Abstract
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.
Highlights
Ras proteins are small GTPases frequently mutated in human cancer
We started by evaluating the necessity of each of the six Ral-guanine nucleotide exchange factors (RalGEF) for the in vitro proliferation and/or survival of four human Non-Small Cell Lung Carcinoma (NSCLC) cell lines harboring different Ras mutations (A549, H23, H1299, H838), as compared to other cell lines where Ral and RalGEF proteins have been studied in the past: HeLa, HEK-HT-HRasG12V and its isogenic pair HEK-HT [10]
Efficiencies of the siRNA oligonucleotides against the RalGEF were confirmed by Quantitative reverse-transcription real-time PCR (qPCR) (S1 Fig): the target mRNA expression was below 30% of endogenous level for all siRNA sequences, at 72 h post-transfection, confirmed in at least two independent cell lines
Summary
Ras proteins are small GTPases frequently mutated in human cancer. They have many downstream effectors, including the small GTPases “Ras-related protein Ral-A” (RalA) and “Rasrelated protein Ral-B” (RalB), which are activated by Guanine nucleotide Exchange Factors (RalGEF). The RalGEF-Ral pathway gained special attention after the finding that the expression of a mutant form of the GTPase HRas that and exclusively activates this signaling pathway is sufficient for Ras-induced transformation of human cells [1]. Despite extensive work on RalA and RalB GTPases contribution to human cancer [6], only recently their role in lung cancer, frequently harboring Ras oncogenic mutations, has been reported [7,8]. RalGEF role in human Non-Small Cell Lung Carcinoma (NSCLC) remains unknown
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