CKS1B基因促进多发性骨髓瘤细胞增殖与病程恶化的非SKP2/P27依赖性机制研究
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摘要
多发性骨髓瘤(multiple myeloma,MM)是一种源于晚期B细胞的恶性肿瘤,其特征为骨髓中单克隆浆细胞无限增生及血清或尿中出现单克隆的免疫球蛋白,常伴有多发性溶骨性损害,高钙血症,贫血,肾脏损害,正常免疫机能受抑,病人预后极差。由于该病的分子病因学以及耐药机理还不十分清楚,目前尚无根治的有效手段。因此,深入多发性骨髓瘤的发病机理研究对其诊断和治疗将具有重要的意义。
多发性骨髓瘤细胞存在广泛的染色体变异,1号染色体1q21区连锁扩增被认为与高危型多发性骨髓瘤的预后密切相关,是目前国际上研究的热点。我们以前的研究中,已经发现70个表达与MM预后紧密联系的基因型,位于1号染色体1q21区的CDC28蛋白激酶调节亚基1B(CDC28 protein kinase regulatory subunit B,CKS1B)为其中之一。而CKS1B的表达水平与1q21区连锁扩增拷贝数呈正相关,骨髓瘤病人的预后呈负相关。有人报道CKS1B高表达与难治型多发性骨髓瘤病人和浆细胞型淋巴瘤疗效不佳有关,是预后不良的一个信号。而本课题组前期的结果显示,在MM肿瘤细胞中敲除CKS1B基因,能引起致命的细胞凋亡和生长抑制。这些证据提示,CKS1B在MM肿瘤细胞的增殖和生存中发挥至关重要的作用,但是CKS1B在骨髓瘤细胞生长和疾病进程中的功能仍然需要更多的实验来验证。
很多实验室已经证实CKS1B参与细胞周期调控。蛋白CKS1是CKS/Suc1家族成员之一,是真核细胞周期素依赖性蛋白激酶(CDKs)的重要调节单位。CKS1B是SCFSKP2-CKS1泛素链接酶复合物的重要组成蛋白,CKS1能够通过与SKP2结合,调节泛蛋白化反应并降解CDKs抑制蛋白p27最后推动细胞周期从G1期进入S期,完成细胞分裂繁殖。我们之前的研究也证实了敲除CKS1B基因后,引起在RNA和蛋白水平SKP2下调,和P27上调。CKS1-/-缺陷小鼠由于CDKs抑制蛋白p27Kip1不能被降解,细胞分裂增殖过程被抑制而导致这种小鼠呈现典型的身体纤小。然而令我们感兴趣的是,敲除基因SKP2只引起了部分的细胞生长受抑制和微弱的MM肿瘤细胞凋亡,其效果远远弱于CKS1B基因敲除引起的细胞凋亡和生长抑制。并且,我们在特异性p27Kip1但等位基因缺失的骨髓瘤细胞系中敲除基因CKS1B,仍然导致细胞严重的凋亡和生长抑制。这提示CKS1B可能通过除SKP2/p27Kip1之外的机制来影响骨髓瘤细胞的生长和增殖。
在本论文中,为了进一步阐述CKS1B影响多发性骨髓瘤细胞生存和增殖的分子机制,以及研究CKS1B在多发性骨髓瘤疾病预后中所起的作用,我们进行了如下研究,并取得了相应的结果：
我们在以前的研究中,已经知道CKS1B是一个骨髓瘤高危基因,其表达水平与疾病的预后呈负相关。在本论文中,用骨髓瘤病人骨髓中肿瘤细胞mRNA做基因芯片分析,我们比较了51个病人从刚确诊到复发后的CKS1B表达水平的变化,发现有76%的病人在复发后CKS1B水平明显增高。在刚确诊为骨髓瘤时这些病人CKS1B表达信号平均值为1398(低至370,高至4433),而复发后的平均值为2174(低至405,高至9867)。并且,在这51名病人中有76%复发后CKS1B水平表达增高,其中51%增高水平超过1.5倍。Kaplan-Meier生存曲线分析结果显示,在以初诊-复发配对比较的51位病人中,和那些从初诊断到复发CKS1B表达水平没有明显增高的病人相比,增高1.5倍以上的36位病人复发后4年的生存率显著性的低。
以上证据进一步证实了CKS1B在多发性骨髓瘤的诊断阶段是个诊断指标,并且也是个预示病人预后不良的指标。
CKS1B在多发性骨髓瘤中随着疾病进程中表达增高,这提示在疾病开始的时候CKS1B可能只在小部分的骨髓瘤细胞中高表达,而目前的治疗手段不能有效的杀死这群CKS1B特异高表达的细胞,因此导致病人的治疗失败和骨髓瘤复发。
为了验证这个假说,进一步阐述CKS1B在多发性骨髓瘤中起的作用,我们利用lentenvirus病毒系统转染骨髓瘤细胞,建立了CKS1B过表达的细胞系。细胞分别在含1%、5%、10%胎牛血清的培养基中培养7天,与转染空白载体的对照组(empty vector, EV)细胞相比,CKS1B高表达的骨髓瘤细胞表现出更高的增殖率。这一结果在在含1%胎牛血清的培养基中更为明显。这部分实验证明,CKS1B能促进骨髓瘤细胞的增殖。
接着,我们进一步验证了CKS1B对骨髓瘤细胞耐药的诱导作用。CKS1B转染骨髓瘤细胞系OCI-MY5和XG1,含5nM的化疗药物硼替佐米(Velcade, vel)的培养基中培养48小时,计数细胞生长和存活率。与EV细胞和无药物处理细胞相比,CKS1B转染的细胞表现出较低的细胞死亡率和生长抑制,对疗药物硼替佐米不敏感。我们继续检测了其他的化疗药物,发现CKS1B的骨髓瘤细胞对化疗药浓度为100nM阿霉素和浓度为100nM的依托泊苷存在明显的药物抵抗。
以上实验证明了CKS1B高表达诱发了骨髓瘤细胞对多种抗肿瘤药物耐药。
本课题组为了研究CKS1B基因促进骨髓瘤生长和疾病进程的机制,前期工作已经建立了可诱导性CKS1B基因敲除、SKP2基因敲除以及p27过表达的骨髓瘤细胞系。在骨髓瘤细胞中,CKS1B基因敲除引起SKP2表达降低和p27表达上调,并且导致强烈的细胞凋亡和生长抑制。然而,敲除SKP2基因后并只引起微弱的部分细胞凋亡,并没有CKS1B敲除引起的细胞凋亡那么严重；而p27过表达使骨髓瘤细胞停滞于细胞周期G1期,细胞分裂增殖受阻。并且,值得注意的是KMS28PE是特异性p27基因缺乏骨髓瘤细胞,CKS1B基因敲除照样引起了严重的细胞凋亡。
这些试验结果提示CKS1B是通过SKP2-p27依赖性和非依赖性途径影响骨髓瘤细胞生长和生存,并且SKP2-p27非依赖性途径可能更重要一些。
为了进一步证实CKS1B影响骨髓瘤细胞生长的机制中的确存在SKP2-p27非依赖性途径,在351个接受TT2治疗方案的多发性骨髓瘤新诊断病人中,我们利用Kaplan-Meier生存曲线分别比较CKS1B高／低表达、SKP2高／低表达以及p27低／高表达病人的生存率。结果显示,CKS1B高表达的病人生存期明显短于低表达的病人,P<0.001；而SKP2高、低表达的病人生存时间没有明显的差异；p27低、高表达的病人生存时间也没有明显的差异。
这个临床证据表明,CKS1B、SKP2和p27对多发性骨髓瘤疾病进程的影响不同,只有CKS1B的表达显著影响疾病的进行和恶化。
对351个新诊断的多发性骨髓瘤病人进行基因普表达分析,通过聚类分析得到了CKS1B高／低表达、SKP2高／低表达以及p27低／高表达病人中表达明显差异的基因群。通过ingenuity信号通路分析软件分别分析这3群基因群参与的信号通路,分析结果显示了CKS1B、SKP2、p27参与的主要信号通路调控图。比较结果证实了,骨髓瘤中CKS1B参与的主要信号通路与SKP2、p27的大不相同,除相同的G1／S期细胞周期调控通路外,CKS 1B主要涉及DNA修复、MAPK信号传导通路。
根据信息学分析的提示,我们在CKS1B基因敲除骨髓瘤细胞系OCI-MY5中,通过Western-Blot筛选了MAPK, NF-kB, TP53, PI3K/AKT, STAT3等重要细胞信号途径的主要信号分子。检测到在骨髓瘤细胞OCI-MY5中,CKS1B基因敲除后引起下列信号分子蛋白表达下调：p-MEK1/2, p-ERK1/2, p-STAT3, MCL-1,和p-BCL2。为了确证上述结果,我们对另外2个骨髓瘤细胞系KMS28PE和XG1进行了重复验证,得到了相同的结果。
为了进一步明确STAT3, MEK/ERK,BCL2是CKS1B的下游调控分子,我们通过Western-Blot检测了CKS1B cDNA转染细胞系OCI-MY5和XG1中以上分子的表达。结果显示,在CKS1B外源性高表达的细胞中,p-MEK1/2, p-ERK1/2, p-STAT3, MCL-1,和p-BCL2表达上调。这些结果证实,STAT3, MEK/ERK,BCL2是CKS1B的下游调控分子,CKS1B可能通过它们影响骨髓瘤细胞生长和生存。
为了验证CKS1B是否通过SKP2-P27kip1激活STAT3, MEK/ERK,BCL2信号通路,我们用Western-Blot检测以上信号通路分子在SKP2基因敲除以及p27过表达骨髓瘤细胞系中的表达情况。结果显示,SKP2基因敲除后,引起p-MEK1/2, p-ERK1/2, p-STAT3,和MCL-1表达上调,而P-BCL2无显著变化。P27KIP1通过稳定转染在骨髓瘤细胞中高表达后,p-MEK1/2, p-ERK1/2, p-STAT3,和MCL-1的表达上调,而p-BCL2无显著变化。实验结果证实,SKP2基因敲除或者P27KIP1基因高表达引起的信号通路改变,与CKS1B基因敲除所导致的信号通路改变,并不完全一致。这证明了CKS1B高表达激活了STAT3, MEK/ERK和BCL2信号途径,并且这种激活并不依赖SKP2-P27KIP1途径。
