CN113853216A - 治疗去势抵抗性前列腺癌的方法 - Google Patents
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Abstract
本发明涉及含CUB结构域的蛋白质1(CDCP1)的降调剂,用于治疗患有去势抵抗性前列腺癌的患者的方法。本发明进一步涉及包含CDCP1降调剂和除衰化合物的药物组合物,以及选择适合用CDCP1降调剂和除衰化合物的组合治疗的前列腺癌患者的方法。
Description
本发明涉及通过诱导癌细胞衰老用于治疗癌症的方法。所述癌症为前列腺癌,特别是去势抵抗性前列腺癌。
1引言
去势抵抗性前列腺癌(CRPC)仍然是西方国家男性死亡的第二大原因。尽管第二代雄激素剥夺疗法(ADT)已经成功用于治疗转移性CRPC(mCRPC)患者,但是患者会产生耐药性并最终屈服于该疾病。mCRPC耐药性机制包括雄激素受体(AR)激活(例如,AR扩增、突变或剪接变体)和促进AR非依赖性生长的信号通路上调,如PI3K/AKT和MAPK通路。尽管在大多数转移性前列腺癌中PI3K信号通路是通过PTEN的缺失或突变激活的,但是MAPK通路激活的机制仍然未知。事实上,该通路的两个主要调节剂K-RAS和BRAF的过表达或突变仅占前列腺癌病例的少数(El Sheikh等,2008.Neoplasia 10:949-953;Reid等,2010.Br J Cancer 102:678-684;Taylor等.,2010.Cancer Cell 18:11-22)。因此,在PTEN无效前列腺癌的情况中,MAPK通路的新调节剂的鉴定将为治疗这些患者的新的潜在有效疗法开辟道路。
2发明简述
本发明提供了含CUB结构域的蛋白质1(CDCP1)的降调剂,用于治疗患有去势抵抗性前列腺癌的患者的方法。
本发明人惊喜地发现CDCP1在前列腺癌患者的治疗过程中变得上调,特别是在抗雄激素疗法的治疗期间。发现CDCP1的降调剂在这些细胞中诱导衰老。
所述降调剂优选为或包括识别含CUB结构域的蛋白质1(CDCP1)的胞外表位的抗体。
根据本发明使用的所述降调剂优选与抗雄激素疗法组合,优选与雄激素受体拮抗剂组合。所述抗雄激素优选选自恩杂鲁胺、阿比特龙、比卡鲁胺和尼鲁米特。
根据本发明使用的所述降调剂优选与除衰(senolytic)化合物和/或遗传毒性剂组合,优选进一步与雄激素受体拮抗剂(例如恩杂鲁胺、阿比特龙、比卡鲁胺和尼鲁米特)组合。
所述除衰化合物优选选自雷帕霉素、ABT263、FOXO4-DRI、CXCR4抑制剂和/或拮抗剂以及达沙替尼。
本发明进一步提供了药物组合物,其包括CDCP1的降调剂和雄激素受体拮抗剂,除衰化合物和/或遗传毒性剂。所述药物组合物优选包括包含CDCP1降调剂的药物制剂,以及包含雄激素受体拮抗剂、除衰化合物和/或遗传毒性剂的药物制剂。
根据本发明的药物组合物中的CDCP1降调剂优选是存在于脂质体中,所述脂质体优选进一步包含蒽环类如多柔比星。
根据本发明的药物制剂优选为用于治疗患有去势抵抗性前列腺癌的患者的方法。
本发明进一步提供了选择患有前列腺癌的适合CDCP1降调剂和除衰化合物组合治疗的患者的方法,包括确定患者体液中的睾酮水平如何;鉴定睾酮水平低于50ng/dL的患者;确定所鉴定的病人的前列腺癌是否正在进展;并选择睾酮水平低于50ng/dL且前列腺癌正在进展的患者作为适合接受CDCP1降调剂和除衰化合物组合治疗的患者。
鉴定的患者中前列腺癌的进展优选通过血清前列腺特异性抗原(PSA)水平的持续上升和/或所述患者中新的转移灶的出现来确定。
本发明进一步提供了用CDCP1降调剂和除衰化合物组合治疗前列腺癌患者的方法,包括确定患者体液中的睾酮水平;鉴定睾酮水平低于50ng/dL的患者;确定所鉴定的患者的前列腺癌是否在进展;以及用CDCP1降调剂和除衰化合物和/或遗传毒性剂的组合治疗睾酮水平低于50ng/dL且前列腺癌正在进展的患者,优选其中在给药除衰化合物和/或遗传毒性剂之前给药所述降调剂。
本发明进一步提供了用于治疗去势抵抗性前列腺癌患者的方法,包括鉴定患有去势抵抗性前列腺癌的患者;用CDCP1降调剂和除衰化合物和/或遗传毒性剂的组合治疗所述被鉴定的患者,优选其中在给药除衰化合物和/或遗传毒性剂之前给药降调剂。
3附图图例
图1:晚期前列腺肿瘤表现出含CUB结构域的蛋白质1(CDCP1)的表达升高,且CDCP1的条件性过表达在转基因小鼠模型中促进前列腺肿瘤发生。A.在正常前列腺、晚期/转移性PCa和人前列腺癌的TMA的远端转移灶中CDCP1的IHC染色代表性图像。B.在正常前列腺/良性、局部HSPC、原发性CRPC、晚期/转移PCa和人前列腺癌的TMA(n=438)的远端转移灶中CDCP1阳性样品的百分比。C.WT和CDCP1小鼠的前列腺组织病理学表征和定量。(BPH:良性前列腺增生)。D.WT和CDCP1小鼠在指定年龄的前部前列腺Ki-67染色的定量(每个基因型n=3)。E.条形图表示与WT前列腺相比,CDCP1前列腺中p-Akt、p-Erk1/2和p-Src相对于其总蛋白的归一化倍数变化(n=4)。误差条指出标准偏差(SD)。*P<0.05;**P<0.01.n.s,不显著。
图2。CDCP1与Pten-缺失协同驱动前列腺癌的完全恶性和转移。A.10月龄的Ptenpc-/-和CDCP1;Ptenpc-/-前部前列腺的H&E染色的代表性图像。原始放大倍数,10x。插图是每种基因型以更高放大倍数(40x)显示的区域。B.指定基因型的前部前列腺Ki-67染色定量(对于每种基因型n=4)。误差条指出SD。*P<0.05.C.WT、CDCP1;Ptenpc-/-和CDCP1;Ptenpc-/-小鼠的累积生存分析。插图代表Ptenpc-/-和CDCP1;Ptenpc-/-的前部前列腺。标度1cm。D.20周龄时来自指定基因型的前列腺前腺中特定蛋白质的蛋白质印迹分析和蛋白质倍数变化定量。E.在去势后8周,Ptenpc-/-和CDCP1;Ptenpc-/-去势和未去势的小鼠的前列腺重量和体积的量化(n=4)。F.20周龄的Ptenpc-/-和CDCP1;Ptenpc-/-去势小鼠的前部前列腺中指定蛋白质的蛋白质印迹分析及蛋白质倍数变化定量。误差条指出SD.n.s,不显著;*P<0.05;**P<0.01;***P<0.001.
图3。CDCP1的过度表达通过激活c-Myc克服了Pten-缺失诱导的细胞衰老。A.在来自指定基因型的前列腺前腺中p21、c-Myc、周期蛋白D1、COUP-TFII、Smad4和p53的蛋白质印迹分析。B.来自12-16周龄小鼠的Ptenpc-/-和CDCP1;Ptenpc-/-前列腺中c-Myc、周期蛋白D、COUP-TF-II、p21、p27和p16表达的定量实时PCR分析(小鼠n=3)。C.用塞卡替尼(100nM)治疗12小时的Pten-/-和CDCP1;Pten-/-MEF的蛋白质印迹分析。D.用塞卡替尼(100nM)治疗或用DMSO处理作为对照的Pten-/-和CDCP1;Pten-/-MEF中通过结晶紫对生长的倍数变化量化(n=3)。
图4。A.表达PLKOsh-CDCP1(sh-CDCP1#1)和多西环素诱导型Tripz-sh-CDCP1(sh-CDCP1#2)的感染PC3细胞中CDCP1蛋白质的蛋白质印迹分析。B.条形图代表在所有组中SA-β-Gal阳性细胞的百分比(n=3)。
图5。左图,在PC3 sh-Cont#2和PC3 sh-CDCP1#2异种移植瘤中CDCP1、p-SRC、SRC、c-MYC、CYCLIN D1、COUP-TFII的蛋白质印迹分析。右图,在PC3 sh-Cont#2和PC3 sh-CDCP1#2异种移植瘤中p27和p21mRNA水平的定量实时PCR(n=3)。
图6。晚期前列腺肿瘤表现出CDCP1 A表达升高。26名前列腺癌患者在HSPC和CRPC阶段的匹配活检中膜CDCP1的表达(H评分)。显示了中位H分数和四分位距。使用Wilcoxon配对符号秩检验计算p值。B.左图,对LNCaP-ADS和LNCaP-ADI中的CDCP1、p-SRC、SRC、c-MYC、p-ERK1/2和ERK1/2进行蛋白质印迹分析。右图,LNCaP-ADS和LNCaP-ADI中CDCP1蛋白水平的倍数变化量化。C.左图,在全培养基中生长的LNCaP中CDCP1 mRNA水平的定量实时PCR分析,完全雄激素剥夺(FAD),并在FAD中生长2天后用双氢睾酮(DHT 1μM,16小时)刺激。右图,在全培养基中、FAD中生长和在FAD中生长2天后用DHT(1μM,16小时)刺激的LNCaP中CDCP1、p-SRC、SRC、p-AKT、AKT、p-ERK1/2和ERK1/2的蛋白质印迹分析。D.左图,表达空载体(PC3-Cont)的PC3和过表达全长雄激素受体(PC3-AR)的PC3中CDCP1 mRNA水平的定量实时PCR。中图,PC3-cont和PC3-AR中CDCP1蛋白水平倍数变化的量化。右图,PC3-Cont和PC3-AR细胞系中CDCP1和AR的蛋白质印迹分析。
图7A.左图,在全培养基中保存的LNCaP和在全培养基中保存并用mAb-CUB4处理的LNCaP中通过结晶紫对生长倍数变化的量化。右图,在FAD中保存的LNCaP和FAD中保存并用mAb-CUB4处理的LNCaP中通过结晶紫对生长倍数变化的量化。B.左图,未处理、用恩杂鲁胺、免疫脂质体处理和用恩杂鲁胺和免疫脂质体组合处理的LNCaP细胞系的异种移植瘤的生长(mm3)。右图,LNCaP异种移植物组中切割的胱天蛋白酶3的蛋白质水平倍数变化的量化。C.LNCaP异种移植物组中CDCP1表达、p-SRC和切割的胱天蛋白酶3的蛋白质印迹分析。
图8。抗-CDCP1抗体序列。A.小鼠和嵌合小鼠-人重链序列,如WO2015/082446中所述。B.人重链序列,如WO2011/023389中及Siva等人于2008年所述(Siva等,2008.J ImmunolMethods 330:109-119)。C.小鼠和嵌合小鼠-人轻链序列,如WO2015/082446中所述。D.人轻链序列,如WO2011/023389中和Siva等人于2008年所述(Siva等,2008.J Immunol Methods330:109-119)。
图9。在癌症基因组图谱(TCGA;左图)和Taylor数据集(右图;Taylor等,2010.Cancer Cell 18:11-22)中PTEN基因组缺失与CDCP1基因表达的关联。误差条指出平均值的标准误差(SEM)统计学检验:Kruskal-Wallis(Kruskal与Wallis,1952.J AmericanStatistical Association 47:583-621)。
图10。PTEN和CDCP1表达水平与指定患者数据集中的无病生存期的关联。在Taylor数据集中,低PTEN表示表达信号低于8.74的患者,高CDCP1表示表达信号高于11.19的患者。在TCGA中,低PTEN表示表达信号低于10.19的患者,高CDCP1表示表达信号高于9.49的患者。HR,风险比。统计学检验:Mantel-Cox检验(Mantel,1966.Cancer Chemotherapy Reports50:163-170)。
