转录因子AtoH1通过调节NLK促进肺癌细胞增殖和转移的机制

Serine/threonine kinase NLK was found to regulate directly/indirectly many transcription factors and signaling pathway controlling genes, such as FOXO、TCF/LEF、SETDB and STAT. NLK therefore was showed to be highly involved in regulating tumour progression and metastases. However, the mechanism is not yet clear on how NLK protein itself is regulated. It is well known that AtoH1 expresses specifically in early stage of nerve system development and play an important role in neuron development and differentiation. Our previous study in Drosophila has revealed that NLK is a downstream target of AtoH1. Overexpression of NLK can rescue AtoH1 mutant phenotype. Recent study on clinical sample from small cell lung cancer (SCLC) has found that AtoH1 expressed unexpectedly in SCLC cell. The reason why and how does AtoH1 express is not known yet. Interestingly, other study on lung cancer showed an abnormal high expression of NLK. Putting all together, we propose: the unexpected expression of AtoH1 in SCLC results a high expression of NLK; blocking the expression of AtoH1 would repress the NLK expression, which in turn repress the SCLC cell progression and metastases. In order to better understand the mechanism of this regulating cascade, we will produce our research work mainly on two aspects: (1) the methylation and phosphorylation of the AtoH1 will be examined. It will help to analysis the regulation and the functional changes of AtoH1 in SCLC cell; (2) mechanism study on how does AtoH1 regulate the NLK and moreover on the role of AtoH1 in SCLC cell progression and metastases. At the end it would open a window for searching new clinical targets.

丝氨酸/苏氨酸激酶蛋白NLK通过调控TCF/LEF等转录因子和信号通路来调节肿瘤增殖和转移，但其上游调控机制并不清楚。转录因子AtoH1在神经前体细胞特异性表达，决定神经细胞的发育和分化。我们在果蝇模型上发现NLK是AtoH1的靶基因，过表达NLK可以改善AtoH1缺陷带来的神经分化障碍。近期在小细胞肺癌（SCLC）活检样本中发现AtoH1异常表达并伴有NLK异常升高，但AtoH1异常表达的机制不明。我们提出假说：AtoH1在SCLC中异常表达导致NLK高表达；抑制AtoH1表达可降低NLK水平，从而抑制SCLC的增殖和迁移。在该课题中我们将（1）在SCLC患者活检样本中检测AtoH1的甲基化、磷酸化水平，从表观遗传学角度揭示AtoH1在SCLC异常表达的机制；（2）在SCLC细胞系和动物模型上探讨AtoH1调控NLK的机制，确认AtoH1在SCLC增殖和迁移中的作用，为治疗提出新靶点。

细胞中遗传物质的变化是致癌的必要条件，而致癌物质及环境危险因素等各种致癌因子也是通过改变细胞中的遗传物质导致癌症发生的。因此尽管肿瘤成因复杂，确定在癌症中起关键作用的基因，研究其在正常和肿瘤细胞中的生理作用，对于明确肿瘤细胞增殖和迁移的机制依然不可或缺。.有研究表明丝氨酸/苏氨酸激酶蛋白 NLK通过调控TCF/LEF等转录因子和信号通路来调节肿瘤增殖和转移，但其上游调控机制并不清楚。而转录因子AtoH1在神经前体细胞中特异性表达，决定神经细胞的发育和分化。我们的前期研究发现NLK是AtoH1的靶基因，过表达NLK可改善AtoH1缺陷带来的神经分化障碍。近期在小细胞肺癌（SCLC）活检样本中发现AtoH1异常表达并伴有NLK异常升高，但发生的机制不明。因此本项目拟用小细胞肺癌作为研究对象，进一步细化研究NLK 和AtoH1在SCLC中的作用，以及他们对肺癌细胞的增殖和转移的影响。拟揭示SCLC中AtoH1和NLK表达水平和分布上的变化；AtoH1如何通过调控NLK而影响肿瘤的增生和迁移；AtoH1蛋白本身的修饰对其调控功能有何影响；NLK会调节那些下游蛋白；从而为小细胞肺癌提出新的有效治疗靶点。.我们的研究发现，正常肺细胞中未检测到AtoH1表达；而四种小细胞肺癌细胞株细胞中可以发现AtoH1表达。NLK蛋白在四种小细胞肺癌细胞株细胞中的表达水平明显高于正常肺细胞。而两种细胞调控蛋白P21和P27在各细胞株中的表达水平则相异。与正常细胞比较，所研究的四种小细胞肺癌株细胞的胞浆内均可测到不同表达水平的NLK 和AtoH1蛋白。同时有不同比例的蛋白质进入细胞核内。说明这四种小细胞肺癌株细胞不仅表达AtoH1和NLK蛋白，而且两种蛋白都具有活性。小细胞肺癌病人的癌组织切片的组化染色结果与四种小细胞肺癌细胞株的结果一致。与小细胞肺癌株细胞比较，敲减NLK 或AtoH1的小细胞肺癌株细胞的增殖受到抑制。该项目通过对AtoH1调控NLK进而参与影响肿瘤进展的研究，有望为癌症的临床诊断和治疗提供可参考的新靶点。