胰岛素样生长因子1(Insulin-like growth factorⅠ, IGF-1)是得到公认的骨髓瘤细胞生长因子,能激活STAT3信号途径,促进STAT3磷酸化。为了研究STAT3在CKS1B促进骨髓瘤细胞增殖和生存中所发挥的作用,我们用含IGF-1(100ng/ml)的培养基培养CKS1B基因敲除的KMS28PE,OCI-MY5,和XG1细胞72小时。Western-Blot证实,在IGF-1培养的细胞p-STAT3, MCL1表达增高。CKS1B基因敲除的细胞,通过IGF-1上调p-STAT3的表达后,细胞凋亡和抑制生长被部分阻止。以上结果证明了CKS1B通过激活STAT3/MCL1影响骨髓瘤细胞的增殖和生存。
信号分子STAT3能调节许多下游基因,参与细胞的生长和增殖。为了寻找CKS1B激活STAT3之后调控的下游基因,我们检测了与细胞生长和凋亡相关的基因MCL1。MCL1基因能通过抑制凋亡过程从而促进细胞增殖,实验结果发现,CKS1B基因敲除引起了MCL1蛋白水平表达下调,并且CKS1B过表达细胞中MCL1蛋白水平明显上调；但是SKP2基因敲除或者P27过表达并没有引起MCL1表达水平的改变。用IGF-1刺激骨髓瘤细胞引起STAT3表达增加后,MCL1的表达也显著增高.这些证据证明,CKS1B激活STAT3通路,然后引起MCL1转录激活,影响了细胞的增殖。
抗生素肖夫齐特(Nifuroxazide, Nif),已被证明是JAK/STAT信号通路的特异性抑制剂,能够在骨髓里细胞中抑制STAT3的磷酸化。为了证明JAK/STAT3是CKS1B下游信号通路并观察靶向阻断STAT3通路对CKS1B高表达骨髓瘤细胞生长的影响,,我们将CKS1B转染的骨髓瘤细胞OCI-MY5和XG1,在含10nM Nif的培养基中培养48小时。WB验证,Nif处理后的细胞p-STAT3蛋白水平下降。与EV细胞相比,CKS1B转染的OCI-MY5和XG1细胞有更高的细胞死亡率。CKS1B高表达的细胞对STAT3特异性抑制剂Nif诱发的细胞凋亡更敏感。
由于市场上没有MEK/ERK特异性激活剂,我们在CKS1B基因敲除后的细胞中,通过Lenti Virus转染MEK1-cDNA,使MEK1基因特异性高表达。WB验证MEK1转染细胞后p-MEK1/2和p-ERK1/2表达大幅度增加,并且磷酸化p-BCL2被激活。说明转染MEK1导致的MEK/ERK通路的激活,抵消了CKS1B基因敲除导致的bcl2下调,从而证明BCL2是MEK/ERK下游调控分子。MEK1基因高表达,同时CKS1B基因敲除的骨髓瘤细胞KMS28PE,OCI-MY5,和XG1,虽然仍存在细胞凋亡和生长受抑制,但是与单纯CKS1B基因敲除的细胞相比较,细胞死亡率显著性降低,存活细胞总数也显著性增多。由此证明,MEK/ERK的外源性高表达和磷酸化激活,部分抵消了CKS1B基因敲除引起的骨髓瘤细胞死亡和生长抑制。以上研究证明,CKS1B通过激活MEKK/ERK通路,以及其下游的BCL2通路,影响骨髓瘤细胞的增殖和生存.
U0126是MAPK通路抑制剂,能特异而强烈的抑制MEK1／2分子的活化。为了观察靶向阻断MEK/ERK通路对CKS1B高表达骨髓瘤细胞生长的影响,我们用含10μM U0126的培养基培养CKS1B转染的骨髓瘤细胞OCI-MY5和XG1,48小时时观察细胞生长状况。WB证实,与对照组相比与U0126共培养的骨髓瘤细胞,p-MEK1/2和p-ERK1/2蛋白水平下降。与对照EV细胞相比,CKS1B转染的OCI-MY5和XG1细胞有更高的细胞死亡率。CKS1B高表达的细胞对MEK特异性抑制剂U0126引起的细胞生长抑制和死亡更敏感。
实验结果已经提示BCL2是CKS1B和MEK/ERK信号通路的下游调控分子,为了确证BCL2在CKS1B促进骨髓瘤细胞生长的机制所起的作用,我们在CKS1B基因敲除后细胞中,通过LentiVirus转染BCL2-cDNA,使BCL2过表达。Western-Blot验证p-BCL2磷酸化被激活。BCL2过表达,同时CKS1B基因敲除的骨髓瘤细胞,虽然仍存在细胞凋亡和生长抑制,但是与单纯CKS1B基因敲除的细胞相比较,细胞死亡率显著性降低,存活细胞总数也显著性增多。由此证明,BCL2的外源性高表达和BCL2磷酸化,部分抵消了CKS1B基因敲除引起的骨髓瘤细胞死亡和生长抑制。以上研究证明了,BCL2在CKS1B促进骨髓瘤细胞的增殖和生存的过程中发挥重要作用。
为了观察靶向阻断BCL2通路对CKS1B高表达骨髓瘤细胞生长的影响,我们用BCL2特异性抑制剂处理CKS1B转染的骨髓瘤细胞OCI-MY5和XG1,在含10nM的BCL2特异性抑制剂(2,9-Dimethoxy-11,12-dihydrodibenzo[c,g][1,2]-diazocine 5,6-dioxide and 5,5'-Dimethoxy-2,2'-dinitrosobenzyl)的培养基中培养48小时。WB验证被BCL2抑制剂处理的细胞p-BCL2蛋白水平下降。与对照EV细胞相比,CKS1B转染的OCI-MY5和XG1细胞有更高的细胞死亡率。CKS1B高表达的细胞对BCL2抑制剂的杀细胞作用尤其敏感。
由于CKS1B通过激活MEK/ERK和STAT3信号通路促进骨髓瘤细胞生长,靶向于这2条通路的药物联合应用可能会对骨髓瘤细胞造成更强有力的杀伤效果。
我们使用STAT3抑制剂联合MEK1抑制剂,或者联合BCL2抑制剂,处理CKS1B高表达的骨髓瘤细胞OCI-MY5Y。结果显示联合应用这2条信号通路抑制剂,都对CKS1B高表达细胞和空白对照细胞造成强烈的细胞毒作用,而CKS1B过表达细胞的死亡率显著更高,效果强于单独用药。我们又在CKS1B高表达的细胞XG1中重复了这一实验,得到相同的结果。这一实验进一步证实了,与CKS1B表达低的骨髓瘤细胞相比,对CKS1B高表达的骨髓瘤细胞应用STAT3和MEK/ERK抑制剂治疗方案,疗效会更好。
CKS1B在多发性骨髓瘤病人中高表达已经被多个研究者所证实,而CKS1B在骨髓瘤细胞中增高的机制,除了与染色体1q21区域扩增外相关,并无更多的研究。
有文献报道在人骨肉瘤中,转录因子FOXM1能上调CKS1的表达,我们的临床观察和实验室资料也发现FOXM1高表达的MM病人预后差。为了研究FOXM1与CKS1B之间的关系,我们建立了FOXM1高表达骨髓瘤细胞系,WB验证FOXM1过表达引起CKS1B蛋白水平上调,和P27蛋白水平下调；而WB也显示,CKS1B基因敲除后并没有引起FOXM1表达水平的变化,而P27过表达则导致FOXM1表达增加。因此FOXM1是CKS1B的上游调控基因,能抑制CKS1B从而减少P27的降解,但是当P27的表达增高时,反过来又促进了FOXM1的表达,FOXM1-CKS1B-P27之间存在相互作用的调控关系。
小结：本课题的研究,探讨了CKS1B在骨髓瘤中过表达的机制,证实了骨髓瘤细胞中CKS1B接受转录因子FOXM1的正向调控；证明了CKS1B基因促进骨髓瘤细胞生长存活和对多种抗肿瘤药物耐药疾病；阐述了CKS1B促进骨髓瘤细胞生长的SKP2-p27非依赖性机制,证实CKS1B通过激活STAT3,MEK/ERK/BCL2信号通路促进骨髓瘤细胞生长和存活,并且证实CKS1B过表达细胞对靶向阻断STAT3,MEK/ERK/BCL2通路的药物特别敏感,为临床治疗难治型特别是CKS1B高表达型多发性骨髓瘤提供了实验室证据和思路。
Multiple myeloma (MM) is a plasma cell malignancy, which remains largely incurable with current therapeutic strategies. The molecular bases of MM progression and drug-resistance are not completely understood. Gain of chromosome 1q21 is one of the most recurrent chromosomal aberrations observed in MM and is correlated with poor prognosis. We previously identified a 70 high-risk signature pattern in MM, with overrepresentation of over-expressed genes mapping to chromosome 1q, further supporting the hypothesis that 1q21 gain is important in MM progression. High expression of CKS1B, a Cdc28 protein kinase regulatory subunit 1B, mapping to the 1q21 amplicon and one of the 70 high-risk signature gene pattern, was inversely associated with survival in MM. Alternatively, knockdown of CKS1B in MM cells potently induced growth inhibition and apoptosis, suggesting that CKS1B plays a crucial role in MM cell survival. However, the functional role of CKS1B in MM cell survival and MM disease progression remains to be elucidated.