4发明详述
4.1定义
本文所用术语“降调剂”是指减少含CUB结构域的蛋白质1(CDCP1)表达的分子,特别是CDCP1在去势抵抗性前列腺癌细胞表面的表达,和/或减少去势抵抗性前列腺癌细胞中CDCP1-介导MAPK激活的分子。
本文所用术语“CUB结构域”是指具有约110个氨基酸残基的结构基序,其在蛋白质补体成分1r/1s、海胆蛋白表皮生长因子(uEGF)和骨形态发生蛋白1中首次被鉴定。CUB结构域是进化保守蛋白结构域,其几乎仅存在于胞外和细胞质膜关联蛋白中。
本文所用术语“含CUB结构域的蛋白质1(CDCP1)”是指位于人染色体3p21.31上的CDCP1基因的蛋白产物。CDCP1基因编码包含三个胞外CUB结构域的跨膜蛋白且作为Src家族激酶的底物。该蛋白在涉及肿瘤侵袭和转移的细胞事件的酪氨酸磷酸化依赖性调控中起作用。可变剪接产生该基因的多种转录变体。该基因在HGNC被称为:24357;在Entrez基因被称为:64866;和/或在Ensembl被称为:ENSG00000163814。其人类蛋白在UniProt被称为:Q9H5V8。CDCP1包含总计836个氨基酸残基,其N-末端氨基酸残基1-29表示信号肽;氨基酸残基30-668为胞外部分且包含3个CUB结构域;氨基酸残基668和688之间的区域表示跨膜区域;从氨基酸残基689开始C-末端区域表示胞质结构域。
本文所用术语“CDCP1的胞外表位”是指CDCP1的N-末端胞外部分的一种或多种表位,包括氨基酸残基30-668。
本文所用术语“去势抵抗性前列腺癌”是指当体内雄激素减少或甚至不存在时前列腺癌细胞继续增殖。许多早期前列腺癌依赖于雄激素来增殖,但去势抵抗性前列腺癌不是。也称作CRPC。
本文所用术语“雄激素”是指与雄激素受体结合的天然或合成的甾体类激素。雄激素在睾丸、卵巢和肾上腺中合成。主要的雄激素是睾酮,但术语雄激素下也包括双氢睾酮和雄烯二酮。
本文所用术语“抗雄激素疗法”是指旨在减少体内雄激素水平或阻止其影响前列腺癌细胞的疗法。替代性术语包括雄激素剥夺疗法(ADT)和雄激素抑制疗法。所述疗法包括使用促黄体激素释放激素(LHRH)激动剂(也称作LHRH类似物或GnRH激动剂)和LHRH拮抗剂,例如亮丙瑞林(L-焦谷氨酰-L-组氨酰-L-色氨酰-L-丝氨酰-L-酪氨酰-D-亮氨酰-L-亮氨酰-L-精氨酰-L-脯氨酸乙胺酰乙酸)、戈舍瑞林(L-焦谷氨酰-L-组氨酰-L-色氨酰-L-丝氨酰-L-酪氨酰-O-叔丁基-D-丝氨酰-L-亮氨酰-L-精氨酰-N′-氨基甲酰-L-脯氨酸酰阱)、曲普瑞林(L-焦谷氨酰-L-组氨酰-L-色氨酰-L-丝氨酰-L-酪氨酰-D-色氨酰-L-亮氨酰-L-精氨酰-L-脯氨酰-甘氨酰胺)、组氨瑞林(L-焦谷氨酰-L-组氨酰-L-色氨酰-L-丝氨酰-L-酪氨酰-1-苄基-D-组氨酰-L-亮氨酰-L-精氨酰-L-脯氨酸乙胺酰)和地加瑞克(N-乙酰基-3-(2-萘基)-D-丙氨酰-4-氯-D-本病氨酰-3-(3-吡啶基)-D-丙氨酰-L-丝氨酰-4-((S)-二氢乳清氨基(dihydroorotamido))-L-苯丙氨酰-4-脲基-D-本病氨酰-L-亮氨酰-N6-异丙基-L-赖氨酰-L-脯氨酰-D-氨基丙酰胺);CIP17抑制剂和/或雄激素合成抑制剂例如阿比特龙((3S,8R,9S,10R,13S,14S)-10,13-二甲基-17-吡啶-3-基-2,3,4,7,8,9,11,12,14,15-十氢-1H-环戊[a]菲-3-醇);雄激素合成抑制剂例如酮康唑(1-[4-[4-[[(2R,4S)-2-(2,4-二氯苯基)-2-(咪唑-1-基甲基)-1,3-二氧戊环-4-基]甲氧基]苯基]哌嗪-1-基]乙酮),TAK-700(奥特龙(orteronel);6-[(7S)-7-羟基-5,6-二氢吡咯并[1,2-c]咪唑-7-基]-N-甲基萘-2-甲酰胺),和TOK-001(盖勒特龙(galeterone);(3S,8R,9S,10R,13S,14S)-17-(苯并咪唑-1-基)-10,13-二甲基-2,3,4,7,8,9,11,12,14,15-十氢-1H-环戊[a]菲-3-醇),和雄激素受体拮抗剂例如氟他胺(2-甲基-N-[4-硝基-3-(三氟甲基)苯基]
丙酰胺)、比卡鲁胺(N-[4-氰基-3-(三氟甲基)苯基]-3-(4-氟苯基)磺酰基-2-羟基-2-甲基丙酰胺)、尼鲁米特(5,5-二甲-3-[4-硝基-3-(三氟甲基)苯基]咪唑啉酮-2,4-二酮)、ARN-509(阿帕鲁胺;4-[7-[6-氰基-5-(三氟甲基)吡啶-3-基]-8-氧代-6-磺酰亚胺(sulfanylidene)-5,7-氮杂螺[3.4]辛-5-基]-2-氟-N-甲基苯甲酰胺)和恩杂鲁胺(4-[3-[4-氰基-3-(三氟甲基)苯基]-5,5-二甲-4-氧代-2-磺酰亚胺咪唑啉酮-1-基]-2-氟-N-甲基苯甲酰胺)。
本文所用术语″遗传毒性剂″是指诱导细胞的基因组DNA损伤的试剂,包括碱基修饰、单链断裂和交联,例如链内和链间交联。
本文所用术语“除衰化合物”是指消除衰老细胞的化合物,这些细胞已经失去功能,但是对细胞死亡具有抗性。所述消除优选为选择性的,意为衰老细胞数目减少,优选减少为零,而非衰老细胞数目不减少,或只是几乎不减少。除衰化合物(也称作除衰剂)的例子为达沙替尼、槲皮素及其组合(Xu等.,2018.Nature Med 24:1246-1256),UBX0101(也称作纳维托卡(Navitoclax)和ABT-263;Jeon等.,2017.Nature Med 23:775-781),荜茇酰胺及其类似物(Zhu等.,2019.Bioorganic Med Chem 26:3925-3938),漆黄素(fisetin),天然天然产生的黄酮(Zhu等.,2917.Aging 9∶955-963);选择性BCL-XL抑制剂例如A1331852和A1155463(Zhu等,2917.Aging 9:955-963),HSP90伴侣抑制剂例如17-DMAG(Fuhrmann-Stroissnigg等.,2017.Nature Comm 8:422(10.1038/s41467-017-00314-z);阿卡波糖,去甲二氢愈创木酸(nordihydroguaiaretic acid)(NDGA)(Harrison等.,2014.Aging Cell13:273-282);和FOXO4-DRI
(H-D-Leu-D-Thr-D-Leu-D-Arg-D-Lys-D-Glu-D-Pro-D-Ala-D-Ser-D-Glu-D-Ile-D-Ala-D-Gln-D-Ser-D-Ile-D-Leu-D-Glu-D-Ala-D-Tyr-D-Ser-D-Gln-D-Asn-D-Gly-D-Trp-D-Ala-D-Asn-D-Arg-D-Arg-D-Ser-D-Gly-D-Gly-D-Lys-D-Arg-D-Pro-D-Pro-D-Pro-D-Arg-D-Arg-D-Arg-D-Gln-D-Arg-D-Arg-D-Lys-D-Lys-D-Arg-D-Gly-OH;Baar等.,2017.Cell 169:132-147)。
本文所用术语抗体包括重链和轻链的经典异二聚体抗体,单重链可变功能域抗体例如骆驼科VHH、鲨鱼免疫球蛋白衍生的可变新抗原受体,以及scFv、串联scFv、scFab和改进的scFab(Koerber等.,2015.J Mol Biol 427:576-86)。该术语还包括特异性结合CDCP1的α-βT细胞受体分子和/或γ-δT细胞受体分子,分离T细胞受体分子和存在于T细胞表面的T细胞受体分子,例如嵌合抗原受体-T细胞(CAR T细胞)。
术语抗体还包括可以特异性结合CDCP1但与抗体结构上不相关的抗体样分子。这种抗体样分子包括设计的锚蛋白重复蛋白,基于蛋白A的Z结构域的结合蛋白,基于纤连蛋白III型结构域的结合蛋白,工程改造的脂质运载蛋白,和基于人Fyn SH3结构域的结合蛋白(Skerra,2007.Current Opinion Biotechnol 18:295-304;等.,2015.TrendsBiotechnol 33:408-418)。
术语抗体还提供抗体-药物偶联物的参考,由此所述药物偶联物为化疗药物、毒性化合物或放射性化合物。有毒化合物的实例是皂草素、异株泻根毒蛋白、农杆菌素(agrostin)、蓖麻毒蛋白(ricin)、白树毒素(gelonin)、石竹素(dianthin)、丝瓜籽蛋白(luffin)、α-苦瓜素、β-苦瓜素、商陆毒蛋白、麦芽凝集素(tritin)、苦瓜蛋白和天花粉蛋白。
术语‘特异性结合’或‘特异性’或其语法变体是指特定抗体可以结合到的不同类型的抗原或其表位的数目。抗体的特异性可以基于亲和力确定。特异性抗体对其表位的结合亲和力Kd优选小于10-7M,优选小于10-8M,最优选小于10-9M。
4.2 CDCP1的上调
本发明提供了含CUB结构域的蛋白质1(CDCP1)的降调剂,用于治疗患有去势抵抗性前列腺癌的患者的方法。
如果在雄激素剥离治疗后,血浆睾酮水平在20至50ng/dL之间,等同于0.7至1.7nmol/L之间,癌症仍然进展,则前列腺癌已经成为去势抵抗性的。必须对单纯的症状性进展提出质疑,并进行进一步调查。症状性进展本身不足以诊断CRPC。去势抵抗性前列腺癌(CRPC)的其他标志物包括生物化学进展,例如前列腺特异性抗原(PSA)连续三次上升,间隔一周,导致两次比最低点增加50%,和/或PSA>2ng/mL,和/或放射学进展,例如出现新的病灶,例如在骨扫描中出现一个、两个或更多新的骨病灶或出现软组织病灶,使用RECIST(实体瘤反应评价标准)(EAU前列腺癌指南,2017.N.Mottet)。
在本发明中发现,CDCP1在抗雄激素疗法时,特别是在雄激素受体拮抗剂治疗时,在前列腺癌细胞中变得上调。在前列腺癌细胞对所述抗雄激素疗法产生抗性时,上调变得突出。不囿于理论,认为CDCP1的上调提供了一种独立的驱动力,刺激MAPK-通路以致于细胞变得不受雄性激素的影响,即对抗雄激素疗法产生抗性。CDCP1的表达和/或CDCP1的MAPK通路刺激活性的降调,将诱导此类去势抵抗性前列腺癌细胞的衰老。
所述降调剂是指使前列腺癌细胞中CDCP1表达降调的分子和/或使前列腺癌细胞中CDCP1介导的MAPK激活失活的分子。
4.3使前列腺癌细胞中CDCP1表达降调的分子
使前列腺癌细胞表面上的含CUB结构域的蛋白质1(CDCP1)表达降调的分子,优选地选自使CDCP1基因的转录降调或废止的分子,例如锌指蛋白转录因子、转录激活因子样效应物(TALE)阻遏物,以及,例如通过转录激活因子样效应核酸酶(TALEN)或通过规律成簇的间隔短回文重复序列(CRISPR)和CRISPR-相关蛋白CAS介导的CDCP1基因的破坏(Gaj等.,2013.Trends Biotechnol 31:397-405);RNA表达产物的降调,通过例如反义RNA分子和siRNA分子;以及CDCP1蛋白的降调,通过例如抗体。
