达LKB1后,位于S期的细胞数目显著降低,DNA复制合成减少,表明细胞生长增殖能力下降。以上结果说明LKB1蛋白抑制宫颈癌细胞HeLa的生长增殖。LKB1稳转细胞系HeLa-LKB1的研究结果表明：LKB1蛋白在稳转细胞系中有效表达,而且可检测至p-AMPK磷酸化水平的升高,说明在HeLa-LKB1细胞中LKB1可磷酸化其下游的MPK使其活化,LKB1具有活性,且LKB1-AMPK的信号通路是完整的。
二.LKB1蛋白在宫颈癌组织中的表达
为探究LKB1蛋白在临床宫颈癌组织中的表达状况,我们对78例宫颈癌(包括25例宫颈腺癌、53例宫颈鳞癌)和25例正常宫颈组织进行了免疫组化检测。结果表明,在44%(11/25)的宫颈腺癌、60.4%(32/53)的宫颈鳞癌组织中LKB1蛋白表达缺失。以上结果说明,宫颈癌组织中LKB1蛋白表达有较高的缺失率,这与公认的LKB1是一个抑癌基因一致。检测结果发现在正常宫颈组织中存在LKB1表达弱阳性或不表达,这与文献中报道的正常结直肠、胰腺组织LKB1的表达情况类似。
三.LKBl转录谱的研究以及LKB1的新靶标INPP4B的发现
尽管已知LKB1可以调控至少14种下游激酶,但是LKB1的作用机制仍然没有完全清楚,为探究宫颈癌细胞中受LKB1调控的基因,我们进行了Microarray基因芯片水平的转录谱研究。通过Microarray实验,在稳定表达LKB1蛋白的HeLa细胞系中,我们识别了222个LKB1调控的差异表达基因,其中下调的基因有117个,上调的基因有105个。qRT-PCR技术对七个差异表达基因LKB1、PLK2、ABCC2、GLP2R、KYNU、MAL2和AKAP12进行了mRNA水平的验证,结果证实Microarray数据真实反映了基因在转录水平的变化。
生物信息学方法进一步分析差异表达基因。基因本体方法(gene ontology,GO)对差异表达基因进行了分类,GO数据库分为生物学过程分类,分子功能分类以及细胞组分分类。分析结果显示,222个差异表达基因可归属在40个生物学过程分类,23个分子功能分类以及20个细胞组分分类中。LKB1调控的基因多参与信号转导,蛋白间相互作用以及定位于膜上。KEGG信号通路数据库对差异表达基因的分析发现了8个有统计学意义的生物学通路。其中两条通路是与代谢有关,分别是精氨酸和脯氨酸代谢通路、肌醇磷酸代谢通路,这与LKB1参与代谢的观点一致。另外一条重要的信号通路是磷脂酰肌醇(PI3K)信号通路,有文献报道LKB1-AMPK信号通路可与PI3K-Akt信号通路汇合在mTOR,参与调控细胞的生长与代谢。另外LKB1还参与了轴突发育通路和造血干细胞系分化通路。
在PI3K信号通路中的分析中我们发现一个重要的磷酸肌醇磷酸酶INPP4B基因的表达在LKB1过表达后明显上调。采用qRT-PCR进一步证实在过表达LKB1的HeLa中INPP4B mRNA水平显著增加。Western blot结果表明：LKB1稳转HeLa细胞中INPP4B蛋白水平升高1.4倍,同时在瞬转LKB1且经历G418短期筛选的HeLa细胞中INPP4B蛋白水平升高1.9倍。进一步采用特异性siRNA,在表达LKB1且LKB1-AMPK通路完整的宫颈癌CaSki细胞中敲低LKB1, Western blot检测INPP4B的表达变化,发现敲低LKB1后,INPP4B蛋白表达水平也随之降低。我们进一步检测了LKB1稳转HeLa细胞系中p-Akt水平改变,结果表明,与空载对照细胞HeLa-vetor相比,HeLa-LKB1细胞中Akt磷酸化水平明显下降。以上结果证明,抑癌蛋白LKB1可正向调控INPP4B的表达,INPP4B是LKB1下游的一个新靶蛋白。在过表达LKB1的HeLa细胞系中INPP4B表达上调,而磷酸化Akt水平下降,提示LKB1通过上调INPP4B参与了PI3K/Akt通路的负向调控,这可能是LKB1抑制细胞增殖的一个新通路。