CKS1B has a well-documented role in the cell-cycle regulation. Cks1 is a member of the Cks/Suc1 family of proteins, which are essential components of cyclin-dependent kinases (CDKs) that regulate mitosis in all eukaryotes. CKS1B was identified as an essential accessory protein to the SCFSKP2-CKS1 ubiquitin ligase complex. In this complex, Cksl enhances the interaction between SKP2 and p27Kip1 (a CDK inhibitor),, resulting in cell proliferation. The role of Cks1 in regulating p27Kip1 proteasome degradation in the cell cycle is particularly evident in CKS1-/-mice. The key feature of these mice is their small body size, which is due to lack of degradation of the G1-/S-phase CDK inhibitor p27Kip1. Interestingly, besides influencing cell growth and survival through regulation of p27Kip1, silencing of CKSIB also induces cell death and inhibits growth in MM cells in the presence of a bi-allelic deletion of the CDKN1B (p27Kip1) locus, indicating that CKSIB mediates its effects on cell growth and survival also through mechanisms that are independent p27Kip1 and SKP2.
In this study, we found that forced expression of CKS1B by lentivirus vector-mediated CKS1B-cDNA transfection in MM cells increased cell proliferation and multidrug-resistance, providing direct evidence of the crucial role of CKSIB in MM progression. Furthermore, we also identified STAT3 and MEK/ERK/BCL2 pathways to be downstream targets of CKS1B activation independent on the complex of SKP2/p27Kip1, responsible for MM cell growth and survival.
1. CKS1B expression increased in relapsed MM and conferred a short post-relapse survival.
Our previous studies showed that CKS1B was one of the 70 high-risk genes, which was inversely associated with survival in newly diagnosed MM. In this study, we compared CKS1B expression from 51 patients with paired baseline (diagnostic) and relapse samples. The median signals of CKS1B from microarray data at diagnosis and at relapse were 1398 (range:370-4433) and 2174 (range:405-9867), respectively. CKSIB expression increased in 76% relapsed MMs and was in more than 1.5 folds in 51%.
As we expected, patients from the 51 paired relapses who had CKS1B expression in quartile 4 (high-risk) at baseline and receiving various salvage therapies had the worst 4-year post-relapse survival compared with those who had CKS1B expression in quartiles 1 to 3 (low-risk) at baseline (P= 0.0012). The quartile 4 reference line is taken from the complete (n=351) sample of arrays at diagnosis. Interestingly, among 38 relapse patients with low CKS1B expression (quartiles 1-3) at baseline, patients increased CKS1B expression at least 1.5 folds at relapse had inferior 4-year postrelapse survival compared with those lacking CKS1B up-regulation at relapse (P=0.032). Furthermore, among 36 relapsed patients with high CKS1B expression at relapse,4-year postrelapse survival in patients with high CKS1B at baseline was significantly poor compared with those with high CKS1B expression only at relapse. These data further confirmed that CKS1B expression is a prognositic marker at the stage of newly diagnosis in MM and also demonstrated that CKSIB expression can be used to predict patient survival in postrelapse MM.
2. CKS1B over-expression promotes MM cell proliferation and drug-resistance.
Increased expression of CKS1B is a progression event suggesting that CKS1B may be heterogeneously expressed in a small population of myeloma cells at diagnosis, and current treatments ineffectively eliminate the small populations of CKS1B high-expression myeloma cells, which results in myeloma relapse and treatment failure.
To further investigate the functional role of CKS1B played in MM cells, CKS1B was over-expressed in OCI-MY5 and XG1 MM cells by lentivirus vector-mediated CKS1B-cDNA transfection. CKS1B-transfected MM cells cultured respectively in medium with 1%、5%、10%FBS showed higher proliferation ratio than MM cells transfected by empty vector(EV).
To test the hypothesis that MM cells with high expression of CKSIB are more drug-resistant and responsible for MM poor prognosis, CKS1B-transfected OCI-MY5 and XG-1 cells were treated with bortezomib (Vel) at a dose of 5 nM for 48 hours. Cell growth and cell survival were examined. Untreated and EV-transfected cells with or without bortezomib were used as controls. Bortezomib treatment induced significantly less growth inhibition and cell death in CKS1B-transfected cells compared with EV-transfected controls (P<0.05). Similarly, treatment of doxorubicin (Dox) 100nM and etoposide (Epo) 100nM for 48 hours, induced significantly less growth inhibition and cell death in CKS1B-transfected cells compared with EV-transfected controls (P< 0.05). Clearly, increased CKS1B expression induced chemotherapeutic resistance in myeloma.
3. CKS1B over-expression activates STAT3 and MEK/ERK through SKP2 and p27Kip1-independent pathways.
To identify these CKSIB-mediated SKP2/p27Kip1-independent signaling pathways, western blots were applied to screen for activation of the key signaling pathways which relate to cell survival and apoptosis, including MAPK, NF-κB, TP53, PI3K/AKT and STAT3 using OCI-MY5 cells after CKS1B-silencing by specific CKS1B-shRNA transfection. Decreased phosphorylated (p)-MEK1/2, p-ERK1/2, p-STAT3 and p-BCL2 were detected in CKS1B silenced OCI-MY5 cells compared with wild-type cells and cells transfected with a non-targeting scramble sequence (SCR). Similar results were also observed in CKS1B silenced KMS28PE and XG-1 cells.We further examined the alteration of STAT3, MEK/ERK and BCL2 in CKS1B-transfected OCI-MY5 and XG1 cells. Increased levels of p-MEK1/2, p-ERK1/2, p-STAT3 and p-BCL2 were observed in CKS1B-transfected OCI-MY5 and XG-1 cells compared with the EV-transfected controls. These results strongly suggest STAT3, MEK/ERK, and BCL2 are the downstream signaling pathways of CKS1B, which may mediate MM cell growth, survival, and disease progression.
To examine the functional relationship between SKP2 and these CKS1B activated STAT3, MEK/ERK and BCL2 signaling pathways, SKP2-shRNA was used to silence SKP2 in KMS28PE,OCI-MY5, and XG-1 cells and protein levels of p-MEK1/2, p-ERK1/2, p-STAT3, and p-BCL2 in these cells were examined. Wild-type (WT) and SCR-transfected cells were used as controls. SKP2-knockdown resulted in an increase rather than a decrease of p-MEK1/2, p-ERK1/2 and p-STAT3 levels in KMS28PE,OCI-MY5 and XG-1 cells, and with no remarkable change in p-BCL2 level. We then examined the functional relationship between p27Kip1 and STAT3, MEK/ERK and BCL2 by over-expressing p27Kipl in KMS28PE,OCI-MY5 and XG-1 cells using a lentivirus system. Western blots detected increased levels of p-MEK1/2 and p-ERK1/2 in p27Kip1-transfected myeloma cells with no remarkable effect on p-STAT3,p-BCL2 compared with WT and EV controls.
4. [Activation of STAT3 is involved in CKS1B-mediated myeloma cell growth and survival]
A specific STAT3 inhibitor Nifuroxazide at the dose of 10 nM for 48 hours was used to treat CKS1B-transfected OCI-MY5 and XG-1 cells. Nifuroxazide treatment decreased p-STAT3 in both EV-and CKS1B-transfected myeloma cells compared with untreated controls by western blot, confirming its specificity. Nifuroxazide-induced cell growth and cell death were also evaluated in CKS1B-transfected OCI-MY5 and XG-1 cells. Cells were treated with 10 nM Nifuroxazide for 48 hours, Nifuroxazide treatment induced more inhibition of cell growth and more increase in cell death in CKS1B-transfected cells compared with EV-transfected cells, indicating that myeloma cells with higher CKS1B-expression levels are more sensitive to STAT3 inhibition than cells with lower CKS1B-expression.