锌指蛋白转录因子是DNA结合基序,由模块化锌指结构域组成,这些结构域与转录激活子或阻遏物偶联。每个结构域可以工程改造为识别特定DNA三联体。三个或更多结构域的组合导致基因特异性序列识别。所述组装的锌指蛋白与转录阻遏物结构域偶联,例如Krüppel-相关盒(KRAB)、ERF阻遏物结构域(ERD)或mSIN3互作结构域(SID)。在前列腺癌细胞中表达所述偶联的锌指蛋白-转录阻遏物结构域会导致CDCp1基因沉默。
类似地,合成转录因子DNA结合结构域(DBD)可以被编程为识别特定DNA基序。这种转录激活因子样效应物(TALE)DNA结合结构域(DBD)包含一些,从7到34个,高度同源的直接重复,每个由33-35个氨基酸组成。特异性包含在每个重复的12位和13位的两个氨基酸残基中。由于RVD的DNA:蛋白质结合密码已经被破译,有可能通过工程改造合适的DBD设计能结合任意所需靶向DNA序列的TALE。通常,TALE被设计为识别15到20个DNA碱基对,平衡特异性和潜在的脱靶(Boettcher和McManus,2015.Mol Cell 58:575-585)。CDCP1-特异的TALE就与转录阻遏物结构域偶联,例如KRAB结构域、ERD结构域或SID结构域。在前列腺癌细胞中表达所述偶联的TALE-转录阻遏物结构域会导致CDCp1基因沉默。
TALEN或CRISPR-CAS介导CDCP1基因的破坏是通过靶向CDCP1基因上至少一种特异性位置的核酸酶介导的,优选至少两个特异性位置。所述靶向是通过TALE-DNA结合结构域介导的,或通过CRISPR单嵌合向导RNA序列。核酸酶介导在CDCP1基因的基因组DNA上双链断裂,在TALEN的情况下是FOK1核酸酶,对于CRISPR是CAS蛋白质,优选CAS9蛋白质。DNA双链断裂的引入通过同源重组增加了基因编辑的效率,在存在合适供体DNA的情况下删除部分或全部的CDCP1基因(Gaj等.,2013.Trends Biotechnol 31:397-405)。
反义RNA或反义寡核苷酸,采用一个或多个与编码CDCP1蛋白质的信使RNA(mRNA)互补的单链RNA分子。所述反义RNA将与mRNA杂交,从而阻断其翻译成蛋白质。由于RNA分子容易被RNA酶或其他降解酶降解,通常需要化学修饰。寡核苷酸上最常见的化学修饰是向骨架上添加硫代磷酸酯连接。
进一步优选的反义RNA分子包括修饰的骨架,例如吗啉代骨架、氨基甲酸酯骨架、硅氧烷骨架、硫化物、亚砜和砜骨架、甲乙酰基(formacetyl)和硫代甲乙酰基骨架,亚甲基甲乙酰基(methyleneformacetyl)骨架、核苷酸乙酰基(riboacetyl)骨架、含烯烃骨架、氨基磺酸盐、磺酸盐和磺酰胺骨架、亚甲基亚氨基(methyleneimino)和亚甲基肼基骨架(methylenehydrazino)以及酰胺骨架。吗啉代反义RNA分子具有不带电的骨架,其中DNA的脱氧核糖被六元环取代,磷酸二酯键被二氨基磷酸酯键取代。吗啉代寡核苷酸对酶促降解有抵抗性。
在进一步优选的反义RNA分子中,骨架中的核糖核苷酸残基之间的连接不包括磷原子,例如由短链烷基或环烷基-核苷间连接、混合杂原子和烷基或环烷基-核苷间连接、或一个或多个短链杂原子或杂环核苷间连接形成的连接。
优选的反义RNA分子包括取代磷酸二酯键中的非桥接氧之一。这种修饰略微使得碱基配对失稳,但添加了显著的核酸酶降解抗性。优选的核苷酸类似物或等同物包括硫代磷酸酯,手性硫代磷酸酯,二硫代磷酸酯,磷酸三酯,氨基烷基磷酸三酯,H-膦酸酯,甲基和其他烷基膦酸酯,其包括3′-亚烷基膦酸酯,5′-亚烷基膦酸酯和手性膦酸酯,次磷酸酯,磷酰胺,其包括3′-氨基磷酰胺和氨基烷基磷酰胺,硫代磷酰胺,硫代烷基膦酸酯,硫代烷基磷酸三酯,硒磷酸酯或硼磷酸酯。这些取代使得反义RNA分子对RNA酶H和核酸酶具有抗性,且增加了对靶RNA的亲和力。
进一步优选的反义RNA分子包括肽核酸(PNA),具有修饰的聚酰胺骨架(Nielsen等.,1991.Science 254:1497-1500),或包括锁核酸(LNA),其中2′-碳原子连接糖环的3′或4′碳原子从而形成双环糖部分。优选的LNA包括2′-O,4′-C-乙烯-桥接核酸(Morita等.2001.Nucleic Acid Res增刊No.1:241-242)。
RNA干扰(RNAi)i是基于短双链RNA(dsRNA)的产生,其激活细胞过程导致高度特异的RNA降解(Zamore等,2000.Cell 101:25-33)和/或抑制翻译。为了本发明的目的,激活RNAi的dsRNA分子和它们在细胞内经加工产生激活RNAi的dsRNA分子的前体被称为“RNAi分子”。RNA干扰是通过产生具有2核苷酸长度的3’突出端的18到23个核苷酸的dsRNA分子介导的,其称为小干扰RNA(siRNA)双链体。RNAi允许基于其序列的基因沉默。优选地,RNAi分子是一种可以直接或间接地激活细胞中RNAi过程的分子,因为它是可以在细胞中激活RNAi过程的分子的前体。所述前体分子优选为shRNA或前-miRNA或初-miRNA或其变体或类似物。
所述RNAi分子,例如,是短发夹RNA(shRNA)或miRNA前体。短发夹RNA(shRNA)通常包括50-100个核苷酸长的RNA分子,其包括两段互补的可碱基配对的核苷酸,由此这两段通过发夹转弯相互连接。shRNA发夹结构被细胞器切割成18-23(通常是19)个核苷酸长的双链siRNA分子,具有2核苷酸长的3’突出端,其中一条链与来自靶基因的mRNA转录本的一部分有广泛的互补同源性。所述siRNA激活RNA干扰(RNAi)通路并通过特异性mRNA降解干扰所述靶基因的表达。shRNA的表达可以由聚合酶II或聚合酶III增强子/启动子驱动。天然miRNA分子通常由聚合酶II转录为初-miRNA,其具有帽和多聚-A尾,并在细胞核中被加工成短的70个核苷酸的茎环结构,称为前-miRNA。这些前-miRNA随后在细胞质中被加工成约18-25个核苷酸的成熟双链miRNA,通过RNA干扰沉默基因表达,部分是通过特异性RNA降解,部分是通过抑制翻译。根据本发明,初-miRNA和前-miRNA分子也是有用的沉默因子。人工miRNA可以从任何启动子转录,例如polIII启动子,其格式类似于shRNA。它们与shRNA的不同之处在于双链区域不是完全互补的。
根据本发明,优选的RNAi分子包括双链区域,每条链18个核苷酸到25个核苷酸,例如18个核苷酸、19个核苷酸、20个核苷酸、21个核苷酸。22个核苷酸、23个核苷酸、24个核苷酸或25个核苷酸。根据本发明,最优选的RNAi分子包括双链区域,加工为成熟siRNA后长度为19个核苷酸。所述优选的RNAi分子包括序列V3THS_329377:
5’-TGAGGGTAGGCAACAACGA和/或V2THS_191307:
5’-TCTTTCTCCAGACTTGATG。
降调RNA表达产物的优选方法的产物包括反义RNA分子的不均匀混合物和/或所有靶向CDCP1 mRNA序列的siRNA的混合物。这种多重沉默导致高特异性和高效的基因沉默。
所述降调前列腺癌细胞表面上含CUB结构域的蛋白质1(CDCP1)的表达的分子优选在载体中提供。所述载体优选还包括用于高表达水平的手段,如强启动子,例如病毒来源(例如人巨细胞病毒)的,或衍生自在前列腺癌细胞中高度表达的基因的启动子例如PSA-、前列腺碱性蛋白(probasin)-或MMTV LTR-启动子(Lu等.,2013.Biomed Res Int624632)。如本领技术人员已知,载体优选地包含选择系统,例如,谷氨酰胺合成酶的表达或二氢叶酸还原酶的表达,用于在合适的受体细胞中扩增载体。
所述载体优选为病毒载体,优选能够转导前列腺癌细胞的病毒载体。所述病毒载体优选为重组腺相关病毒载体,基于单纯疱疹病毒的载体或基于逆转录病毒的载体例如基于慢病毒的载体,例如基于人免疫缺陷病毒的载体。所述病毒载体最优选是基于逆转录病毒的载体,例如基于慢病毒的载体,例如基于人免疫缺陷病毒的载体,或基于γ-逆转录病毒的载体例如基于莫洛尼鼠白血病病毒(MoMLV)的载体,脾脏病灶形成病毒(SFFV),骨髓增生性肉瘤病毒(MPSV)或基于鼠干细胞病毒(MSCV)的载体。优选的逆转录病毒载体是SFGγ逆转录病毒载体(Rivière等.,1995.PNAS92:6733-6737)。
逆转录病毒,包括基于γ-逆转录病毒的载体,可以被包装在一个合适的补充细胞中,该细胞提供组抗原多聚蛋白(Gag)-聚合酶(Pol)和/或包膜(Env)蛋白质。合适的包装细胞为人类胚胎肾衍生293T细胞、菲尼克斯(Phoenix)细胞(Swift等.,2001.Curr ProtocImmunol,第10章:10 17C单元)、PG13细胞(Loew等.,2010.Gene Therapy 17:272-280)和Flp293A细胞(Schucht等.,2006.Mol Ther 14:285-92)。
作为替代,非病毒性基因疗法可用于转导使前列腺癌细胞表面CDCP1表达降调的分子。非病毒载体包括裸DNA、脂质体、聚合剂和分子偶联物。无质粒细菌DNA序列的小环DNA载体可以在细菌中生成,并可表达编码使前列腺癌细胞中CDCP1表达降调的分子的核酸。
优选的非病毒基因疗法包括携带CDCP1降调剂的双隐性免疫脂质体(dualstealth immunoliposome),CDCP1降调剂优选抗-CDCP1抗体。所述免疫脂质体优选还包括遗传毒性剂,例如烷基化剂,例如氮芥,例如,环磷酰胺,甲氯乙胺或啶芥,尿嘧啶芥(uramustine)和/或乌拉莫司汀(uracil mustard),美法仑,苯丁酸氮芥(chlorambucil),异环磷酰胺;亚硝基脲,包括卡莫司汀,洛莫司汀,链脲菌素;烷基磺酸盐,例如白消安,乙烯利(ethylenime),如噻替帕及其类似物,肼/三嗪,例如达卡巴嗪,六甲蜜胺,米托唑胺,替莫唑胺,六甲蜜胺,甲基苄肼,达卡巴嗪和替莫唑胺;嵌入剂例如基于铂的化合物,如顺铂,卡铂,奈达铂,奥沙利铂,赛特铂;蒽环类药物,例如多柔比星,柔红霉素,表柔比星和伊达比星;丝裂霉素-C,放线菌素D,博来霉素,阿霉素和光神霉素。
所述免疫脂质体优选使用胆碱制备,优选使用磷脂酰胆碱,如氢化大豆磷脂酰胆碱,更优选胆碱和胆固醇的组合,更优选胆碱、胆固醇和磷脂的组合,磷脂如与聚乙二醇(PEG)偶联的二硬脂酰磷脂酰乙醇胺(distearoylphophatidylethanolamine),如本领域技术人员所熟知(Immordino等.,2006.Int J Nanomedicine 1:297-315)。为了纳入免疫脂质体,抗-CDCP1抗体优选与PEG偶联,更优选与PEG-磷脂衍生物,例如,与PEG偶联的二硬脂酰磷脂酰乙醇胺偶联。
优选的降调前列腺癌细胞表面CDCP1表达的分子是抗体,优选识别含CUB结构域的蛋白质1(CDCP1)的胞外表位的抗体。优选的抗体识别CDCP1胞外表位并导致细胞表面CDCP1降调诱发衰老。
