CN115814056A - 靶向癌症治疗的egfr-sglt1相互作用 - Google Patents
靶向癌症治疗的egfr-sglt1相互作用 Download PDFInfo
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Abstract
化合物可使表皮生长因子受体(EGFR)与钠/葡萄糖共转运蛋白1(SGLT 1)之间的结合相互作用不稳定。在一实施方案中，所述化合物为来源于EGFR的相互作用结构域的肽。在另一实施方案中，将所述肽施用于患者以治疗癌症。
Description
相关申请的交叉引用：
本申请要求于2013年5月8日提交的美国临时申请号61/821,028的优先权，所述申请通过引用整体并入本文。
政府资助：
美国癌症学会(RSG-09-206-01)
国防部前列腺癌研究项目(W91ZSQ8334N607)
发明背景
表皮生长因子受体(EGFR)为在大多数上皮来源的癌症中高度活化/过表达的受体酪氨酸激酶(Hynes NE等, ERBB receptors and cancer: thecomplexity of targetedinhibitors, Nature Reviews Cancer, 5(5):341-354(2005))。抑制EGFR的酪氨酸激酶活性已经成为基于EGFR的癌症疗法的主要策略。然而，通过受体酪氨酸激酶的小分子抑制剂靶向EGFR并未产生令人满意的治疗功效。各种人恶性肿瘤的总体应答率为10-20％(WeissJ., First line erlotinib for NSCLC patients not selected by EGFRmutation:keep carrying the TORCH or time to let the flame die？ Transl. LungCancerRes., 1(3):219-223 (2012)；Cohen SJ等, Phase II andpharmacodynamic study ofthe farnesyltransferase inhibitor R115777 asinitial therapy in patients withmetastatic pancreatic adenocarcinoma, J.Clinical Oncology, 21(7):1301-1306(2003)；Dancey JE等, Targetingepidermal growth factor receptor—are we missingthe mark？, Lancet362(9377):62-64(2003))。换言之，存在对EGFR酪氨酸激酶抑制剂无应答的较多癌症患者群体。例如，虽然EGFR在超过80％的晚期前列腺癌中过表达并与预后负相关，但前列腺癌耐受EGFR 抑制剂(Hernes E等,Expression of the epidermal growthfactor receptor family in prostatecarcinoma before and during androgen-independence, British J. Cancer,90(2):449-454 (2004)；Pu YS等, Epidermalgrowth factor receptor inhibitor(PD168393) potentiates cytotoxic effects ofpaclitaxel againstandrogen-independent prostate cancer cells, BiochemicalPharmacology,71(6):751-760 (2006)；Sherwood ER等, Epidermal growth factor-relatedpeptides and the epidermal growth factor receptor in normal andmalignantprostate, World J. Urology, 13(5):290-296 (1995)；Zellweger T等,Expressionpatterns of potential therapeutic targets in prostate cancer,International J.Cancer, 113(4):619-628 (2005)； Canil CM等, Randomized phaseII study oftwo doses of gefitinib in hormone-refractory prostate cancer: atrial of theNational Cancer Institute of Canada-Clinical Trials Group, J.ClinicalOncology, 23(3):455-460 (2005)；Gross M等, A phase II trial ofdocetaxeland erlotinib as first-line therapy for elderly patientswithandrogen-independent prostate cancer, BMC Cancer 7:142 (2007))。
证据表明EGFR具有独立的酪氨酸激酶功能。例如，敲除EGFR的动物在出生后很快死亡(Threadgill DW等,Targeted disruption of mouseEGF receptor: effect ofgenetic background on mutant phenotype, Science,269(5221):230-234 (1995))。然而，具有严重受损的 EGFR 酪氨酸激酶活性的小鼠完全能够存活并仅显示出一些上皮缺陷(Luetteke NC,等, Themouse waved-2 phenotype results from a point mutation inthe EGF receptortyrosine kinase, Genes & Development, 8(4):399-413 (1994))。作为另一实例，野生型和激酶死亡的EGFR增强EGFR阴性32D造血细胞的存活率(Ewald JA等,Ligand- and kinase activity-independent cell survival mediatedby theepidermal growth factor receptor expressed in 32D cells, ExperimentalCellResearch 282(2):121-131 (2003))。同样已经发现EGFR通过与不依赖于EGFR酪氨酸激酶活性的钠-葡萄糖共转运蛋白1 (SGLT1)相互作用并使其稳定来参与维持癌细胞的细胞内葡萄糖基础水平(Weihua Z等,Survival of cancer cells is maintained by EGFRindependent of its kinaseactivity, Cancer Cell,13(5):385-393 (2008))。
