CN103068403A - 用于含有癌干细胞的或由其起源的癌的治疗、预防和诊断的方法和组合物 - Google Patents
用于含有癌干细胞的或由其起源的癌的治疗、预防和诊断的方法和组合物 Download PDFInfo
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- CN103068403A CN103068403A CN2011800398691A CN201180039869A CN103068403A CN 103068403 A CN103068403 A CN 103068403A CN 2011800398691 A CN2011800398691 A CN 2011800398691A CN 201180039869 A CN201180039869 A CN 201180039869A CN 103068403 A CN103068403 A CN 103068403A
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Abstract
提供用于含有癌干细胞的或由其起源的癌的治疗、预防和诊断的方法和组合物。
Description
技术领域
本发明涉及以RPN2基因为靶标的用于含有癌干细胞的或由其起源的癌的治疗、预防和诊断的方法和组合物。
背景技术
证据的积累已经暗示:肿瘤并不是由一样细胞型构成的,经常是由不均一的细胞型构成,其中的细胞亚群承担着肿瘤的抑制、抗药性(drug resistance)和转移。这样的细胞被称为癌干细胞(或肿瘤起始细胞)。如果以该癌干细胞为靶标,则可以期待能够对癌的发生、转移、复发进行治疗和预防。但是,癌干细胞表型的分子基础的大部分是不明确的,因此用来确定癌干细胞的标志物尚不明确,此外,尚未建立以癌干细胞为靶标的治疗或预防的方法。
本发明人等此前已经证实,作为寡糖转移酶(OST)复合物的构成成分的核糖体结合糖蛋白II(RPN2)调节乳癌细胞的抗药性,并且,RPN2的沉默(silencing)是有望战胜抗药性(drug resistance)肿瘤的途径(专利文献1)。但是,通过抑制RPN2表达而抑制癌细胞的增殖等的机理尚不明确。
现有技术文献
专利文献
专利文献1:国际特开第2007/144985号
发明内容
发明要解决的问题
本发明的目的在于提供用于含有癌干细胞的或由其起源的癌的治疗、预防和诊断的方法和组合物。
用于解决问题的方案
本申请中,本发明人等公开了:RPN2在乳癌细胞的癌干细胞级分中高表达,并且RPN2的敲低在体外抑制癌干细胞的集落形成和浸润能力。进一步的分析显示:RPN2的敲低使体内肿瘤形成下降、并且抑制转移能力。利用综合性蛋白质组学分析(Comprehensive proteome analysis),明确了RPN2敲低改变了已知调节TGF-β/Smad通路的14-3-3ζ的表达。因此,本发明人等提供RPN2对于癌干细胞表型的维持重要、并且RPN2可以成为适合于癌干细胞治疗的有希望的靶标的遗传学和生物学证据。
本发明涉及:
[1]一种用于含有癌干细胞的或由其起源的癌的治疗或预防的药物组合物,其包含RPN2抑制剂;
[2]根据[1]的药物组合物,其中,癌干细胞具有突变型p53基因;
[3]根据[1]或[2]的药物组合物,RPN2抑制剂为针对RPN2基因的siRNA;
[4]一种癌干细胞的检测方法,其包括确定RPN2有无表达或表达水平的步骤;
[5]根据[4]的方法,其还包括检测突变型p53基因的步骤。
发明的效果
