Cx43介导的间隙连接通讯调控Rac1维持LSC干性特征的作用机制

Leukemia stem cell has been recognized as a key contributor to tumor initiation,progression and recurrece. Our previous studies have found gap junctional intercellular communication (GJIC) induced by Cx43 is essential for differentiation of hematopoietic stem cell, malfunction of GJIC can suppress leukemia proliferation, inhibit leukemia apoptosis, and our perliminary studies find that expression of Cx43 can inhibit self-renewal ability and stemness associated biomarker, suggesting that GJIC induced by Cx43 may play an improtant role in regulating the stemness of LSC. Rac1, identified as critical factor in maintainance of stemness of cancer stem cell, our pre-experments show that activation of Rac1 might be regulated by Cx43, but the detailed underlying mechanism is poorly known. In this study, we aim to demonstrate that up-regulation of GJIC can inhibit stemness of LSCs by inhibiting activation of Rac1 in vivo and vitro. Meanwhile, Combining single-cell molecular profiling with bioinformatics and functional assays, we will investigate that Cx43 inhibit stemness of LSC by inactivation Rac1 in channel-dependent mechanism (interaction of C terminal of Cx43 and GEF) and channel independent mechanism (enhancement of calcium influx). Further, we will reveal how Rac1 supports stemness of LSC. Our studies will help the field to understand the molecular mechanism of stemness of LSCs induced by GJIC dysregulation, and highlight the significance of novel and potential targets for further therapeutic approaches in cancer.

白血病干细胞(LSC)是白血病发生、发展的根源。前期自然基金课题研究发现：Cx43介导的间隙连接细胞间通讯（GJIC）是骨髓基质调控造血干细胞增殖分化的必要条件；骨髓基质GJIC功能缺失可促进白血病细胞的增殖、抑制凋亡；Cx43的表达上调可抑制LSC的自我更新及干性相关因子的表达，提示Cx43介导的GJIC功能在LSC的干性维持中发挥重要作用。后续预实验发现：Cx43可能通过肿瘤干性特征的重要分子Rac1的激活调节LSC的干性，但具体机制不明。本研究拟采用微流体单细胞基因测序、生物信息学、激光共聚焦、膜片钳、免疫共沉淀等实验技术阐明Cx43通过非通道依赖（其胞内C末端与GDP-Rac1竞争性结合GEF）或/和通道依赖（通过细胞内钙浓度增加）途径抑制Rac1的活化，进而抑制LSC自我更新、多向分化及体内成瘤等干性特征的作用机制，为白血病治疗提供新的思路和靶点。

Cx43介导的间隙连接细胞间通讯（GJIC）是骨髓基质调控造血干细胞增殖分化的必要条件。骨髓基质GJIC功能缺失可促进白血病细胞的增殖、抑制凋亡。白血病干细胞(LSC)是白血病发生、发展的根源。课题组前期研究发现：骨髓基质细胞Cx43的表达上调可抑制LSC的自我更新及干性相关因子的表达，提示Cx43介导的GJIC功能在LSC的干性维持中发挥重要作用。后续预实验发现：白血病细胞中Cx43可能通过肿瘤干性特征的重要分子Rac1的激活调节LSC的干性，但具体机制不明。实验表明敲降Rac1的白血病细胞系的活性降低，增殖减弱，凋亡增加，维持干性的相关基因的表达下降，表明Rac1在维持白血病细胞增殖，激活白血病细胞干性中有重要作用；但同时我们发现，过表达CX43的白血病细胞系中，Rac1的表达明显上调，维持干性的相关基因同样表达升高，说明CX43在白血病细胞内，可能通过非通道依赖的方式，调控白血病细胞干性。进一步研究发现CX43可以与Rac1结合，并通过Akt/GSK3β信号通路，促进白血病细胞增殖，抑制其凋亡，激活LSC的干性基因的表达，发挥LSC的调节作用。成瘤实验也证实，CX43过表达与Rac1敲降，都能使肿瘤生长速度更快。后续实验我们将深入研究CX43在白血病细胞中通过非通道依赖方式参与LSC调控的具体机制，探索相关信号通路的表达在白血病干细胞维持中的作用。