Insulin-like growth factorⅠ(IGF-1) is a well-recognized myeloma cell survival factor activating the STAT3 signaling pathway. To further confirm the functional role of STAT3 signaling in CKS1B-mediated myeloma cell growth and survival, CKS1B-silenced KMS28PE, OCI-MY5 and XG-1 cells were treated with IGF-1 (100ng/ml) for 72 hours. Western-blots detected increased p-STAT3 in these CKS1B-silenced myeloma cells compared with untreated, SCR- or CKS1B-shRNA-transfected controls, confirming activation of STAT3 in IGF-1-treated cells. The effects of IGF-1 treatment on CKS1B-shRNA-induced myeloma cell death and growth inhibition were evaluated. IGF-1 treatment partially abrogated growth inhibition and cell death induced by CKS1B-knockdown (P<0.05).
STAT3 regulates the expression of many target genes involved in cell survival and growth. To investigate whether CKSIB activates STAT3 signaling through regulation of STAT3 target genes, the protein of cell growth and apoptosis related gene MCL1, was measured by western blot. The level of MCL1, a gene that enhances cell survival by inhibiting apoptosis, was decreased in CKSIB silencing MM cells and increased in CKS1B over-expressing MM cells; whereas MCL1 expression did not show alteration in MM cells manipulated by inhibition of SKP2 and over-expression of p27Kip1. Furthermore, MCL1, following STAT3 expression pattern, was significantly increased in IGF1 treated MM cells inhibited CKS1B expression by CKS1B-shRNA.These data provide evidence that CKSIB regulates STAT3 signaling dependent on the STAT3 transcription of MCL1.
5. Activation of the MEK/ERK signaling pathway is also involved in CKS1B-mediated myeloma cell growth and survival
A specific MEK inhibitor U0126 was used to treat CKS1B-transfected OCI-MY5 and XG-1 cells for 48 hours at the dose of 10μM. Cell lysates were prepared and subjected to western analysis using antibodies recognizing p-MEK1/2 and p-ERK1/2 proteins. U0126 decreased p- MEK1/2 and p-ERK1/2 in both EV-and CKS1B-transfected myeloma cells, confirming its specificity. Interestingly, U0126 treatment down-regulated BCL2 in myeloma cells, suggesting that BCL2 represents a downstream target of the MEK/ERK signaling pathway. We also evaluated U0126-induced cell growth and cell viability in CKS1B-transfected OCI-MY5 and XG-1 cells. Cells were treated with 10μM U0126 for 48 hours, U0126-treatment induced more cell growth inhibition and cell death in CKS1B-transfected cells than those in EV-transfected cells (P< 0.05).
Since inhibitors of MEK/ERK signaling pathway may induce some non-specific effects on myeloma cell growth and survival, we constitutively activated the MEK/ERK signaling pathway by lentivirus-vector-mediated MEK1-cDNA transfection in CKS1B-silenced KMS28PE,OCI-MY5 and XG1 cells. Western blots showed a larger increase of p-MEK1/2 and p-ERK1/2 levels in MEK1-cDNA and CKS1B-shRNA double-transfected cells compared with SCR-and CKS1B-transfected controls. MEK1-transfection also increased protein levels of p-BCL2 in CKS1B-silenced cells, indicating that MEK1-transfection could overcome CKS1B-knockdown-mediated inhibition of BCL2 signaling pathway, further demonstrating that BCL2 is the downstream target of CKS1B and MEK/ERK signaling. Although MEK1-cDNA and CKS1B-shRNA doubly-transfected cells clearly showed increased cell death and growth inhibition compared with SCR-transfected control cells (P< 0.05), these double-transfected cells exhibited significantly less cell death and growth inhibition than CKS1B-silenced myeloma cells (P<0.05), indicating that MEK1-transfection partially abrogated cell death and growth inhibition induced by CKS1B-knockdown.
Our results indicate that BCL2 is a downstream target of the CKS1B and MEK/ERK signaling pathway. Therefore, a specific BCL2 inhibitor (2,9-Dimethoxy-11,12-dihydrodibenzo[c,g][1,2]-diazocine 5,6-dioxide and 5,5'-Dimethoxy-2,2'-dinitrosobenzyl) at the dose of 10 nM for 48 hours was used to treat CKS1B-transfected OCI-MY5 and XG-1 cells. Treatment with this BCL2-inhibitor resulted in significantly more cell growth inhibition and cell death in CKS1B-transfected cells compared with EV-transfected cells, indicating that myeloma cells with higher CKS1B-expression are more sensitive to BCL2 inhibition than cells with lower CKS1B-expression. We subsequently over-expressed BCL2 by lentivirus-mediated BCL2 cDNA transfection in CKS1B-silenced KMS28PE, OCI-MY5 and XG-1 cells. Cells were cultured for 4 days and western blots confirmed increased p-BCL2 levels in BCL2 cDNA and CKS1B-shRNA doubly-transfected cells. The effects of BCL2-transfection on CKS1B-shRNA induced myeloma cell growth inhibition and death were also evaluated. Although BCL2-cDNA and CKS1B-shRNA doubly-transfected cells clearly showed cell growth inhibition and death compared with SCR-transfected control cells (P<0.05), BCL2 transfection partially abrogated myeloma cell growth inhibition and death induced by CKS1B-silencing (P< 0.05).
6. Combination of STAT3 and MEK1 inhibitors induced synergistic cytotoxicity on myeloma cells with high CKS1B expression
Since the STAT3 and MEK/ERK signaling pathways were both involved in CKS1B-induced myeloma cell growth and survival, a combined targeting of these signaling pathways might induce more cytotoxicity in myeloma cells. CKS1B-transfected OCI-MY5 cells were treated with combination of STAT3 and MEK1 inhibitors (i.e. U0126 [10μM] plus Nifuroxazide [10 nM]), or combination of STAT3 and BCL2 inhibitors (i.e. U0126 [10μM] plus BCL2 inhibitor [10 nM]) for 48 hours. EV-transfected cells treated with the same combinations were used as controls. Untreated CKS1B-and EV-transfected cells also served as controls. The combinations of the STAT3 with MEK1 and the STAT3 with BCL2 inhibitors induced significant less cell growth and more cell death in CKS1B-transfected OCI-MY5 compared with EV-transfected control cells (P<0.05). CKS1B-transfected XG-1 cells were also treated with the same combinations for 48 hours. Similar to the CKS1B-transfected OCI-MY5 cells, the combinations of the STAT3 with MEK1 and STAT3 with BCL2 inhibitors induced significant less cell growth and more cell death in CKS1B-transfected XG-1 cells compared with EV-transfected control cells (P< 0.05). These results further demonstrate that myeloma cells with higher CKS1B-expression are more sensitive to combinations therapy with STAT3 and MEK/ERK inhibitors than cells with lower CKS1B-expression.
7. FOXM1 inhibited expression of CKS1B and was up-regulated by p27kip1
Kaplan-meior analysis showed that high expression of Foxm1 was associated with shorter survival in MM patients. To investigate the functional relation between FOXM1 and CKS1B, we transfected FOXM1 cDNA into MM cells. WB was used to examine protein expression of FOXM1, CKS1B and p27. Expression of CKS1B was increased, while expression of p27 was decreased in FOXM1-high expressed MM cells. Furthermore, FOXM1 was up-regulated in p27-cDNA transfected MM cells but was not significantly regulated in CKS1B-knockdown MM cells. Our datas confirmed that FOXM1 up-regulated expression of gene CKS1B and down-regulated expression of p27, while over-expression of p27 positively regulated the expression of FOXM1.
[Conclusion]
The role of the present study is to further clarify the functional role of CKS1B in myeloma cell survival and drug-resistance and investigate CKS1B induced SKP2-and p27Kip1-independent signaling pathways. Our results show that forced-expression of CKSIB in MM cells induced multidrug-resistance, providing direct evidence of the crucial role of CKS1B in myeloma progression. Using western blots, we found that over-expression of CKS1B stimulated STAT3 and MEK/ERK, whereas SKP2 knockdown or p27Kip1 over-expression activated rather than suppressed STAT3 and MEK/ERK pathways, suggesting that SKP2 over-expression or p27Kip1 inhibition exerted the opposite effect of CKS1B over-expression on STAT3 and MEK/ERK. Further investigation showed that BCL2 is a downstream target of CKS1B-induced MEK/ERK signaling. Moreover, stimulation of STAT3 and MEK/ERK/BCL2 signaling pathways partially abrogated MM cell death and growth inhibition induced by CKS1B-knockdown. Targeting STAT3 and MEK/ERK/BCL2 activity by specific inhibitors resulted in significant MM cell death and growth inhibition and their combinations had a synergistic effect on cytotoxicity of myeloma cells. Furthermore, feedback of inter-regulation of FOXM1-CKS1B-P27 was involved in mechanisms of MM cell growth and survival induced by CKS1B.
