肿瘤靶标蛋白TNIK磷酸化促乳腺癌生长转移机制及其靶向抑制剂研究

Accumulating observations support the notion that TNIK exerts a central role in tumor progression, and closely related to the clinical staging and poor prognosis in the many kinds of the malignant tumors. As a new target of tumor is one of the important targets for cancer drugs. However, the molecular function of TNIK promotes proliferation and migration potentials of tumour that is still unclear. Our previous study found that p-TNIK only highly expressed in the cytoplasm, not in the nucleus of breast carcinomas cells and its activation of cytoskeletal protein-associated complexes（F-action/β-catenin/E-cadherin）are possible for the oncogenesis of BC. Ground on this view, a new CRISPR/Cas9 technology will be employed to obtain completely knockout TNIK-/-, and highly express TNIK+/+ breast cancer lines that explore the supposed mechanism of p-TNIK as a potential target for oncogenesis of breast cancer and develop effectively targeted inhibitors. IF, CoIP-MS and GST pull-down technologies will study the interacted cytoskeletal proteins with TNIK. In addition, the natural benzophen anthridine alkaloid derivative (CP-03) targeting p-TNIK regulating interacted cytoskeletal proteins that impact on inhibition of proliferation and migration/invasion in BC will be detailed studied; Orthotopic transplantation tumor with TNIK-/- and TNIK+/+ mouse model will be built for further research its targeting p-TNIK treatment for BC. Our study will not only provide new insight and mechanism that p-TNIK is essential for proliferation and migration/invasion in breast oncogenesis but also indicate that targeting TNIK may further yield therapeutic efficacy for novelty of the natural small molecules derivatives-targeted in treating BC.

Traf-2和Nck互作蛋白激酶(TNIK)在实体恶性肿瘤中高度表达，作为肿瘤新靶标是癌症药物治疗的重要靶点，但其促进癌症发生发展的分子机制仍不明确。申请人前期研究发现，磷酸化TNIK（p-TNIK）仅在胞质中高度表达，其调控细胞骨架蛋白F-action/β-catenin/E-cadherin复合体可能是促进乳腺癌生长转移的主要原因。本项目拟以CRISPR/Cas9技术敲除TNIK(TNIK-/-)和高表达TNIK (TNIK+/+)乳腺癌细胞为模型，IF、CoIP-MS和GST pull-down等技术鉴定TNIK互作蛋白；基于天然苯并菲啶生物碱结构分子模拟衍生TNIK抑制剂，阐明其作用p-TNIK抑制乳腺癌生长转移可能分子机制；原位移植瘤模型体内评价其靶向抑癌效果。本项目将揭示TNIK促乳腺癌生长转移新的磷酸化作用机制和探寻TNIK新型靶向抑制剂，为乳腺癌的靶向药物治疗提供理论基础。

乳腺癌是严重危害女性健康的恶性肿瘤，术后转移是其较难治愈的主要原因。肿瘤坏死因子受体相关因子-2与Nck互作的丝氨酸蛋白激酶（TNIK）是一种多效能的肿瘤靶蛋白，在乳腺癌组织中异常高度表达，TNIK可参与激活Wnt/β-catenin通路，对于乳腺癌的侵袭转移至关重要。药根碱属于原小檗碱型异喹啉类生物碱，该类生物碱对多种癌细胞均具有较强的细胞毒性作用。通过分子模拟对接显示TNIK可能是药根碱作用癌细胞的靶标抑制蛋白。因此研究TNIK调控乳腺癌转移的分子机制，探寻抑制乳腺癌侵袭转移的靶向药物，对于乳腺癌的治疗具有重要的意义。.本研究利用基因编辑CRISPR/Cas9技术靶向敲除TNIK，建立稳定转染的癌细胞株MDA-MB-231/TNIK-KO及MDA-MB-231/TNIK-Control，通过免疫荧光及免疫印迹检测TNIK敲除后癌细胞内TNIK、p-TNIK及Wnt/β-catenin通路关键蛋白表达情况，从而证明敲除TNIK可调控Wnt /β-catenin通路关键蛋白表达及逆转上皮-间质转化（EMT），影响癌细胞的转移侵袭能力，且其效果可通过加入药根碱处理加强。MTT、流式、免疫荧光及Western Blot实验结果表明药根碱可显著抑制乳腺癌细胞MDA-MB-231及MCF-7的细胞活性，调控细胞生长周期，诱导细胞线粒体膜电位发生改变，从而发生早期凋亡。再通过克隆形成、划痕愈合、免疫荧光及Western Blot实验证明药根碱能够破坏细胞膜骨架蛋白F-actin，调控TNIK蛋白表达破坏乳腺癌细胞的完整性，从而降低癌细胞的运动迁移能力。对于Wnt信号通路，药根碱能够调控关键蛋白β-catenin的表达使其下调，上调表达GSK-3β蛋白，并调控EMT上皮表型E-cadherin蛋白表达显著增加，而间质表型标志物N-cadherin蛋白表达呈降低趋势，从而逆转EMT。以上结果在体内实验中得到进一步证实，药根碱能够明显抑制乳腺肿瘤组织的生长和转移，且对荷瘤小鼠无明显毒性。.通过本研究得出药根碱作为一种极有前景的新型TNIK抑制剂，有望用于乳腺癌的临床靶向治疗。

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