综上所述,本研究探讨了抑癌蛋白LKB1在宫颈癌中的作用；并首次应用Microarray技术对表达LKB1的宫颈癌细胞进行了基因转录谱分析,进一步对发现的新靶标INPP4B进行了初步研究。研究证实,过表达LKB1对宫颈癌细胞生长增殖有抑制作用,在超过50%的宫颈癌组织中LKB1蛋白表达缺失,说明LKB1的抑癌活性在宫颈癌发生发展中具有重要作用。基于Microarray分析我们识别了222个受LKB1调控的基因,发现了8个有统计学意义的生物学通路。在PI3K信号通路中一个重要的抑癌基因INPP4B在LKB1过表达后明显上调,细胞学实验进一步证明INPP4B是LKB1下游的一个新靶标。在过表达LKB1的HeLa细胞系中INPP4B上调,而磷酸化Akt水平下降,提示LKB1通过上调INPP4B参与了P13K/Akt通路的负向调控,这可能是LKB1抑制细胞增殖的一个新通路。以上发现为深入研究LKB1调控的分子信号通路奠定了实验基础,为宫颈癌的治疗干预提供了新的分子靶标。第二部分CIP2A调控宫颈腺癌多药耐药的研究
宫颈癌是世界范围内最常见的妇科肿瘤疾病之一,严重威胁女性的身体健康,死亡率位居发展中国家女性肿瘤的第二位。近年来,全球宫颈癌的发病率呈现上升和年轻化的趋势。流行病学研究结果表明,高危型人乳头瘤病毒(Human papillomavirus,HPV)感染是妇女宫颈癌和宫颈上皮内瘤变的高危因素。
虽然宫颈癌筛查方法的普及和HPV预防性疫苗的出现有效降低了全球宫颈癌的发病率。然而,在发展中国家宫颈癌仍然是影响和威胁女性健康的重要因素。手术和放疗是宫颈癌的主要治疗方法,早期以手术治疗为主,中晚期多采用放射治疗。过去,一直认为宫颈癌属于化疗不敏感的肿瘤,仅在晚期及复发的患者中将化疗作为综合治疗的一部分。近年来,国内外学者对化疗在宫颈癌中的应用进行了大量的基础和临床研究,发现经化疗后患者5年生存率明显提高,因此确立了化疗在宫颈癌治疗中的重要地位。化疗的优势在于可以治疗肿瘤周围肉眼看不见的微小转移灶以及可能存在的全身亚临床转移和复发的病人。近10年来对宫颈癌的综合治疗日益受到关注,综合治疗已成为现代治疗宫颈癌的一个重要策略。
然而,尽管人们已经认识到化疗在宫颈癌中的作用,并且已经将化疗作为宫颈癌治疗的重要手段,但是宫颈癌多药耐药的产生却大大限制了化疗药物的疗效和应用,导致治疗失败,肿瘤复发。宫颈癌细胞产生多药耐药的机制分为两大类：①对天然化疗药物的耐药机制,主要包括P糖蛋白的过度表达及DNA拓扑异构酶酶含量与活性的改变；②对铂类化合物和烷化剂耐药的机制,主要包括药物在细胞内积聚减少,巯基化合物(GSH、GST、MT)对药物的解毒力增加,DNA损伤修复能力增加等。
P糖蛋白(P-glycoprotein, P-gp)是一种跨膜糖蛋白,由多药耐药基因(MDR1)编码。P糖蛋白是ABC转运蛋白家族的一员,通过与抗癌药物结合后再与ATP结合,经ATP供能将细胞内药物逆浓度梯度运出胞外,使细胞内药物浓度不断下降从而使之达不到有效杀伤浓度,最终导致肿瘤细胞耐药性的产生。在使用天然化疗药物后,几乎50%的肿瘤都有P-gp蛋白的表达增加。宫颈癌的耐药问题也与P-gp的表达增加密切相关。
Cancerous inhibitor of protein phosphatase2A (CIP2A)是一种2007年发现的癌蛋白,CIP2A可抑制PP2A对c-Myc62位丝氨酸(S62)的去磷酸化,从而增加细胞中c-Myc的蛋白水平。研究发现,CIP2A在很多人类恶性肿瘤中过表达,例如胃癌、乳腺癌、非小细胞肺癌、急性白血病、前列腺癌、宫颈癌等。CIP2A还可以促进细胞的恶性生长增殖,并与化疗药物导致肝细胞癌、乳腺癌、白血病细胞的凋亡有关。那么,CIP2A是否也和宫颈癌的耐药性相关?进而是否参与了宫颈癌多药耐药机制的产生?CIP2A在宫颈癌中与P-gp蛋白的表达有无关联?这些问题是我们此项研究的重点。
一.敲低CIP2A提高了宫颈癌细胞对化疗药物的敏感性
我们在宫颈腺癌细胞系HeLa中特异性敲除CIP2A后,使用临床常用的三种肿瘤化疗药物多柔比星(Dox)、顺铂(Cis)和紫杉醇(Pac)处理细胞,通过MTT实验观察药物对HeLa细胞的杀伤作用。