Thus, our findings clarify the SKP2/p27Kip1-independent mechanisms of CKS1B activity in maintaining myeloma cell growth and survival, and also provide a rationale for specifically targeting STAT3 and MEK/ERK/BCL2 in aggressive CKS1B-overexpressing MM.
多发性骨髓瘤细胞存在广泛的染色体变异,1号染色体1q21区连锁扩增被认为与高危型多发性骨髓瘤的预后密切相关,是目前国际上研究的热点。我们以前的研究中,已经发现70个表达与MM预后紧密联系的基因型,位于1号染色体1q21区的CDC28蛋白激酶调节亚基1B(CDC28 protein kinase regulatory subunit B,CKS1B)为其中之一。而CKS1B的表达水平与1q21区连锁扩增拷贝数呈正相关,骨髓瘤病人的预后呈负相关。有人报道CKS1B高表达与难治型多发性骨髓瘤病人和浆细胞型淋巴瘤疗效不佳有关,是预后不良的一个信号。而本课题组前期的结果显示,在MM肿瘤细胞中敲除CKS1B基因,能引起致命的细胞凋亡和生长抑制。这些证据提示,CKS1B在MM肿瘤细胞的增殖和生存中发挥至关重要的作用,但是CKS1B在骨髓瘤细胞生长和疾病进程中的功能仍然需要更多的实验来验证。
很多实验室已经证实CKS1B参与细胞周期调控。蛋白CKS1是CKS/Suc1家族成员之一,是真核细胞周期素依赖性蛋白激酶(CDKs)的重要调节单位。CKS1B是SCFSKP2-CKS1泛素链接酶复合物的重要组成蛋白,CKS1能够通过与SKP2结合,调节泛蛋白化反应并降解CDKs抑制蛋白p27最后推动细胞周期从G1期进入S期,完成细胞分裂繁殖。我们之前的研究也证实了敲除CKS1B基因后,引起在RNA和蛋白水平SKP2下调,和P27上调。CKS1-/-缺陷小鼠由于CDKs抑制蛋白p27Kip1不能被降解,细胞分裂增殖过程被抑制而导致这种小鼠呈现典型的身体纤小。然而令我们感兴趣的是,敲除基因SKP2只引起了部分的细胞生长受抑制和微弱的MM肿瘤细胞凋亡,其效果远远弱于CKS1B基因敲除引起的细胞凋亡和生长抑制。并且,我们在特异性p27Kip1但等位基因缺失的骨髓瘤细胞系中敲除基因CKS1B,仍然导致细胞严重的凋亡和生长抑制。这提示CKS1B可能通过除SKP2/p27Kip1之外的机制来影响骨髓瘤细胞的生长和增殖。
在本论文中,为了进一步阐述CKS1B影响多发性骨髓瘤细胞生存和增殖的分子机制,以及研究CKS1B在多发性骨髓瘤疾病预后中所起的作用,我们进行了如下研究,并取得了相应的结果：
我们在以前的研究中,已经知道CKS1B是一个骨髓瘤高危基因,其表达水平与疾病的预后呈负相关。在本论文中,用骨髓瘤病人骨髓中肿瘤细胞mRNA做基因芯片分析,我们比较了51个病人从刚确诊到复发后的CKS1B表达水平的变化,发现有76%的病人在复发后CKS1B水平明显增高。在刚确诊为骨髓瘤时这些病人CKS1B表达信号平均值为1398(低至370,高至4433),而复发后的平均值为2174(低至405,高至9867)。并且,在这51名病人中有76%复发后CKS1B水平表达增高,其中51%增高水平超过1.5倍。Kaplan-Meier生存曲线分析结果显示,在以初诊-复发配对比较的51位病人中,和那些从初诊断到复发CKS1B表达水平没有明显增高的病人相比,增高1.5倍以上的36位病人复发后4年的生存率显著性的低。
以上证据进一步证实了CKS1B在多发性骨髓瘤的诊断阶段是个诊断指标,并且也是个预示病人预后不良的指标。
CKS1B在多发性骨髓瘤中随着疾病进程中表达增高,这提示在疾病开始的时候CKS1B可能只在小部分的骨髓瘤细胞中高表达,而目前的治疗手段不能有效的杀死这群CKS1B特异高表达的细胞,因此导致病人的治疗失败和骨髓瘤复发。
为了验证这个假说,进一步阐述CKS1B在多发性骨髓瘤中起的作用,我们利用lentenvirus病毒系统转染骨髓瘤细胞,建立了CKS1B过表达的细胞系。细胞分别在含1%、5%、10%胎牛血清的培养基中培养7天,与转染空白载体的对照组(empty vector, EV)细胞相比,CKS1B高表达的骨髓瘤细胞表现出更高的增殖率。这一结果在在含1%胎牛血清的培养基中更为明显。这部分实验证明,CKS1B能促进骨髓瘤细胞的增殖。
接着,我们进一步验证了CKS1B对骨髓瘤细胞耐药的诱导作用。CKS1B转染骨髓瘤细胞系OCI-MY5和XG1,含5nM的化疗药物硼替佐米(Velcade, vel)的培养基中培养48小时,计数细胞生长和存活率。与EV细胞和无药物处理细胞相比,CKS1B转染的细胞表现出较低的细胞死亡率和生长抑制,对疗药物硼替佐米不敏感。我们继续检测了其他的化疗药物,发现CKS1B的骨髓瘤细胞对化疗药浓度为100nM阿霉素和浓度为100nM的依托泊苷存在明显的药物抵抗。
以上实验证明了CKS1B高表达诱发了骨髓瘤细胞对多种抗肿瘤药物耐药。
本课题组为了研究CKS1B基因促进骨髓瘤生长和疾病进程的机制,前期工作已经建立了可诱导性CKS1B基因敲除、SKP2基因敲除以及p27过表达的骨髓瘤细胞系。在骨髓瘤细胞中,CKS1B基因敲除引起SKP2表达降低和p27表达上调,并且导致强烈的细胞凋亡和生长抑制。然而,敲除SKP2基因后并只引起微弱的部分细胞凋亡,并没有CKS1B敲除引起的细胞凋亡那么严重；而p27过表达使骨髓瘤细胞停滞于细胞周期G1期,细胞分裂增殖受阻。并且,值得注意的是KMS28PE是特异性p27基因缺乏骨髓瘤细胞,CKS1B基因敲除照样引起了严重的细胞凋亡。
这些试验结果提示CKS1B是通过SKP2-p27依赖性和非依赖性途径影响骨髓瘤细胞生长和生存,并且SKP2-p27非依赖性途径可能更重要一些。
为了进一步证实CKS1B影响骨髓瘤细胞生长的机制中的确存在SKP2-p27非依赖性途径,在351个接受TT2治疗方案的多发性骨髓瘤新诊断病人中,我们利用Kaplan-Meier生存曲线分别比较CKS1B高／低表达、SKP2高／低表达以及p27低／高表达病人的生存率。结果显示,CKS1B高表达的病人生存期明显短于低表达的病人,P<0.001；而SKP2高、低表达的病人生存时间没有明显的差异；p27低、高表达的病人生存时间也没有明显的差异。
这个临床证据表明,CKS1B、SKP2和p27对多发性骨髓瘤疾病进程的影响不同,只有CKS1B的表达显著影响疾病的进行和恶化。
对351个新诊断的多发性骨髓瘤病人进行基因普表达分析,通过聚类分析得到了CKS1B高／低表达、SKP2高／低表达以及p27低／高表达病人中表达明显差异的基因群。通过ingenuity信号通路分析软件分别分析这3群基因群参与的信号通路,分析结果显示了CKS1B、SKP2、p27参与的主要信号通路调控图。比较结果证实了,骨髓瘤中CKS1B参与的主要信号通路与SKP2、p27的大不相同,除相同的G1／S期细胞周期调控通路外,CKS 1B主要涉及DNA修复、MAPK信号传导通路。
根据信息学分析的提示,我们在CKS1B基因敲除骨髓瘤细胞系OCI-MY5中,通过Western-Blot筛选了MAPK, NF-kB, TP53, PI3K/AKT, STAT3等重要细胞信号途径的主要信号分子。检测到在骨髓瘤细胞OCI-MY5中,CKS1B基因敲除后引起下列信号分子蛋白表达下调：p-MEK1/2, p-ERK1/2, p-STAT3, MCL-1,和p-BCL2。为了确证上述结果,我们对另外2个骨髓瘤细胞系KMS28PE和XG1进行了重复验证,得到了相同的结果。
为了进一步明确STAT3, MEK/ERK,BCL2是CKS1B的下游调控分子,我们通过Western-Blot检测了CKS1B cDNA转染细胞系OCI-MY5和XG1中以上分子的表达。结果显示,在CKS1B外源性高表达的细胞中,p-MEK1/2, p-ERK1/2, p-STAT3, MCL-1,和p-BCL2表达上调。这些结果证实,STAT3, MEK/ERK,BCL2是CKS1B的下游调控分子,CKS1B可能通过它们影响骨髓瘤细胞生长和生存。
为了验证CKS1B是否通过SKP2-P27kip1激活STAT3, MEK/ERK,BCL2信号通路,我们用Western-Blot检测以上信号通路分子在SKP2基因敲除以及p27过表达骨髓瘤细胞系中的表达情况。结果显示,SKP2基因敲除后,引起p-MEK1/2, p-ERK1/2, p-STAT3,和MCL-1表达上调,而P-BCL2无显著变化。P27KIP1通过稳定转染在骨髓瘤细胞中高表达后,p-MEK1/2, p-ERK1/2, p-STAT3,和MCL-1的表达上调,而p-BCL2无显著变化。实验结果证实,SKP2基因敲除或者P27KIP1基因高表达引起的信号通路改变,与CKS1B基因敲除所导致的信号通路改变,并不完全一致。这证明了CKS1B高表达激活了STAT3, MEK/ERK和BCL2信号途径,并且这种激活并不依赖SKP2-P27KIP1途径。