SGLT1为主动的葡萄糖转运蛋白，其依靠细胞外钠浓度将葡萄糖转运至细胞而不依赖于葡萄糖浓度(Wright EM,等, Biology of human sodiumglucose transporters,Physiological Reviews, 91(2):733-794 (2011)。SGLT1在体内的葡萄糖吸收和保留中起关键作用(Castaneda-Sceppa C等,Sodium-dependent glucose transporter protein asa potential therapeutic targetfor improving glycemic control in diabetes,Nutrition Reviews,69(12):720-729 (2011))。癌症的其中一个标志为癌细胞显示出改变的能量代谢，即癌细胞消耗的营养物和能量底物的量大大高于其正常对应物(Hanahan D等, Hallmarks of cancer: the next generation, Cell,144(5):646-674 (2011)。该增强的能量消耗要求高比率的营养物摄取，这通过过表达质膜转运蛋白来实现(GanapathyV,等, Nutrient transporters incancer: relevance to Warburg hypothesis andbeyond, Pharmacology &Therapeutics 121(1):29-40 (2009)。研究已经发现SGLT1在不同类型的癌症中过表达，所述癌症包括卵巢癌、口腔鳞状细胞癌、结直肠癌、胰腺癌和前列腺癌(Lai B等, Overexpression of SGLT1 is correlated with tumordevelopment andpoor prognosis of ovarian carcinoma, Archives ofGynecology and Obstetrics,285(5):1455-1461 (2012)；Hanabata Y等,Coexpression of SGLT1 and EGFR isassociated with tumor differentiation inoral squamous cell carcinoma,Odontology/the Society of the Nippon DentalUniversity, 100(2):156-163 (2012)；Guo GF等, Overexpression of SGLT1and EGFR in colorectal cancer showing acorrelation with the prognosis,Medical Oncology 28 Suppl 1:S197-203 (2011)；Casneuf VF等, Expressionof SGLT1, Bcl-2 and p53 in primary pancreatic cancerrelated to survival,Cancer Investigation 26(8):852-859 (2008)；Blessing A等,Sodium/GlucoseCo-transporter 1 Expression Increases in Human DiseasedProstate, J. CancerSci. Ther. 4(9):306-312 (2012))。作为例子，晚期前列腺癌表达升高水平的EGFR并摄取大量的葡萄糖(Hernes E等, Expression of theepidermalgrowth factor receptor family in prostate carcinoma before andduringandrogen-independence, British J. Cancer, 90(2):449-454 (2004)； Pu YS等,Epidermal growth factor receptor inhibitor (PD168393) potentiatescytotoxiceffects of paclitaxel against androgen-independent prostate cancercells,Biochemical Pharmacology, 71(6):751-760 (2006)；Sherwood ER等,Epidermalgrowth factor-related peptides and the epidermal growth factorreceptor innormal and malignant prostate, World J. Urology, 13(5):290-296(1995)；Lee ST等, PET in prostate and bladder tumors, Seminars in NuclearMedicine 42(4):231-246 (2012)；Oyama N等, The increased accumulation of[18F]fluorodeoxyglucose in untreated prostate cancer, Japanese J.ClinicalOncology, 29(12):623-629 (1999))。较好理解EGFR与SGLT1的功能关系可产生鉴别癌症疗法的新型治疗靶标。
因此，本领域需要可充分治疗耐受EGFR酪氨酸激酶抑制剂的癌细胞的方法和组合物。
附图简要说明
当结合附图阅读时，前述概要以及以下详述将得到更好的理解。仅出于说明的目的，图中显示了某些实施方案。然而，应理解本文公开的发明构思不限于图中显示的精确排列和手段。