实验结果显示,多柔比星(Dox)、顺铂(Cis)和紫杉醇(Pac)三种化疗药物抑制HeLa细胞增殖,在特异性敲低CIP2A后,三种化疗药物对HeLa细胞增殖的抑制能力显著增加。这表明,在HeLa细胞中CIP2A与化疗药物疗效有关,敲低CIP2A能够提高HeLa细胞对化疗药物的敏感性,因此本部分实验提示CIP2A可能参与肿瘤的多药耐药(MDR)。二. CIP2A和P-gp蛋白的表达在宫颈癌组织中密切相关
我们在山东大学齐鲁医院收集了103例石蜡包埋组织块,其中包括15例正常宫颈组织、16例CIN I、17例CIN II、12例CIN III、43例宫颈腺癌,利用免疫组化技术检测CIP2A和P-gp的蛋白表达情况。实验结果显示CIP2A和P-gp在15例正常组织、16例CIN I、17例CIN II组织中均不表达；12例CIN III组织中有1例(8.3%)CIP2A阳性,在43例宫颈腺癌组织中有16例(37.2%)有CIP2A的表达；P-gp在12例CIN III组织中表达阴性,在43例宫颈腺癌组织中有13例(30.2%)有P-gp的表达。CIP2A主要表达于胞浆,而P-gp主要位于细胞膜上。免疫组化结果显示,CIP2A和P-gp的表达在同一个宫颈癌标本上强度一致。并且统计学分析显示在宫颈腺癌组织中,CIP2A和P-gp表达正相关((r2=0.617,p<0.001)),P-gp表达和病人年龄、肿瘤大小、临床分期、肿瘤淋巴结转移、淋巴转移无相关性,但与肿瘤分化成度有关(p=0.029)。以上结果显示,在宫颈腺癌组织标本中,CIP2A和P-gp表达成正相关。三. CIP2A通过调控P-gp蛋白影响宫颈癌的多药耐药
为了进一步验证CIP2A与P-gp蛋白之间的关系,我们进行了如下实验：多柔比星(Dox)、顺铂(Cis)和紫杉醇(Pac)三种化疗药物处理HeLa细胞,检测化疗药物作用后CIP2A与P-gp蛋白的表达水平；同时检测抗Dox HeLa细胞系HeLa/Dox中CIP2A与P-gp蛋白的表达水平,同时在该细胞系中特异敲低CIP2A后,检测P-gp蛋白的水平变化；在HeLa、HeLa/Dox细胞系中特异性敲低CIP2A后,罗丹明外排实验检测P-gp蛋白的功能。
研究结果显示,化疗药物处理HeLa细胞后,CIP2A与P-gp蛋白的表达水平均明显上升；耐药细胞系HeLa/Dox中CIP2A与P-gp蛋白的表达水平较HeLa细胞显著增高,在干扰CIP2A后,P-gp蛋白的表达水平随之降低；在HeLa、 HeLa/Dox细胞系干扰CIP2A后,罗丹明外排实验结果显示细胞内荧光信号显著增强,表明P-gp蛋白的药物外排功能受到抑制。上述结果提示,CIP2A通过P-gp蛋白实现对宫颈腺癌耐药的调控。
综上,本研究利用体内外实验首次验证了CIP2A参与宫颈腺癌多药耐药的形成,这种作用主要是CIP2A通过上调P-gp蛋白的表达来实现的。在宫颈腺癌组织中CIP2A和P-gp蛋白的表达呈正相关；敲低CIP2A可提高宫颈腺癌细胞对多种化疗药物的敏感性,P-gp蛋白的表达水平也随之降低。因此,我们的研究结果提示,CIP2A作为一个新的基因治疗靶标,在宫颈腺癌的综合治疗中有着潜在的应用前景。
Part One:Transcription profiling and molecular mechanism of the LKB1tumor suppressor in cervical cancer
Cervical cancer is the second most common cancer in women worldwide with an annual incidence of530,000cases. Although the incidence of cervical cancer in developed countries has decreased due to improved screening methods, it is still a main mortality factor for women in developing countries.