根据本发明,提供一种以RPN2基因为靶标的用于含有癌干细胞的或由其起源的癌的治疗、预防和诊断的方法和组合物。附图说明
图1:(A)是表示CD44+CD24-癌干细胞的不等分裂(unequal division)的图。(B)是表示利用RT-PCR进行的RPN2的表达解析的图。
图2是表示RPN2敲低对CD44+CD24-癌干细胞数的效果的图。
图3:(A)是表示RPN2敲低对集落形成能力的效果的图。(B)是表示RPN2敲低对形成集落数的效果的图。
图4是表示RPN2敲低对肿瘤形成能力的效果的图。
图5是表示RPN2敲低对肿瘤转移的效果的图。
图6是表示RPN2敲低对致死性的效果的图。
图7:(A)是表示RPN2敲低对突变型p53的表达的效果的图。(B)是表示RPN2敲低对E-钙黏着蛋白的表达的效果的图。
图8是表示RPN2敲低对14-3-3ζ的表达的效果的图。
图9是表示在动物中移植MM231-LN细胞而形成的肿瘤的免疫组织染色的结果图。
图10是表示人乳癌患者的乳癌组织中的RPN2和突变型p53的表达的图。
图11是表示利用TUNEL分析评价而得的肿瘤凋亡(apoptosis)图。通过投与3天后的RPN2-siRNA/A6K的肿瘤内送达证明了高度诱导了乳癌的肿瘤凋亡。
图12是表示狗乳癌中的RPN2的敲低分析的图。与RPN2mRNA的对照(生理盐水)相比,RPN2-siRNA/A6K可见约50%的抑制(n=3,p<0.001)。
具体实施方式
本发明提供一种用于含有癌干细胞的或由其起源的癌的治疗或预防的药物组合物,其包含RPN2抑制剂。此外,本发明提供一种用于含有癌干细胞的或由其起源的癌的治疗或预防的方法,其包括对对象投与本发明的药物组合物的步骤。
本说明书中使用的术语“癌干细胞”是指具有多潜能性和自我复制能力(本说明书中也将这些称为自我复制分化能力)的癌细胞。癌细胞包括乳癌细胞、胃癌细胞、大肠癌细胞、肺癌细胞、前列腺癌细胞、造血细胞癌细胞等任意的癌。癌干细胞除了自我复制分化能力外,还可能具有对抗癌剂的耐受性(抗药性)、向周边组织浸润和/或向体内的远端部位转移的能力(浸润转移能力)。认为若以癌干细胞为靶标,则能够治疗和预防癌的发生、转移、复发。本发明在以这样的癌干细胞为靶标的癌的治疗和预防中有用。
1个实施方式中,癌干细胞的核糖体结合糖蛋白II(RPN2)的表达水平增大。“RPN2”为存在于粗糙内质网、具有向新合成的多肽链添加N结合型糖链的功能的寡糖转移酶(OST)复合物的构成成分(亚基)之一。人RPN2是包含631个氨基酸的碱性膜蛋白。对RPN2的来源没有限制,例如为动物、优选哺乳动物、更优选灵长类、进而优选人。“RPN2基因”是编码RPN2的基因。序列号1中示出人RPN2基因的碱基序列。
本发明人等发现,在CD44+/CD24-的乳癌的癌干细胞级分中通过短发夹RNA(shRNA)敲低RPN2的表达时,在体外癌干细胞的集落形成和浸润能力得到抑制,并且免疫缺陷动物中的(即体内的)肿瘤形成能力和浸润转移能力消失。如上所述,癌干细胞由于可能具有自我复制分化能力、抗药性、浸润转移能力,因此,认为与上述全部均相关的RPN2为癌干细胞的标志物,此外,若以RPN2为靶标,能够治疗和预防含有癌干细胞的或由其起源的癌。
其它实施方式中,癌干细胞具有突变型p53基因。“p53”为抗癌基因产物,已在多种人癌中发现p53基因中的突变(Adorno,M.et al.,Cell,137:87-98(2009);Wang,S.P.et al.,Nat.Cell Biol.,11:694-704(2009);Muller,P.A.et al.,Cell,139:1327-1341(2009);Morton,J.P.et al.,Proc.Natl.Acad.Sci.USA,107:246-251(2010))。对p53基因中的突变没有限制,突变优选为不发生移码(frameshift)的置换型点突变(错义突变)或缺失突变(密码子缺失突变)。