图1A示出根据实施方案，人EGFR的不同构建体的示意图。图1B示出根据实施方案，突变的EGFR与SGLT1之间的相互作用的免疫沉淀结合蛋白质印记分析。
图2A示出根据实施方案，用WT-EGFR、KD-EGFR和ΔAutophos-EGFR共转染的HEK293细胞中的SGTL1表达水平的蛋白质印记分析。图2B示出根据实施方案，图2A的蛋白质印记中条带的光密度定量。图2C示出根据实施方案，蛋白酶体抑制剂MG132对通过ΔAutophos-EGFR下调的SGLT1的作用的蛋白质印记分析。图2D示出根据实施方案，图2C的蛋白质印记中条带的光密度定量。
图3A示出根据实施方案，在用EGF或AEE788处理的HEK293细胞中EGFR-HA与SGLT1-Flag之间的相互作用的免疫沉淀结合蛋白质印记分析。图3B示出根据实施方案，在用EGF或AEE788处理的PC3细胞中内源EGFR与SGLT1之间的相互作用的 免疫沉淀结合蛋白质印记分析。
图4A示出根据实施方案，在来自前列腺癌组织阵列的前列腺癌组织中SGLT1和EGFR的共定位。图4B示出根据实施方案，在PC3和LNCaP细胞中内源EGFR和 SGLT1的表达的蛋白质印记分析。图4C示出根据实施方案，显示SGLT1对吉非替尼和埃罗替尼在PC3细胞上的生长抑制作用的抑制作用的MTT测定。图4D示出根据实施方案，显示SGLT1对吉非替尼和埃罗替尼在LNCaP细胞上的生长抑制作用的抑制作用的MTT测定。
图5A示出根据实施方案，使两种蛋白都不稳定的小分子肽破坏EGFR-SGLT1相互作用。图5B示出根据实施方案，用EGFR-SGLT1破坏肽(MTG-01)处理PC3细胞显著下调EGFR和SGLT1蛋白质。
图6A至图6E示出根据实施方案，用MTG-01处理时，癌性PC3、Du145、HCT116和 MDA-MB-231细胞以及处理的非癌性HEK293细胞的活力。
发明概述
以下示出本文公开的某些实施方案的概述。应理解，呈现该部分仅为读者提供某些实施方案的简述，并且这些描述不旨在限制本申请的范围。事实上，本公开可以涵盖此处未示出的多种实施方案。
本申请公开了治疗癌症的方法和组合物。在一个实施方案中，化合物使表皮生长因子受体(EGFR)与钠/葡萄糖共转运蛋白1(SGLT 1)之间的结合相互作用不稳定。在另一实施方案中，所述化合物为根皮苷样化合物或来源于EGFR的相互作用结构域的肽。
在实施方案中，将使EGFR与SGLT 1的结合相互作用不稳定的化合物经瘤内、经静脉内或经全身施用于对象。所述化合物可以为根皮苷样化合物或来源于EGFR的相互作用结构域的肽。在又一个实施方案中，可以将化合物与酪氨酸激酶抑制剂共同施用。
发明详述
在详细解释至少一个实施方案之前，应理解本文示出的发明构思并非将其申请限于以下描述中示出或附图中阐明的构建细节或组分配置。也应理解本文使用的用语和术语仅出于描述目的且不应被视为限制性的。
还应理解所述特征中的任一个可以单独使用或与其它特征组合使用。在查阅本文附图和详述后，其它发明的系统、方法、特征和优势对本领域技术人员将为或将变为显而易见的。本发明旨在使所有此类另外的系统、方法、特征和优势受所附权利要求的保护。
本申请中引用的所有参考文献通过引用整体并入。
表皮生长因子受体(EGFR)的过表达与恶性肿瘤的不良预后相关。钠/葡萄糖共转运蛋白1(SGLT1)为在癌症中过表达的主动葡萄糖转运蛋白，所述癌症包括前列腺癌。已经发现在癌细胞中EGFR与SGLT1相互作用并使其稳定。
如以下大量细节所解释，在本申请中已经确定了以下实施方案：
■EGFR的关键微结构域，其对EGFR与SGLT1的充分相互作用以及相互稳定是必需的；
■EGFR对EGFR-SGLT1相互作用的激活/失活作用；
■在前列腺癌组织和细胞系中测量EGFR和SGLT1的表达；
■SGLT1对于前列腺癌细胞对EGFR酪氨酸抑制剂的敏感性的抑制作用；
■合成的肽ESD-01的氨基酸序列；
■ESD-01对癌细胞中EGFR和SGLT1蛋白质的稳定性的影响；以及
■ESD-01对体外培养的非癌细胞(HEK293)和若干类型的癌细胞的存活性的影响。
■由L-氨基酸或D-氨基酸组成的ESD-01肽在体外杀伤癌细胞中同样有效。
在一个实施方案中，EGFR的自磷酸化区域(978-1210个氨基酸)对于EGFR与SGLT1的充分相互作用是必需的。该相互作用不依赖于EGFR的酪氨酸激酶活性。最重要地是，在另一实施方案中，EGFR-SGLT1相互作用对EGFR酪氨酸激酶调节剂(EGF和酪氨酸激酶抑制剂)无反应。在又一个实施方案中，EGFR和SGLT1共定位于前列腺癌组织中。在又一实施方案中，通过SGLT1抑制剂(根皮苷)对SGLT1的抑制使前列腺癌细胞(PC3和LNCaP)对EGFR 抑制剂(吉非替尼和埃罗替尼)敏感。在进一步实施方案中，ESD-01 (SEQ ID NO: 1) 使EGFR-SGLT1相互作用不稳定。所有这些数据表明，癌细胞中的EGFR可以以两种类型的状态存在——酪氨酸激酶调节剂反应状态和无反应状态。因此，在实施方案中，SGLT1为参与EGFR的功能的蛋白质，所述功能对EGFR 酪氨酸激酶抑制剂无反应，以及EGFR-SGLT1相互作用可为前列腺癌治疗的新型靶标。与SGLT1相互作用所需的EGFR蛋白区域
在一个实施方案中，为测定与SGLT1相互作用所需的EGFR蛋白区域，可以建立具有多种突变的flag标记的SGLT123 和HA标记的EGFR(Blessing A等, Sodium/Glucose Co-transporter 1 Expression Increases inHuman Diseased Prostate, J. Cancer Sci.Ther. 4(9):306-312 (2012))。图1A仅以举例的方式说明了人EGFR构建体的示意图，所述人EGFR构建体可以用于测定与SGLT1相互作用所需的EGFR蛋白质区域。例如，在一些实施方案中，该构建体可以包括：野生型EGFR (“WT”)；激酶死亡的EGFR (R817M) (“KD”) (SEQ IDNO: 12)；跨膜结构域缺失(645-670aa)(“ΔTM”) (SEQ ID NO: 13)；细胞外结构域缺失(1-644aa) (“ΔExtra”) (SEQID NO: 14)；细胞内结构域缺失(671-1210aa) (“ΔIntra”)(SEQ ID NO: 15)；酪氨酸激酶结构域缺失(670-977aa) (“ΔTK”) (SEQ ID NO: 16)；或自磷酸化结构域缺失(978-1210aa) (“ΔAutophos”) (SEQ ID NO: 17)。
