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

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.

Fig 1. RALGPS2 silencing induces pronounced anchorage-dependent and anchorage-independent cell population growth inhibition.

(A—F) Cell population growth in adherence was measured using the resazurin reduction assay and is expressed as percentage of untreated (non-transfected) cells (mean ± SEM,). It was evaluated from 72 to 120 h after cell transfection, depending on the optimized condition for each individual cell line. Each panel depicts the effect of silencing one RalGEF with two or three independent siRNA oligonucleotides: (A) RGL1; (B) RGL2; (C) RGL3; (D) RALGDS; (E) RALGPS1; (F) RALGPS2. The effect of each siRNA was statistically compared with the effect obtained with siNT by Two-way ANOVA and Bonferroni posttests (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; n = 2 to 4 mean values from independent experiments, each done in quadruplicate wells). (G) Anchorage-independent growth of H1299 cells was evaluated over 14 days and is expressed as mean relative colony number with respect to siNT-treated cells. Data were collected from independent experiments where siPLK1 (positive control) originated none or very few colonies. (H) Illustrative images of H1299 colonies in agarose at 14 days of growth after siNT, siPLK1, siRalGPS2 #231, and siRalGPS2 pool, as indicated. Statistical comparison was done by One-way ANOVA with Dunnett’s posttest. (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; n = 2 (siRGL1 and siRALGPS1), 3 (siRalGPS1 and siRalGPS2) or 4 (siRGL2, siRGL3 and siRalGDS) independent experiments, each performed in triplicate wells.

Fig 2. Cell survival is impaired by RalGPS2 depletion, especially in HRas^G12V transformed cells.

(A) The number of viable H1299 cells and (B) the percentage of viability after induction of RalGPS2-depletion were obtained daily for four days with the Trypan Blue exclusion assay and statistically compared by Two-way ANOVA and Bonferroni posttests (*p ≤ 0.05, ***p ≤ 0.001; n = 2 independent experiments, each in triplicate). (C) The proportion of cells harbouring active Caspase-3 was evaluated 72 h post-transfection with siRalGPS2, by flow cytometry analysis, in H1299 cells and (D) in the isogenic pair of the immortalized BEAS-2B and HRas^G12V-transformed BZR cells. Statistical significance was evaluated respectively by one-way ANOVA with Dunnett’s posttest, **p ≤ 0.01 and ***p ≤ 0.001, n = 4 or 5 independent experiments (C); and two-way ANOVA with Bonferroni posttest, ^### p ≤ 0.001, n = 2 or 3 independent experiments (D).

Fig 3. RalB, not RalA, is required for cell population growth, and survival does not explain it all.

(A) Cell population growth was evaluated in the cell lines indicated in the graph by resazurin reduction 72–120 h post-transfection, and expressed as percentage of resazurin reduction relatively to untreated cells. The effect of each siRNA was statistically compared with the effect obtained with siNT (negative control) by Two-way ANOVA and Bonferroni posttests (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; n = 2 or 3 independent experiments, each the mean obtained from quadruplicate wells). (B) Caspase-3 activation was evaluated by flow cytometry analysis 72 h post-transfection and is expressed as the percentage of the cell population with positive staning. Statistical significance was evaluated by one-way ANOVA with Dunnett’s posttest but no significance was obtained (n = 3 independent experiments). (C) Western blots of the indicated cell lines and time points show RalA and RalB protein downregulation, cleaved Poly [ADP-ribose] polymerases (Cl. PARP) immunodetection and Actin or Adaptin loading control.

Fig 4. Ral-GTP levels in H1299 cells after RalGPS2 depletion.

H1299 “starved” cells were obtained by overnight (~19 h) culture without serum before lysis at 48 h post-transfection. (A) Pull-down was performed as independent duplicates from the same lysates at 0.4 mg protein/pull-down/lane (0.5 mg/ml). The input corresponds to 25-fold less protein (16 μg/lane). 20 μg GST-Sec5-RBD and 40 μl beads were used per pull-down. (B) Quantification of immunoblots (IB) Ral-GTP/total Ral ratios from “starved” cells and expressed relatively to siNT (mean ± SD, n = 2 or, in the case of siRalGPS2_ft10, 3 independent pull-downs). (C) IB signal quantification from the experiment with cells “not starved” (mean ± SD, n = 2 independent pull-downs). No significant differences were found among means at a significance level of 0.05 (Two-way ANOVA with Bonferroni posttests).

Fig 5. RALGPS2 or RALB silencing cause G0/G1 cell cycle arrest.

Flow cytometry analysis of cell cycle distribution was performed in singlet events negative for active Caspase-3, 72 h post-transfection of (A and C) H1229 and (B and D) A549 cells. The effect of each siRNA was statistically compared with the effect obtained with the control siRNA siNT by Two-way ANOVA and Bonferroni posttests (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, n = 3 or 4 independent experiments).

Fig 6. RalGPS2 depletion increases p21 and p27 protein expression.

(A and B) Representative Western blots and quantification (mean ± SEM) of up to 4 independent experiments of the expression of Cyclin D1 and Cyclin D3, and (C and D) of the Cyclin dependent kinase inhibitors p21 and p27. (E and F) RalA and RalB protein downregulation efficiency is shown together with Cyclin E expression and its respective quantification data (mean ± SEM) corresponding to 1 and 2 independent experiments in H1299 and A459, respectively. Data were collected 72 h after siRNA transfection. Light-grey bars—siRalA, medium-grey bars—siRalB, and dark-grey bars—siRalGPS2. Statistics: the difference from the reference (theoretical value 1) was evaluated by One sample t test, two-tailed, *p ≤ 0.05, **p ≤ 0.01. The vertical white lines in the Western blots indicate positions were gel images were cut in order to juxtapose non-adjacent lanes coming from the same gel.

Fig 7. Skp2 protein levels decrease upon RalGPS2 depletion.

Representative Western blots and respective quantification (mean ± SEM) of 3–5 independent experiments of the expression of Skp2 (upper band, arrow) in H1299 (A) and A549 (B) cells, 72 h after siRNA transfection. The Skp2 faster migrating band (*), at the level of the 40 KD standard, has not been described so far but accompanied the variation of the upper band. Light-grey bars—siRalA, medium-grey bars—siRalB, and dark-grey bars—siRalGPS2. Statistics: the difference from the theoretical reference value 1 was evaluated by One-sample t-test, two-tailed, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.