FRK靶向竞争性结合cyclin D1/ pRb在脑胶质瘤中的作用及机制的研究

Cyclin D1 and pRb play a key role in cell cycle regulation. Studies have shown that the abnormal regulation of them is intimately associated with origination, development, and prognosis of tumors; Moreover, it has relevance to tumor cell resistance to chemotherapy drug. FRK, as a member of the oncogene Src family, plays a unique tumor suppressor function. The laboratory of applicant first found and put forward that FRK may play a protective role against the progression of glioma. Recently, the pilot experiments showed that FRK over-expression can prevent the nuclear accumulation of cyclin D1, improve the activity of nucleoprotein pRb, induce G1 phase arrest, and suppress glioma cell proliferation. The project will apply the glioma specimens, the cultured glioma cells and the exenograft nude mouse model to clarify the growth inhibition mechanism FRK involved by competitively combining with cyclinD1 and pRb inside and outside the nucleus, and blocking pRb-E2Fs signaling pathway, and study the effect of FRK on the sensitivity of chemotherapy drug and the relevance of FRK , cyclin D1,pRb in glioma . Our results will not only provide potential targets for molecular targeting therapy of brain gliomas, but also open new ideas to the development of efficient cell cycle inhibitors and the treatments of gliomas.

Cyclin D1与pRb在细胞周期调控中起关键性作用。研究表明其调控异常与肿瘤的发生、发展及预后密切相关；并且与肿瘤细胞对化疗药物的抗拒性有关。作为原癌基因Src家族成员之一， FRK却发挥着独特的肿瘤抑制功能。申请者实验室率先发现并提出FRK在胶质瘤中扮演抑癌角色。新近又通过预实验观察到过表达FRK可以阻止cyclin D1的入核，提高核蛋白pRb的活性，诱导G1期的阻滞，抑制胶质瘤细胞的增殖。本项目应用胶质瘤标本、培养的胶质瘤细胞与裸鼠移植瘤模型，从整体、细胞和分子水平研究并阐明FRK在核内外分别竞争性结合于cyclin D1、pRb，进而阻断pRb-E2Fs信号通路，抑制脑胶质瘤生长的机制；并研究FRK对化疗药物敏感性的影响及FRK、cyclin D1、pRb在胶质瘤组织中的相关性，为胶质瘤的分子靶向治疗提供潜在的靶点，为研发高效的细胞周期抑制剂、胶质瘤病的治疗开启提供新的思路。

Fyn相关激酶（FRK）是Src非受体酪氨酸激酶家族成员之一。越来越多的证据表明FRK参与多种肿瘤的发生发展。起初我们发现FRK可以通过调控 JNK-c-Jun信号通路抑制胶质瘤迁移与侵袭能力。本研究继续通过体内外实验观察FRK与胶质瘤的关系。结果发现，过表达FRK明显得抑制了胶质瘤的增殖。相比，通过siRNA下调FRK促进了胶质瘤的的生长。过表达FRK还可以导致胶质瘤细胞停滞于G1期，并且可以促进胶质瘤细胞的凋亡。进一步研究表明FRK诱导的G1期停滞与过度磷酸化的成视网膜细胞瘤蛋白（pRb）有关，进而抑制了转录因子E2F1的表达。更重要是的我们发现，FRK可以抑制增殖性胶质瘤细胞核里cyclin D1的聚集。此外我们拓展研究了FRK对细胞周期相关的上游信号通路的作用。过表达FRK可以促进JAK2、STAT1的活化，但没有影响JAK2、STAT1总蛋白的表达。下调STAT1可以促进胶质瘤细胞的增殖。在胶质瘤细胞中共同转染FRK与STAT1小干扰质粒，可以减弱过表达FRK对胶质瘤U251细胞增殖的抑制作用。总之，我们的结果揭示了FRK抑制胶质瘤增殖的联合机制，可能是通过调控细胞周期相关的特定分子发挥作用。这些为我们进一步研究FRK介导的抑癌功能及临床治疗进展指明了方向。