在实施方案中，可将flag标记的SGLT1和以上鉴别的HA 标记的EGFR瞬时共转染HEK293细胞。在另一实施方案中，可使用抗-flag抗体使SGLT1免疫沉淀。在又一个实施方案中，可以对HA 标记的EGFR进行蛋白质印记分析。图1B仅以举例的方式说明了突变的EGFR与SGLT1的相互作用的蛋白质印记分析，其中IP＝免疫沉淀，IB＝免疫印迹并且Input＝在用于免疫沉淀的HEK293全细胞裂解物中指定的外源蛋白的表达水平。如图1B中显示，EGFR的整个细胞内结构域或自磷酸化结构域的缺失基本上使EGFR与SGLT1的相互作用减少。在一个实施方案中，EGFR的自磷酸化结构域对于其与SGLT1的充分相互作用是必需的。
先前，使用EGFR的细胞外结构域以及不含有EGFR TM结构域的细胞内结构域，发现EGFR的细胞外结构域与SGLT1的相互作用优于EGFR的细胞内结构域(Weihua Z等,Survival of cancer cells is maintained byEGFR independent of its kinaseactivity, Cancer Cell 13(5):385-393 (2008))。在实施方案中，为进一步表征质膜处EGFR-SGLT1相互作用，可以将TM结构域包含于截短的EGFR的构建体。在一个实施方案中，含有TM的EGFR的细胞内结构域(尤其是自磷酸化结构域)与SGLT1的相互作用强于EGFR的细胞外结构域。该实施方案与先前报道中显示的数据的不一致很可能是由于在先前研究中使用的细胞内结构域构建体无TM结构域。
防止蛋白酶体介导的SGLT1降解所需的EGFR的自磷酸化结构域
在实施方案中，为确定是否需要EGFR的自磷酸化结构域来维持SGLT1的稳定性，可以测量用WT-EGFR、KD-EGFR和ΔAutoPhos-EGFR共转染HEK293细胞的SGLT1的表达水平。例如，图2A仅以举例的方式说明了用WT-EGFR、KD-EGFR和ΔAutophos-EGFR共转染的HEK293细胞中SGTL1的表达水平的蛋白质印记分析。在每个处理组中可以使用相同量的SGLT1和EGFR的DNA质粒。此外，对照细胞可用相同量的空载体的DNA转染。可将肌动蛋白用作上样对照。图2B仅以举例的方式说明了图2A的蛋白质印记中条带的光密度定量。星号标记表明来自一式三份实验的关联代表性群组间的统计显著性。
如图2A至图2B所示，在一个实施方案中，在WT-EGFR和KD-EGFR转染的细胞中SGLT1的水平远高于在对照载体或ΔAutoPhos-EGFR转染的细胞中SGLT1的水平。在另一实施方案中，EGFR的自磷酸化结构域可保持SGLT1的表达水平。在又一实施方案中，在ΔAutoPhos-EGFR转染的细胞中SGLT1的水平显著低于对照细胞中SGLT1的水平。在又一个实施方案中，SGLT1与EGFR相互作用的减少可促进SGLT1的下调。
在实施方案中，为确定蛋白酶体是否参与与诱导SGLT1下调的EGFR的相互作用的减少，可用蛋白酶体抑制剂处理SGLT1和ΔAutoPhos-EGFR共转染的HEK293细胞，所述蛋白酶体抑制剂诸如但不限于MG231。例如，图2C说明了蛋白酶体抑制剂MG132对通过ΔAutophos-EGFR下调的SGLT1的影响的蛋白质印记分析。可将肌动蛋白用作上样对照。图2D仅以举例的方式说明了图2C的蛋白质印记中条带的光密度定量。星号标记表明来自一式三份实验的关联代表性群组间的统计显著性。
如图2C至图2D中显示，仅以举例的方式，在一个实施方案中，MG231可抑制ΔAutoPhos-EGFR转染的细胞中的SGLT1的下调。在另一实施方案中，蛋白酶体细胞器参与与诱导SGLT1下调的EGFR的相互作用的减少。换言之，当EGFR-SLGT1相互作用受损时，蛋白酶体可引起SGLT1的最终降解。在又一个实施方案中，蛋白酶体细胞器可为单独的或与EGFR-SLGT1相互作用组合的潜在治疗靶标。
在一个实施方案中，在EGFR中缺失SGLT1相互作用结构域经由蛋白酶体细胞器促进SGTL1的下调。在另一实施方案中，破坏在EGFR阳性癌细胞中的EGFR-SGLT1相互作用可导致SGLT1的下调。此外，先前的数据显示通过shRNA敲低SGLT1导致前列腺癌细胞的细胞自吞噬死亡(Weihua Z等, Survival of cancer cells is maintained by EGFRindependentof its kinase activity, Cancer Cell 13(5):385-393 (2008))。在实施方案中，EGFR-SGLT1相互作用可为改善基于EGFR的癌症治疗的有效靶标。
EGFR-SGLT1相互作用对EGFR 酪氨酸激酶抑制剂无反应
在实施方案中，可测定EGFR酪氨酸激酶抑制剂对EGFR与SGLT1相互作用的影响。在实施方案中，WT-EGFR和SGLT1共转染的HEK293细胞可用 EGF或EGFR 酪氨酸激酶抑制剂AEE788处理。可以免疫沉淀SGLT1，以及可测量与SGLT1共免疫沉淀的EGFR的水平。例如，图3A 仅以举例的方式说明了在用EGF或AEE788处理的HEK293细胞中EGFR-HA与SGLT1-Flag之间的相互作用的免疫沉淀结合蛋白质印记分析，其中EGFR ＝总EGFR，pEGFR ＝ 磷酸化的EGFR，IP ＝ 免疫沉淀，IB ＝免疫印迹，以及Input＝在用于免疫沉淀的HEK293全细胞裂解物中指定的外源蛋白的表达水平。如图3A中显示，在一个实施方案中，EGF或AEE788均未显著影响EGFR-SGLT1相互作用。在又一个实施方案中，与SGLT1共沉淀的EGFR未被磷酸化。
在一个实施方案中，为确定EGF和AEE788对内源EGFR-SGLT1相互作用的影响以及与SGLT1相互作用的内源 EGFR的磷酸化状态，可以免疫沉淀用EGF或AEE788处理的PC3细胞的内源 SGLT1。在另一实施方案中，可测量与SGLT1共沉淀的EGFR的磷酸化状态。仅以举例的方式，图3B说明了在用EGF或AEE788处理的PC3细胞中内源EGFR与SGLT1之间的相互作用的免疫沉淀结合蛋白质印记分析。在实施方案中，EGF或AEE788都不影响EGFR-SGLT1相互作用。在又一个实施方案中，内源 SGLT1相互作用的EGFR未被磷酸化。在又一实施方案中，EGFR-SGLT1相互作用对EGFR的酪氨酸激酶活性调节剂无反应。在又一实施方案中，EGFR可以其非激酶功能靶向癌症治疗，包括但不限于其与SGLT1的相互作用。