Cervical cancer has a viral infectious etiology. Infection with high-risk types of human papillomavirus (HPVs) is strongly-associated with the development of cervical cancer. This fact provides a great opportunity for primary prevention of cervical cancer by prophylactic vaccination to prevent cervical HPV infection. Although HPV vaccine has become available, they are less likely to be effective for those who have already been infected by HPV and those who are immunosuppressed.
Papillomaviruses are small DNA nonenvelopedviruses that replicate in epidermal or mucosal epithelial cells. HPVs can be classified into high-or low-risk types depending on their clinical associations. Persisting high-risk HPV infections are the most significant risk factor for development of cervical cancer. HPV types16and18are responsible for about70%of cervical cancers worldwide. The two oncogenes E6and E7of high-risk HPVs possess proliferation-stimulating activity and play a critical role during malignant transformation of cervical cancer. E6and E7interact with p53and pRB respectively and promote their degradation. Though HPVs play significant roles in the progression to cervical neoplasia, only a small percentage of high-risk HPV-infected women develop cancer eventually and it often takes decades after the initial infection, suggesting that infection by high-risk HPVs is not sufficient for cervical carcinogenesis. In addition, some cervical cancers are negative for HPVs. Therefore, there must be other cofactors or genetic events involved in cervical carcinogenesis.
The liver kinase LKB1(also known as serine threonine kinase, STK11) was originally identified as the causative gene of Peutz-Jeghers syndrome (PJS), an autosomal dominant disease. Patients with PJS have increased cancer predisposition in GI tract, breast, and gynecological organs. Importantly, somatic LKB1mutations are frequently found in significant number of sporadic non-small cell lung carcinomas (approximately30%)、colon cancer、breast cancer and cervical carcinomas (approximately20%). In recent years, LKB1has become more established as a tumor suppressor. However, the molecular mechanism of LKB1has not been fully understood. LKB1is a master upstream kinase that forms a complex with a pseudokinase STRAD and the scaffold protein MO25. This complex can phosphorylate at least14serine-threonine kinases with conserved threonine in the "T-loop" activation site. A well-known substrate of LKB1is AMP-Activated Protein Kinase (AMPK), which is the master regulator of cellular and organismal metabolism. AMPK subsequently activates the TSC2complex or mTOR-binding partner raptor, which negatively regulates mTOR signaling. LKB1is involved in response to diverse signals under physiological and pathological conditions via AMPK, e.g.:skeletal muscle contraction, hypoxia, starvation, H2O2, metformin and phenformin. In addition, LKB1exerts its effects on cell polarity, proliferation, migration, senescence, apoptosis, DNA damage response, differentiation and aerobic glycolysis.
Mutation of LKB1in cervical cancer was originally studied in1999. The frequency of LKB1mutation in cervical cancer is between2%to20%according to different reports. But the expression level of LKB1protein in cervical cancer is not known.
At present, the molecular mechanism of LKB1as a tumor suppressor in cervical cancer is mainly focused on LKB1/AMPK pathway. It was reported that overexpression of LKB1in HeLa and SiHa could inhibit the growth of cells depending on its kinase activity. Moreover, LKB1can inhibit the anchorage-independent growth of HeLa not SiHa. AMPK activators, such as:AICAR, Metformin etc., are able to inhibit the growth of cervical cancer cell lines which have intact LKB1/AMPK/mTOR pathway. However, there are still a lot of questions about LKB1as a tumor suppressor. Are there more downstream targets of LKB1? Any new pathways involved into tumor-suppressing activity of LKB1? The expression of LKB1protein in normal cervix and cervical cancer?Detailed study of the molecular mechanisms of LKB1will help find more key molecules, provide new insights and targets for the treatment of cervical cancer.
