整合素αⅤβ1介导的细胞粘附在PSCA阳性前列腺癌骨转移中的作用机制

Prostate stem cell antigen (PSCA) expression was highly associated with carcinogenesis and development of human prostate cancer(PCa). The overexpression of PSCA has been identified in all bone metastases from PCa. Our prior data showed that PSCA mRNA was detected in peripheral blood of patients with metastatic PCa, and that both of PSCA-positive PCa LNCaP-PSCA and PC-3M cells metastasized much more frequently to the distant sites in SCID mice in vivo. The further studies of our group demonstrated that LNCaP-PSCA and PC-3M cells had firm adhesion to human HUVECs and fibronectin (a main component of extracellular matrix) as compared to the PSCA-negative PCa cells, and most importantly, along with the positive expression of integrin αⅤβ1. It is well-known that integrins have the key roles of mediating the cell-cell and cell- extracellular matrix adhesion. Therefore, we infer that PSCA may combine with integrin αⅤβ1 to form a complex, which enhanced the adhesion of PCa cells to bone marrow endothelial cells and extracellular matrix by activating cells adhesion pathways. On the platform of establishing human PCa cells models with the upregulated- and silenced- PSCA in vitro and corresponding SCID mice models with bone metastases in vivo, the present study is to investigate the effects of PSCA/ integrin αⅤβ1 complex on activation of FAK-centered downstream signaling network, subsequent re-arrangement of cytoskeleton, direct link between integrinβ1 subunit and fibronectin, and transcriptional activity of VCAM-1 and ICAM-1 genes promoters. The goal of this study is to elucidate the mechanisms of PSCA-positive PCa cells metastasis to bone, which will provide the novel strategies for prevention and treatment of human PCa metastasis to bone.

PSCA表达与前列腺癌发生发展密切相关，前列腺癌骨转移灶高表达PSCA。课题组前期研究发现：晚期前列腺癌患者外周血中PSCA mRNA表达为阳性；表达PSCA的人前列腺癌LNCaP-PSCA、PC-3M细胞在SCID鼠体内易早期发生远处转移。进一步研究发现：LNCaP-PSCA、PC-3M细胞与人脐静脉内皮细胞和纤连蛋白的粘附显著增强，同时伴有整合素αⅤβ1的阳性表达。整合素具有介导细胞粘附的重要作用。据此我们首次提出：PSCA与整合素αⅤβ1结合，依赖性激活细胞粘附通路，促进癌细胞与骨髓内皮细胞和细胞外基质的粘附形成转移。本项目以上调、下调表达PSCA的前列腺癌细胞模型和骨转移动物模型为平台，研究PSCA/αⅤβ1对FAK为中枢的下游信号网络的活化及其细胞骨架重排、β1与纤连蛋白联接、VCAM-1和ICAM-1转录水平的影响，阐明PSCA阳性前列腺癌骨转移的机制，为其防治提供新的切入点。

临床上前列腺癌易发生远处转移，尤其是亲骨性转移。这也是导致前列腺癌患者死亡的最主要和最重要原因。然而，目前前列腺癌骨转移的发生机制仍不清楚。前列腺癌细胞具有的与骨髓内皮细胞（bone marrow endothelial cells，BMECs）独特的粘附作用可导致其发生亲骨性转移。申请人前期研究结果表明，前列腺干细胞抗原（prostate stem cell antigen，PSCA）表达阳性前列腺癌细胞发生远处转移的潜能明显高于PSCA阴性前列腺癌细胞。本项目在前期研究工作的基础上，重点研究了PSCA基因表达在前列腺癌细胞通过与BMECs粘附介导骨转移中的作用及其机制。研究中应用细胞粘附实验分析细胞间的粘附以及跨内皮移动；qRT-PCR, 蛋白印迹（western blot）, 荧光素酶报告者基因（promoter-luciferase activity）和染色质免疫共沉淀（ChIP）检测分析整合素的表达及其调控。在功能学上，运用免疫共沉淀（Co-IP）和质谱（MS）分析法鉴定与PSCA相互作用的蛋白分子。在前列腺癌动物模型以及临床前列腺癌患者水平，进一步研究PSCA基因表达水平与骨转移发生的关系。本研究结果发现：PSCA基因过表达可通过上调整合素-α4表达而显著增加前列腺癌细胞与BMECs的粘附，同时伴有转录因子NF-κB转录活性的增高；在人前列腺癌细胞PSCA相互作用的蛋白分子为颗粒蛋白前体progranulin （PGRN），属生长因子家族。利用siRNAs分别沉默或下调PSCA和PGRN，在功能上可以显著抑制前列腺癌细胞整合素-α4表达以及与BMECs的粘附；而外源性重组人PGRN可以重新恢复这一功能。本研究项目在前列腺癌动物模型的一个重要发现是，抑制或下调PSCA表达可以显著抑制裸鼠前列腺癌骨转移灶的形成。对临床前列腺癌患者组织标本检测进一步发现：PSCA与PGRN的表达水平与Gleason 评分增高、临床及病理分期增加、骨转移发生以及不良的临床预后和结局均呈显著正相关。本研究项目发现PSCA/PGRN - NF-κB – 整合素-α4信号轴增加前列腺癌细胞与BMECs的粘附从而促进骨转移。研究结果为前列腺癌骨转移的治疗提供了新的策略和靶点。