特异性结合CDCP1胞外结构域的合适抗体是本领域已知的。例如,2004年,Bühring等(Bühring等.,2004.Stem Cells 22:334-343)描述了针对CDCP1胞外结构域的小鼠单克隆抗体。2008年,Siva等(Siva等,2008.J Immunol Methods 330:109-119)和已经公开的国际专利申请WO2011/023389、WO2015/082446中已经描述了其他适合的抗体。
所述抗体优选包括如图8所示的CDR1、CDR2和CDR3氨基酸序列,更优选如图8所示的可变结构域氨基酸序列。
4.4使前列腺癌细胞中CDCP1介导MAPK激活失活的分子
降调MAPK-通路刺激CDCP1活性的分子优选为小化合物分子。所述分子优选抑制CDCP1通过Src激酶的磷酸化,和/或抑制蛋白质激酶C-8(PKCd)和磷酸化的CDCP1之间的相互作用,更优选为PKCd的C2结构域和磷酸化的CDCP1之间的相互作用。
合适的分子已被描述,例如,2017年由Nakashima等(Nakashima等.,2017.CancerSci 108:1049-1057)。所述分子优选包括糖偶联钯复合物(Pd-Oqn)。优选分子包括氯(chloror){N-(配位羟(hydroxo)-喹啉-2-基亚甲基)-b-D-葡萄糖胺}钯(II)}和氯{N-(配位羟-喹啉-2-基亚甲基)-b-D-葡萄糖胺}钯(II),最优选氯{N-(配位羟-喹啉-2-基亚甲基)-b-D-葡萄糖胺}钯(II)}。
4.5治疗方法
本发明提供了CDCP1的降调剂,用于治疗患有去势抵抗性前列腺癌的患者的方法。
所述CDCP1的降调剂,是降调前列腺癌细胞表面的含有CUB结构域的蛋白质1(CDCP1)的表达的分子,或可全身给药降调MAPK通路刺激CDCP1活性的分子。全身给药包括口服、静脉内、肌内、关节内、动脉内、髓内、鞘内、硬膜外、心室内、透皮、皮下、腹膜内、鼻内、肠内、局部、舌下、吸入、眼内、耳内或直肠注射或输液,优选静脉内或肌肉内注射或输液。
根据与个体需要治疗相关的因素,CDCP1降调剂的给药剂量将由被给药的个体决定。所述剂量优选为每次给药0.5微克/公斤至10毫克/公斤。调整剂量和给药以提供足够水平的活性剂或维持所需的治疗效果。可以考虑的因素包括前列腺癌的严重程度和其他因素,包括对象的一般健康状况、年龄、对象的体重和性别、饮食、给药时间和频率、一种或多种药物组合、反应敏感性和对治疗的耐受性/反应,如本领域技术人员已知的。
所述抑制剂的合适的剂量为1-50mg/kg体重,对于反义RNA分子和siRNA分子优选约10mg/kg体重;对于抗体,0.1-50mg/kg体重,优选约0.5-2.5mg/kg体重。
CDCP1的降调剂可以每天给药一次、两次或三次,隔天一次和/或每周一次。治疗方案可以持续1-13周,并且可以中断1-4周不给药。如果需要,可以重复治疗方案,直到获得前列腺癌负荷的减少。
根据本发明,CDCP1降调剂可以与抗雄激素疗法、除衰化合物和/或遗传毒性剂组合使用。
因此,本发明提供了一种用抗体-药物偶联物和雄激素受体拮抗剂的组合治疗患有前列腺癌的患者的方法,由此抗体识别CDCP1的细胞外表位,由此药物偶联物是化疗药物、毒性化合物或放射性化合物,其中抗雄激素优选为恩杂鲁胺。
根据本发明,CDCP1降调剂可以与抗雄激素疗法组合使用。抗雄激素疗法的组合导致前列腺癌细胞中CDCP1的持续上调,通过CDCP1降调剂的共同给药,所述细胞变得衰老。本领域技术人员将理解抗雄激素疗法优选地至少部分地在CDCP1降调剂给药之前。这意味着该组合导致连续的、可能重叠的抗雄激素和CDCP1降调剂的给药,其中抗雄激素给药开始于CDCP1降调剂给药开始前至少一天。
所述抗雄激素疗法优选是或包括雄激素受体拮抗剂,选自:恩杂鲁胺,口服50-300mg,每日一次,优选口服约160mg,每日一次;比卡鲁胺,口服25-300mg,每日一次,优选口服约150mg,每日一次,以及尼鲁米特,口服100-450mg,每日一次,优选口服约150mg,每日一次。
根据本发明CDCP1降调剂优选与除衰化合物组合使用。所述与除衰化合物的组合选择性地诱导已经变得衰老的去势抵抗性前列腺癌细胞的死亡。
根据本发明,CDCP1降调剂优选与遗传毒性剂组合使用,例如烷基化剂,例如氮芥,例如,环磷酰胺,甲氯乙胺或啶芥,尿嘧啶芥(uramustine)和/或乌拉莫司汀(uracilmustard),美法仑,苯丁酸氮芥(chlorambucil),异环磷酰胺;亚硝基脲,包括卡莫司汀,洛莫司汀,链脲菌素;烷基磺酸盐,例如白消安,乙烯利(ethylenime),如噻替帕及其类似物,肼/三嗪,例如达卡巴嗪,六甲蜜胺,米托唑胺,替莫唑胺,六甲蜜胺,甲基苄肼,达卡巴嗪和替莫唑胺;嵌入剂例如基于铂的化合物,如顺铂,卡铂,奈达铂,奥沙利铂,赛特铂;蒽环类药物,例如多柔比星,柔红霉素,表柔比星和伊达比星;丝裂霉素-C,放线菌素D,博来霉素,阿霉素和光神霉素。
根据本发明CDCP1的降调剂优选与抗雄激素疗法组合使用,优选与雄激素受体拮抗剂和除衰化合物组合。
所述除衰化合物优选选自雷帕霉素(每天0.1-20mg,优选每天约2mg),阿卡波糖(每天5-150mg,优选每天约50mg),去甲二氢愈创木酸(NDGA;(每天500-5000mg,优选每天约2000mg),ABT263(每天10-500mg,优选每天约150mg),FOXO4-DRI(每天或每两天0.2-50mg优选每天或每两天约20mg,CXCR4抑制剂和/或拮抗剂例如TG-0054(2-[4-[6-氨基-2-[[4-[[3-(环己氨基)丙氨基]甲基]环己基]甲氨基]嘧啶-4-基]哌嗪-1-基]乙基膦酸),AMD070(~{N}′-(1~{H}-苯并咪唑-2-基甲基)-~{N}′-[(8~{S})-5,6,7,8-四氢喹啉-8-基]丁烷-1,4-二胺),AMD3100或普乐沙弗(1-[[4-(1,4,8,11-四氮杂环十四烷(tetrazacyclotetradec)-1-基甲基)苯基]甲基]-1,4,8,11-四氮杂环十四烷(tetrazacyclotetradecane);每天0.5-50mg),环肽CXCR4拮抗剂例如LY2510924和和达沙替尼(每天10-500mg,优选每天约140mg)。
优选在CDCP1降调剂的给药开始后一天,优选两天,优选3天,优选多于3天例如1-2周开始给药除衰化合物。
本发明进一步提供了用CDCP1降调剂和除衰化合物组合治疗前列腺癌患者的方法,该方法包括确定患者体液中的睾酮水平如何;鉴定睾酮水平低于50ng/dL的患者;确定所鉴定的患者的前列腺癌是否在进展;以及用CDCP1降调剂和除衰化合物组合治疗睾酮水平低于50ng/dL且前列腺癌正在进展的患者。
本发明进一步提供治疗患有去势抵抗性前列腺癌的患者的方法,包括鉴定患有去势抵抗性前列腺癌的患者;并用CDCP1降调剂和除衰化合物的组合治疗所述已鉴定的患者。
根据本发明的治疗的优选方法中,在除衰化合物给药之前给药所述降调剂。
4.6组合物
本发明进一步提供了药物组合物,其包括CDCP1的降调剂和除衰化合物和/或遗传毒性剂。
所述优选的药物组合物是等渗无菌溶液。所述缓冲液优选为基于柠檬酸盐的缓冲液,优选为柠檬酸-锂、钠、钾或钙一水合物、三水合物、四水合物、五水合物或七水合物;乳酸锂、钠、钾或钙;磷酸锂、钠、钾或钙;马来酸锂、钠、钾或钙;酒石酸锂、钠、钾或钙;琥珀酸锂、钠、钾或钙;或醋酸锂、钠、钾或钙,或上述两种或更多种的组合。所述缓冲液的pH值可以通过盐酸、氢氧化钠、柠檬酸、磷酸、乳酸、酒石酸、琥珀酸或上述两种或更多种的组合来调节,优选调节至7.27-7.37的pH值。体积可以在0.5ml到5ml的范围内。所述赋形剂优选选自但不限于尿素、L-组氨酸、L-苏氨酸、L-天冬酰胺、L-丝氨酸、L-谷氨酰胺、聚山梨醇酯、聚乙二醇、丙二醇、聚丙二醇,或上述两种或更多种的组合。
根据本发明的药物组合物优选地包括试剂盒,包括包括CDCP1降调剂的药物制剂和包括除衰化合物和/或遗传毒性剂的药物制剂。所述试剂盒优选进一步包括在给药除衰化合物之前给药CDCP1降调剂的说明书,在除衰化合物给药之前优选一天,优选两天,优选3天,优选多于3天例如1-2周。
其他优选的药物组合物包括如上文所述的脂质体,所述脂质体包括CDCP1的降调剂。所述脂质体优选进一步包括遗传毒性剂,优选蒽环类例如多柔比星。
根据本发明的药物组合物优选为用于治疗患有去势抵抗性前列腺癌的患者的方法。
4.7选择适合CDCP1降调剂和除衰化合物组合治疗的患者的方法
本发明进一步提供了一种选择适合用降调前列腺癌细胞表面上含CUB结构域的蛋白1(CDCP1)表达的分子和除衰化合物的组合治疗的前列腺癌患者的方法,包括确定患者体液中的睾酮水平如何;鉴定睾酮水平低于50ng/dL的患者;确定已鉴定的患者的前列腺癌是否正在进展;并选择睾酮水平低于50ng/dL且前列腺癌正在进展的患者作为适合用CDCP1降调剂和除衰化合物的组合治疗的患者。
所述前列腺癌的进展优选通过血清前列腺特异性抗原(PSA)水平的持续上升和/或所述患者中新的转移灶的出现来确定。
5实施例
实施例1
材料和方法
小鼠
所有小鼠都在贝林佐纳生物医学研究所的动物设施中保持在无特定病原体的条件下,并根据州指导方针进行实验并获得当地伦理委员会的批准。PtenloxP条件敲除等位基因已有描述(Chen,等,2005.Nature 436:725-730)。如结果中所述,产生了CDCP1条件性过表达。为了获得所有基因型,将雌性CDCP1和/或PtenloxP/loxP小鼠与雄性前列腺碱性蛋白(Probasin)-Cre4(Pb-Cre4)转基因小鼠(Trotman等.,2003.PLoS Biol 1,E59)杂交,以获得前列腺特异性过表达CDCP1和Pten缺失的小鼠。为了基因分型,用下述引物对尾DNA进行聚合酶链式反应分析。
对于PtenloxP/loxP,使用引物1(5′-AAAAGTTCCCCTGATGATGATTTGT-3′)和引物2(5′-TGTTTTTGACCAATTAAAGTAGGCTGTG-3′)。为了检测前列腺中缺失的等位基因,使用引物3(5′-TTCTCTTGAGCACTGTTTCACAGGC-3′)和引物1。对于前列腺碱性蛋白-Cre4(Pb-Cre4),使用引物1(5′-TGATGGACATGTTCAGGGATC-3′)和引物2(5’-GCCACCAGTCTGCATGA-3′)进行基因分型或检测前列腺中的等位基因。对于CDCP1小鼠,使用引物1(5′-CAAGGGAGAAGAGAGTGCGG-3′)和引物2(5′-CCCAACAATGGGGATGTAAG-3′)。
对于异种移植实验,感染Tripz-shCDCP1或Tripz-shRNA对照的1×106个PC3细胞被皮下注射(s.c.)给SCID-NOD。肿瘤开始发作时,用补充有5%蔗糖的多西环素(0.2g/L)水喂养小鼠。每三天监测来自每个单独注射的肿瘤形成直到实验终止。对动物进行尸检,并检查所有组织,无论其病理状态如何。正常组织和肿瘤组织样品在10%中性缓冲的福尔马林(Catalog#HT501128,西格玛公司(Sigma))中固定过夜。根据标准方案通过乙醇脱水处理组织并包埋在石蜡中。制备切片(5μm)用于抗体检测和苏木精和伊红染色。