Given the importance of LKB1in tumor suppression and the potential role in development of cervical cancer, we characterized the biological activities of LKB1, performed the immunohistochemical (IHC) study for LKB1in cervical cancers and initiated the first microarray experiment to identify LKB1-regulated genes. We used the bioinformatics to analyze the differentially expressed genes and verified the data at the mRNA and protein levels. More importantly, we identified inositol polyphosphate4-phosphatase type Ⅱ, INPP4B from PI3K pathway was upregulated after LKB1overexpression. And it was further confirmed that INPP4B is an important downstream target of LKB1. INPP4B is also a potential tumor suppressor and the regulation of LKB1on INPP4B is not reported untill now.
1. Characterization of LKB1expressing cervical cancer cells
To examine the effects of LKB1expression on cervical cancer cells, we transfected a plasmid encoding LKB1into HeLa cells, which contains a deletion in the gene for LKB1, followed with short period of G418selection. Upon transfection of the plasmid encoding LKB1, it becomes readily detectable by western blotting. We then examined the proliferation of LKB1expressing HeLa cells using the CCK8assay. The data showed that,HeLa cells expressing LKB1proliferate significantly slower than the vector control cells. We also examined cell cycle profiles in HeLa cells transfected with LKB1by BrdU assay. We found that overexpression of LKB1significantly reduced the number of cells in S-phase. These results demonstrate that, after transient transfection of LKB1in HeLa, proliferation and percentage in S phase significantly decreased, so expression of LKB1inhibits cervical cancer cell proliferation. Next we also examined p-AMPK of HeLa cells stably expressing LKB1. We can detect the expression of LKB1and increase of p-AMPK in HeLa-GFP-LKB1cell. The data indicated that LKB1can activate AMPK and the pathway was intact.
2. LKB1expression in cervical cancer
To characterize the expression of LKB1protein in cervical cancer, we performed immunohistochemical analysis on a series of cervical carcinoma tissues as well as normal controls. Paraffin-embedded tissue blocks were obtained (78cervical cancer:25cervical adenocarcinoma and53cervical squamous carcinoma samples, and25normal cervical tissues) from Qilu Hospital of Shandong University. For cervical adenocarcinoma tissues,56%showed positive LKB1stain while the other44%stained negative. For squamous cell carcinoma of the cervix,60.4%(32/53) of the samples were negative for LKB1, only39.6%(21/53) showed positive stain. Among the cervical cancer tissues, more than50%of samples did not express LKBl. The results were consistent with the notion for LKB1as a tumor suppressor. The observation of weak or no LKB1protein expression in normal cervix was consistent with the reports of LKB1expression in normal epithelia of colorectum and pancreas.
3. Transcriptional profiling of LKB1and INPP4B as a LKB1-regulated target
Although it's well known that LKB1can activate at least14downstream kinases, molecular mechanism of LKB1was still not fully understood. Therefore we performed Microarray analysis to study transcriptional profiling of LKB1. We took advantage of the HeLa cell line stably expressing LKB1, to advoid the alteration of a large number of genes by transfection. We identified222LKB1-regulated genes by microarray,117of them were down-regulated while105were up-regulated after LKBl overexpression. We then performed real-time PCR (qRT-PCR) experiment to confirm the results obtained by microarray. LKB1and the other six randomly selected genes PLK2, ABCC2, GLP2R, KYNU, MAL2and AKAP12were verified at mRNA level. Thus the qRT-PCR data suggest that probe sets used in the microarray experiments are likely to accurately measure the levels of transcripts.
Next we performed bioinformatic analysis of genes differentially expressed in LKB1expressing HeLa cells. We first analyzed the differentially expressed genes using gene ontology (GO) system. In the GO database, the genes are classified into three ontologies, i.e., biological process, molecular function and cellular component. Based on the GO annotation, the222genes whose expression was significantly altered in LKB1expressing HeLa cells can be classified into40,23,20functional groups in the biological process ontology, molecular function ontology and cellular component ontology, respectively. Most LKB1-regulated genes were involved in signal transduction, protein binding and relocated on the membrane. Then we searched the differentially expressed genes against KEGG pathway maps. Eight biological pathways were identified to be significantly regulated. Two pathways involved in metabolism, i.e., arginine and proline metabolism and inositol phosphate metabolism were represented, which was consistent with a role of LKB1in metabolism. Notably, the phosphatidylinositol signaling system is also identified. It is known that the PI3K-AKT pathway and LKB1-AMPK pathway converge at mTOR. In addition, LKB1involved in axon guidance pathway and the hematopoietic cell lineage pathway.