本发明人等发现,当在具有突变型p53的细胞株中敲低RPN2的表达时,突变型p53蛋白质水平降低、抑制了E-钙黏着蛋白的表达降低。进而,本发明人等发现,在高表达RPN2的癌细胞中,14-3-3ζ的表达降低,而通过RPN2的敲低,其表达增加。已知14-3-3ζ在mdm2介导下发挥将突变型p53分解的作用。
癌细胞已知呈现一种称为上皮间质转化(EMT)的现象。EMT时,与细胞粘附相关的E-钙黏着蛋白的表达降低,认为其结果是癌细胞进行浸润或转移。上述结果表明,癌细胞中RPN2使14-3-3ζ的表达降低,其结果是突变型p53稳定化、引起EMT,也带来了癌细胞的浸润或转移。
已经阐明突变型p53与癌细胞的自我复制分化能力和浸润转移能力相关,存在众多与p53相关的研究。但是,截至目前还没有过以p53为靶标的癌的治疗或预防的成功例子被报道。RPN2除了与自我复制分化能力和浸润转移能力相关,还显示与抗药性相关,因此认为:如果能够抑制RPN2,则不仅能够使癌细胞的抗药性消失,还能够在其上游抑制突变型p53的作用,抑制癌细胞的自我复制分化能力和浸润转移能力。如果在检测RPN2的表达的基础上检测突变型p53的存在,则能够更准确地鉴定具有抑制RPN2有效的突变型p53的癌细胞。此外,通过抑制RPN2,则能够比以突变型p53为靶标更有效地治疗和预防与突变型p53相关的癌。
本说明书中使用的术语“RPN2抑制剂”是指具有抑制RPN2基因的表达、抑制RPN2基因产物的作用的任意物质。RPN2已知在胎盘以外的正常组织中是几乎不表达的。因此,RPN2抑制剂基本不会作用于孕妇以外的对象中的癌细胞以外的细胞,作为无副作用的特异性癌治疗药是有用的。对对象没有限定,例如为动物、优选哺乳动物、更优选灵长类、进一步优选人。
作为RPN2抑制剂,可以使用例如抑制RPN2基因转录的物质、与RPN2转录产物结合或分解其的物质、与RPN2蛋白质结合的物质等。作为与RPN2蛋白质结合的物质的例子,可以列举抗RPN2抗体或其片段(Fab、F(ab’)2等)、寡糖转移酶(OST)复合物中与RPN2结合的其它构成成分等。作为与RPN2转录产物结合或分解其的物质的例子,可以列举出针对RPN2基因的反义RNA、核糖酶、低分子干扰RNA(siRNA)、微小RNA(miRNA)等。
优选使用针对RPN2基因引发RNA干扰(RNAi)的siRNA、miRNA等作为RPN2抑制剂。RNA干扰是指利用双链(ds)RNA分子序列特异性地抑制基因表达的现象。例如,siRNA引起的靶标mRNA的切断,siRNA引起的靶标DNA区域的异染色质化所介导的基因沉默(silencing),miRNA引起的翻译抑制、转录抑制、mRNA分解等从而引发RNA干扰。siRNA可以基于作为对象基因的RPN2基因的序列进行序列设计并人工合成,因此在本发明中优选使用。
可以通过本技术领域中公知的任意方法获得这样的siRNA。例如,可以利用也用于DNA化学合成中的亚磷酰胺法从3'末端起向着5'末端一个碱基一个碱基地依次进行缩合反应从而化学合成。在合成过程中,为了防止RNase所致的分解,优选对各核糖核苷酸的2'末端的羟基进行保护。作为这样的保护基,可以列举出2’-O-叔丁基二甲基硅基(2’-tBDMS)、2’-O-三异丙基硅氧基甲基(2’-TOM)、5’-硅基-2’-乙酰氧基乙氧基(2’-ACE)等。