细胞培养和试剂
人前列腺癌细胞系,包括PC3,购自ATCC并根据生产商的说明书进行培养。使用购自美国马萨诸塞州沃尔瑟姆的赛默飞世尔科技公司(Thermo Scientific)的空载体和针对人CDCP1基因的PLKO或TRIPZ多西环素可诱导慢病毒构建体(CloneIDs:V3THS_329377和V2THS_191307)转导细胞。LNCaP-abl和LAPC4细胞由Jean-Philippe Theurillat博士(贝林佐纳肿瘤研究所(IOR))赠与获得。PC3-AR是通过用表达全长人AR的逆转录病毒感染它们产生的(由Jean-Philippe Theurillat博士提供。PC3-ΔAR是通过表达缺失氨基酸538-614,缺失AR的DNA结合结构域的人AR(艾德基因公司(Addgene),目录号:89107)产生的。LNCaP-ADI细胞是通过在包含10%活性炭剥离的FBS的RPMI 1640中生长亲本LNCaP产生的。使用1nM的5α-双氢睾酮(DHT)(西格玛公司,目录号:521-18-6)进行雄激素刺激实验。在含有活性炭剥离的FBS和恩杂鲁胺的RPMI中培养细胞进行完全雄激素剥夺(FAD)实验。恩杂鲁胺(APExBIO目录号:A3003)以10uM溶解于DMSO。
原代MEF来源于同窝仔胚胎且通过杂交CDCP1;PtenWT/loxP和PtenWT/loxP动物获得。在交配后13.5天收获胚胎,产生并培养个体的MEF,如之前所述(Chen等,2005.Nature436:725-730)。用表达pMSCV-CRE-PURO-IRES-GFP的逆转录病毒感染原代MEF持续48小时并用浓度为2μg/mL的嘌呤霉素选择。简言之,通过将雄性野生型和Ptenlox-lox型与雌性CDCP1lox-stop-lox小鼠杂交获得所有基因型的MEF。交配后13-或14-天的怀孕小鼠通过颈脱位法处死。收获胚胎,且个体MEF被培养在含有10%胎牛血清和1%青链霉素的DMEM中。用表达pMSCV-CRE-PURO-IRES-GFP或pMSCV-PURO-IRES-GFP的逆转录病毒感染原代Ptenlox/lox MEF,持续48小时,并用浓度为3ug ml的嘌呤霉素选择,如前所述。所有小鼠都在IRB研究所的动物设施中保持在无特定病原体的条件下,并根据州指导方针进行实验并获得当地伦理委员会的批准。
下述抗体用于蛋白质印迹:Tag-Myc(目录号:551101;BD法敏进公司(BDPharmingen),1∶1000);微管蛋白(DSHB,E7,1∶1000);pCDCP1(目录号:13111;细胞信号技术公司(Cell Signaling Technology),1∶1000);PTEN(目录号:9552S;细胞信号技术公司,1∶1000);HSP90(目录号:4877S;细胞信号技术公司,1∶1000);c-Myc(目录号:A713(G-4),圣克鲁兹生物技术公司(Santa Cruz Biotechnology),1∶500);p21(目录号:F1013(C-19),圣克鲁兹生物技术公司,1∶500);β-肌动蛋白(目录号:A5316;西格玛公司,1∶5000);周期蛋白D1(目录号:2978S,细胞信号技术公司,1∶1000);COUP-TFII(目录号#PP-H7147-00;珀尔修斯蛋白质组学(Perseus Proteomics),1∶1000)。SMAD4(目录号:E0615,圣克鲁兹生物技术公司,1∶500).p-SRC-Tyr416(目录号:6943S,细胞信号技术公司,1∶1000);SRC(目录号:2123S,细胞信号技术公司,1∶1000);AKT(目录号:9272S,细胞信号技术公司,1∶1000);p-AKT-S473(目录号:9171S,细胞信号技术公司,1∶1000);p53(目录号#MEDRPR025-1,精确化学(Accuratechemical),1∶1000);CDCP1(目录号:4115,细胞信号技术公司,1∶1000);Erk1/2(目录号:4695S,细胞信号技术公司,1∶1000).p-Erk1/2-Thr202/Tyr204(目录号:9106S,Cell细胞信号技术公司,1∶1000);S6(目录号:2317S,细胞信号技术公司,1∶1000);pS6-Ser235/236(目录号#4857,细胞信号技术公司,1∶1000);p27(目录号:K0413,圣克鲁兹生物技术公司,1∶500);AR(N-20)(目录号:SC-816圣克鲁兹生物技术公司,1∶500)。
以下抗体被用于IHC:Ki-67(克隆SP6,目录号#RT-9106-R7;多克隆兔抗;未掩蔽的水浴锅98℃pH6 20′;实验室版本,稀释备用);CDCP1(目录号#4115,多克隆兔抗;未掩蔽的水浴锅98℃pH6 20′;细胞信号技术公司,1:50);p-HP1γ-Ser83(目录号#2600,未掩蔽的水浴锅98℃pH6 20′;细胞信号技术公司,1∶50),周期蛋白D1(目录号:2978S,细胞信号技术公司);AR(N-20)(目录号:SC-816,多克隆兔抗;未掩蔽的水浴锅98℃pH6 20′;圣克鲁兹生物技术公司,1∶300);广谱细胞角蛋白(泛角蛋白(Pankeratin))(目录号#Z0622;多克隆兔抗;未掩蔽的水浴锅98℃pH9 20′;DAKO,1∶2000)。
以下抗体被用于IF:E钙粘蛋白(克隆26)(目录号:610181;单克隆小鼠抗;未掩蔽的水浴锅98℃pH9 20′;BD,1∶700);CK5(目录号:ab52635;多克隆兔抗;未掩蔽的水浴锅98℃pH9 20′;阿柏堪穆公司(Abcam),1∶500);CK8(目录号#ab59400;多克隆兔抗;未掩蔽的水浴锅98℃pH9 20′;阿柏堪穆公司,1∶150),CDCP1(目录号:4115,细胞信号技术公司,1∶100)和DE-钙粘蛋白(杂交瘤发展研究库(Developmental Studies Hybridoma Bank)[DSHB],DCAD2,1∶100)。
c-Myc siRNA和阴性对照siRNA购自西格玛公司(目录号:8024873724-000050;#8024873724-000060)。我们根据制造商的方案使用Lipofectamin RNAiMAX(目录号:13778-030;英杰公司(Invitrogen))转染了细胞。
生成GAL4-UAS-CDCP1-wt和GAL4-UAS-CDCP1-δ黑腹果蝇(Drosophilamelanogaster)谱系和免疫荧光分析(IF)。
从布卢明顿果蝇库存中心(Bloomington Drosophila Stock Centre)获得UAS-egfr.B(5368)、src64BP1(7379)、Src42AK10108(10969)、GMR-ga14(1104)和ptc-gal4(2017)谱系。在谷物粉/琼脂饮食上进行培养,(6.65%谷物粉、7.15%葡聚糖、5%酵母、0.66%琼脂,补充有2.2%对羟基苯甲酸甲酯(nipagin)和3.4ml 1-1丙酸)。培养在25℃和29℃进行。为了过表达人CDCP1-wt和CDCP1-δ,从人CDCP1-wt和CDCP1-δcDNA中产生UAS转基因谱系,其采用下述引物对:
(5′-GATATCCACCATGGCCGGCCTGAACTGCGGG-3′)和(5′-ACTAGTTCAATGGTGATGGTGATGATG-3`)。使用来自新英格兰生物实验室公司(New EnglandBiolabs)的Q5高保真聚合酶(M0491S)进行PCR。使用Zero Blunt TOPO PCR克隆试剂盒(生物技术公司(Life Technologies),K2800-20)克隆PCR产物,然后克隆进pUAST-attB载体(Bischof等.,2007.PNAS USA 104:3312-3317)中。所述构建体经序列验证并通过PhiC31整合酶-介导转基因(最佳基因公司(BestGene),attP位点:VK27)建立转基因谱系。唾液腺被浸入PBS中,用PBS中的4%多聚甲醛(PFA)固定,在PBT(含有0.1%Triton X-100的PBS)中洗涤并用PAXDG中的一抗(含有1%BSA、0.3%Triton X-100、0.3%脱氧胆酸和5%山羊血清的PBS)在4℃孵育过夜。用PBT洗涤组织并用PAXDG中的二抗在4℃孵育5小时,并在Vectashield封固剂(载体实验室公司(Vector Laboratories))中封固。Alexa-568-偶联抗-兔抗体和Alexa-488-偶联抗-大鼠抗体被用作二抗(分子探针)。成体眼睛和刚毛的图像是用配备SXY-I30 3M像素彩色相机的Leica M165 FC显微镜拍摄的。唾液腺的荧光图像是用配备Leica DFC 3000G数码相机的Leica M165 FC荧光显微镜拍摄的。
CDCP1蛋白质在人前列腺癌中表达
第一个TMA由处于不同阶段的良性前列腺组织和PCa组成(n=237),如先前报道(Zellweger等.,2013.Endocr Relat Cancer 20:403-413)。分析中不包括具有转移的斑点以避免由于组织固定不良(主要来自尸检材料)而导致假阴性结果。第二个TMA(n=192)由局部晚期、无法手术、主要是转移性PCa组成,其包括CRPC和激素原初(未经治疗)PCa,如先前报道(Zellweger等.,2013.Endocr Relat Cancer 20:403-413)。对于远端转移灶,在6个远端和淋巴结PCa转移的常规组织切片上进行CDCP1染色。H-评分:0(无染色)、1+(弱染色)、2+(中度染色)和3+(强染色)等级上的染色强度乘以阳性肿瘤细胞的百分比。仅胞质染色认为是阴性。列联表分析和卡方检验用于研究标志物表达与组织学亚组之间的关系。p值<0.05,值之间的差异被认为具有统计学显著性。使用JMP 12软件(美国北卡罗来纳州凯瑞SAS研究所)进行分析。使用临床样品进行TMA构建得到了巴塞尔大学伦理委员会的批准。
配对诊断(HSPC)和CRPC活检
患者是从皇家马斯登NHS基金会信托医院治疗的患有转移性去势抵抗性前列腺癌(CRPC)的男性人群中鉴定的。所有的患者都有书面的知情同意书,并在皇家马斯登NHS基金会信托医院(英国伦敦)伦理审查委员会批准的机构方案中入组(参考编号:04/Q0801/60)。选择了25名诊断为前列腺腺癌的患者,他们有足够的福尔马林固定、石蜡包埋(FFPE)、匹配的诊断(档案)激素敏感的前列腺癌(HSPC)和CRPC组织用于进行CDCP1免疫组织化学分析。HSPC组织显示为腺癌,获取自前列腺针头活检(21例)、经尿道前列腺切除术(TURP;3例)或骨活检(1例)。CRPC组织是通过骨活检(19例)、淋巴结(5例)或肝(1例)从相同的患者获取。所有的组织块都是新鲜切片,只有在有足够的材料(≥50个肿瘤细胞;由Daniel NavaRodrigues审查)时才考虑进行IHC分析。
