O Santos Adriana, Parrini Maria Carla, Camonis Jacques
INSERM U830, Institut Curie, PSL Research University, Paris, France.
The Biophenics Project, Translational Research Department, Institut Curie, Paris, France.
PLoS One. 2016 May 5;11(5):e0154840. doi: 10.1371/journal.pone.0154840. eCollection 2016.
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.
人类基因组包含六个编码蛋白质的基因，这些蛋白质在体外被验证为小GTP酶“Ras相关蛋白Ral - A”和“Ras相关蛋白Ral - B”的特异性激活剂，统称为Ral - 鸟嘌呤核苷酸交换因子（RalGEF）。Ral蛋白是Ras致癌信号的重要贡献者，而RAS癌基因在人类非小细胞肺癌（NSCLC）中很重要。因此，在这项研究中，研究了RalGEF对人NSCLC细胞系致癌和非致癌特征的贡献，如贴壁依赖性和非依赖性生长、细胞存活和增殖。在所有人类RalGEF中，RGL1和RALGPS1的沉默没有可检测到的影响。然而，RGL2、RGL3、RALGDS的沉默，或者在更大程度上，RALGPS2的沉默，在贴壁依赖性和非依赖性条件下抑制细胞群体生长（分别高达90%和80%）。RALGPS2的沉默还导致凋亡细胞数量增加，在转化的支气管BZR细胞中高达细胞群体的45%。在两种NSCLC细胞系H1299和A549中，RALGPS2的沉默导致细胞在细胞周期的G0/G1期停滞。此外，它与重要细胞周期调节因子的调节有关：E3泛素蛋白连接酶S期激酶相关蛋白2（Skp2）在mRNA和蛋白质水平均被强烈下调，其靶点细胞周期抑制剂p27和p21被上调。沉默RALA、RALB或两者均未模拟这些分子效应。然而，RALB的沉默对细胞周期进程有适度抑制，在H1299细胞中这与细胞周期蛋白D1的调节有关。总之，RALGPS2参与了NSCLC细胞系体外生长中细胞周期进程和存活的控制。该功能在很大程度上独立于Ral GTP酶，并与Skp2、p27和p21细胞周期调节因子的调节有关。
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