胰岛素样生长因子1(Insulin-like growth factorⅠ, IGF-1)是得到公认的骨髓瘤细胞生长因子,能激活STAT3信号途径,促进STAT3磷酸化。为了研究STAT3在CKS1B促进骨髓瘤细胞增殖和生存中所发挥的作用,我们用含IGF-1(100ng/ml)的培养基培养CKS1B基因敲除的KMS28PE,OCI-MY5,和XG1细胞72小时。Western-Blot证实,在IGF-1培养的细胞p-STAT3, MCL1表达增高。CKS1B基因敲除的细胞,通过IGF-1上调p-STAT3的表达后,细胞凋亡和抑制生长被部分阻止。以上结果证明了CKS1B通过激活STAT3/MCL1影响骨髓瘤细胞的增殖和生存。
信号分子STAT3能调节许多下游基因,参与细胞的生长和增殖。为了寻找CKS1B激活STAT3之后调控的下游基因,我们检测了与细胞生长和凋亡相关的基因MCL1。MCL1基因能通过抑制凋亡过程从而促进细胞增殖,实验结果发现,CKS1B基因敲除引起了MCL1蛋白水平表达下调,并且CKS1B过表达细胞中MCL1蛋白水平明显上调；但是SKP2基因敲除或者P27过表达并没有引起MCL1表达水平的改变。用IGF-1刺激骨髓瘤细胞引起STAT3表达增加后,MCL1的表达也显著增高.这些证据证明,CKS1B激活STAT3通路,然后引起MCL1转录激活,影响了细胞的增殖。
抗生素肖夫齐特(Nifuroxazide, Nif),已被证明是JAK/STAT信号通路的特异性抑制剂,能够在骨髓里细胞中抑制STAT3的磷酸化。为了证明JAK/STAT3是CKS1B下游信号通路并观察靶向阻断STAT3通路对CKS1B高表达骨髓瘤细胞生长的影响,,我们将CKS1B转染的骨髓瘤细胞OCI-MY5和XG1,在含10nM Nif的培养基中培养48小时。WB验证,Nif处理后的细胞p-STAT3蛋白水平下降。与EV细胞相比,CKS1B转染的OCI-MY5和XG1细胞有更高的细胞死亡率。CKS1B高表达的细胞对STAT3特异性抑制剂Nif诱发的细胞凋亡更敏感。
由于市场上没有MEK/ERK特异性激活剂,我们在CKS1B基因敲除后的细胞中,通过Lenti Virus转染MEK1-cDNA,使MEK1基因特异性高表达。WB验证MEK1转染细胞后p-MEK1/2和p-ERK1/2表达大幅度增加,并且磷酸化p-BCL2被激活。说明转染MEK1导致的MEK/ERK通路的激活,抵消了CKS1B基因敲除导致的bcl2下调,从而证明BCL2是MEK/ERK下游调控分子。MEK1基因高表达,同时CKS1B基因敲除的骨髓瘤细胞KMS28PE,OCI-MY5,和XG1,虽然仍存在细胞凋亡和生长受抑制,但是与单纯CKS1B基因敲除的细胞相比较,细胞死亡率显著性降低,存活细胞总数也显著性增多。由此证明,MEK/ERK的外源性高表达和磷酸化激活,部分抵消了CKS1B基因敲除引起的骨髓瘤细胞死亡和生长抑制。以上研究证明,CKS1B通过激活MEKK/ERK通路,以及其下游的BCL2通路,影响骨髓瘤细胞的增殖和生存.
U0126是MAPK通路抑制剂,能特异而强烈的抑制MEK1／2分子的活化。为了观察靶向阻断MEK/ERK通路对CKS1B高表达骨髓瘤细胞生长的影响,我们用含10μM U0126的培养基培养CKS1B转染的骨髓瘤细胞OCI-MY5和XG1,48小时时观察细胞生长状况。WB证实,与对照组相比与U0126共培养的骨髓瘤细胞,p-MEK1/2和p-ERK1/2蛋白水平下降。与对照EV细胞相比,CKS1B转染的OCI-MY5和XG1细胞有更高的细胞死亡率。CKS1B高表达的细胞对MEK特异性抑制剂U0126引起的细胞生长抑制和死亡更敏感。
实验结果已经提示BCL2是CKS1B和MEK/ERK信号通路的下游调控分子,为了确证BCL2在CKS1B促进骨髓瘤细胞生长的机制所起的作用,我们在CKS1B基因敲除后细胞中,通过LentiVirus转染BCL2-cDNA,使BCL2过表达。Western-Blot验证p-BCL2磷酸化被激活。BCL2过表达,同时CKS1B基因敲除的骨髓瘤细胞,虽然仍存在细胞凋亡和生长抑制,但是与单纯CKS1B基因敲除的细胞相比较,细胞死亡率显著性降低,存活细胞总数也显著性增多。由此证明,BCL2的外源性高表达和BCL2磷酸化,部分抵消了CKS1B基因敲除引起的骨髓瘤细胞死亡和生长抑制。以上研究证明了,BCL2在CKS1B促进骨髓瘤细胞的增殖和生存的过程中发挥重要作用。
为了观察靶向阻断BCL2通路对CKS1B高表达骨髓瘤细胞生长的影响,我们用BCL2特异性抑制剂处理CKS1B转染的骨髓瘤细胞OCI-MY5和XG1,在含10nM的BCL2特异性抑制剂(2,9-Dimethoxy-11,12-dihydrodibenzo[c,g][1,2]-diazocine 5,6-dioxide and 5,5'-Dimethoxy-2,2'-dinitrosobenzyl)的培养基中培养48小时。WB验证被BCL2抑制剂处理的细胞p-BCL2蛋白水平下降。与对照EV细胞相比,CKS1B转染的OCI-MY5和XG1细胞有更高的细胞死亡率。CKS1B高表达的细胞对BCL2抑制剂的杀细胞作用尤其敏感。
由于CKS1B通过激活MEK/ERK和STAT3信号通路促进骨髓瘤细胞生长,靶向于这2条通路的药物联合应用可能会对骨髓瘤细胞造成更强有力的杀伤效果。
我们使用STAT3抑制剂联合MEK1抑制剂,或者联合BCL2抑制剂,处理CKS1B高表达的骨髓瘤细胞OCI-MY5Y。结果显示联合应用这2条信号通路抑制剂,都对CKS1B高表达细胞和空白对照细胞造成强烈的细胞毒作用,而CKS1B过表达细胞的死亡率显著更高,效果强于单独用药。我们又在CKS1B高表达的细胞XG1中重复了这一实验,得到相同的结果。这一实验进一步证实了,与CKS1B表达低的骨髓瘤细胞相比,对CKS1B高表达的骨髓瘤细胞应用STAT3和MEK/ERK抑制剂治疗方案,疗效会更好。
CKS1B在多发性骨髓瘤病人中高表达已经被多个研究者所证实,而CKS1B在骨髓瘤细胞中增高的机制,除了与染色体1q21区域扩增外相关,并无更多的研究。
有文献报道在人骨肉瘤中,转录因子FOXM1能上调CKS1的表达,我们的临床观察和实验室资料也发现FOXM1高表达的MM病人预后差。为了研究FOXM1与CKS1B之间的关系,我们建立了FOXM1高表达骨髓瘤细胞系,WB验证FOXM1过表达引起CKS1B蛋白水平上调,和P27蛋白水平下调；而WB也显示,CKS1B基因敲除后并没有引起FOXM1表达水平的变化,而P27过表达则导致FOXM1表达增加。因此FOXM1是CKS1B的上游调控基因,能抑制CKS1B从而减少P27的降解,但是当P27的表达增高时,反过来又促进了FOXM1的表达,FOXM1-CKS1B-P27之间存在相互作用的调控关系。
小结：本课题的研究,探讨了CKS1B在骨髓瘤中过表达的机制,证实了骨髓瘤细胞中CKS1B接受转录因子FOXM1的正向调控；证明了CKS1B基因促进骨髓瘤细胞生长存活和对多种抗肿瘤药物耐药疾病；阐述了CKS1B促进骨髓瘤细胞生长的SKP2-p27非依赖性机制,证实CKS1B通过激活STAT3,MEK/ERK/BCL2信号通路促进骨髓瘤细胞生长和存活,并且证实CKS1B过表达细胞对靶向阻断STAT3,MEK/ERK/BCL2通路的药物特别敏感,为临床治疗难治型特别是CKS1B高表达型多发性骨髓瘤提供了实验室证据和思路。
Multiple myeloma (MM) is a plasma cell malignancy, which remains largely incurable with current therapeutic strategies. The molecular bases of MM progression and drug-resistance are not completely understood. Gain of chromosome 1q21 is one of the most recurrent chromosomal aberrations observed in MM and is correlated with poor prognosis. We previously identified a 70 high-risk signature pattern in MM, with overrepresentation of over-expressed genes mapping to chromosome 1q, further supporting the hypothesis that 1q21 gain is important in MM progression. High expression of CKS1B, a Cdc28 protein kinase regulatory subunit 1B, mapping to the 1q21 amplicon and one of the 70 high-risk signature gene pattern, was inversely associated with survival in MM. Alternatively, knockdown of CKS1B in MM cells potently induced growth inhibition and apoptosis, suggesting that CKS1B plays a crucial role in MM cell survival. However, the functional role of CKS1B in MM cell survival and MM disease progression remains to be elucidated.