相关性分析
使用斯皮尔曼相关性进行PCa数据集中CDCP1和PTEN之间的相关性分析(Taylor等.,Cancer Cell 18:11-22;Chandran等.,2007.BMC Cancer 7:64;Varambally等.,2005.Cancer Cell 8:393-406;Grasso等.,2012.Nature487:239-243;Lefort等.,2016.Oncotarget 7:48011-48026),估计相关系数值‘R’和显著性P值。
免疫印迹
用含有PMSF(苯基甲磺酰氯;目录号:329-98-6,西格玛公司)的RIPA缓冲液(目录号:9806,细胞信号技术公司)制备组织和细胞裂解物。通过Pierce BCA蛋白质测定试剂盒(目录号:23225,赛默飞世尔科技公司)测量裂解物的蛋白质浓度。然后用SDS-PAGE解析裂解物并用指定的抗体进行免疫印记。为了分析蝇组织,将游荡的三龄幼虫在PBS中冲洗,解剖出唾液腺,在PBS中清洗并在SDS样品缓冲液中匀浆。
实时RT-PCR
使用Plus RNA纯化试剂盒(目录号:12183555,生物技术公司)提取RNA。使用III一步式qRT-PCR试剂盒(目录号:11732-020,生物技术公司),用1μg的总RNA合成cDNA。如前所述进行定量实时PCR(q-RT PCR)(Chen等.,2005.Nature 436:725-730)。表1和2列出了使用的引物。所有示出的qRT-PCR数据是使用GAPDH、HRPT或18S rRNA归一化的。
表1:RT-PCR引物(小鼠)
ChIP试验
细胞被培养到90-95%汇合度,用1%福尔马林交联10分钟,然后在室温下加入2.5M甘氨酸持续5分钟。吸出培养基,用冰冷的磷酸盐缓冲盐水洗涤细胞两次。用Misonix3,000型超声机对核提取物进行超声处理,以剪切交联的DNA使其平均片段大小为~500个碱基对。超声处理过的染色质与γ-结合Plus Sepharose珠(目录号:17-0886-01,GE医疗生命科学公司(GE Healthcare))在4℃孵育16小时,通过在转子上4℃孵育过夜与抗-c-Myc((9E10)x L0815)抗-SMAD4((B-8)目录号:E0615;圣克鲁兹生物技术公司)或小鼠-IgG抗体(目录号:92590密理博公司(Millipore))偶联。孵育后,彻底清洗珠然后离心。染色质被从珠上洗脱,并通过在56℃孵育12小时去除交联。然后使用QIA快速PCR纯化试剂盒(目录号:28104,凯杰公司(Qiagen))纯化DNA。使用SABiosciences的DNA元件的专有数据库DECipherment(DECODE)确定转录因子c-Myc与周期蛋白D1启动子的结合。
表2:RT-PCR引物(人)
用于ChIP试验的引物混合物是EpiTect ChIP qPCR小鼠Ccnd1的测试引物,NM_007631.2(-)04Kb(目录号:GPM1053924(-)04A)。设计用于ChIP试验的引物序列,为了1)检测周期蛋白D1启动子上的Smad4结合位点(SBE)的是:SBEChIPf 5’-CCGCTTAGTCCCCATTCTAAAG-3’和SBEChIPr:5’-GGCATCTCCATTCTTAATCCAG-3’;2)检测Coup-tfII启动子上的c-Myc结合的是:COUP-TFIIChIPf5’-GTGCGGGGACAAGTCGAGCGG-3’和COUP-TFIIChIPr5’-GCGGTGGTGCTGGTCGATGGG-3’。使用一步式实时PCR系统(应用生物系统公司(Applied Biosystems))上的KAPA SYBR FAST ABI qPCR主混合物溶液(目录号:07959389001,KAPA生物系统公司(KAPA Biosystem),罗氏公司(Roche))进行ChIP qPCR。
增殖和衰老试验
MEF的增殖试验是通过在24孔板的每个孔接种104个细胞进行,一式三份,而人PCa细胞系是通过在24孔板的每个孔接种1-2×104个细胞进行,一式三份。在第0、3、6和9天监测细胞增殖,由此将细胞在用磷酸缓冲盐水(pH7.2)洗涤过的10%缓冲福尔马林溶液中固定15分钟,然后用0.01%结晶紫溶液染色。通过用蒸馏水洗涤除去多余的染色,将板干燥过夜。将结晶紫染色的细胞溶解在10%乙酸溶液中,在振荡器上30分钟,然后用分光光度计在590nm处读取提取的染料。根据制造商的说明,使用衰老β-半乳糖苷酶染色试剂盒(目录号:9860;细胞信号技术公司)进行体外细胞衰老并使用Hoechst 3342,Trilhydrochloride,三水合物(目录号:953557;英杰公司)对细胞总数进行计数来定量。
隐形脂质体
用HSPC∶CHOL∶mPEG5kDa-DSPE以18∶9∶1摩尔比制备隐形脂质体。通过蒸发所述组分的氯仿溶液获得脂质膜,用250mM硫酸铵(pH 5.5)溶液水合,然后在60℃挤出直到达到~100nm的囊泡大小。通过PD-10脱盐柱将外部缓冲液更换为pH 7.4的PBS。多柔比星(DXR)通过远程装载(DXR∶HSPC 0.2∶1w/w)在60℃封装。使用PD-10脱盐柱去除游离的DXR,并在甲醇中通过分光光度法(=477nm)确认载药量。如下所述通过将CUB4的Fab’和马来酰亚胺-PEG-DSPE反应,然后CUB4 Fab’-PEG-DSPE通过后插入(post-insertion)被引至脂质体表面制备CUB4 Fab’-偶联的PEG-磷脂衍生物以提供隐形免疫脂质体(SIL)。简言之,在pH 3.8的0.1M乙酸钠中用胃蛋白酶酶促消化CUB4(1∶50w/w E/S,在37℃,3小时),随后在Superose 1210/300GL柱上使用pH 7.4的PBS(流速0.5ml/分钟)进行FPLC分析。收集F(ab’)2片段并用10mM半胱胺在室温处理30分钟以获得Fab’片段,然后用50mM磷酸盐缓冲液、150mM NaCl和10mM EDTA,pH 5通过FPLC纯化。通过利用其游离巯基,Fab’立即偶联(室温过夜,pH 7.0-7.5)至混合胶束的马来酰亚胺基团,其由马来酰亚胺-PEG5kDa-DSPE∶mPEG5kDa-DSPE 4∶1mol/mol以Maleimide∶Fab’的10∶1最终摩尔比构成。最后,这些胶束与SL以PEG∶HSPC摩尔比0.05∶1在60℃孵育1小时,以获得SIL,随后在Sepharose CL-4B柱上使用pH7.4的PBS纯化并通过BCA试验定量Fab’。
统计学
所有数据点表示定量数据,并用SD和SEM叠加平均值(在附图图例中指定)。所有统计分析使用Graph Pad Prism 8或微软Excel 2016或R-studio进行。使用的统计检验是1或2尾的T检验(如附图图例中指定的)。其他使用的统计分析在附图图例中指定。
结果
CRPC和转移前列腺肿瘤表现出CDCP1表达升高和CDCP1过表达与PTEN缺失相关
含CUB结构域的蛋白质1(CDCP1)是跨膜蛋白,其作为Src家族酪氨酸激酶(SFKs)的底物,可被单克隆抗体或小分子抑制剂抑制(Nakashima等,2017.Cancer Sci 108:1049-1057;Kollmorgen等.,2013.Mol Oncol 7:1142-1151;Siva等.,2008.Cancer Res 68:3759-3766)。为了评估CDCP1是否在人前列腺癌(PCa)中过表达,我们在不同组织微阵列(TMA)中评估了CDCP1蛋白表达,包括435个良性、局部晚期和转移性PCa的PCa病例。免疫组织化学(IHC)分析显示,当与来自激素敏感性患者的良性前列腺肿瘤相比时,CDCP1主要在来自CRPC患者的晚期和转移性肿瘤样品中过表达(图1A,B)。然而这些数据指出CDCP1是相关的致癌基因,但最近在不同肿瘤模型中的发现显示CDCP1在癌症中作为肿瘤抑制因子。
为了评估CDCP1的致癌潜力,我们生成了在小鼠前列腺中过表达人CDCP1的转基因小鼠模型和过表达正常和突变人CDCP1的黑腹果蝇(Drosophila melanogaster)模型。
首先,我们构建了pGACCS载体,其转录终止序列侧接CDCP1-cDNA上游的loxP位点。将所得的PGACCS-loxP-STOP-loxP-CDCP1-载体与PGK-FlpO质粒一起共电穿孔到ColA基因座修饰的胚胎干细胞KH2中(Beard等.,2006.Genesis 44:23-28;数据未显示)。PCR和Southern印迹分析证实了基因整合和重组事件(数据未显示),而蛋白质印迹分析证实了CDCP1在源自这些小鼠4的小鼠胚胎成纤维细胞(MEF)中的表达(数据未显示)。随后,我们将CDCP1动物和PB-Cre4小鼠杂交以进行CDCP1的前列腺特异性表达。值得注意的是,在一组人前列腺肿瘤细胞系、源自患者的前列腺癌异种移植物(PDX)和从CDCP1+小鼠收集的肿瘤中,CDCP1的表达在CDCP1水平上未显示显著差异(数据未显示)从而证明CDCP1在小鼠模型中的过度表达与CDCP1在人肿瘤中的水平相似。在CDCP1;Pb-Cre小鼠(CDCP1pcLSL/+,下文称之为CDCP1)的10周龄前列腺组织上进行IHC分析,以确认CDCP1的前列腺特异性表达(数据未显示)。值得注意的是,与背外侧前列腺(DLP)相比,在前部前列腺(AP)和腹侧前列腺(VP)中CDCP1 mRNA和蛋白质水平高表达。然后,我们检测了24个月期间CDCP1小鼠模型的肿瘤发生率。在小鼠队列中,在4-6月龄之间我们观察到50%外显率(penetrance)的前列腺增生,以及7-9月龄之间观察到前列腺上皮内肿瘤(PIN)病灶,其特征在于前列腺含有多层上皮细胞,具有核异型特征(75%外显率)。CDCP1小鼠在14月龄后发展出高级别PIN(HGPIN),外显率100%(图1C),位于前部、背外侧和腹侧前列腺(数据未显示)。与其野生型年龄匹配的同窝仔相比,通过Ki-67阳性染色观察到CDCP1小鼠前列腺上皮增殖显著增加(图1D)。同时,蛋白质印迹分析显示在CDCP1过表达的MEF中和前列腺上皮中Src和Erk1/2磷酸化显著增加(图1E)。
