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. 2021 Sep 16;19(1):95.
doi: 10.1186/s12964-021-00773-4.

R-Ras subfamily proteins elicit distinct physiologic effects and phosphoproteome alterations in neurofibromin-null MPNST cells

Shannon M Weber #  1   2   3 , Nicole M Brossier #  1   2   3   4 , Amanda Prechtl  1   2   3 , Stephen Barnes  1   2   3 , Landon S Wilson  1   2   3 , Stephanie N Brosius  1   2   3   5   6 , Jody Fromm Longo  1   2   3 , Steven L Carroll  7   8   9
Affiliations

R-Ras subfamily proteins elicit distinct physiologic effects and phosphoproteome alterations in neurofibromin-null MPNST cells

Shannon M Weber et al. Cell Commun Signal. .

Abstract

Background: Loss of the Ras GTPase-activating protein neurofibromin promotes nervous system tumor pathogenesis in patients with neurofibromatosis type 1 (NF1). Neurofibromin loss potentially hyperactivates classic Ras (H-Ras, N-Ras, K-Ras), M-Ras, and R-Ras (R-Ras, R-Ras2/TC21) subfamily proteins. We have shown that classic Ras proteins promote proliferation and survival, but not migration, in malignant peripheral nerve sheath tumor (MPNST) cells. However, it is unclear whether R-Ras, R-Ras2 and M-Ras are expressed and hyperactivated in MPNSTs and, if so, whether they contribute to MPNST pathogenesis. We assessed the expression and activation of these proteins in MPNST cells and inhibited them to determine the effect this had on proliferation, migration, invasion, survival and the phosphoproteome.

Methods: NF1-associated (ST88-14, 90-8, NMS2, NMS-PC, S462, T265-2c) and sporadic (STS-26T, YST-1) MPNST lines were used. Cells were transfected with doxycycline-inducible vectors expressing either a pan-inhibitor of the R-Ras subfamily [dominant negative (DN) R-Ras] or enhanced green fluorescent protein (eGFP). Methodologies used included immunoblotting, immunocytochemistry, PCR, Transwell migration, 3H-thymidine incorporation, calcein cleavage assays and shRNA knockdowns. Proteins in cells with or without DN R-Ras expression were differentially labeled with SILAC and mass spectrometry was used to identify phosphoproteins and determine their relative quantities in the presence and absence of DN R-Ras. Validation of R-Ras and R-Ras2 action and R-Ras regulated networks was performed using genetic and/or pharmacologic approaches.

Results: R-Ras2 was uniformly expressed in MPNST cells, with R-Ras present in a major subset. Both proteins were activated in neurofibromin-null MPNST cells. Consistent with classical Ras inhibition, DN R-Ras and R-Ras2 knockdown inhibited proliferation. However, DN R-Ras inhibition impaired migration and invasion but not survival. Mass spectrometry-based phosphoproteomics identified thirteen protein networks distinctly regulated by DN R-Ras, including multiple networks regulating cellular movement and morphology. ROCK1 was a prominent mediator in these networks. DN R-Ras expression and RRAS and RRAS2 knockdown inhibited migration and ROCK1 phosphorylation; ROCK1 inhibition similarly impaired migration and invasion, altered cellular morphology and triggered the accumulation of large intracellular vesicles.

