Department of Medicine, the Acute Lung Injury Center of Excellence, University of Pittsburgh, Pittsburgh, PA, 15213, USA.
Department of Medicine, Pulmonary, Allergy, & Critical Care Medicine, The University of Pittsburgh, UPMC Montefiore, NW 628, Pittsburgh, PA, 15213, USA.
Respir Res. 2018 Oct 25;19(1):206. doi: 10.1186/s12931-018-0910-0.
The ubiquitin-proteasome pathway, mediated in part, by ubiquitin E3 ligases, is critical in regulating cellular processes such as cell proliferation, apoptosis, and migration. FBXO17 was recently identified as an F-box protein that targets glycogen synthase kinase-3β to the E3 ubiquitin ligase protein complex for polyubiquitination and proteasomal degradation. Here, we identified that in several lung adenocarcinoma cell lines, FBXO17 cellular protein was detected at relatively high levels, as was expression in a subset of lung cancers. Hence, we investigated the effects of FBXO17 on cell proliferation.
Single cell RNA sequencing analysis was performed on a resection of a non-small cell lung carcinoma tumor to examine FBXO17 expression. Multiple lung cancer cell lines were immunoblotted, and The Cancer Genome Atlas was analyzed to determine if FBXO17 expression was amplified in a subset of lung cancers. A549 cells were transfected with empty vector or FBXO17-V5 plasmid and immunoblotted for Akt pathway mediators including PDK1, ERK1/2, ribosomal protein S6, and CREB. Cell proliferation and viability were analyzed by trypan blue exclusion, BrdU incorporation and an MTS-based fluorometric assay. Studies were also performed after transfecting with sifbxo17. Samples were used in an RNA microarray analysis to evaluate pathways affected by reduced FBXO17 gene expression.
We observed that overexpression of FBXO17 increased A549 cell proliferation coupled with Akt activation. Ectopically expressed FBXO17 also increased ERK1/2 kinase activation and increased phosphorylation of RPS6, a downstream target of mTOR. We also observed an increased number of cells in S-phase and increased metabolic activity of lung epithelial cells expressing FBXO17. FBXO17 knockdown reduced Akt Ser 473 phosphorylation approaching statistical significance with no effect on Thr 308. However, ERK1/2 phosphorylation, cellular metabolic activity, and overall cell numbers were reduced. When we analyzed RNA profiles of A549 cells with reduced FBXO17 expression, we observed downregulation of several genes associated with cell proliferation and metabolism.
These data support a role for FBXO17 abundance, when left unchecked, in regulating cell proliferation and survival through modulation of Akt and ERK kinase activation. The data raise a potential role for the F-box subunit in modulating tumorigenesis.
泛素-蛋白酶体途径部分由泛素 E3 连接酶介导，在调节细胞增殖、凋亡和迁移等细胞过程中至关重要。FBXO17 最近被鉴定为一种 F 盒蛋白，可将糖原合酶激酶-3β靶向 E3 泛素连接酶蛋白复合物进行多泛素化和蛋白酶体降解。在这里，我们发现，在几种肺腺癌细胞系中，FBXO17 细胞蛋白水平相对较高，一部分肺癌中也有表达。因此，我们研究了 FBXO17 对细胞增殖的影响。
对非小细胞肺癌肿瘤切除标本进行单细胞 RNA 测序分析，以检测 FBXO17 的表达情况。对多种肺癌细胞系进行免疫印迹分析，并对癌症基因组图谱进行分析，以确定 FBXO17 在部分肺癌中是否扩增。用空载体或 FBXO17-V5 质粒转染 A549 细胞，并对 Akt 通路调节剂，包括 PDK1、ERK1/2、核糖体蛋白 S6 和 CREB 进行免疫印迹分析。通过台盼蓝排斥、BrdU 掺入和基于 MTS 的荧光测定法分析细胞增殖和活力。在转染 sifbxo17 后也进行了研究。使用 RNA 微阵列分析评估 FBXO17 基因表达降低对通路的影响。
我们观察到，FBXO17 的过表达增加了 A549 细胞的增殖，并伴有 Akt 的激活。异位表达的 FBXO17 还增加了 ERK1/2 激酶的激活，并增加了 mTOR 的下游靶点 RPS6 的磷酸化。我们还观察到表达 FBXO17 的肺上皮细胞中 S 期细胞数量增加，代谢活性增加。FBXO17 敲低减少了 Akt Ser 473 的磷酸化，接近统计学意义，但对 Thr 308 没有影响。然而，ERK1/2 磷酸化、细胞代谢活性和细胞总数减少。当我们分析 FBXO17 表达降低的 A549 细胞的 RNA 谱时，我们观察到与细胞增殖和代谢相关的几个基因下调。
这些数据支持 FBXO17 丰度的作用，当不受控制时，通过调节 Akt 和 ERK 激酶的激活来调节细胞增殖和存活。数据提出了 F 框亚基在调节肿瘤发生中的潜在作用。