我们接下来在黑腹果蝇(Drosophila melanogaster)中过表达野生型人CDCP1(CDCP1-WT)和CDCP1-δ,人CDCP1的缺乏Src-磷酸化位点的突变体(CDCP1-δ)(Alajati等.,2015.Cell Rep 11:564-576;Liu等.,2011.PNAS 108:1379-1384)。果蝇幼虫的成虫盘是单层上皮,被认为在形态上与哺乳动物上皮相当,因此构成在其中模拟体内癌症进展的理想系统(Brumby和Richardson,2005.Nat Rev Cancer 5:626-639)。增加的EGFR/Ras信号先前已显示促进位于蝇胸部分位于背部(背板)的刚毛的形成(也称作长毫(macrochaetae)形成),一种肿瘤样表型(Culi等,2001.Development 128:299-308;Khare等,2017.PLoS One12:e0173565)。我们发现CDCP1-WT的过表达,而不是CDCP1-δ,促进蝇背板的额外长毫形成(数据未显示)。有趣的是,src42A和src64B的一个拷贝缺失(50%减少)抑制了由CDCP1-WT过表达驱动的额外长毫形成,证明这种表型是Src依赖的(数据未显示)。此外,CDCP1的过表达引发了蝇眼的异常增殖(数据未显示)。总之,这些结果使用跨物种遗传方法,证明CDCP1体内过表达通过激活Src-MAPK信号通路起始肿瘤发生。
CDCP1与Pten-缺失协同驱动前列腺癌进展和转移性去势抵抗性前列腺癌
为了进一步评估CDCP1作为CRPC驱动因素的作用,我们将所述CDCP1小鼠与发生去势敏感性前列腺肿瘤的Pten-无效的前列腺条件小鼠(Ptenpc-/-)杂交,我们获得了CDCP1;Ptenpc-/-双突变小鼠。
虽然PTEN中的单等位基因缺失或突变与良性前列腺肿瘤有关(Trotman等,2003.PLoS Biol 1:E59;Alimonti等,2010.Nat Genet 42:454-458),但是在人转移性前列腺癌中经常观察到完全缺失PTEN(Taylor等,2010.Cancer Cell18:11-22)。然而,在小鼠中Pten完全缺失不足以促进转移性前列腺癌,需要其他基因命中(genetic hits)以促进转移的发生(Chen等.,2005.Nature 436:725-730)。
我们检查了CDCP1的过表达在Pten-缺陷肿瘤中是否会加剧前列腺肿瘤发生。注意,完全Pten缺失导致高级别前列腺上皮内肿瘤病灶(HGPIN),其发展为无转移的局部浸润性癌(focally invasive adenocarcinoma)(Alimonti等,2010.Nat Genet 42:454-458;Chen,等,2005.Nature 436:725-730;Trotman等,2006.Cell 128:141-156)。引人注目的是,在16周龄时出现了CDCP1;Ptenpc-/-发展为局部浸润性癌,其在36周龄进展为高侵袭性癌,该表型从未在Ptenpc-/-肿瘤中观察到(图2A)。此外,与其对应小鼠相比,CDCP1;Ptenpc-/-小鼠中Ki-67阳性细胞的百分比显著增加(图2B)。重要的是,CDCP1;Ptenpc-/-小鼠的病理分析显示,在50%的分析的病例(n=4/8)上皮肿瘤结节的转移性传播为泛-角蛋白(PanK)、CDCP1和雄激素受体(AR)阳性,以使腰淋巴结枯竭,以及12%的病例(n=1/9)中肺转移(数据未显示)。这些转移瘤的组学特征类似于原发性前列腺肿瘤的那些特征(数据未显示)。相比之下,Ptenpc-/-小鼠未如先前报道的那样发生转移(Chen,等,2005.Nature 436:725-730;Ding等,2011.Nature 470:269-273;Qin等,2013.Nature 493:236-240)。与Pten-/-细胞相比,CDCP1;Pten-/-MEF还具有增加的增殖和迁移能力。
此外,Kaplan-Meier累积生存分析显示CDCP1;Ptenpc-/-小鼠在60-80周龄时由于广泛的肿瘤负荷不得不被安乐死或死亡(图2C)。值得注意的是,在同样的年龄没有一只Ptenpc-/-小鼠死亡,证明CDCP1的过表达与Pten-缺陷结合对转基因小鼠的生存有深远影响。此外,与其对应小鼠相比,CDCP1;Ptenpc-/-小鼠中Ki-67阳性细胞的百分比显著更高(数据未显示)。这在人前列腺癌中也得到了证实,其中PTEN水平与CDCP1过表达水平呈负相关。此外,在受前列腺肿瘤影响的具有PTEN水平降低和CDCP1水平增加的患者中,我们观察到短的无病生存期(DFS)和总生存期(OS),从而验证了小鼠模型中发现的相关性(数据未显示)。
肿瘤中的蛋白质印迹(WB)分析显示,Ptenpc-/-肿瘤中的CDCP1过表达促进SRC激活和后续的p-ERK1/2上调,而与Ptenpc-/-肿瘤相比,p-Akt无变化。因此,在这些肿瘤中,虽然Pten缺失驱动p-AKT的激活,但CDCP1促进MAPK通路的上调(图2D)。由于已知激活的Src调控c-Myc水平(Furstoss等,2002.EMBO J 21:514-524;Jain等,2015.Cancer Res 75:4863-4875;Barone和Courtneidge,1995.Nature 378:509-512),我们认为CDCP1过表达可以通过Src驱动c-Myc过表达。实际上,CDCP1过表达肿瘤表现出c-Myc表达水平增加(数据未显示)。此外,IHC分析显示,与Ptenpc-/-肿瘤相比,CDCP1;Ptenpc-/-肿瘤中c-Myc和pErk1/2水平高(数据未显示)。
我们接下来检查了在Pten无效肿瘤中CDCP1过表达是否也可以促进对雄激素剥夺疗法(ADT)的抵抗性。为此,我们对Ptenpc-/-和CDCP1;Ptenpc-/-小鼠平行进行了手术去势。宏观分析显示,虽然Ptenpc-/-肿瘤对去势有反应(Lunardi等,2013.Nat Genet 45:747-755),但CDCP1;Ptenpc-/-肿瘤没有,正如肿瘤重量、体积、组织病理学分析和Ki-67的IHC的定量所示(图2E)。与Ptenpc-/-肿瘤相比,CDCP1;Ptenpc-/-肿瘤中对去势的抵抗性与p-SRC/MAPK水平增加相关,因此解释了在这种遗传背景下CRPC的出现(图2F)。这些数据在另一个前列腺癌的异种移植模型中被验证,其中CDCP1在TRAMPC1小鼠前列腺上皮细胞中过表达。在这个模型中,CDCP1的过表达显著增加了pSRC和pERK的水平,与对照组肿瘤相比,CRPC的出现加速。此外CDCP1的过表达缩短了荷瘤小鼠的生存期(数据未显示)。
CDCP1的过表达通过激活Src/MAPK/Myc通路克服了Pten-缺失诱导的细胞衰老并导致CRPC
先前的证据证明Ptenpc-/-小鼠发展出以衰老反应为特征的惰性肿瘤,该反应作为限制前列腺癌进展的内在屏障(Chen,等,2005.Nature 436:725-730;Alimonti等,2010.JClin Invest 120:681-693)。由于CDCP1加速了Ptenpc-/-小鼠的肿瘤进展,我们测试了CDCP1过表达在这种遗传背景下能否促进体外和体内的衰老逃逸,导致mCRPC。通过进行SA-β-gal和p-HP1γ染色体内衰老的额外标志物,分析不同基因型(WT、CDCP1、Ptenpc-/-和CDCP1;Ptenpc-/-)的前列腺切片对衰老的反应(Di Mitri等,2014.Nature 515:134-137)。虽然在Ptenpc-/-肿瘤中观察到强烈的细胞衰老反应,CDCP1;Ptenpc-/-肿瘤染色切片对SA-β-gal和p-HP1γ均为阴性,对周期蛋白D1呈阳性(数据未显示)证明CDCP1绕过Pten-缺失驱动的衰老。
与Pten-/-MEF相比,CDCP1;Pten-/-MEF对SA-β-gal染色也是阴性并表现出细胞增殖增加,表型延长(数据未显示)。
最近的两份独立报告表明,由PTEN缺失引发的TGF-β/Smad4通路上调通过阻断周期蛋白D1转录限制前列腺癌进展(Ding等,2011.Nature470:269-273;Qin等,2013.Nature493:236-240)。有趣的是,抑制Smad4-依赖转录的COUP-TFII过表达通过在PTEN无效细胞中释放周期蛋白D1促进衰老逃逸(Ding等,2011.Nature 470:269-273;Qin等,2013.Nature493:236-240)。因此,我们在Ptenpc-/-和CDCP1;Ptenpc-/-肿瘤样品中比较了参与这些通路(例如p53、p21、Smad4、周期蛋白D1和COUP-TFII)的几种组分的状态。与对照组相比,虽然我们的分析显示在CDCP1;Pten-无效MEF和肿瘤中Smad4和p53表达没有变化,但是p21、周期蛋白D1和COUP-TFII水平显著改变(图3A、B)。这些数据表明CDCP1允许PTEN无效的良性肿瘤通过增加COUP-TFII的水平逃逸TGF-β-诱导的衰老屏障,从而获得转移潜能。接下来我们试图理解CDCP1是如何控制COUP-TFII的表达的。对COUP-TFII启动子的分析表明存在多个MYC-结合位点(数据未显示)。由于CDCP1控制Src激活,其反过来调控c-Myc水平(Furstoss等,2002.EMBO J 21:514-524;Jain等,2015.Cancer Res 75:4863-4875;Barone和Courtneidge,1995.Nature 378:509-512),我们检查了CDCP1和CDCP1;Pten无效的MEF和肿瘤中的MYC水平,我们发现c-Myc在mRNA和蛋白质水平的显著诱导(图3A、B)。有趣的是,在塞卡替尼(Src20抑制剂)治疗下,CDCP1;Pten-/-MEF中,c-Myc、COUP-TFII和周期蛋白D1的mRNA和蛋白质水平显著减少(图3C)。值得注意的是,用塞卡替尼治疗导致CDCP1;Pten-/-MEF中衰老重激活和增殖的严重阻滞(图3D)。在CDCP1;Pten-/-MEF中沉默c-Myc,表型模拟Src抑制剂的结果,从而验证我们的发现,Myc调控CDCP1;Ptenpc-/-肿瘤中COUP-TFII水平(数据未显示)。染色质免疫沉淀(ChIP)试验验证了,与Pten-/-MEF相比,在CDCP1;Pten-/-MEF中c-Myc特异性结合周期蛋白D1和COUP-TFII的启动子。其他ChIP分析显示,与Pten-/-相比,在CDCP1;Pten-/-MEF中周期蛋白D1启动子上的c-Myc结合增加且Smad4对周期蛋白D1的启动子结合亲和力减少(数据未显示)。结合这些数据证明,在CDCP1;Ptenpc-/-肿瘤中,c-Myc启动子水平增加促进COUPTF-II激活,其阻止Smad4结合周期蛋白D1的启动子。结果,这些肿瘤绕过衰老并朝着转移性和CRPC表型进展。
接下来我们认为CDCP1的抑制可以驱动具有CDCP1水平升高的前列腺癌中衰老激活。因此,通过使用两条独立的shRNA,我们在PC3(PTEN;TP53-缺陷的人前列腺癌细胞系)中耗竭了CDCP1(图4A)。值得注意的是,通过SA-β-gal染色阳性评估,CDCP1的沉默抑制了PC3细胞的3D增殖和迁移(数据未显示)并促进衰老(图4B)。与此证据一致,我们在体内皮下注射了表达sh-CDCP1及其杂乱对照的PC3细胞。与对照相比,在sh-CDCP1肿瘤中肿瘤大小和重量均显著降低(数据未显示)。CDCP1耗竭的PC3-肿瘤也显示,平行于SRC磷酸化的减少,c-Myc COUP-TFII和周期蛋白D1的表达显著降低。此外,在缺乏CDCP1的肿瘤中,衰老的两个标志物例如p21和p27稳健地上调(Lin等,2010.Nature 464:374-379)(图5)。综合这些数据证明CDCP1抑制通过抑制前列腺癌细胞中的c-MYC水平促进衰老。这些结果也在LNCaP-abl(雄激素敏感细胞系,衍生自与LNCaP亲本相比表现出更高水平CDCP1的LNCaP)中被验证了(数据未显示)。在这一细胞系中CDCP1的下调减少了增殖并增加了衰老。