已有文件证明，在EGFR的自磷酸化结构域内酪氨酸磷酸化后，自磷酸化结构域用作募集反式激活下游信号传导的衔接蛋白/效应蛋白的主要停靠(docking)位点(BazleyLA等, The epidermal growth factor receptorfamily, Endocrine-Related Cancer,12 Suppl 1:S17-27 (2005))。基于图3A的结果，在实施方案中，EGFR-SGLT1相互作用不依赖于EGFR激活/失活。EGFR的自磷酸化结构域起到不依赖于EGFR的酪氨酸激酶活性的蛋白质-蛋白质相互作用结构域的作用。在一个实施方案中，EGFR具有不依赖于其酪氨酸激酶活性的促存活功能。例如，EGFR可以以两种类型的状态存在——酪氨酸激酶反应状态和酪氨酸激酶无反应状态。在通过EGFR的配体激活后，激酶反应的EGFR的自磷酸化结构域可被磷酸化并且可募集效应物以引发下游信号。可选地，激酶无反应的EGFR可以不断地与蛋白质相互作用，而与是否存在EGFR配体无关以及与EGFR的酪氨酸激酶的激活或失活无关。在又一个实施方案中，SGLT1可以为这样的蛋白质之一，所述蛋白质结合于EGFR并保持EGFR处于其激酶无反应状态。在又一个实施方案中，可以将EGFR的非磷酸化的自磷酸化结构域用作鉴别治疗靶标的工具。
通过SGLT1抑制剂抑制SGLT1使前列腺癌细胞对EGFR 抑制剂敏感
在实施方案中，为确定EGFR-SGLT1相互作用的临床相关性，可以在前列腺癌(n＝44)的组织微阵列上进行EGFR与SGLT1的免疫荧光共染色。例如，图4A仅以举例的方式说明了来自前列腺癌组织阵列的三个代表性前列腺癌组织的结果。在实施方案中，SGLT1(绿色)和EGFR(红色)可以在前列腺组织(例如，橙色或黄色，如通过箭头所指示)中共定位。 在另一实施方案中，基质细胞可为SGLT1阳性但为EGFR阴性的(例如，通过箭头所指示)。在又一个实施方案中，在EGFR阳性癌样品(n＝41)中，SGLT1与EGFR共定位于癌细胞而非基质细胞中。
在又一实施方案中，EGFR-SGLT1相互作用可以促成前列腺癌的发病。例如，在实施方案中，前列腺癌组织中EGFR与 SGLT1的共定位可表明EGFR-SGLT1相互作用为癌症相关的。当前，在临床上，尚未表明EGFR酪氨酸激酶抑制剂对前列腺癌令人满意的治疗效果(Canil CM等,Randomized phase II study of two doses of gefitinib in hormone-refractoryprostate cancer: a trial of the National Cancer Institute ofCanada-ClinicalTrials Group, J. Clinical Oncology, 23(3):455-460 (2005)；GrossM等, Aphase II trial of docetaxel and erlotinib as first-line therapy forelderly patientswith androgen-independent prostate cancer, BMC Cancer 7:142(2007))。在一个实施方案中，考虑到如下事实，EGFR表达与前列腺癌的疾病进展相关以及前列腺癌对EGFR 酪氨酸激酶抑制剂的临床无反应性，EGFR可以促进不依赖于其酪氨酸激酶活性的前列腺癌的疾病进展。另外，先前的发现推断(1)前列腺癌组织具有增加的SGLT1表达，23 (2)EGFR蛋白质而非其酪氨酸激酶活性的损失使前列腺癌细胞对化学治疗剂敏感，29以及(3)EGFR诱导的自噬性细胞死亡的损失受SGLT1蛋白质的下调介导(Blessing A等,Sodium/Glucose Co-transporter 1 Expression Increases inHuman DiseasedProstate, J. Cancer Sci. Ther. 4(9):306-312 (2012)；Xu S等,Loss of EGFRinduced autophagy sensitizes hormone refractory prostatecancer cells toadriamycin, The Prostate. 17:1216-1224 (2011)；Weihua Z等,Survival of cancercells is maintained by EGFR independent of its kinaseactivity, Cancer Cell,13(5):385-393 (2008))。在又一个实施方案中，EGFR可经由使SGLT1稳定来促进前列腺癌进展以维持晚期癌细胞对葡萄糖的高度需求。该实施方案不仅得到本文所述的所有实施方案的支持，而且也得到SGLT1的过表达防止肾上皮细胞和肠上皮细胞凋亡的早先数据的支持(Ikari A等, Sodium-dependent glucose transporter reduces peroxynitriteandcell injury caused by cisplatin in renal tubular epithelial cells,Biochimicaet Biophysica Acta 1717(2):109-117 (2005)；Yu LC等, SGLT-1-mediatedglucose uptake protects human intestinal epithelial cells againstGiardiaduodenalis-induced apoptosis, International journal for parasitology38(8-9):923-934 (2008))。