CKS1B has a well-documented role in the cell-cycle regulation. Cks1 is a member of the Cks/Suc1 family of proteins, which are essential components of cyclin-dependent kinases (CDKs) that regulate mitosis in all eukaryotes. CKS1B was identified as an essential accessory protein to the SCFSKP2-CKS1 ubiquitin ligase complex. In this complex, Cksl enhances the interaction between SKP2 and p27Kip1 (a CDK inhibitor),, resulting in cell proliferation. The role of Cks1 in regulating p27Kip1 proteasome degradation in the cell cycle is particularly evident in CKS1-/-mice. The key feature of these mice is their small body size, which is due to lack of degradation of the G1-/S-phase CDK inhibitor p27Kip1. Interestingly, besides influencing cell growth and survival through regulation of p27Kip1, silencing of CKSIB also induces cell death and inhibits growth in MM cells in the presence of a bi-allelic deletion of the CDKN1B (p27Kip1) locus, indicating that CKSIB mediates its effects on cell growth and survival also through mechanisms that are independent p27Kip1 and SKP2.
In this study, we found that forced expression of CKS1B by lentivirus vector-mediated CKS1B-cDNA transfection in MM cells increased cell proliferation and multidrug-resistance, providing direct evidence of the crucial role of CKSIB in MM progression. Furthermore, we also identified STAT3 and MEK/ERK/BCL2 pathways to be downstream targets of CKS1B activation independent on the complex of SKP2/p27Kip1, responsible for MM cell growth and survival.
1. CKS1B expression increased in relapsed MM and conferred a short post-relapse survival.
Our previous studies showed that CKS1B was one of the 70 high-risk genes, which was inversely associated with survival in newly diagnosed MM. In this study, we compared CKS1B expression from 51 patients with paired baseline (diagnostic) and relapse samples. The median signals of CKS1B from microarray data at diagnosis and at relapse were 1398 (range:370-4433) and 2174 (range:405-9867), respectively. CKSIB expression increased in 76% relapsed MMs and was in more than 1.5 folds in 51%.
As we expected, patients from the 51 paired relapses who had CKS1B expression in quartile 4 (high-risk) at baseline and receiving various salvage therapies had the worst 4-year post-relapse survival compared with those who had CKS1B expression in quartiles 1 to 3 (low-risk) at baseline (P= 0.0012). The quartile 4 reference line is taken from the complete (n=351) sample of arrays at diagnosis. Interestingly, among 38 relapse patients with low CKS1B expression (quartiles 1-3) at baseline, patients increased CKS1B expression at least 1.5 folds at relapse had inferior 4-year postrelapse survival compared with those lacking CKS1B up-regulation at relapse (P=0.032). Furthermore, among 36 relapsed patients with high CKS1B expression at relapse,4-year postrelapse survival in patients with high CKS1B at baseline was significantly poor compared with those with high CKS1B expression only at relapse. These data further confirmed that CKS1B expression is a prognositic marker at the stage of newly diagnosis in MM and also demonstrated that CKSIB expression can be used to predict patient survival in postrelapse MM.
2. CKS1B over-expression promotes MM cell proliferation and drug-resistance.
Increased expression of CKS1B is a progression event suggesting that CKS1B may be heterogeneously expressed in a small population of myeloma cells at diagnosis, and current treatments ineffectively eliminate the small populations of CKS1B high-expression myeloma cells, which results in myeloma relapse and treatment failure.
To further investigate the functional role of CKS1B played in MM cells, CKS1B was over-expressed in OCI-MY5 and XG1 MM cells by lentivirus vector-mediated CKS1B-cDNA transfection. CKS1B-transfected MM cells cultured respectively in medium with 1%、5%、10%FBS showed higher proliferation ratio than MM cells transfected by empty vector(EV).
To test the hypothesis that MM cells with high expression of CKSIB are more drug-resistant and responsible for MM poor prognosis, CKS1B-transfected OCI-MY5 and XG-1 cells were treated with bortezomib (Vel) at a dose of 5 nM for 48 hours. Cell growth and cell survival were examined. Untreated and EV-transfected cells with or without bortezomib were used as controls. Bortezomib treatment induced significantly less growth inhibition and cell death in CKS1B-transfected cells compared with EV-transfected controls (P<0.05). Similarly, treatment of doxorubicin (Dox) 100nM and etoposide (Epo) 100nM for 48 hours, induced significantly less growth inhibition and cell death in CKS1B-transfected cells compared with EV-transfected controls (P< 0.05). Clearly, increased CKS1B expression induced chemotherapeutic resistance in myeloma.
3. CKS1B over-expression activates STAT3 and MEK/ERK through SKP2 and p27Kip1-independent pathways.
To identify these CKSIB-mediated SKP2/p27Kip1-independent signaling pathways, western blots were applied to screen for activation of the key signaling pathways which relate to cell survival and apoptosis, including MAPK, NF-κB, TP53, PI3K/AKT and STAT3 using OCI-MY5 cells after CKS1B-silencing by specific CKS1B-shRNA transfection. Decreased phosphorylated (p)-MEK1/2, p-ERK1/2, p-STAT3 and p-BCL2 were detected in CKS1B silenced OCI-MY5 cells compared with wild-type cells and cells transfected with a non-targeting scramble sequence (SCR). Similar results were also observed in CKS1B silenced KMS28PE and XG-1 cells.We further examined the alteration of STAT3, MEK/ERK and BCL2 in CKS1B-transfected OCI-MY5 and XG1 cells. Increased levels of p-MEK1/2, p-ERK1/2, p-STAT3 and p-BCL2 were observed in CKS1B-transfected OCI-MY5 and XG-1 cells compared with the EV-transfected controls. These results strongly suggest STAT3, MEK/ERK, and BCL2 are the downstream signaling pathways of CKS1B, which may mediate MM cell growth, survival, and disease progression.
To examine the functional relationship between SKP2 and these CKS1B activated STAT3, MEK/ERK and BCL2 signaling pathways, SKP2-shRNA was used to silence SKP2 in KMS28PE,OCI-MY5, and XG-1 cells and protein levels of p-MEK1/2, p-ERK1/2, p-STAT3, and p-BCL2 in these cells were examined. Wild-type (WT) and SCR-transfected cells were used as controls. SKP2-knockdown resulted in an increase rather than a decrease of p-MEK1/2, p-ERK1/2 and p-STAT3 levels in KMS28PE,OCI-MY5 and XG-1 cells, and with no remarkable change in p-BCL2 level. We then examined the functional relationship between p27Kip1 and STAT3, MEK/ERK and BCL2 by over-expressing p27Kipl in KMS28PE,OCI-MY5 and XG-1 cells using a lentivirus system. Western blots detected increased levels of p-MEK1/2 and p-ERK1/2 in p27Kip1-transfected myeloma cells with no remarkable effect on p-STAT3,p-BCL2 compared with WT and EV controls.
4. [Activation of STAT3 is involved in CKS1B-mediated myeloma cell growth and survival]
A specific STAT3 inhibitor Nifuroxazide at the dose of 10 nM for 48 hours was used to treat CKS1B-transfected OCI-MY5 and XG-1 cells. Nifuroxazide treatment decreased p-STAT3 in both EV-and CKS1B-transfected myeloma cells compared with untreated controls by western blot, confirming its specificity. Nifuroxazide-induced cell growth and cell death were also evaluated in CKS1B-transfected OCI-MY5 and XG-1 cells. Cells were treated with 10 nM Nifuroxazide for 48 hours, Nifuroxazide treatment induced more inhibition of cell growth and more increase in cell death in CKS1B-transfected cells compared with EV-transfected cells, indicating that myeloma cells with higher CKS1B-expression levels are more sensitive to STAT3 inhibition than cells with lower CKS1B-expression.
