Research ArticleCancer

Oncogenic RAS isoforms show a hierarchical requirement for the guanine nucleotide exchange factor SOS2 to mediate cell transformation

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Science Signaling  04 Sep 2018:
Vol. 11, Issue 546, eaar8371
DOI: 10.1126/scisignal.aar8371
  • Fig. 1 Oncogenic mutant RAS isoforms show a hierarchical requirement for SOS2 to drive transformation in MEFs.

    (A) Sos2+/+ and Sos2−/− MEFs were transduced with lentiviruses expressing either empty vector (V) or the indicated HA-tagged mutant RAS isoform (HRAS, NRAS, and KRAS: HRASG12V, NRASG12V, and KRASG12V, respectively). Whole-cell lysates (WCLs) were analyzed by Western blotting with antibodies specific for EGFR, SOS1, SOS2, HA (for RASG12V), total RAS, or β-actin to assess total protein. Glutathione S-transferase (GST)–RAS binding domain (RBD) pulldowns (PDs) were analyzed by Western blotting with an antibody specific for the HA epitope to assess activation of mutant HA-RASG12V. Blots are representative of three independent experiments. (B to D) Sos2+/+ and Sos2−/− MEFs expressing the indicated mutant RAS isoform were assessed for (B) proliferation in 2D culture plates, (C) colony growth in soft agar to assess anchorage-independent growth, and (D) loss of contact inhibition as assessed by a focus-forming assay. Data are means ± SD from three independent experiments. **P < 0.01 by analysis of variance (ANOVA) using the Tukey’s method to correct for multiple comparisons. (E) Representative 10× images of post-confluent MEFs from (D). Scale bar, 100 μm. See also fig. S1 for an overlay of the proliferation curves in (B).

  • Fig. 2 SOS2 critically mediates mutant KRAS-driven transformation in MEFs.

    (A) Sos2+/+ (+) and Sos2−/− (−) MEFs were transduced with lentiviruses expressing either empty vector, wild-type (WT) KRAS, or the indicated HA-tagged mutant KRAS constructs. WCLs were analyzed by Western blotting with antibodies specific for SOS2, HA (KRAS), or β-actin. Blots are representative of three independent experiments. (B and C) Sos2+/+ and Sos2−/− MEFs expressing the indicated KRAS constructs were assessed for (B) colony growth in soft agar to assess anchorage-independent growth, and (C) loss of contact inhibition as assessed by focus-forming assay. Data are means ± SD from three independent experiments. ** P < 0.01, *** P < 0.001 by ANOVA using the Tukey’s method to correct for multiple comparisons.

  • Fig. 3 SOS2 RASGEF activity contributes to KRAS-driven transformation.

    (A) Schematic showing potential routes of SOS2-dependent WT RAS activation in the presence of mutant KRAS. SOS2 point mutants block either RASGEF activity (F927A) or putative allosteric SOS2 activation by KRAS (W727E). (B) Sos2−/− MEFs expressing KRASG12C, KRASG12V, or KRASQ61R were transduced with lentiviruses expressing either empty vector, WT SOS2, RASGEF-deficient (F927A) SOS2, or feedback-defective (W727E) SOS2. WCLs were analyzed by Western blotting with antibodies specific for SOS2 or β-actin. (C and D) Sos2+/+ MEFs, Sos2−/− MEFs, or Sos2−/− MEFs expressing the indicated SOS2 constructs along with either KRASG12C (closed), KRASG12V (hashed), or KRASQ61R (open) were assessed for (C) colony growth in soft agar to assess anchorage-independent growth, and (D) loss of contact inhibition by a focus-forming assay. (E) Representative 10× images of post-confluent MEFs from (D). Scale bar, 100 μm. (F and G) Western blotting for activated V5-HRAS from GST-RBD PDs (middle, quantified above) or for total V5-HRAS from WCLs (below) from (F) Sos2+/+ or Sos2−/− MEFs expressing HA-KRASG12C and V5-WT HRAS in either actively cycling cells (left) or cells serum-starved overnight and then lysed or stimulated with EGF (100 μg/ml) for 5 min (right) or (G) Sos2−/− MEFs expressing HA-KRASG12C, V5-WT HRAS, and the indicated SOS2 construct. All data are means ± SD from three independent experiments; all blots and images are representative of three independent experiments. *P < 0.05, **P < 0.01 [for (C), versus Sos2+/+ and WT; for (G), versus vector and SOS2F927A] by ANOVA using the Tukey’s method to correct for multiple comparisons.