We found inositol polyphosphate4-phosphatase type II (INPP4B) in PI3K pathway was up-regulated after LKB1overexpression. We confirmed the increase of INPP4B at mRNA level and protein level by qRT-PCR and Western blot. Western blot results demonstrated that INPP4B increased1.4fold in HeLa stably-expressing LKBl and1.9fold in HeLa cells transfected with LKB1and followed with short period of G418selection. Moreover, knock-down of LKB1in the cervical carcinoma CaSki cells, which have an intact LKB1-AMPK pathway, reduced the steady state level of INPP4B. We further examined the level of p-Akt in HeLa stably-expressing LKB1and found the significant decrease in the level of p-Akt compared to the control cell of HeLa-vector. These results provide further evidence for INPP4B upregulation by LKB1and being a target for LKB1. INPP4B upregulation and decrease in level of p-Aktin HeLa stably-expressing LKB1suggest that LKB1regulates PI3K/Akt pathway negatively by upregulation of INPP4B and maybe this is a new signaling pathway for inhibition of cell growth by LKB1.
In summary, we characterized the biological activities of LKB1in cervical cancer, performed transcriptional profiling of LKB1and found INPP4B as a new target in this study. The results showed that, transiently transfected LKB1inhibits the growth of cervical cancer cell, loss of LKB1expression was found in more than50%cervical cancer samples and identified LKB1-regulated222genes and8pathways. We performed bioinformatics analysis and therefore identified a target of LKB1in PI3K signaling pathway, INPP4B. INPP4B upregulation and decrease in level of p-Aktin HeLa stably-expressing LKB1suggest that LKB1regulates PI3K/Akt pathway negatively by upregulation of INPP4B and maybe this is a new signaling pathway for inhibition of cell growth by LKB1. This study sheds light on the novel signaling pathways regulated by LKB1and provides new therapeutic targets in cervical cancer in the future.
Part Two:Regulation of multidrug resistance of cervical adenocarcinoma by CIP2A
Cervical cancer is one of the most common cancer in women worldwide. It is a major problem for women's health and is the second mortality factor among female tumors in the developing countries. In recent years, the incidence of cervical cancer worldwide rises and patients become younger. Epidemiological studies suggest that infection of high-risk human papillomavirus (hr-HPV) is a risk factor for cervical cancer and cervical intraepithelial neoplasia.
The incidence of cervical cancer worldwide has decreased due to improved screening methods and HPV prophylactic vaccines. However, prevention and early screening for cervical cancer has not been popularized in developing countries, due to economic factors and other medical conditions. Cervical cancer is still a mortality factor to women's health. It's well known that surgery and radiation therapy are the main treatments for cervical cancer, surgical treatment for early disease, radiation therapy for advanced. In the past, cervical cancer was considered to be not sensitive to chemotherapy and used in patients with advanced and recurrent tumors as part of a multimodality therapy.However, people performed a lot of basic and clinical research about chemotherapy in cervical cancer, and found that5-year survival rate was significantly improved in patients, thus establishing chemotherapy as an important part in cervical cancer treatment. Chemotherapy can be used in patients with tumor micrometastases, subclinical metastasis and recurrence. During the past10years, multimodality therapy has been used including surgery, radiation, and adjuvant and neoadjuvant chemotherapy for management of cervical cancer.
Although the importance of chemotherapy in cervical cancer has been established, the crucial factor that influences the chemotherapeutic response is the limited sensitivity of carcinoma of the cervix to cytotoxic agents and acquired drug resistance frequently following chemotherapeutic regimens. During the formation of the drug resistance, the worst is the development of multidrug resistance which can severely impede the efficacy of chemotherapy treatment. There are two categories of mechanisms of multidrug resistance for cervical cancer cells:①natural resistance, including the overexpression of P-glycoprotein and change in DNA topoisomerase enzymes activity;②cquired resistance, including the reduction in the intracellular accumulation of the drug, increase of the drug detoxification capacity of mercapto compounds (GSH, GST, MT) and increased DNA repair capacity.
