RalGPS2 Is Essential for Survival and Cell Cycle Progression of Lung Cancer Cells Independently of Its Established Substrates Ral GTPases

The human genome contains six genes coding for proteins validated in vitro as specific activators of the small GTPases “Ras-related protein Ral-A” and “Ras-related protein Ral-B”, generically named Ral-guanine nucleotide exchange factors (RalGEF). Ral proteins are important contributors to Ras oncogenic signaling, and RAS oncogenes are important in human Non-Small Cell Lung Carcinoma (NSCLC). Therefore in this work, RalGEF contribution to oncogenic and non-oncogenic features of human NSCLC cell lines, as anchorage-dependent and independent growth, cell survival, and proliferation, was investigated. Among all human RalGEF, silencing of RGL1 and RALGPS1 had no detectable effect. However, silencing of either RGL2, RGL3, RALGDS or, to a larger extent, RALGPS2 inhibited cell population growth in anchorage dependent and independent conditions (up to 90 and 80%, respectively). RALGPS2 silencing also caused an increase in the number of apoptotic cells, up to 45% of the cell population in transformed bronchial BZR cells. In H1299 and A549, two NSCLC cell lines, RALGPS2 silencing caused an arrest of cells in the G0/G1-phase of cell cycle. Furthermore, it was associated with the modulation of important cell cycle regulators: the E3 Ubiquitin Protein Ligase S-phase kinase-associated protein 2 (Skp2) was strongly down-regulated (both at mRNA and protein levels), and its targets, the cell cycle inhibitors p27 and p21, were up-regulated. These molecular effects were not mimicked by silencing RALA, RALB, or both. However, RALB silencing caused a modest inhibition of cell cycle progression, which in H1299 cells was associated with Cyclin D1 regulation. In conclusion, RALGPS2 is implicated in the control of cell cycle progression and survival in the in vitro growth of NSCLC cell lines. This function is largely independent of Ral GTPases and associated with modulation of Skp2, p27 and p21 cell cycle regulators.

人类基因组中存在6个编码经体外验证的、可作为小GTP酶（small GTPases）“Ras相关蛋白Ral-A”与“Ras相关蛋白Ral-B”特异性激活因子的蛋白质的基因，其通用名称为Ral鸟苷酸交换因子（Ral-guanine nucleotide exchange factors, RalGEF）。Ral蛋白是Ras致癌信号通路的重要介导因子，而RAS致癌基因在人类非小细胞肺癌（Non-Small Cell Lung Carcinoma, NSCLC）中发挥关键作用。因此本研究探讨了RalGEF对人NSCLC细胞系致癌与非致癌特性（包括锚定依赖性生长与非依赖性生长、细胞存活及增殖）的影响。
在所有人类RalGEF中，沉默RGL1与RALGPS1未检测到明显生物学效应。然而，沉默RGL2、RGL3、RALGDS，尤其是RALGPS2，可在锚定依赖性与非依赖性培养条件下抑制细胞群体生长（抑制率分别可达90%与80%）。RALGPS2基因沉默还可诱导细胞凋亡数量增加，在转化支气管BZR细胞中凋亡细胞占细胞群体的比例可达45%。
在两种NSCLC细胞系H1299与A549中，RALGPS2基因沉默可使细胞周期阻滞于G0/G1期。此外，该现象伴随关键细胞周期调控因子的表达改变：E3泛素连接酶（E3 Ubiquitin Protein Ligase）S期激酶相关蛋白2（S-phase kinase-associated protein 2, Skp2）在mRNA与蛋白水平均显著下调，而其靶标——细胞周期抑制因子p27与p21则出现上调。
沉默RALA、RALB或二者共沉默均无法模拟上述分子效应。不过，RALB沉默可轻度抑制细胞周期进程，在H1299细胞中该效应与细胞周期蛋白D1（Cyclin D1）的调控相关。
综上，RALGPS2参与调控NSCLC细胞系体外生长过程中的细胞周期进程与细胞存活，该功能在很大程度上不依赖于Ral GTP酶，且与Skp2、p27及p21等细胞周期调控因子的表达调控密切相关。