众所周知，葡萄糖水平的增加可激活EGFR，SGLT1在前列腺癌组织中过表达以及前列腺癌耐受EGFR抑制剂。(Han L等, High glucosepromotes pancreatic cancer cellproliferation via the induction of EGFexpression and transactivation of EGFR,PloS One 6(11):e27074 (2011)；Blessing A等, Sodium/Glucose Co-transporter 1Expression Increases inHuman Diseased Prostate, J. Cancer Sci. Ther. 4(9):306-312 (2012)；CanilCM等, Randomized phase II study of two doses of gefitinibinhormone-refractory prostate cancer: a trial of the National CancerInstitute ofCanada-Clinical Trials Group, J. Clinical Oncology, 23(3):455-460(2005)；Gross M等, A phase II trial of docetaxel and erlotinib as first-linetherapy forelderly patients with androgen-independent prostate cancer, BMCCancer7:142 (2007))。在实施方案中，SGLT1和EGFR可协同地促进前列腺癌生长。在一个实施方案中，为测试SGLT1的抑制是否可使前列腺癌细胞对EGFR抑制剂敏感，在有/无SGLT1抑制剂(例如，根皮苷和根皮苷衍生物，如卡那列嗪(Canagliflozin)和达格列嗪(Dapagliflozin))的情况下，可以用EGFR 酪氨酸激酶抑制剂(例如，吉非替尼(Gefitnib)、埃罗替尼(Erlotinib)、埃克替尼(Icontinib)、木利替尼(Mubritinib)、凡德他尼(Vandertanib)、拉帕替尼(Lapatinib)、培利替尼(Pelitinib)、卡奈替尼(Canertinib)、来那替尼(Neratinib)、阿法替尼(Afatinib)和达克替尼(Dacomitinib))来处理前列腺癌细胞系(例如，PC3和LNCaP (均为EGFR和SGLT1阳性)) (Ehrenkranz JR等, Phlorizin: areview,Diabetes/Metabolism Research and Reviews 21(1):31-38 (2005))。在一个实施方案中，可以测定治疗的生长抑制作用。例如，在实施方案中，在进行MTT测定之前，可将细胞用SGLT1抑制剂根皮苷(50μM)和/无EGFR抑制剂(吉非替尼, 10μM；埃罗替尼, 10μM)处理 48小时。可将对照细胞的OD值人工设定为1。可以将所有实验重复至少3次。星号标记表明关联组之间的统计显著性。
图4B仅以举例的方式说明了在PC3和LNCaP细胞中内源 EGFR和SGLT1表达的蛋白质印记分析。在一个实施方案中，乳腺癌和结肠癌细胞表达EGFR和SGLT1。图4C 显示SGLT1对吉非替尼和埃罗替尼在PC3细胞上的生长抑制作用的抑制作用的MTT测定。图4D显示SGLT1对吉非替尼和埃罗替尼在LNCaP细胞上的生长抑制作用的抑制作用的MTT测定。基于这些结果，在一个实施方案中，SGLT1和EGFR功能的共抑制可更有效地抑制癌细胞生长。在又一实施方案中，根皮苷可显著地使前列腺癌细胞对吉非替尼和埃罗替尼的生长抑制作用敏感。在实施方案中，可将根皮苷或根皮苷样化合物通过静脉内、瘤内或全身施用于患者以治疗癌症。在又一实施方案中，可将根皮苷或根皮苷样化合物与酪氨酸激酶抑制剂一起施用于患者以治疗癌症。
在另一实施方案中，肽可使癌细胞的EGFR和SGLT1蛋白质不稳定。图5A仅以举例的方式说明了被称为ESD-01的肽(SEQ ID NO: 1)的实施方案，所述肽可使癌细胞的EGFR和SGLT1蛋白质不稳定。在一个实施方案中，ESD-01肽可选自EGFR的SGLT1相互作用结构域。在另一实施方案中，ESD-01包含人EGFR蛋白质的氨基酸1049-1062(GenBank:AAH94761.1)。在又一实施方案中，可通过蛋白质印记分析来测定ESD-01对培养的细胞(例如，PC3、MDA-MB-231和HCT116细胞)中EGFR和SGLT1稳定性的影响。图5B仅以举例的方式显示ESD-01处理(200μM保持6小时)显著下调PC3细胞中的EGFR和 SGLT1水平，这受蛋白酶体抑制剂MG132的抑制。在实施方案中，通过MG132抑制由ESD-01下调的SGLT1和EGFR表明MTG-01经由蛋白水解使EGFR和SGLT1不稳定。在又一实施方案中，ESD-01对培养的癌细胞(例如，PC3、MDA-MB-231和HCT116细胞)的存活性的影响可通过台盼蓝摄取测定来确定。图6A至图6E仅以举例的方式显示ESD-01处理(100μM保持 24小时)显著降低多种癌细胞的存活性，所述癌细胞包括前列腺癌 PC3和Du145细胞、乳腺癌MDA-MB-231细胞和结肠癌HCT116细胞。在又一个实施方案中，ESD-01处理不能降低非癌性HEK293细胞的存活性。
众所周知，在蛋白质/肽中用具有类似化学性质的不同氨基酸置换蛋白/肽中的氨基酸可生成具有相同功能的蛋白质/肽。在实施方案中，ESD-01(SEQ ID NO: 1)可被具有类似化学性质的氨基酸或将使癌细胞的EGFR和SGLT1蛋白质不稳定的任何其它突变取代。例如，在一个实施方案中，氨基酸可为L-型或D-型手性异构体。在另一实施方案中，ESD-01中的丝氨酸(S)可被苏氨酸(T)、酪氨酸(Y)或任何其它非天然的含有羟基的氨基酸取代(例如，SEQ ID NO: 2)。在又一个实施方案中，ESD-01中的苏氨酸(T)可被丝氨酸(S)、酪氨酸(Y)或任何其它非天然的含有羟基的氨基酸取代(例如，SEQ ID NO: 3)。在又一实施方案中，ESD-01中的赖氨酸(K)可被苏氨酸(T)取代(例如，SEQ ID NO: 4)。在实施方案中，ESD-01中的谷氨酰胺(Q)可被组氨酸(H)取代(例如，SEQ ID NO: 5)。在另一实施方案中，ESD-01的位点10处的苏氨酸(T)可被丙氨酸(A)取代(例如，SEQID NO: 6)。在又一实施方案中，其中ESD-01的位点12处的丝氨酸(S)可被亮氨酸(L)取代(例如，SEQ ID NO: 7)。在又一实施方案中，ESD-01中的极性带正电荷的氨基酸、赖氨酸(K)和组氨酸(H)可为任何其它天然或非天然的带正电荷的氨基酸(例如，SEQ ID NO: 8)。在一个实施方案中，ESD-01中的极性氨基酸、谷氨酰胺(Q)和半胱氨酸(C)可为任何其它天然和非天然的极性氨基酸(例如，SEQ IDNO: 9)。在另一实施方案中，ESD-01中的半胱氨酸(C)可为任何其它天然和非天然的含有硫醇侧链(-SH)的氨基酸(例如，SEQ ID NO: 10)。在又一实施方案中，ESD-01中的非极性氨基酸、亮氨酸(L)、缬氨酸(V)和色氨酸(W)可为任何其它天然和非天然的非极性氨基酸(例如，SEQ ID NO: 11)。