针对RPN2基因的siRNA具有与RPN2基因的规定序列对应的序列、即与作为靶标的mRNA的一部分序列对应的序列。例如,对于序列号1所示的RPN2基因序列中的第1194位~第1212位的序列,可以使用包含作为正义链的序列号2的RNA和作为反义链的序列号3的RNA的dsRNA(序列A)作为siRNA。该dsRNA中,各链的3'末端存在2个碱基的突出(overhang),因此双链部分为19碱基长。序列号4~25中示出专利文献1公开的针对RPN2基因的siRNA(序列B~L)的正义链和反义链的序列。可以将这些对(dsRNA)作为本发明中RPN2抑制剂来使用。
此外,miRNA为不编码蛋白质的低分子RNA,已知在基因组中存在数百种。miRNA以数百~数千碱基的核苷酸形式转录后,接受加工,最终成为19~24个碱基的2聚核苷酸,通过具有与该miRNA互补的碱基序列的mRNA的翻译抑制、mRNA的分解、转录的控制等来抑制基因表达。由于已知RPN2也可以被多个miRNA控制表达,因此通过人工合成这样的miRNA,作为本发明中RPN2抑制剂使用,从而能够抑制RPN2基因表达。有可能抑制RPN2基因的表达的已知miRNA的序列可以由公开数据库(TargetScan Release3.1等)检索而得。
本发明的药物组合物的投与可以是全身投与、局部投与中的任一种。投与途径可以是静脉内、皮下、腹腔内、肌肉内、鼻腔内等任意途径。本发明的药物组合物还可以含有制剂领域中使用的赋形剂、稀释剂、稳定化剂等任意成分。例如,RPN2抑制剂为抗体等蛋白质的情况下,药物组合物还可以含有蛋白质制剂领域中通常使用的成分。此外,例如,RPN2抑制剂为siRNA等核酸的情况下,可以含有用于导入核酸的任意物质(例如脂质体)。国际公开第2010/024262号中记载的含有肽表面活性剂的转染剂显示出低毒性、高的送达患部效率和靶标基因的抑制效率,能够全身投与,因此可以优选用于本发明。
本发明的药物组合物中包含的RPN2抑制剂的使用量可根据投与方法、肿瘤的种类、大小、患者的状态、并用的药物等而改变,本领域技术人员可以适当确定。例如,使用siRNA作为RPN2抑制剂时,在局部投与的情况下,期望为1nmol/kg以上且10nmol/kg以下,全身投与的情况下期望为2nmol/kg以上且50nmol/kg以下。
本发明提供一种癌干细胞的检测方法,其包括确定RPN2有无表达或表达水平的步骤。
RPN2存在于细胞质内,因此,在检测时,从由对象获得的细胞或组织中制备包含RNA、蛋白质等的提取物,并检测其中的转录产物(RPN2mRNA)、翻译产物(RPN2蛋白质)。RPN2mRNA的检测中,可以使用RNA印迹法、逆转录聚合酶链反应(RT-PCR)等本技术领域中公知的任意方法。RPN2蛋白质的检测中,可以使用本技术领域中公知的任意方法,例如使用了抗RPN2抗体的免疫学方法(蛋白质印迹法、ELISA等)。由于已知RPN2在胎盘以外的正常组织中几乎不表达,因此存在RPN2的表达或高水平表达则成为与RPN2的癌相关的指标。这样的癌的治疗和预防中,使用包含RPN2抑制剂的本发明的药物组合物进行处置是有效的。
1个实施方式中,本发明的检测方法还包括检测突变型p53基因的步骤。突变型p53基因的检测可以使用本技术领域中公知的利用了杂交、电泳、核酸扩增、测序等的任意核酸检测·分析方法来进行。如上所述,当表达RPN2的癌细胞中存在突变型p53时,通过抑制RPN2可以有效治疗和预防与突变型p53相关的癌。因此,本发明的检测方法作为用于确定有效的治疗和预防方法的诊断方法是有用的。
