骨源性RANKL通过上调泛素连接酶cIAP介导的NF-κB通路使乳腺癌细胞豁免的机制研究

Skeleton related events (SREs) induced by breast cancer bone metastasis, like pathological bone fracture, are one leading inducement for mortality of breast cancer patients. The clinical approaches, including bisphospholate and anti-RANKL antibody, aim to inhibit enhanced osteoclastic bone resorption caused by metastatic tumor cells, while the outcome of these unitary approaches are limited. Therefore, to study the mechanism by which breast cancer cells metastasize to bone at early stage, and to develop effective approaches to inhibit the ocurrance of SREs would be extremely urgent and clinically significant. Preliminary data from our group indicates that breast cancer patients with bone metastases have OPG decreased and IL-4 increased in serum; besides, exogenous RANKL is capable of markedly stimulating E3 ligase cIAP expression, total protein ubiquintination, RelB nuclear translocation and IL-4 gene transcription in MDA-231 cells. These preliminary data indicated that RANKL might promote IL-4 expression and induce suppression of immune response through ubiquitin-proteasome system regulation. In this study, various molecular biology methods and experimental animal approaches will be employed to investigate if RANKL, in the bone microenvironment, regulates ubiquitin E3 ligase cIAP-mediated NF-κB pathway activation, promotes BCCs bone metastases and produces of IL-4 by BCCs to induce myeloid-derived suppressor cell generation, which thereby effectively protect bone metastatic BCCs from host immune system attack. Besides, we also will determine if cIAP inhibitor SM-164 and ubiquitin-proteasome inhibitor Bortezomib, through inhibiting bone metastatic BCCs, thereby reduce the occurrence of osteolytic SREs.

乳腺癌骨转移引起的骨相关事件，如病理性骨折，是乳腺癌患者死亡重要诱因。目前临床治疗采用双膦酸盐及RANKL单克隆抗体, 旨在抑制破骨吸收，治疗手段单一效果尚不理想。因此，研究乳腺癌骨转移的分子机制，防治骨相关事件极为重要。本课题组预实验表明，乳腺癌骨转移患者血清OPG降低且IL-4升高；重组RANKL促进MDA-231细胞泛素连接酶cIAP表达，泛素化水平升高，NF-κB分子RelB入核和IL-4基因转录增加；RANKL诱导的IL-4基因转录可被蛋白酶体抑制剂有效抑制。这暗示RANKL可能通过泛素化促进乳腺癌细胞表达IL-4，诱导免疫抑制，以促进其骨转移。本课题将通过分子生物学和实验动物学方法，证实在骨微环境中RANKL通过调节cIAP介导的NF-κB通路激活，促进乳腺癌细胞产生IL-4,诱导髓源性免疫抑制细胞生成，以发挥对癌细胞的保护作用；证实cIAP及蛋白酶体的抑制剂改善骨相关事件。

乳腺癌骨转移引起的骨相关事件是乳腺癌患者预后不良的重要因素，目前临床上乳腺癌骨转移的治疗手段单一且效果尚不理想。本课题组通过本项课题的研究表明，乳腺癌骨转移患者血清中炎症因子IL-4水平显著升高；重组RANKL促进MDA-231乳腺癌细胞系NF-κB炎症通路中分子RelB分子入核和IL-4基因转录增加；我们进一步在小鼠模型体内证实了骨髓微环境中，合成并分泌RANKL的主要细胞亚群为间充质干细胞（MSC）。综合以上结果表明，在骨转移微环境中MSC来源的RANKL通过调节NF-κB通路的激活，促进乳腺癌细胞产生炎症因子IL-4，进而诱导髓源性免疫抑制细胞（MDSC）生成，最终发挥了对微环境中乳腺癌细胞的保护作用。进一步的分子机制研究发现，当RANKL作用于MDA-231乳腺癌细胞后，泛素连接酶cIAP及泛素化总蛋白的表达量均显著升高，伴随着NF-κB非经典通路分子RelB入核的增加，当应用蛋白酶体抑制剂MG-132后这一现象可被逆转。在未来的研究中，有必要进一步探索cIAP抑制剂SM-164或者蛋白酶体抑制剂硼替佐米（两种临床应用药物）在治疗乳腺癌骨转移的潜在价值。