应理解以上说明旨在说明而非限制。已经呈现的材料以使得本领域任何技术人员能够完成和使用本文所述的发明构思，并且该材料提供于特定实施方案的上下文中，该特定实施方案的变化对于本领域技术人员是显而易见的(例如，所公开实施方案中的一些可以彼此组合使用)。在参考以上描述后，许多其它实施方案对于本领域技术人员是显而易见的。因此，本发明的范围应参考所附权利要求连同与该权利要求所赋予的全部等同范围一起来确定。在所附权利要求中，术语“包括”和“其中”被用作各自的术语“包含”和“其中所述”的简明英文等同物。
实施例
细胞和试剂。最初从美国典型培养物保藏中心(ATCC)获得 HEK293细胞系、前列腺癌细胞系PC3、LNCaP、Du145、MDA-MB-231和HCT116细胞，并在37℃下于5％ CO2中，将所述细胞系维持在补充有10％胎牛血清和 1％青霉素/链霉素的DMEM中。小鼠抗-Flag-标签抗体(F1804)、蛋白酶体抑制剂MG231和根皮苷二水合物获自Sigma-Aldrich (St. Louis,MO)。AEE788、吉非替尼和埃罗替尼获自Selleckchem (Houston, TX)。抗pEGFR的抗体(Y1173)(目录号2434L)获自Cell Signaling(Danvers,MA)。抗C225的单克隆抗体获自Lee Elis博士 (M.D. 安德森癌症中心)。兔抗肌动蛋白(目录号sc-7210)、兔抗-HA-标签抗体(sc-805)、用辣根过氧化物酶标记的抗兔和小鼠二抗，以及蛋白质A/G偶联的琼脂糖小球(目录号sc-2003)获自Santa Cruz Biotechnology (Santa Cruz, CA)。MTT试剂盒(目录号30-1010K)获自ATCC。先前已经描述了用于免疫组织化学分析(SGLT1-IHC)和蛋白质印记分析(SGLT1-WB)的表达flag 标记的人SGLT1和兔抗人-SGLT1多克隆抗体的质粒 (Blessing A等,Sodium/Glucose Co-transporter 1 Expression Increases in HumanDiseasedProstate, J. Cancer Sci. Ther. 4(9):306-312 (2012))。
质粒构建。将人野生型EGFR克隆至pcDNA3.1载体(Clontech, CA)，其被用作亲代载体，以生成所有其它EGFR构建体。将具有c-末端HA标签的pRK5表达质粒(Clontech, CA)用于所有HA 标记的EGFR的构建。用正向引物EGFR-F (ATTCTCGAGCGGGGAGCAGCGATG (SEQID NO:18))和反向引物EGFR-R(CCTAAGCTTTGCTCCAATAAATTCACTG(SEQ ID NO: 19))来扩增全长人EGFR。将DNA片段通过 Xho I和Hind III消化并克隆至pRK5载体的相应位点。用于克隆具有细胞外结构域缺失的EGFR (ΔExtra, 1-644aa)的引物为ΔExtra-F：ATTCTCGAGATGTCCATCGCCACTGGGATG (SEQ ID NO: 20)和ΔExtra-R:CCTAAGCTTTGCTCCAATAAATTCACTGC (SEQ ID NO: 21)；用于克隆具有细胞内缺失的EGFR(ΔIntro, 671-1210aa) 的引物为ΔIntra-F:TATCTCGAGATGCGACCCTCCGGGACGGC (SEQ IDNO: 22)和ΔIntra-R: CCTAAGCTTCC TTCGCATGAAGAGGCC (SEQ ID NO: 23)；用于克隆具有自磷酸化结构域缺失的EGFR (ΔAutophos, 978-1210aa)的引物为ΔAutophospho-F:ATTCTCGAGATGTCCATCGCCACTGGGATG(SEQ ID NO: 20)和ΔAutophospho-R:CCTAAGCTTGTAGCGCTGGGGGTCTCGG (SEQ ID NO: 24)；用于克隆具有细胞内结构域缺失的EGFR (645-1210aa)的引物为ΔIntra-F:TATCTCGAGATGCGACCCTCCGGGACGGC (SEQ ID NO:22)和ΔIntra-R: CCTAAGCTTCCTTCGCATGAAGAGGC (SEQ ID NO: 25)。根据制造商的方案，使用QuikChange Lightning定点诱变试剂盒(Agilent,CA)，由通过定点诱变pRK5-WT-EGFR-HA来构建EGFR的激酶死亡突变体(KD-EGFR, R817M)，跨膜结构域缺失(ΔTM, 645-670个aa)和酪氨酸激酶结构域缺失(ΔTK, 670-977aa)质粒。引物为：KD-EGFR-F:GCACCGCGACCTGGCAGCC ATGAACGTACTGGTGAAAACACC(SEQ ID NO: 26) 和 KD-EGFR-R:GGTGTTTTCACCAGTACGTTCATGGCTGCCAGGTCGCGGTGC (SEQID NO: 27)；ΔTM-F:CGAGACCCCCAGCGCTACCGGACTCCCCTCCTGAGC (SEQ ID NO:28)和ΔTM-R: CGAGACCCCCAGCGCTACCGGACTCCCCTCCTGAGC(SEQ ID NO: 28)；ΔTK-F: CGCTGCGGAGGCTGCTGCAGTACCTTGTCATTCAGGGGG (SEQ IDNO: 29)和ΔTK-R:CCCCCTGAATGCAAGGTACTGCAGCAGCCTCCGCAGCG (SEQ IDNO: 30)。所有构建体产生具有C-末端HA标签的融合蛋白。通过测序验证所有质粒。
