背景:结直肠癌(CRC)是全球第三大常见癌症类型,也是导致癌症相关死亡的第二大原因,在晚期诊断时存活率接近10%。因此,迫切需要鉴定新的分子靶标,以设计更有选择性和有效的治疗方法。丝裂原活化蛋白激酶激酶3(MKK3)是一种双特异性苏氨酸/酪氨酸蛋白激酶,响应细胞应激和炎症刺激而激活,调节过多的生物过程。先前的研究揭示了MKK3在支持肿瘤恶性肿瘤方面的新作用,因为它的消耗在不同肿瘤类型的癌细胞系中诱导自噬和细胞死亡,包括CRC。因此,MKK3可能代表晚期CRC的一个有趣的新治疗靶点,然而,目前尚无选择性MKK3抑制剂。
方法:该研究涉及基于转录组学的药物再利用方法和CRC行的确认试验,原代结肠细胞和CRC患者来源的类器官(PDO)的子集。进行了体外和体内研究。
结果:再利用方法将多靶向激酶抑制剂AT9283鉴定为具有MKK3耗竭模拟活性的推定化合物。的确,AT9283在测试的CRC模型中降低了磷酸和总MKK3蛋白水平。可能是MKK3沉默,AT9283治疗:i)在测试的CRC细胞系和PDO中抑制细胞增殖,促进自噬和细胞死亡;ii)导致CCD-18Co结肠细胞具有良好的耐受性;iii)降低癌细胞运动性,抑制CRC细胞迁移和侵袭;iv)抑制COLO205异种移植肿瘤生长。机械上,AT9283主要通过抑制极光激酶A(AURKA)消除MKK3蛋白水平,影响MKK3/AURKA蛋白质-蛋白质相互作用和蛋白质稳定性,因此揭示了MKK3/AURKA串扰在维持CRC恶性肿瘤的体外和体内的相关性。
结论:总体而言,我们证明了AT9283治疗引发的抗肿瘤作用在体外和体内所有测试的CRC模型中概括了MKK3消耗效应,表明AT9283是一种再利用的药物。根据其良好的耐受性,当用原代结肠细胞(CCD-18CO)测试时,AT9283是一种有前途的药物,用于开发针对晚期和转移性CRC患者的MKK3致癌功能的新型治疗策略。
BACKGROUND: Colorectal cancer (CRC) is the third most common type of cancer and the second leading cause of cancer-related deaths worldwide, with a survival rate near to 10% when diagnosed at an advanced stage. Hence, the identification of new molecular targets to design more selective and efficient therapies is urgently required. The Mitogen activated protein kinase kinase 3 (MKK3) is a dual-specificity threonine/tyrosine protein kinase that, activated in response to cellular stress and inflammatory stimuli, regulates a plethora of biological processes. Previous studies revealed novel MKK3 roles in supporting tumor malignancy, as its depletion induces autophagy and cell death in cancer lines of different tumor types, including CRC. Therefore, MKK3 may represent an interesting new therapeutic target in advanced CRC, however selective MKK3 inhibitors are currently not available.
METHODS: The study involved transcriptomic based drug repurposing approach and confirmatory assays with CRC lines, primary colonocytes and a subset of CRC patient-derived organoids (PDO). Investigations in vitro and in vivo were addressed.
RESULTS: The repurposing approach identified the multitargeted kinase inhibitor AT9283 as a putative compound with MKK3 depletion-mimicking activities. Indeed, AT9283 drops phospho- and total-MKK3 protein levels in tested CRC models. Likely the MKK3 silencing, AT9283 treatment: i) inhibited cell proliferation promoting autophagy and cell death in tested CRC lines and PDOs; ii) resulted well-tolerated by CCD-18Co colonocytes; iii) reduced cancer cell motility inhibiting CRC cell migration and invasion; iv) inhibited COLO205 xenograft tumor growth. Mechanistically, AT9283 abrogated MKK3 protein levels mainly through the inhibition of aurora kinase A (AURKA), impacting on MKK3/AURKA protein-protein interaction and protein stability therefore uncovering the relevance of MKK3/AURKA crosstalk in sustaining CRC malignancy in vitro and in vivo.
CONCLUSIONS: Overall, we demonstrated that the anti-tumoral effects triggered by AT9283 treatment recapitulated the MKK3 depletion effects in all tested CRC models in vitro and in vivo, suggesting that AT9283 is a repurposed drug. According to its good tolerance when tested with primary colonocytes (CCD-18CO), AT9283 is a promising drug for the development of novel therapeutic strategies to target MKK3 oncogenic functions in late-stage and metastatic CRC patients.