Insulin-like growth factorⅠ(IGF-1) is a well-recognized myeloma cell survival factor activating the STAT3 signaling pathway. To further confirm the functional role of STAT3 signaling in CKS1B-mediated myeloma cell growth and survival, CKS1B-silenced KMS28PE, OCI-MY5 and XG-1 cells were treated with IGF-1 (100ng/ml) for 72 hours. Western-blots detected increased p-STAT3 in these CKS1B-silenced myeloma cells compared with untreated, SCR- or CKS1B-shRNA-transfected controls, confirming activation of STAT3 in IGF-1-treated cells. The effects of IGF-1 treatment on CKS1B-shRNA-induced myeloma cell death and growth inhibition were evaluated. IGF-1 treatment partially abrogated growth inhibition and cell death induced by CKS1B-knockdown (P<0.05).
STAT3 regulates the expression of many target genes involved in cell survival and growth. To investigate whether CKSIB activates STAT3 signaling through regulation of STAT3 target genes, the protein of cell growth and apoptosis related gene MCL1, was measured by western blot. The level of MCL1, a gene that enhances cell survival by inhibiting apoptosis, was decreased in CKSIB silencing MM cells and increased in CKS1B over-expressing MM cells; whereas MCL1 expression did not show alteration in MM cells manipulated by inhibition of SKP2 and over-expression of p27Kip1. Furthermore, MCL1, following STAT3 expression pattern, was significantly increased in IGF1 treated MM cells inhibited CKS1B expression by CKS1B-shRNA.These data provide evidence that CKSIB regulates STAT3 signaling dependent on the STAT3 transcription of MCL1.
5. Activation of the MEK/ERK signaling pathway is also involved in CKS1B-mediated myeloma cell growth and survival
A specific MEK inhibitor U0126 was used to treat CKS1B-transfected OCI-MY5 and XG-1 cells for 48 hours at the dose of 10μM. Cell lysates were prepared and subjected to western analysis using antibodies recognizing p-MEK1/2 and p-ERK1/2 proteins. U0126 decreased p- MEK1/2 and p-ERK1/2 in both EV-and CKS1B-transfected myeloma cells, confirming its specificity. Interestingly, U0126 treatment down-regulated BCL2 in myeloma cells, suggesting that BCL2 represents a downstream target of the MEK/ERK signaling pathway. We also evaluated U0126-induced cell growth and cell viability in CKS1B-transfected OCI-MY5 and XG-1 cells. Cells were treated with 10μM U0126 for 48 hours, U0126-treatment induced more cell growth inhibition and cell death in CKS1B-transfected cells than those in EV-transfected cells (P< 0.05).
Since inhibitors of MEK/ERK signaling pathway may induce some non-specific effects on myeloma cell growth and survival, we constitutively activated the MEK/ERK signaling pathway by lentivirus-vector-mediated MEK1-cDNA transfection in CKS1B-silenced KMS28PE,OCI-MY5 and XG1 cells. Western blots showed a larger increase of p-MEK1/2 and p-ERK1/2 levels in MEK1-cDNA and CKS1B-shRNA double-transfected cells compared with SCR-and CKS1B-transfected controls. MEK1-transfection also increased protein levels of p-BCL2 in CKS1B-silenced cells, indicating that MEK1-transfection could overcome CKS1B-knockdown-mediated inhibition of BCL2 signaling pathway, further demonstrating that BCL2 is the downstream target of CKS1B and MEK/ERK signaling. Although MEK1-cDNA and CKS1B-shRNA doubly-transfected cells clearly showed increased cell death and growth inhibition compared with SCR-transfected control cells (P< 0.05), these double-transfected cells exhibited significantly less cell death and growth inhibition than CKS1B-silenced myeloma cells (P<0.05), indicating that MEK1-transfection partially abrogated cell death and growth inhibition induced by CKS1B-knockdown.
Our results indicate that BCL2 is a downstream target of the CKS1B and MEK/ERK signaling pathway. Therefore, a specific BCL2 inhibitor (2,9-Dimethoxy-11,12-dihydrodibenzo[c,g][1,2]-diazocine 5,6-dioxide and 5,5'-Dimethoxy-2,2'-dinitrosobenzyl) at the dose of 10 nM for 48 hours was used to treat CKS1B-transfected OCI-MY5 and XG-1 cells. Treatment with this BCL2-inhibitor resulted in significantly more cell growth inhibition and cell death in CKS1B-transfected cells compared with EV-transfected cells, indicating that myeloma cells with higher CKS1B-expression are more sensitive to BCL2 inhibition than cells with lower CKS1B-expression. We subsequently over-expressed BCL2 by lentivirus-mediated BCL2 cDNA transfection in CKS1B-silenced KMS28PE, OCI-MY5 and XG-1 cells. Cells were cultured for 4 days and western blots confirmed increased p-BCL2 levels in BCL2 cDNA and CKS1B-shRNA doubly-transfected cells. The effects of BCL2-transfection on CKS1B-shRNA induced myeloma cell growth inhibition and death were also evaluated. Although BCL2-cDNA and CKS1B-shRNA doubly-transfected cells clearly showed cell growth inhibition and death compared with SCR-transfected control cells (P<0.05), BCL2 transfection partially abrogated myeloma cell growth inhibition and death induced by CKS1B-silencing (P< 0.05).
6. Combination of STAT3 and MEK1 inhibitors induced synergistic cytotoxicity on myeloma cells with high CKS1B expression
Since the STAT3 and MEK/ERK signaling pathways were both involved in CKS1B-induced myeloma cell growth and survival, a combined targeting of these signaling pathways might induce more cytotoxicity in myeloma cells. CKS1B-transfected OCI-MY5 cells were treated with combination of STAT3 and MEK1 inhibitors (i.e. U0126 [10μM] plus Nifuroxazide [10 nM]), or combination of STAT3 and BCL2 inhibitors (i.e. U0126 [10μM] plus BCL2 inhibitor [10 nM]) for 48 hours. EV-transfected cells treated with the same combinations were used as controls. Untreated CKS1B-and EV-transfected cells also served as controls. The combinations of the STAT3 with MEK1 and the STAT3 with BCL2 inhibitors induced significant less cell growth and more cell death in CKS1B-transfected OCI-MY5 compared with EV-transfected control cells (P<0.05). CKS1B-transfected XG-1 cells were also treated with the same combinations for 48 hours. Similar to the CKS1B-transfected OCI-MY5 cells, the combinations of the STAT3 with MEK1 and STAT3 with BCL2 inhibitors induced significant less cell growth and more cell death in CKS1B-transfected XG-1 cells compared with EV-transfected control cells (P< 0.05). These results further demonstrate that myeloma cells with higher CKS1B-expression are more sensitive to combinations therapy with STAT3 and MEK/ERK inhibitors than cells with lower CKS1B-expression.
7. FOXM1 inhibited expression of CKS1B and was up-regulated by p27kip1
Kaplan-meior analysis showed that high expression of Foxm1 was associated with shorter survival in MM patients. To investigate the functional relation between FOXM1 and CKS1B, we transfected FOXM1 cDNA into MM cells. WB was used to examine protein expression of FOXM1, CKS1B and p27. Expression of CKS1B was increased, while expression of p27 was decreased in FOXM1-high expressed MM cells. Furthermore, FOXM1 was up-regulated in p27-cDNA transfected MM cells but was not significantly regulated in CKS1B-knockdown MM cells. Our datas confirmed that FOXM1 up-regulated expression of gene CKS1B and down-regulated expression of p27, while over-expression of p27 positively regulated the expression of FOXM1.
[Conclusion]
The role of the present study is to further clarify the functional role of CKS1B in myeloma cell survival and drug-resistance and investigate CKS1B induced SKP2-and p27Kip1-independent signaling pathways. Our results show that forced-expression of CKSIB in MM cells induced multidrug-resistance, providing direct evidence of the crucial role of CKS1B in myeloma progression. Using western blots, we found that over-expression of CKS1B stimulated STAT3 and MEK/ERK, whereas SKP2 knockdown or p27Kip1 over-expression activated rather than suppressed STAT3 and MEK/ERK pathways, suggesting that SKP2 over-expression or p27Kip1 inhibition exerted the opposite effect of CKS1B over-expression on STAT3 and MEK/ERK. Further investigation showed that BCL2 is a downstream target of CKS1B-induced MEK/ERK signaling. Moreover, stimulation of STAT3 and MEK/ERK/BCL2 signaling pathways partially abrogated MM cell death and growth inhibition induced by CKS1B-knockdown. Targeting STAT3 and MEK/ERK/BCL2 activity by specific inhibitors resulted in significant MM cell death and growth inhibition and their combinations had a synergistic effect on cytotoxicity of myeloma cells. Furthermore, feedback of inter-regulation of FOXM1-CKS1B-P27 was involved in mechanisms of MM cell growth and survival induced by CKS1B.
Thus, our findings clarify the SKP2/p27Kip1-independent mechanisms of CKS1B activity in maintaining myeloma cell growth and survival, and also provide a rationale for specifically targeting STAT3 and MEK/ERK/BCL2 in aggressive CKS1B-overexpressing MM.
引文
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