P-glycoprotein (P-gp) is a transmembrane glycoprotein and is encoded by multidrug resistance gene (MDR1). P-gp is a member of sub-family B of the ATP-binding cassette (ABC) transporter superfamily, The protein displays basal ATPase activity, which is often (but not always) stimulated by substrates, and it hydrolyzes ATP to power active transport, generating a drug concentration gradient across the membrane and extrudes the drug out of the cells leading to the multidrug resistance (MDR). Almost50%of cancers have increased P-gp expression after treatment of natural chemotherapeutic regimens. Overexpression of P-gp is related with MDR in cervical cancer.
CIP2A is a newly identified oncoprotein that stabilizes c-Myc protein level by inhibiting the activity of protein phosphatase2A (PP2A) dephosphorylating c-Myc S62in cancer cells. We and other researchers have reported that CIP2A is overexpressed in many carcinomas such as gastric cancer, breast cancer, non small-cell lung cancer, acute myeloid leukemia, prostate cancer and cervical cancer. Overexpression of CIP2A promotes the malignant growth of cancer cells. Furthermore, CIP2A has been demonstrated as a major determinant mediating chemotherapeutic drug-induced apoptosis in hepatocellular carcinoma, breast cancer andleukemia cells. It is still unknown whether CIP2A is related with P-gp and involved in the multi-drug resistance of cervical cancer cells in vitro and in vivo.
1. CIP2A knockdown increased the sensitivity of HeLa cells to chemotherapeutics
After efficient depletion of CIP2A protein expression in HeLa cell,3chemotherapeutic agents, Dox, Cis and Pac, were used to observe the cytotoxic effect by MTT assay.
Dox, Cis and Pac, dose-dependently inhibited cell proliferation of HeLa cells. Such inhibition was facilitated after CIP2A siRNA knockdown. The data indicates that CIP2A might be associated with MDR of HeLa cells.
2. Expression of CIP2A and P-gp in cervical caner
We obtained103paraffin-embedded tissue blocks (43cervical adenocarcinoma,16CIN I,17CIN II,12CIN III and15normal cervical tissues) from Qilu Hospital for immunohistochemistry staining of CIP2A and P-gp. CIP2A and P-gp expression were absent in15normal cervical tissue samples and16CIN I and17CIN II samples. CIP2A was present in1of12CIN III samples (8.3%) and in16of43adenocarcinoma samples (37.2%). Meanwhile, P-gp expression was not detected in any CIN III samples but in13of43adenocarcinoma samples (30.2%). CIP2A was positively stained in cytoplasm and P-gp in the cell membrane. P-gp and CIP2A immunoreactivity were uniformly both in location and intensity on the same cancer specimens. Additionally, P-gp and CIP2A expression levels were positively correlated in cervical adenocarcinoma (r2=0.617, p<0.001). P-gp expression was associated with differentiation grade (p=0.029) of tumors but not age, tumor size, grade, tumor-node-metastasis stage or lymph-node metastasis. The data showed that CIP2A and P-gp expression might be linked in cervical adenocarcinoma.
3. CIP2A contributes to MDR of HeLa cells via P-gp
To further confirm that upregulation of P-gp by CIP2A, we performed the following experiments:examined P-gp, CIP2A protein levels in HeLa cells treated with Dox, Cis and Pac; examined P-gp, CIP2A protein levels in HeLa/Dox cells and P-gp protein levels after CIP2A knockdown by specific siRNA in HeLa/Dox cells; conducted Rh123efflux assay after CIP2A knockdown by specific siRNA in HeLa, HeLa/Dox cells.
The results demonstrated that protein levels of P-gp and CIP2A increase in drug-treated HeLa cells; the P-gp and CIP2A levels in the drug-resistant HeLa/Dox cells were remarkably elevated compared with the HeLa cell line, P-gp protein level was significantly decreased after CIP2A knockdown by specific siRNA in HeLa/Dox cells; Flow cytometry results revealed that when the CIP2A expression was knocked down by specific siRNA, the Rh123fluorescence signal in HeLa, HeLa/Doxcells was significantly increased and indicated that the function of P-gp was inhibited.
CIP2A might enhance the P-gp function via up-regulating the P-gp expression, and thus induce the drug resistance of cervical adenocarcinoma cells.
In summary, our study first demonstrated CIP2A is involved in multidrug resistance of cervical adenocarcinoma via upregulation of P-gp and CIP2A.CIP2Aand P-gp are positively related in cervical adenocarcinoma.CIP2A knockdown maybe increase the sensitivity of cervical adenocarcinoma cells to chemotherapeutics. Our study suggests, CIP2A is a good target for gene therapy for clinical application.
引文
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