【作者】 刘娟；

【导师】 干文娟；

【作者基本信息】 苏州大学 ， 临床病理学（专业学位）， 2025， 硕士

【摘要】 背景:结直肠癌（colorectal cancer,CRC）是消化道常见肿瘤,死亡率约9.3%。CRC发病涉及多种基因突变,其中KRAS是最常见的突变之一。KRAS突变后会激活下游的信号通路,导致细胞异常增殖并促进肿瘤的发生发展,同时增强对包括瑞戈非尼在内的抗癌药物的耐药性。因此,KRAS突变型CRC的患者预后往往较差。TRIM21作为一种E3泛素连接酶,在肿瘤的发生、发展及耐药中发挥重要作用。我们课题组前期研究发现:一方面,抗抑郁药维拉佐酮可以通过靶向TRIM21抑制CRC的侵袭和转移;另一方面,在KRAS突变型CRC中,磷酸化的TRIM21能够激活c-Myc/ENO2/糖酵解信号轴产生促癌作用。虽然瑞戈非尼是转移性CRC的标准三线治疗药物,但其对KRAS突变型CRC的疗效有限。因此,本研究重点探讨两个关键问题:一是KRAS突变型CRC对瑞戈非尼耐药的分子机制,二是维拉佐酮单用或联合瑞戈非尼治疗的潜在抗肿瘤效果。方法:培养肠癌细胞,构建肠癌类器官模型,对细胞/类器官模型进行加药处理并进行半数抑制浓度（IC50）分析,观察KRAS突变（KRAS/MT）和KRAS野生型（KRAS/WT）的细胞/类器官模型对瑞戈非尼的耐药性差异;通过q-PCR和免疫印迹（Western blot）分别检测KRAS突变（KRAS/MT）和KRAS野生型（KRAS/WT）的CRC组织中磷酸化TRIM21及下游信号通路因子表达情况;利用Eno2^ΔIEC和Eno2^fl/fl小鼠构建AOM/DSS诱导肠癌的动物模型,观察ENO2基因缺失对瑞戈非尼疗效的影响;联合免疫共沉淀（CO-IP）和Western blot研究维拉佐酮靶向TRIM21后对信号通路下游相关蛋白表达的影响;构建类器官模型及人源异种移植模型（PDX）,评估瑞戈非尼和维拉佐酮单药治疗及联合治疗的疗效;对小鼠的PDX肿瘤进行包埋、切片、HE染色以及免疫组化（IHC）,分析各组肿瘤的p-TRIM21表达情况及细胞增殖情况。结果:研究发现,KRAS突变型CRC细胞对瑞戈非尼治疗表现出明显的耐药性。机制研究表明,KRAS突变通过激活TRIM21/c-Myc/ENO2/糖酵解信号通路,显著增强了CRC类器官对瑞戈非尼的耐药特性。在动物实验中,ENO2条件性敲除小鼠的肠道肿瘤更小、更少,特别是在联合瑞戈非尼治疗后效果更为突出,这提示抑制糖酵解通路能够有效增强瑞戈非尼的抗肿瘤作用。进一步研究发现,维拉佐酮在KRAS突变型CRC细胞中能够特异性靶向TRIM21,从而抑制KRAS突变激活的c-Myc/ENO2/糖酵解信号通路。在类器官模型和PDX中,维拉佐酮与瑞戈非尼联合治疗对KRAS突变型CRC显示出显著的协同抗肿瘤效应。结论:本研究阐明了KRAS突变型CRC通过c-Myc/ENO2/糖酵解轴介导瑞戈非尼耐药的分子机制,通过抑制ENO2活性进而抑制糖酵解可以增强瑞戈非尼的疗效。维拉佐酮通过靶向TRIM21抑制KRAS突变驱动的c-Myc/ENO2/糖酵解信号轴,与瑞戈非尼联合应用展现出显著的协同抗肿瘤作用,为KRAS突变型CRC的精准治疗提供了新的策略和实验依据。更多还原

【Abstract】 Background:Colorectal cancer(CRC)is a common malignant tumor of the digestive tract,with a mortality rate of approximately 9.3%.The pathogenesis of CRC involves multiple genetic mutations,among which KRAS is one of the most frequently mutated genes.KRAS mutations activate downstream signaling pathways,leading to abnormal cell proliferation,tumor progression,and increased resistance to anticancer drugs,including regorafenib.Consequently,patients with KRAS-mutant CRC often exhibit poor prognosis.TRIM21,an E3 ubiquitin ligase,plays a crucial role in tumor initiation,progression,and drug resistance.Our previous studies demonstrated that:(1)the antidepressant vilazodone can inhibit CRC invasion and metastasis by targeting TRIM21;and(2)in KRAS-mutant CRC,phosphorylated TRIM21 activates the c-Myc/ENO2/glycolysis axis,promoting tumorigenesis.Although regorafenib is a standard third-line treatment for metastatic CRC,its efficacy in KRAS-mutant CRC remains limited.Therefore,this study focuses on two key questions:(1)the molecular mechanisms underlying regorafenib resistance in KRAS-mutant CRC,and(2)the potential antitumor effects of vilazodone alone or in combination with regorafenib.Methods:CRC cell lines were cultured,and organoid models were established.Drug treatments and half-maximal inhibitory concentration(IC50)analyses were performed to compare regorafenib resistance between KRAS-mutant(KRAS/MT)and KRAS wild-type(KRAS/WT)cell/organoid models.q PCR and Western blotting were used to assess the expression of phosphorylated TRIM21 and downstream signaling factors in KRAS/MT and KRAS/WT CRC tissues.An AOM/DSS-induced CRC mouse model was generated using Eno2ΔIEC and Eno2fl/fl mice to evaluate the impact of ENO2 deletion on regorafenib efficacy.Co-immunoprecipitation(Co-IP)and Western blotting were employed to investigate how vilazodone-mediated TRIM21 targeting affects downstream signaling proteins.Organoid and patient-derived xenograft(PDX)models were established to assess the therapeutic effects of regorafenib and vilazodone,either alone or in combination.PDX tumors were embedded,sectioned,and subjected to H&E staining and immunohistochemistry(IHC)to analyze p-TRIM21 expression and cell proliferation.Results:KRAS-mutant CRC cells exhibited significant resistance to regorafenib.Mechanistically,KRAS mutations enhanced regorafenib resistance in CRC organoids by activating the TRIM21/c-Myc/ENO2/glycolysis axis.In vivo,ENO2 conditional knockout mice developed fewer and smaller intestinal tumors,particularly when treated with regorafenib,suggesting that glycolysis inhibition enhances regorafenib’s antitumor effects.Further studies revealed that vilazodone specifically targets TRIM21 in KRAS-mutant CRC cells,suppressing the KRAS-driven c-Myc/ENO2/glycolysis pathway.In both organoid and PDX models,the combination of vilazodone and regorafenib demonstrated marked synergistic antitumor activity against KRAS-mutant CRC.Conclusion:This study elucidates the molecular mechanism by which KRAS-mutant CRC develops regorafenib resistance via the c-Myc/ENO2/glycolysis axis.KRAS mutations enhance drug resistance by activating this pathway,while ENO2 inhibition improves regorafenib’s efficacy.Vilazodone counteracts KRAS-driven signaling by targeting TRIM21,and its combination with regorafenib exhibits potent synergistic antitumor effects.These findings provide a novel therapeutic strategy and experimental basis for the precision treatment of KRAS-mutant CRC.更多还原