甲基转移酶NSD1对头颈部鳞癌吉西他滨耐药的调控作用及其机制

Gemcitabine has a wonderful application prospect in therapy of head and neck squmous cancer (HNSCC), but tumor drug resistance impedes its healing efficiency. So it is meaningful to research the mechansims of tumor resistance to gemcitabine. The current acknowledgement on it mainly focusing on the gemcitabine metabolic pathway, and little on the signaling pathway, is far from fully understanding the mechanism. The applicant found the NSD1 Ser726Pro mutation relates to HNSCC resistance to gemcitabine, and moreover, the muation blocks phosophrylation at this site to change NSD1 activity, and in turn impact the downstream NF-kB signaling pathway. NF-kB signaling pathway play an important role in regulting gemcitabine resistance. All togheter, we hypothesize that NSD1 S726P mutation modulates tumor gemcitabine resistance by regulating NF-kB signaling pathway. we will use the methods of mass spectrometry, co-immunoprecipitation, and so on, at levels of molecules, cells, tissues and animals, to define the functions of NSD1 S726P mutation in HNSCC gemcitabine resistance, elucidate the site mutation regulates gemictabine resistance through NF-kB siganling pathway, identify the kinase to NSD1 Ser726. The project provides biomarkers for clinically reversing gemitabine resistance, and gives new theories and clues for new drug targets for HNSCC and other tumors.

吉西他滨在中晚期头颈鳞癌治疗中应用前景良好，但肿瘤的耐药影响其治疗效果，故研究耐药机制对于提高其临床疗效具有重要意义。目前吉西他滨耐药研究主要集中在代谢途径，信号通路研究较少，尚不能完全解释机制。申请人发现NSD1 Ser726Pro（S726P）突变与吉西他滨耐药相关，而该位点突变可影响NSD1磷酸化，从而改变其活性，进而影响下游NF-kB通路。而NF-kB通路可调控肿瘤吉西他滨耐药，由此我们推测NSD1 S726P突变可通过NF-kB信号通路调控肿瘤吉西他滨药物敏感性。为此，我们拟在分子、细胞、组织、动物多个层面，利用蛋白质谱、免疫共沉淀等方法，明确NSD1 S726P突变在头颈鳞癌吉西他滨耐药中的作用；阐明该位点突变通过激活NF-kB通路调控耐药；鉴定获得NSD1 Ser726的作用激酶。旨在为临床逆转吉西他滨耐药提供分子标志物，为寻找头颈癌乃至其他肿瘤的治疗新靶点提供新理论新思路。

本研究中我们发现NSD1是吉西他滨耐药的关键基因，并通过基因敲减稳定株建立、裸鼠成瘤、药物毒性等体内外实验明确NSD1在头颈鳞癌细胞吉西他滨耐药产生中的关键作用。Western Bloting发现，敲减NSD1后，H3K36me2显著降低。考虑到H3K36me2对转录水平的调控虽弱，但H3K36me2能招募DNA甲基转移酶DNMT3A促进DNA甲基化修饰，而DNA甲基化在基因转录调控中起着重要的作用。这也与NSD1表达失活导致DNA甲基化降低并与良好的化疗治疗效果的报道一致。因此我们用DNA甲基化芯片分析了NSD1敲减对DNA甲基化的影响。甲基化芯片发现敲减NSD1后引起广泛的DNA甲基化降低，包括在与药物敏感性相关的FOXA2基因上。Western Bloting、QPCR 结果表明NSD1敲减后细胞FOXA2表达上调。因此敲减NSD1可能通过低甲基化修饰上调了FOXA2的表达。而吉西他滨具有DNA甲基转移酶抑制作用，吉西他滨预处理细胞后，Western Bloting、QPCR结果表明吉西他滨亦可上调FOXA2表达。综上，NSD1通过修饰H3K36me2提高FOXA2基因上的DNA甲基化修饰，从而抑制吉西他滨诱导FOXA2的表达，从而导致肿瘤细胞对吉西他滨的耐药作用。本研究结果阐明了吉西他滨临床耐药分子机理，并为耐药逆转提供了新的靶点，为开展个体化靶向药物治疗提供理论依据，也可为其他肿瘤的耐药机制研究提供可靠平台及新思路。