Rac1和cAMP/PKA 信号通路对2型神经纤维瘤病肿瘤形成的作用机制研究

Neurofibromatosis type 2 is amultiple neoplasia syndrome that results from a mutation in the NF2 tumoursuppressor gene on chromosome 22q12.From studies about the pro-tumorigenicpathways in which loss of Merlin function is implicated,several cellulartargets are identified that may respond to therapeutics.NF2 negativelyregulates Rac1 small GTPase as loss of NF2 in fibroblasts and NF2 mutation inhuman schwannoma cells is associated with activation of the Rac1.In the absenceof functional Merlin, Rac becomes activated and allows integrin-RTK signalingresulting in tumor proliferation. Importantly, a study reported increased Rac1activity is critical for schwannoma formation in a mouse model with increasedPKA activity.Inhibition of Rac1 in these tumor mice significantly reduced tumorsize. However, the mechanism by which Rac1 and cAMP/PKA signaling contributesto tumor formation is unclear. Our preliminary research found that the level ofcAMP changes positively with Rac1 and negatively with Merlin. In that case, we hypothesisthat there was crosstalk relationship, among Rac1,merlin and Merlin, Playing acritical role in the tumor formation of NF2.With the study of NF2 tumor cells,NF2 mutant/loss cell lines and xenograft mouse model,our purposes of this subjectare 1.To identify the mechanism ofaction between Rac1 and cAMP/PKA; 2.to validate the activation mechanism ofRac1 by merlin and cAMP/PKA; 3.to explore the molecular mechanism and criticalgene of cAMP/PKA signaling pathway in NF2. This project can reveal themechanism in NF2 tumorigenesis and provide a novel target for clinicalpharmacotherapy.

2型神经纤维瘤病（NF2）表现为全身多发肿瘤形成，系NF2基因突变引起编码蛋白Merlin失活所致。人NF2肿瘤中Merlin缺失后，可测得Rac1活性升高。在小鼠施旺细胞中，激活的PKA通过提高Rac活性，进一步降低Merlin表达并导致肿瘤发生。我们研究发现：小鼠施旺细胞中敲除Rac1基因，会引起cAMP水平的降低。由于PKA为cAMP下游主要效应物，因此我们推测：Merlin、Rac1和cAMP/PKA通路三者存在交互作用，在NF2肿瘤形成中发挥重要作用。本项目拟在NF2肿瘤标本、原代培养瘤细胞及裸鼠移植瘤模型上研究：1.Rac1和cAMP/PKA通路间的相互影响及在成瘤中的作用；2.调控Rac1活性和cAMP/PKA通路表达，对Merlin表达及肿瘤增殖活性的影响；3.cAMP/PKA信号通路中参与肿瘤形成的关键基因。本项目将为明确NF2成瘤机制，寻找新治疗靶点提供重要依据。

2 型神经纤维瘤病（NF2）表现为全身多发肿瘤形成，系NF2 基因突变引起编码蛋白Merlin 失活所致。人NF2 肿瘤中Merlin 缺失后，可测得Rac1 活性升高。在小鼠施旺细胞中，激活的PKA 通过提高Rac 活性，进一步降低Merlin 表达并导致肿瘤发生。我们研究发现：NF2肿瘤样本中NF2基因突变以无义突变为主要类型，突变主要集中在1-8号外显子。基因突变导致肿瘤中NF2基因表达及Merlin蛋白表达降低。通过对正常神经样本及NF2前庭神经鞘瘤样本进行全基因转录表达谱分析及western blot检测发现：NF2肿瘤中，RAC1及PAK通路上调但是cAMP通路下调。通过对文献中散发鞘瘤原代培养细胞条件进行优化，我们建立了适合NF2鞘瘤原代培养细胞的条件，通过此方法培养5例NF2原代细胞。将生长状态良好的原代细胞注射进免疫功能缺陷的裸鼠右下肢坐骨神经中，建立了NF2鞘瘤PDX小鼠模型。分离移植瘤进行细胞培养，成功建立NF2蛋白功能缺失的施万细胞永生化细胞系。该细胞系已申请发明专利1项，目前已经通过初步审查进入实质审查阶段。我们在体外实验证明Rac1抑制剂能明显抑制NF2肿瘤细胞增殖，cAMP抑制剂无明显作用。体外及体内实验证明降低RAC1活性可以抑制NF2神经鞘瘤的细胞增殖并诱导细胞凋亡。Rac1抑制剂可作为潜在的药物用于NF2相关神经鞘瘤的治疗。目前，本课题组该项目研究已发表SCI文章3篇，中文文章9篇。申请发明专利1项并通过初步审查进入实质审查阶段。