Conclusions: R-Ras proteins function distinctly from classic Ras proteins by regulating distinct signaling pathways that promote MPNST tumorigenesis by mediating migration and invasion. Mutations of the NF1 gene potentially results in the activation of multiple Ras proteins, which are key regulators of many biologic effects. The protein encoded by the NF1 gene, neurofibromin, acts as an inhibitor of both classic Ras and R-Ras proteins; loss of neurofibromin could cause these Ras proteins to become persistently active, leading to the development of cancer. We have previously shown that three related Ras proteins (the classic Ras proteins) are highly activated in malignant peripheral nerve sheath tumor (MPNST) cells with neurofibromin loss and that they drive cancer cell proliferation and survival by activating multiple cellular signaling pathways. Here, we examined the expression, activation and action of R-Ras proteins in MPNST cells that have lost neurofibromin. Both R-Ras and R-Ras2 are expressed in MPNST cells and activated. Inhibition of R-Ras action inhibited proliferation, migration and invasion but not survival. We examined the activation of cytoplasmic signaling pathways in the presence and absence of R-Ras signaling and found that R-Ras proteins regulated 13 signaling pathways distinct from those regulated by classic Ras proteins. Closer study of an R-Ras regulated pathway containing the signaling protein ROCK1 showed that inhibition of either R-Ras, R-Ras2 or ROCK1 similarly impaired cellular migration and invasion and altered cellular morphology. Inhibition of R-Ras/R-Ras2 and ROCK1 signaling also triggered the accumulation of abnormal intracellular vesicles, indicating that these signaling molecules regulate the movement of proteins and other molecules in the cellular interior. Video Abstract.