  • Fig. 4 SOS2 mediates RTK-dependent AKT phosphorylation in cells expressing mutant RAS.

    Sos2+/+ and Sos2−/− MEFs expressing the indicated mutant RAS isoforms were placed in serum-free media overnight and then stimulated with EGF (100 μg/ml) for the indicated times. (A) WCLs were analyzed by multiplex Western blotting for pERK1/2, ERK1/2, pAKT (Ser473), pAKT (Thr308), AKT, and β-actin on a LI-COR Odyssey machine. Blots are representative of three independent experiments. (B) Quantification of pERK1/2, pAKT (Ser473), and pAKT (Thr308) abundance versus a weighted average of total proteins (ERK1/2, AKT, and β-actin). Data are means ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by ANOVA using the Tukey’s method to correct for multiple comparisons.

  • Fig. 5 Mutant RAS isoforms show a hierarchical requirement for PI3K signaling to drive transformation in MEFs.

    (A to C) Sos2+/+ MEFs expressing the indicated mutant RAS isoforms were seeded onto either tissue culture–treated 96-well plates to assess anchorage-dependent growth or low-attachment 96-well plates to assess anchorage-independent growth. Cells were treated with the indicated concentrations of (A) the PI3K inhibitor LY294002, (B) the AKT inhibitor AZD5363, or (C) the MEK1/2 inhibitor trametinib for 4 days, and cell number was assessed. IC50 values and AUC measurements are shown. Data are means ± SD from four independent experiments, presented relative to vehicle-treated controls. **P < 0.01 versus HRASG12V. HRASG12V (salmon triangles), NRASG12V (dark blue inverted triangles), and KRASG12V (light blue diamonds) by ANOVA using the Tukey’s method to correct for multiple comparisons. (D) Sos2+/+ MEFs expressing the indicated mutant RAS isoform were seeded in low-attachment 96-well round-bottomed plates and treated with the indicated concentrations of the PI3K inhibitor LY294002 to assess the effects of PI3K inhibition on RAS-induced cancer spheroid formation (left). Sos2−/− MEFs expressing the indicated mutant RAS isoforms were seeded in parallel and left untreated for comparison (right). Images of spheroids were taken 16 hours after plating (day 0) and again 7 days later, and are representative of three independent experiments. Scale bar, 100 μm. The outlined image for each cell line represents the LY294002 concentration where cancer spheroid size did not increase relative to day 0. Images are representative of three independent experiments. See fig. S4 for inhibition of downstream protein phosphorylation by specific inhibitors, and fig. S5 for quantification of spheroid growth between Sos2+/+ and Sos2−/− MEFs expressing mutant RAS.

  • Fig. 6 Activated PI3K (p110α) cooperates with mutant KRAS to transform Sos2−/− MEFs.

    (A) Sos2+/+ and Sos2−/− MEFs were transduced with lentiviruses expressing KRASG12V ± p110αH1047R. WCLs were analyzed by Western blotting with antibodies specific for SOS2, p110α, HA (KRASG12V), or β-actin. Blots are representative of three independent experiments. (B and C) Sos2+/+ and Sos2−/− MEFs expressing KRASG12V ± p110αH1047R were assessed for loss of contact inhibition by focus-forming assay (stained dishes below, quantified above). Images are representative from three independent experiments. See fig. S6 for 10× images of cells from (B).

  • Fig. 7 SOS2 critically mediates transformation of KRAS mutant tumor cells.