以下通过实施例更详细地说明本发明,但本发明不受这些实施例限定。
实施例
实施例1
将人乳癌细胞MCF7-ADR分成2种细胞级分CD44+CD24-和CD44+CD24+,培养7天。仅CD44+CD24-的级分具有作为癌干细胞性质的不等分裂(图1A)。
利用RT-PCR解析核糖体结合糖蛋白II(RPN2)的表达。其结果是,CD44+CD24-(癌干细胞)中的表达比CD44+CD24+(非癌干细胞)中的表达亢进约20倍以上(图1B)。
实施例2
使用shRPN2载体,制作RPN2的表达被恒常敲低的人乳癌细胞。使用的细胞为MCF7、MCF7-ADR、MDA-MB-231LN(MM231-LN)这三种人乳癌细胞株。MCF7为MCF7-ADR(抗药性株)的母株,为非恶性乳癌细胞,为激素受体(hormonereceptor)阳性,对药物具有敏感性。MM231-LN为激素受体阴性、高转移性的恶性度高的细胞株。
对获得的各细胞,在10nM多西紫杉醇(docetaxel)存在下孵育96小时后,测定CD44+CD24-癌干细胞数目。其结果是,导入了shRPN2载体的MCF7-ADR细胞和MM231-LN细胞与导入了对照载体(shNC)的细胞相比,CD44+CD24-癌干细胞数量显著减少(图2)。
实施例3
作为癌干细胞的特征之一,具有在平面培养(培养皿)中形成集落的能力。如图3A上图(MM231-LN-shNC)所示,MM231-LN细胞形成多个集落,在导入shRPN2载体时,集落形成能力显著受到抑制(图3A下图,MM231-LN-shRPN2)。图3B示出所形成的集落数。
实施例4
癌干细胞的另一特征是:即使在动物中移植较少的细胞数,也会形成明显的肿瘤。在此,对2种人乳癌细胞(MCF7-ADR和MM231-LN),比较了敲低RPN2的情况(shRPN2)和未敲低的情况(shNC)。将导入了shRPN2载体的细胞移植到小鼠(NOD-Scid小鼠、雌性、6周龄)背部的右侧,将导入了shNC载体的细胞移植到左侧。就移植后的细胞数而言,在MCF7-ADR的情况下为1×104个/部位,在MM231-LN的情况下为1×102个/部位。
其结果是,如小鼠的影像图(图4中间图(MCF7-ADR-luc)和右图(MM231-LN-luc))所示,导入了shRPN2载体的细胞均丧失了肿瘤形成能力。表1中总结了上述结果。
表1
实施例5
MM231-LN细胞为恶性度高的高转移性株,当将该细胞移植到小鼠的乳腺时,转移至腋下、胸部淋巴结,100%致死。比较了在该株系中导入了shRPN2载体的情况和导入了shNC载体的情况。其结果是,在shRPN2的组中,淋巴结转移显著受到抑制(图5)。表2中总结了上述结果。
表2
实施例6
对实施例5所述的小鼠进一步长期观察的结果是,shNC组中,移植细胞数1×102个、1×103个时均为全部致死,shRPN2组中,移植1×102个、1×103个均为全部生存(图6)。图6中,纵轴表示无转移生存率(%),横轴表示时间(天)。
实施例7
对通过shRPN2载体的导入而将RPN2敲低的MCF7-ADR和MM231-LN这2种细胞,通过利用蛋白质印迹法的蛋白质解析调查了突变型p53的表达。其结果是,发现任一细胞的情况下突变型p53的表达均显著降低(图7A)。
进而,明确了利用shRPN2进行的RPN2敲低诱导了E-钙黏着蛋白的表达(图7B)。图7B左图表示关于shNC的结果,图7中间图表示关于shRPN2的结果,图7B右图表示E-钙黏着蛋白阳性细胞率(%)。