雄激素剥夺疗法(ADT)诱导CDCP1表达,抑制CDCP1改进ADT的功效
由于CDCP1在来自mCRPC患者的肿瘤样品中过表达,我们检查了CDCP1在癌症中的过表达是否由ADT引发。因此,我们在一项涉及接受不同线的ADT治疗的患者的纵向研究中,通过检查连续切片CDCP1水平来扩展我们的检查。IHC分析证明,CDCP1水平随着患者从HSPC(中位HS 0;四分位间距[IQR]0.0至22.5)进展到mCRPC(30;0.0至82.5)显著增加(图6A)。为了在体外进一步验证这些数据,我们在完全雄激素剥夺(FAD)的条件下培养雄激素敏感性LNCaP细胞系40天以上,并等待直到这些细胞产生抗性。值得注意的是,与恩杂鲁胺敏感细胞(ADS)相比,对恩杂鲁胺(ADI)产生抗性的细胞中的CDCP1表达和蛋白质水平增加。这种上调与p-SRC、c-Myc和p-ERK1/2的同时激活以及逃避由恩杂鲁胺处理驱动的衰老有关(图6B)。
这些结果促使我们研究AR信号是否调控CDCP1的表达。实际上,FAD处理增强了CDCP1水平,而双氢睾酮(DHT)刺激降低了其在LNCaP ADS细胞中的表达和蛋白质水平(图6C)。此外,AR的过表达降低了AR阴性前列腺癌细胞系PC3中CDCP1的表达(图6D)。当DNA结合域缺失的突变型AR在相同细胞中过表达时,这种表型就被消除了(数据未显示)。最后,ChIP-定量PCR(ChIp-qPCR)分析表明AR被直接募集到CDCP1近端启动子并可能抑制了CDCP1转录。
我们接下来研究了FAD中保存的细胞中CDCP1的上调是否需要PTEN的缺失。事实上,CDCP1水平在PTEN无效LNCaP细胞中增加,但在FAD中保存的PTEN野生型LAPC4和VCaP细胞系中没有增加(数据未显示)。与这些发现一致,我们发现在ADT不敏感细胞系PC3和22RV1中,FAD没有上调CDCP1水平。有趣的是,LNCaP细胞中,而不是22RV1细胞中的PI3K的抑制,促进了FAD中保存的细胞中CDCP1的下调。这与相同细胞中AR水平的伴随上调有关。这些数据与先前的发现一致,证明PTEN缺失导致AR活性的相互反馈抑制(Carver等,2011.CancerCell19:575-586)。因此,PI3K的抑制导致AR水平增加,其促进CDCP1的后续下调。
CDCP1-靶向改进ADT的功效
这些发现在LNCaP-ab1细胞(雄激素不敏感细胞系)中进一步验证。在这些细胞中,我们发现与LNCap亲本细胞相比,CDCP1水平上调。值得注意的是,ChIP-qPCR显示在这些细胞中AR不能结合CDCP1启动子。有趣的是,患者的AR活性受CRPC影响,其与CDCP1表达反相关。鉴于CDCP1表达因雄激素剥夺而升高,我们假定靶向CDCP1可以作为治疗经恩杂鲁胺治疗的前列腺癌的有力工具。为了支持这一观点,用靶向CDCP1的人源化单克隆抗体(Kollmorgen等,2013.Mol Oncol 7:1142-1151)处理,与未经恩杂鲁胺处理的细胞相比,在经恩杂鲁胺处理的细胞中强烈地影响了增殖诱导的衰老和细胞死亡(图7A)。注意,在经恩杂鲁胺处理的LNCaP细胞中,用CDCP1-mAb处理强烈地减少了CDCP1水平(图7C)。这些数据也在与亲代细胞相比过表达CDCP1的LNCaP-abl中得到验证(数据未显示)。由于衰老是一把双刃剑,在某些条件下可能会刺激癌细胞生长,因此我们决定开发一种新型抗-CDCP1双隐形免疫脂质体,其携带多柔比星(抗CDCP1 TL)以消除过表达CDCP1的抗性细胞。恩杂鲁胺组合抗CDCP1 IL治疗诱导强烈的细胞凋亡反应并阻断CDCP1+ADI细胞的出现。为了在体内验证这些数据,将LNCaP细胞皮下注射到SCID-小鼠体内,当肿瘤形成时,用有或没有抗-CDCP1IL的恩杂鲁胺(10mg/kg)治疗小鼠一周。重要的是,虽然单独使用恩杂鲁胺和抗CDCP1 IL对肿瘤生长的影响很小,但恩杂鲁胺和抗-CDCP1的组合通过诱导细胞凋亡显著阻断了肿瘤的生长和进展(图7B)。注意,蛋白质印迹分析显示,恩杂鲁胺治疗后CDCP1水平显著增加,而组合处理后,这一现象被消除了(图7C)。这些数据共同表明,CDCP1靶向剂与ADT组合使用时是有效的。
总之,我们的研究提供证据表明,CDCP1过表达在几种转基因模型中启动了肿瘤发生,并与其他致癌事件(例如肿瘤抑制基因Pten的缺失)协同以驱动肿瘤进展。此外,我们证明了Pten-无效前列腺肿瘤中CDCP1的过表达绕过Pten-无效诱导的衰老屏障,从而通过激活MAPK通路允许肿瘤进展和转移性去势抵抗性前列腺癌。值得注意的是,我们证明ADT增强了癌细胞中CDCP1的水平,导致CRPC。通过CDCP1_shRNA或治疗性抗体在前列腺癌细胞中灭活CDCP1废除了c-Myc-Src轴,从而引发细胞衰老。最后,我们首次报道了抗-CDCP1免疫脂质体可以增强恩杂鲁胺治疗的功效,开启了这种化合物组合可用于临床以预防恩杂鲁胺抗性CDCP1+肿瘤细胞发生的可能性。
鉴于CDCP1是一种细胞表面蛋白,其可以被多种单克隆抗体和小分子抑制剂靶向(Nakashima等,2017.Cancer Sci 108:1049-1057;Kollmorgen等,2013.Mol Oncol 7:1142-1151;Siva等,2008.Cancer Res 68:3759-3766),我们的研究还证实了新的治疗方法,以靶向具有升高的CDCP1水平的去势抵抗性和转移性前列腺肿瘤。实际上我们关于突变AR不能高效结合并抑制CDCP1水平的发现表明,携带AR突变或剪接变体的肿瘤具有更高水平的CDCP1,并且可能受益于采用CDCP1单克隆抗体或抗CDCP1 IL的治疗。
因此,靶向CDCP1也可以代表在ADT失败后靶向CRPC患者的替代治疗策略。
实施例2
为了进一步评估CDCP1在人类前列腺癌(PCa)中的临床相关性,肿瘤微阵列(TMA)的数量扩大到总计990个病例,涵盖良性、原发性和转移性PCa。免疫组织化学(IHC)分析表明,虽然分析的前列腺肿瘤大部分不表达CDCP1,但一部分(48%)CRPC和转移性肿瘤样品表达高水平的CDCP1(数据未显示)。与这些发现一致,对来自纵向研究的连续肿瘤样品的分析显示,在PCa患者中,CDCP1在从激素敏感到CRPC的过渡期间上调(数据未显示)。有趣的是,在原发性、CRPC和转移性前列腺肿瘤样品中,CDCP1的高水平与PTEN水平降低相关(表3、4)。
表3。
原发性肿瘤
卡方统计值为7.246。P-值为.007106。
当p<.05时结果显著。
表4CRPC/转移瘤
卡方统计值为7.6471。P-值为.005686。
当p<.05时结果显著。
与原发性肿瘤相比,在CRPC和转移性肿瘤中显示低水平PTEN和高水平CDCP1的肿瘤的频率增加,从而验证了这种反相关性的临床相关性。此外,评估不同数据集的生物信息学分析证实了PTEN和CDCP1 mRNA水平之间存在反相关性(数据未显示)。在PCa的不同独立数据集中,CDCP1表达水平的升高也与PTEN基因缺失和低CDCP1启动子甲基化显著相关(图9)。尽管受高水平CDCP1的前列腺肿瘤影响的患者的无病生存期(DFS)与低CDCP1的患者相似,但表达低水平PTEN和增加水平CDCP1的肿瘤的患者比其他类别的患者具有显著短的DFS(图10)。综上所述,这些数据验证了CDCP1的临床相关性,并表明CDCP1可以与PTEN的缺失协同促进高度侵袭性前列腺癌。
Claims (16)
1.一种含CUB结构域的蛋白质1(CDCP1)的降调剂,用于治疗患有去势抵抗性前列腺癌的患者的方法。
2.如权利要求1所述使用的降调剂,其包括识别含CUB结构域的蛋白质1(CDCP1)的胞外表位的抗体。
3.如权利要求1或权利要求2所述使用的降调剂,其中所述降调剂与抗雄激素疗法组合。
4.如权利要求3所述使用的降调剂,其中所述抗雄激素疗法为或包括雄激素受体拮抗剂。
5.如权利要求3或权利要求4所述使用的降调剂,其中所述抗雄激素疗法为或包括抗雄激素,选自恩杂鲁胺、阿比特龙、比卡鲁胺和尼鲁米特。
6.如前述权利要求中任一项所述使用的降调剂,其中所述降调剂与除衰化合物和/或遗传毒性剂组合。
7.如权利要求6所述使用的降调剂,其中所述除衰化合物选自雷帕霉素、ABT263、FOXO4-DRI、CXCR4抑制剂和/或拮抗剂和达沙替尼。
8.一种药物组合物,其包括CDCP1的降调剂和雄激素受体拮抗剂,除衰化合物和/或遗传毒性剂。
9.如权利要求8所述的药物组合物,其包括包含所述CDCP1降调剂的药物制剂,和包含所述雄激素受体拮抗剂、除衰化合物和/或遗传毒性剂的药物制剂。
10.如权利要求8或权利要求9所述的药物组合物,其中所述CDCP1降调剂存在于脂质体中,所述脂质体优选进一步包含蒽环类如多柔比星。
11.如权利要求8-10中任一项所述的药物组合物,用于治疗患有去势抵抗性前列腺癌的患者的方法。
12.一种选择适合用CDCP1降调剂和除衰化合物组合治疗的患者的方法,其包括
确定所述患者的体液中睾酮水平如何;
鉴定所述睾酮水平低于50ng/dL的患者;
确定所述鉴定的患者中所述前列腺癌是否正在进展;以及
选择其中前列腺癌正在进展的睾酮水平低于50ng/dL的患者作为适合用CDCP1降调剂和除衰化合物组合治疗的患者。
13.如权利要求12所述的方法,其中所述经鉴定的患者中前列腺癌的进展通过血清前列腺特异性抗原(PSA)水平的持续上升和/或所述患者中新的转移灶的出现来确定。
14.一种用CDCP1降调剂和除衰化合物组合治疗患前列腺癌的患者的方法,其包括
确定所述患者的体液中睾酮水平;
鉴定所述睾酮水平低于50ng/dL的患者;
确定所述经鉴定的患者中所述前列腺癌是否正在进展;以及
用CDCP1降调剂和除衰化合物和/遗传毒性剂的组合治疗所述睾酮水平低于50ng/dL且前列腺癌正在进展的患者,优选其中在所述除衰化合物和/或遗传毒性剂给药之前给药所述降调剂。
15.一种用抗体-药物偶联物和雄激素受体拮抗剂的组合治疗患有前列腺癌的患者的方法,由此所述抗体识别CDCP1的细胞外表位,由此所述药物偶联物是化疗药物、毒性化合物或放射性化合物,由此所述抗雄激素优选为恩杂鲁胺。
16.一种治疗患有去势抵抗性前列腺癌患者的方法,其包括
鉴定患有去势抵抗性前列腺癌的患者;和
用CDCP1降调剂和除衰化合物和/或遗传毒性剂的组合治疗所述经鉴定的患者,优选其中在给药所述除衰化合物和/或遗传毒性剂之前给药所述降调剂。
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