瞬时转染和免疫沉淀。用表达flag标记的SGLT1的质粒单独转染或与指定的HA 标记的EGFR 构建体一起转染HEK293细胞。在转染24小时之后，将细胞在1× 磷酸盐缓冲液中洗涤，并在摇床于4℃下，用补充有蛋白酶抑制剂混合物的RIPA缓冲液(50 mM Tris-HCl,pH 8.0, 具有150 mM氯化钠, 1.0％ Igepal CA-630 (NP-40), 0.5％脱氧胆酸钠和0.1％十二烷基硫酸钠)裂解 6小时。然后，将细胞裂解物以12000× rpm 离心2分钟。然后，将上清液用偶联有抗-flag或抗-SGLT1抗体的琼脂糖蛋白质A/G珠粒于4℃孵育过夜。然后，将样品离心，用 RIPA缓冲液洗涤三次，然后在Laemmle缓冲液(Biorad, CA)中煮沸，以及进行蛋白质印记分析。为测定EGFR的酪氨酸激酶在EGFR-SGLT1相互作用中的作用，用SGLT1和野生型EGFR转染HEK293细胞。在18小时之后，细胞在无血清培养基中挨饿 6小时，然后用EGF处理 (10 ng/ml)或EGF + AEE788 (5μM)处理30-60 分钟。将对照细胞用相等体积的媒介物二甲基亚砜(DMSO)处理。然后，对细胞裂解物进行以上所述的免疫沉淀。
蛋白质印记分析。对于蛋白质印记(WB)分析，将细胞用RIPA缓冲液(150 mM NaCl,50 mM Tris-HCl, pH 7.4, 0.1％ SDS, 1％ TritonX-100, 1mM EDTA, 1 mM PMSF, 20 μg/ml抑肽酶, 20 μg/ml亮抑酶肽, 20 μg/ml胃蛋白酶抑制剂(pepstatine), 1％脱氧胆酸钠, 1 mM NaF, 1 mM Na3VO4,于H2O中)裂解。将通过8％ SDS-PAGE分离的蛋白质转移至PVDF膜，随后用5％脱脂奶粉封闭，然后在4℃下与一抗在最佳浓度过夜孵育。将膜用0.1％TBS/T (1XTBS, 0.1％ 吐温-20) 洗涤3次，每次5分钟，然后与二抗在室温孵育1小时。通过增强化学发光使信号可视化。
免疫荧光共染色。对于SGLT1和EGFR的免疫荧光共染色，将前列腺癌组织阵列的载玻片脱蜡并再水化，然后在煮沸的柠檬酸盐缓冲液中保持10 分钟恢复抗原。然后，将冷却的组织载玻片在室温下于封闭溶液(含5％驴血清的PBS)中孵育1小时，接着在4℃下用抗SGLT1(SGLT1-IHC) (1:200稀释)和C225 (1:200)的兔多克隆抗体在含有10％驴血清的PBS中孵育过夜 (Blessing A等, Sodium/Glucose Co-transporter 1Expression Increasesin Human Diseased Prostate, J. Cancer Sci. Ther.,4(9):306-312 (2012))。在用PBS洗涤三次之后，将组织用溶解在含有10％驴血清的PBS中的Alexa Fluor 488-偶联的驴抗兔IgG和Alexa Flour 594-偶联的驴抗鼠IgG的混合物在室温孵育30分钟。 然后，将染色的样品用PBS在室温洗涤三次(每次洗涤5分钟)。拍摄荧光图像并用共聚焦显微镜(Olympus)分析。将细胞核用4',6-二氨基-2-苯茚二酮(DAPI)染色。
细胞生长测定。根据制造商提供的方案，在96孔板中通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四氮唑(MTT)测定来确定细胞生长。简言之，在96孔板的每个孔中，接种悬浮于100 μL培养基的5000个细胞。第二天，将培养基用含有根皮苷 (50μM)和 EGFR 抑制剂(易瑞沙: 20μM；埃罗替尼: 20μM)的培养基更换。在与药物孵育 24或48小时之后，将10μL MTT试剂加入至每个孔中并孵育4小时。在去除培养基之后，将细胞中的甲臜沉淀物溶解在100 μL DMSO中。通过MultiSkan板读数仪(Thermo Fisher Scientific, NC)在570 nm下测量吸光度。在每组中使用一式三份样品。通过台盼蓝摄取测定来确定细胞活力。在10x放大率下，一式三份，于随机选择的区域中(对于每个样品，n＝3)对活细胞的百分比进行计数。
统计分析。将学生t检验用于评估在有/无SGLT1抑制剂时用EGFR抑制剂处理的细胞的生长差异。P值小于0.05被定义为具有统计显著性。
Claims (7)
1.用于治疗癌症细胞的组合物，其包含能够使表皮生长因子受体(EGFR)蛋白与钠/葡萄糖共转运蛋白1(SGLT 1)之间的相互作用不稳定的肽；
其中所述肽选自SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ IDNO:9、SEQ ID NO:10和SEQ ID NO:11中的一项或多项。
2.肽在制备用于治疗对象中癌细胞的药物中的用途：
其中所述肽能够使表皮生长因子受体(EGFR)蛋白与钠/葡萄糖共转运蛋白1(SGLT 1)之间的相互作用不稳定；以及
所述述肽选自SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10和SEQ ID NO:11中的一项或多项。
3.如权利要求2所述的用途，其中所述药物还包括向所述对象共同施用的酪氨酸激酶抑制剂。
4.如权利要求3所述的用途，其中所述酪氨酸激酶抑制剂为以下中的至少一种：吉非替尼、埃罗替尼、埃克替尼、木利替尼、凡德他尼、拉帕替尼、培利替尼、卡奈替尼、来那替尼、阿法替尼和达克替尼。
5.如权利要求2所述的用途，其中所述药物还包括向所述对象共同施用的靶向蛋白酶体细胞器的化合物。
6.如权利要求2所述的用途，其中所述癌细胞为乳腺癌细胞、前列腺癌细胞和结肠癌细胞中的至少一种。
7.鉴别癌症治疗靶标的方法，其包括通过使用肽来鉴别受表皮生长因子受体(EGFR)蛋白与钠/葡萄糖共转运蛋白1(SGLT 1)的相互作用控制的下游存活途径而鉴别癌症治疗的靶标，
其中所述肽能够使表皮生长因子受体(EGFR)蛋白与钠/葡萄糖共转运蛋白1(SGLT 1)之间的相互作用不稳定；以及
所述肽选自SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ IDNO:5、SEQ ID NO:6、SEQID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10和SEQ ID NO:11中的一项或多项。
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