Department of Family Medicine, Chang Bing Show Chwan Memorial Hospital, Changhua 50544, Taiwan.
Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, Taichung 40601, Taiwan.
Cells. 2020 Feb 5;9(2):371. doi: 10.3390/cells9020371.
The specialized cholesterol/sphingolipid-rich membrane domains termed lipid rafts are highly dynamic in the cancer cells, which rapidly assemble effector molecules to form a sorting platform essential for oncogenic signaling transduction in response to extra- or intracellular stimuli. Density-based membrane flotation, subcellular fractionation, cell surface biotinylation, and co-immunoprecipitation analyses of bichalcone analog ((E)-1-(4-Hydroxy-3-((4-(4-((E)-3-(pyridin-3-yl)acryloyl)phenyl)piperazin-1-yl)methyl)phenyl)-3-(pyridin-3-yl)prop-2-en-1-one (TSWU-BR4)-treated cancer cells showed dissociation between GRP78 and p85α conferring the recruitment of PTEN to lipid raft membranes associated with p85α. Ectopic expression of GRP78 could overcome induction of lipid raft membrane-associated p85α-unphosphorylated PTEN complex formation and suppression of GRP78PI3KAktGTP-Rac1-mediated and GRP78-regulated PERKNrf2 antioxidant pathway and cancer cell invasion by TSWU-BR4. Using specific inducer, inhibitor, or short hairpin RNA for ASM demonstrated that induction of the lipid raft membrane localization and activation of ASM by TSWU-BR4 is responsible for perturbing homeostasis of cholesterol and ceramide levels in the lipid raft and ER membranes, leading to alteration of GRP78 membrane trafficking and subsequently inducing p85α-unphosphorylated PTEN complex formation, causing disruption of GRP78PI3KAktGTP-Rac1-mediated signal and ER membrane-associated GRP78-regulated oxidative stress balance, thus inhibiting cancer cell invasion. The involvement of the enrichment of ceramide to lipid raft membranes in inhibition of NF-κB-mediated MMP-2 expression was confirmed through attenuation of NF-κB activation using C2-ceramide, NF-κB specific inhibitors, ectopic expression of NF-κB p65, MMP-2 promoter-driven luciferase, and NF-κB-dependent reporter genes. In conclusion, localization of ASM in the lipid raft membranes by TSWU-BR4 is a key event for initiating formation of ceramide-enriched lipid raft membrane platforms, which causes delocalization of GRP78 from the lipid raft and ER membranes to the cytosol and formation of p85α-unphosphorylated PTEN complexes to attenuate the GRP78-regulated oxidative stress balance and GRP78p85αAktGTP-Rac1NF-κBMMP-2-mediated cancer cell invasion.
特化的胆固醇/神经鞘脂丰富的膜域称为脂筏，在癌细胞中高度动态，它们迅速组装效应分子，形成一个分选平台，对于响应细胞外或细胞内刺激的致癌信号转导至关重要。密度梯度膜浮选、亚细胞分级分离、细胞表面生物素化和双查尔酮类似物（（E）-1-（4-羟基-3-（（4-（（E）-3-（吡啶-3-基）丙烯酰基）苯基）哌嗪-1-基）甲基）苯基）-3-（吡啶-3-基）丙-2-烯-1-酮（TSWU-BR4））处理的癌细胞中的 GRP78 和 p85α 的共免疫沉淀分析表明，GRP78 与 p85α 解离，导致 PTEN 募集到与 p85α 相关的脂筏膜。GRP78 的异位表达可以克服 TSWU-BR4 诱导的脂筏膜相关 p85α 未磷酸化 PTEN 复合物形成和抑制 GRP78PI3KAktGTP-Rac1 介导的和 GRP78 调节的 PERKNrf2 抗氧化途径以及 TSWU-BR4 诱导的癌细胞侵袭。使用特定的诱导剂、抑制剂或短发夹 RNA 对 ASM 进行研究表明，TSWU-BR4 诱导脂筏膜定位和 ASM 激活负责扰乱胆固醇和神经酰胺水平在脂筏和内质网膜中的平衡，导致 GRP78 膜运输的改变，随后诱导 p85α 未磷酸化的 PTEN 复合物形成，破坏 GRP78PI3KAktGTP-Rac1 介导的信号和内质网膜相关的 GRP78 调节的氧化应激平衡，从而抑制癌细胞侵袭。通过使用 C2-神经酰胺、NF-κB 特异性抑制剂、NF-κB p65 的异位表达、MMP-2 启动子驱动的荧光素酶和 NF-κB 依赖性报告基因来衰减 NF-κB 激活，证实了神经酰胺在 NF-κB 介导的 MMP-2 表达中的富集到脂筏膜中的参与。总之，TSWU-BR4 将 ASM 定位于脂筏膜中是启动形成富含神经酰胺的脂筏膜平台的关键事件，导致 GRP78 从脂筏和内质网膜到细胞质的定位，并形成 p85α 未磷酸化的 PTEN 复合物，从而减弱 GRP78 调节的氧化应激平衡和 GRP78p85αAktGTP-Rac1NF-κBMMP-2 介导的癌细胞侵袭。