    (A) YAPC pancreatic cancer cells (harboring a KRASG12V mutation) were transduced with lentiviruses expressing Cas9 and an NT sgRNA, an sgRNA targeting KRAS, or one of three different sgRNAs targeting SOS2. WCLs were analyzed by Western blotting with antibodies specific for KRAS, SOS2, SOS1, or tubulin (left). The SOS2 protein abundance relative to the NT sgRNA control in the SOS2 CRISPR samples is given. Cells were assessed for colony growth in soft agar 21 days after plating to assess anchorage-independent growth (right), and 10× images showing transformed colonies growing in soft agar were taken (bottom). Scale bars, 100 μm. (B) H358 non–small cell lung cancer (NSCLC) cells (harboring a KRASG12C mutation) were transduced with lentiviruses expressing Cas9 and an NT sgRNA, an sgRNA targeting KRAS, or one of two different sgRNAs targeting SOS2. WCLs were analyzed by Western blotting with antibodies specific for KRAS, SOS2, SOS1, or tubulin (left). The SOS2 protein abundance relative to the NT sgRNA control in the SOS2 CRISPR samples is given. Cells were assessed for anchorage-independent growth by cancer spheroid assay (right) and cancer spheroid growth 16 hours after plating (day 0) or 14 days later (10× images below). Scale bar, 100 μm. (C and D) YAPC cells from (A) were either lysed actively cycling (C) or starved overnight and then stimulated with EGF (100 ng/ml) for 5 min (D) before lysis. Multiplex Western blotting for pERK1/2, ERK1/2, pAKT (Ser473), AKT, and β-actin was performed on a LI-COR Odyssey machine. Quantification of pERK1/2 and pAKT (Ser473) abundance versus a weighted average of total proteins (ERK1/2, AKT, and β-actin) is shown above. All data are means ± SD from three independent experiments; all blots and images are representative of three independent experiments. *P < 0.05, ***P < 0.001, ****P < 0.0001 by ANOVA using the Tukey’s method to correct for multiple comparisons.

  • Fig. 8 SOS2 deletion synergizes with MEK inhibition to revert the transformed phenotype of KRAS mutant tumor cells.

    (A to D) KRAS mutant YAPC pancreatic cancer cells (A and C) or H358 NSCLC cells (B and D) transduced with lentiviruses expressing Cas9 and an NT sgRNA, an sgRNA targeting KRAS, or an sgRNAs targeting SOS2 were seeded onto either tissue culture–treated 96-well plates to assess anchorage-dependent growth (left) or low-attachment 96-well plates to assess anchorage-independent growth (right). Cells were treated with the indicated concentrations of the PI3K inhibitor buparlisib (A and B) or the MEK1/2 inhibitor trametinib (C and D) for 5 days, and cell number was assessed. For (C) and (D), NT cells were treated either with the indicated concentration of trametinib alone or in the presence of buparlisib (100 ng/ml). IC50 values and AUC measurements are shown. Data are means ± SD from three independent experiments, presented relative to vehicle-treated controls. *P < 0.05 (versus NT), #P < 0.05 [versus KRAS deletion; NT (gray squares), KRAS-deleted (black circles), SOS2-deleted (blue triangles), NT + buparlisib (100 ng/ml) (red diamonds)] by ANOVA using the Tukey’s method to correct for multiple comparisons.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/546/eaar8371/DC1

    Fig. S1. Sos2 deletion does not alter cellular proliferation in MEFs expressing oncogenic RAS.

    Fig. S2. Sos2 deletion does not alter GTP loading of mutant KRAS in MEFs.

    Fig. S3. Introduction of SOS2 into Sos2−/− MEFs using different promoters to drive Sos2 expression.

    Fig. S4. Inhibitor treatment of MEFs expressing mutant HRAS, NRAS, and KRAS blocks their corresponding downstream signaling pathways.

    Fig. S5. There is a hierarchical requirement for SOS2 in promoting mutant RAS-driven cancer spheroid growth.

    Fig. S6. Activated PI3K (p110α) cooperates with KRASG12V to transform Sos2−/− MEFs.

    Fig. S7. Deletion of SOS2 using CRISPR/Cas9.

    Fig. S8. Inhibitor treatment of YAPC cells blocks their corresponding downstream signaling pathways.

    Table S1. sgRNA sequences used in this study.

  • This PDF file includes:

    • Fig. S1. Sos2 deletion does not alter cellular proliferation in MEFs expressing oncogenic RAS.
    • Fig. S2. Sos2 deletion does not alter GTP loading of mutant KRAS in MEFs.
    • Fig. S3. Introduction of SOS2 into Sos2−/− MEFs using different promoters to drive Sos2 expression.
    • Fig. S4. Inhibitor treatment of MEFs expressing mutant HRAS, NRAS, and KRAS blocks their corresponding downstream signaling pathways.
    • Fig. S5. There is a hierarchical requirement for SOS2 in promoting mutant RAS-driven cancer spheroid growth.
    • Fig. S6. Activated PI3K (p110α) cooperates with KRASG12V to transform Sos2−/− MEFs.
    • Fig. S7. Deletion of SOS2 using CRISPR/Cas9.
    • Fig. S8. Inhibitor treatment of YAPC cells blocks their corresponding downstream signaling pathways.
    • Table S1. sgRNA sequences used in this study.

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