E-钙黏着蛋白的表达在癌细胞显示上皮间质转化(EMT)时丧失。E-钙黏着蛋白减少的细胞具有更容易转移的性质。当利用shRPN2将RPN2高表达的细胞中的RPN2的表达敲低时,E-钙黏着蛋白的表达上升,这一事实可以支持RPN2诱导了EMT。
实施例8
为了研究突变型p53的表达受RPN2控制的机理,利用蛋白质组方法,对由于向细胞中导shRPN2载体从而表达发生变化的蛋白质进行了解析。其结果是,显示了与14-3-3ζ有关(图8)。14-3-3ζ在mdm2的介导下发挥使突变型p53分解的作用。因此认为:在RPN2亢进的细胞中,14-3-3ζ的表达降低,因此发生突变型p53的稳定化,细胞的命运注定走向E-钙黏着蛋白减少等与转移相关的EMT方向。
实施例9
将MM231-LN细胞移植到动物中,对所形成的肿瘤,通过使用对核特异性染色的DAPI(蓝)、抗RPN2抗体(绿)、抗突变型p53抗体(红)的3色免疫组织染色进行了调查(图9左图)。RPN2阳性细胞和突变型p53阳性细胞呈现一致。进而,利用ABC法染色调查了RPN2的表达(图9中间图)。图9中染成深色的RPN2高表达癌细胞中,E-钙黏着蛋白的表达减少(图9右图)。
实施例10
对2个人乳癌患者的乳癌组织,通过使用对核特异性染色的DAPI(蓝)、抗RPN2抗体(绿)、抗突变型p53抗体(红)的3色荧光免疫染色进行了调查。组织为原发灶,均为淋巴结转移阳性。任一种情况下,RPN2阳性细胞和突变型p53阳性细胞均呈现一致,这可以支持实施例9中的体外结果(图10)。
实施例11
狗的自然发病乳癌中的、针对RPN2基因的siRNA投与所引起的乳癌细胞的凋亡和乳癌肿瘤的缩小
通过对狗的自然发病癌组织(包括乳腺癌)进行PCR解析,发现狗的乳癌组织中也具有RPN2高表达的倾向。针对狗RPN2设计siRNA,与核酸移送载体水溶液(作为核酸移送载体,以终浓度0.5%使用了含有肽表面活性剂的移送载体(A6K:日本专利公开2010-222338)。)混合,制备siRNA-载体复合物(终浓度1mg/mL)。
对狗(金毛猎犬、体重约40kg)的自然发病乳癌肿瘤,将上述siRNA-载体复合物每隔2天局部投与2次,自最后投与起第3天通过外科方式切除肿瘤组织。作为对照,仅投与生理盐水、仅投与载体(A6K)。
就肿瘤的外观尺寸而言,在siRNA-载体复合物投与前为长径36.3mm、短径17.1mm,在最后投与起第3天变成长径24.9mm、短径15.6mm,肿瘤体积(GTV)可见43%的缩小。
对外科方式切除的肿瘤组织进行切片观察和TUNEL分析,确认肿瘤细胞通过siRNA-载体复合物的投与而凋亡,投与后的组织中看不到肿瘤的特征性结构(图11右图)。另一方面,仅投与A6K的情况下,肿瘤组织未见大的变化(图11左图)。
通过siRNA-载体复合物的投与,RPN2的mRNA被敲低约50%(图12)。
产业上的可利用性
根据本发明,可以提供以RPN2基因为靶标的用于含有癌干细胞的或由其起源的癌的治疗、预防和诊断的方法和组合物。
Claims (5)
1.一种用于含有癌干细胞的或由其起源的癌的治疗或预防的药物组合物,其包含RPN2抑制剂。
2.根据权利要求1所述的药物组合物,其中,癌干细胞具有突变型p53基因。
3.根据权利要求1或2所述的药物组合物,其中,RPN2抑制剂是针对RPN2基因的siRNA。
4.一种癌干细胞的检测方法,其包括确定RPN2有无表达或表达水平的步骤。
5.根据权利要求4所述的方法,其还包括检测突变型p53基因的步骤。
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