PSMB5 和NRF2 拮抗骨髓瘤细胞硼替佐米耐药的机制研究

In the therapy of multiple myeloma (MM) with bortezomib (BTZ) as a primary agent, the major challenge is to enhance the sensitivity of MM cells to BTZ by reducing or disrupting the capacity of their resistant machinery.Two important targeting mechanisms involved in the resistance of MM cells to BTZ are high level of proteasome activity of catalytic beta 5 subunit (PSMB5),which is the chymotrypsin-like enzyme dominated in ubiquitin-proteasome response pathway (UPP),and responsive activation of transcription factor NF-E2-related factor 2 (NRF2), which is an activator of antioxidant response element (ARE)-bearing genes, including those encoding endogenous antioxidants, phase II detoxifying enzymes, and most importantly the proteasome catalytic subunits PSMB5.They control the response of MM cells by up-regulating activities against the effects of BTZ. Our preliminary studies showed that by means of RNA interference to down-regulate expression of NRF2 and PSMB5 either individually or simultaneously, there came out with synergistically increased MM cell sensitivity to BTZ. In this proposed project, we will commit to the following studies: 1) to establish human MM cell strains with PSMB5 and NRF2 knockdown by stably expression of interfering shRNAs through constructs of pLK-shNRF2-RFP and pLK-shPSMB5-GFP using modified lentiviral vectors based transfection system in MM cell lines and BTZ adapted respective cell lines(BTZ-resistant daughter cells) from parental MM cells,2) to observe and compare the effects of down-regulating PSMB5 and NRF2 pathway on reversing cell resistance to BTZ and enhancement of sensitivity between parent and resistant daughter cell strains in vitro and in vivo, 3)to achieve an explanation of signal pathway involvement based on featured gene expression and functional measurement of tested cell lines in the context of PSMB5 and NRF2 pathway,4) we will also explore potential target genes in association with regulation of PSMB5 pathway by analyzing gene expression profiles (GEP) and validating experiments.Thus,we might be able to elucidate the mechanism of BTZ resistance and sensitivity difference in MM cells to BTZ, and provide an experimental basis with profound insights for the development of next generation of proteasome inhibitors.

降低和延缓硼替佐米在骨髓瘤治疗中出现的耐药性是提高疗效的关键。在耐药细胞株中过表达的蛋白酶体β5亚单位（PSMB5），是增敏和克服耐药的靶点；稳定地降低PSMB5 的表达会增强硼替佐米的抗肿瘤作用，其中转录因子NRF2参与PSMB5的调控。我们的前期研究证实：RNA干扰分别或同时下调NRF2 和PSMB5的表达可提高骨髓瘤细胞对硼替佐米的敏感性，且具有协同作用。本项目拟采用RNA干扰技术稳定下调骨髓瘤细胞系及硼替佐米诱导的耐药细胞株中NRF2及PSMB5的表达，通过体外和体内试验观察骨髓瘤细胞对硼替佐米的敏感性变化和PSMB5在增敏及逆转耐药中的作用；利用表达谱分析寻找参与PSMB5调节的新基因，并进一步探索PSMB5在硼替佐米耐药及增敏中的作用机制，为提高硼替佐米临床治疗骨髓瘤的疗效，同时为新蛋白酶体抑制剂的研发提供实验依据和新思路，有潜在的应用前景。

硼替佐米（Bortezomib)是多发性骨髓瘤治疗的里程碑。但是硼替佐米的快速耐药，限制了它的临床应用。因此，降低和延缓硼替佐米在骨髓瘤治疗中出现的耐药性是提高疗效的关键。在耐药细胞株中过表达的蛋白酶体β5 亚单位（PSMB5），是增敏和克服耐药的靶点；稳定地降低PSMB5的表达增强硼替佐米的抗肿瘤作用，其中转录因子NRF2 参与PSMB5 的调控。结果：①耐药细胞株和亲代细胞株中敲减NRF2导致蛋白酶体活性降低，而敲减PSMB5导致β5 降低；联合敲减二者，则beta 5降低更明显，增强细胞对硼替佐米的敏感性，caspase3/7 活性明显增加，诱导细胞凋亡作用增强。通过基因芯片的方法，我们筛选到了与蛋白酶体、ER应激反应、蛋白-泛素酶体以及凋亡相关的多个基因并进行了功能验证。②成功构建了骨髓瘤的小鼠成瘤模型，体内试验提示下调NRF2和PSMB5的表达提高细胞对硼替佐米的敏感性，抑制细胞的增殖、促进细胞的凋亡，下调细胞周期蛋白Cyclin D和CDK4的表达，降低蛋白酶体的活性，且两者同时下调具有协同作用。通过基因芯片的方法，我们筛选到了基本上与体外实验相同的变化基因，提示上述基因在骨髓瘤耐药中起作用。③荜茇酰胺主要通过降低细胞周期蛋白cyclin D、cyclin E、CDK2和CDK4的表达使骨髓瘤细胞处于G1期；通过影响ERK的磷酸化影响MM细胞的凋亡；可破坏骨髓微环境影响MM细胞的增殖和生存。④ 初步明确了Sprouty2、USP7以及Blimp-1分别通过影响MAPK信号通路、蛋白酶体PSMB5亚基与USP7的相互作用以及影响浆细胞的发育和ER的应激反应参与硼替佐米的耐药和多发性骨髓瘤的发生发展中的作用机制，为进一步探索MM硼替佐米耐药机制提供了新视角。⑤通过尾静脉注射的方法在NSG小鼠中注射NRF2和PSMB5同时或分别下调亲代或者耐药细胞株建立骨髓瘤发病小鼠模型，该模型可以更真实的模拟骨髓瘤的发病进程，为体内探索骨髓瘤硼替佐米耐药机制提供了新工具。意义：基本阐明了NRF2和PSMB5在骨髓瘤硼替佐米耐药中的作用机制，且两者同时下调具有协同作用的发现为进一步深入探索骨髓瘤硼替佐米耐药的机制提供了新方向和新的研究视角；发现了参与硼替佐米耐药的新基因，为分析其耐药提供了新的切入点，获得了新的研究增长点。