Keywords: Malignant peripheral nerve sheath tumor; Neurofibromatosis; R-Ras; Ras; TC21.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Human MPNST cell lines variably express R-Ras subfamily members. a Lysates from eight human MPNST cell lines probed with antibodies recognizing R-Ras or both R-Ras and R-Ras2 proteins. Blots were probed with an anti-GAPDH antibody to confirm equal loading. Antibody dilutions were: R-Ras 1:10,000, R-Ras plus R-Ras2 (Abnova H000022800_M01) 1:10,000 and GAPDH 1:100,000. b R-Ras and R-Ras2 transcript levels in MPNST lines were quantitated using real-time PCR and compared to the level of expression evident for each transcript in normal human cerebellum tissue, Daoy medulloblastoma cells, MCF-7 breast adenocarcinoma cells, SK-N-MC neuroepithelioma cells, SK-OV-3 ovarian adenocarcinoma cells and U87-MG glioblastoma cells. c Raf-1 RBD pulldown assays performed on ST88-14 cells transiently transfected with plasmids directing the expression of Myc-tagged wild-type R-Ras, Myc-tagged wild-type R-Ras2, HA-epitope tagged dominant negative H-Ras plasmid, HA-epitope tagged dominant negative R-Ras, or eGFP. Only activation of wild-type R-Ras and R-Ras2 is evident in these cells. Upper panel is the material captured with Raf-1 RBD probed with antibodies recognizing the HA or Myc epitope tags on the recombinant proteins. The bottom two panels are clarified lysates probed for the epitope tags or GAPDH, respectively
Fig. 2
Fig. 2
Assessment of the regulation of doxycycline-inducible plasmids expressing eGFP or DN R-Ras stably tranfected into MPNST cells. a, b Expression of eGFP (a) or HA-tagged DN R-Ras (b) in stably transfected ST88-14, STS-26T and T265-2c cells 72 h after the cells were challenged with 2 μg/ml doxycycline. c Raf-1 RBD pulldown assays in ST88-14 MPNST cells stably transfected with doxycycline-inducible vectors expressing DN R-Ras or DN H-Ras and transiently transfected with a plasmid expressing Myc-tagged H-Ras. DN H-Ras, but not DN R-Ras, inhibits H-Ras activation
Fig. 3
Fig. 3
Induction of DN R-Ras expression inhibits MPNST proliferation, migration and invasion but not survival. a Induction of DN R-Ras expression, but not expression of eGFP, reduces 3H-thymidine incorporation in the ST88-14, STS-26T, and T265-2c doxycycline-inducible stable cell lines relative to uninduced controls. 16 replicates were performed for each condition. b Induction of DN R-Ras expression, but not eGFP expression, reduces Transwell migration in ST88-14, STS-26T, and T265-2c cells. c Immunofluorescence microscopy of phalloidin-stained ST88-14 MPNST cells grown in 3D Matrigel-matrix cultures. Left images are of ST88-14 cells stably transfected with a doxycycline-inducible eGFP expression vector and right images are the same cell line stably transfected with a doxycycline-inducible DN R-Ras vector. Upper panels are uninduced cells and lower panels are cells with expression induced by doxycycline. Scalebar = 400 µm (d) Quantification of percentage of spheroids in 3D culture with invasive phenotype, defined by circularity < .65. e Calcein AM cleavage assays in ST88-14 cells grown in the absence (left) or presence (right) of serum. Induction of eGFP and DN R-Ras expression had no statistically significant effect on the number of viable cells. For all panels, * indicates p-value < .0001 for comparison to controls
Fig. 4
Fig. 4
Distinct networks of phosphorylated proteins are affected by DN R-Ras expression. a The global distribution of heavy (DN R-Ras) to light (uninduced) ratios of detected phosphoproteins in ST88-14 MPNST cells. Plotted values are log2(H:L). Note that the phosphorylation of the vast majority of detected proteins are unaffected by DN R-Ras expression. b IPA analysis identified thirteen networks affected by DN R-Ras induction with IPA scores ≥ 16; the seven top ranked networks are shown. The proteins in the top seven networks whose phosphorylation is altered by DN R-Ras expression are presented in Table 2
Fig. 5
Fig. 5
DN R-Ras inhibits phosphorylation of ROCK1 and MYPT1 and activation of RhoA while ROCK inhibition, like DN R-Ras expression, impedes migration. a Levels of total and phosphorylated ROCK1 and MYPT1 in ST88-14 and STS-26T cells stably transfected with doxycycline-inducible plasmids expressing DN R-Ras in the presence (+) and absence (−) of DN R-Ras expression. Blots were probed with an anti-actinin antibody as a loading control. Quantification indicated below the blots represents the ratio of the normalized phosphorylated to total protein levels. b Rhotekin pull-down assays in ST88-14 and STS-26T cells stably transfected with doxycycline-inducible plasmids expressing DN R-Ras. Induction of DN R-Ras expression (+) decreases RhoA activation. c Induction of DN R-Ras expression and treatment with the ROCK1 inhibitor Y27632 similarly reduce the migration of ST88-14, STS-26T, and T265 cells in Transwell migration assays. A combination of DN R-Ras expression and Y27632 treatment produces an additional small, but statistically significant reduction in migration. **, p-value < .0001; *, p-value < .05
Fig. 6
Fig. 6
DN R-Ras decreases phosphorylation of ROCK1 and alters migration and cell morphology. a Effect of DN R-Ras expression on the actin cytoskeleton in ST88-14 cells as assessed by phalloidin staining. The enlarged image demonstrates actin-rimmed vesicular structures (white arrows) that accumulate in ST88-14 cells following induction of DN R-Ras expression. b, c Total ROCK1 (b; red) or phospho-ROCK1 immunoreactivity (c; red) in ST88-14 MPNST cells in the presence (+) or absence (−) of DN R-Ras expression and treatment with vehicle (−) or the ROCK inhibitor Y27632 (+). Cells were counterstained with phalloidin counterstain (green) to demonstrate cell morphology as highlighted by the actin cytoskeleton and bisbenzimide (blue) to label nuclei. Arrows denote notable changes in the actin cytoskeleton, including a loss of filamentous structures and the accumulation of actin-rich puncta (white arrows) following induction of DN R-Ras expression or Y27632 treatment. A non-immune isotype matched primary antibody was used as a negative control. Scale bars = 25 μm. d Semi-quantitative analysis of normalized total and phosphorylated ROCK1 levels relative to uninduced or untreated controls. **, p-value < 0.0001
Fig. 7
Fig. 7
RRAS and RRAS2 differentially promote proliferation and migration in ST88-14 cells. a Real-time PCR assays for RRAS (left panel) and RRAS2 (right panel) mRNAs in ST88-14 cells transduced with lentiviruses expressing a non-targeting control shRNA (NT), RRAS shRNAs (RRasA, RRasB) or RRAS2 shRNAs (RRas2A, RRas2B). b RRAS and RRAS2 knockdown both potently inhibit the migration of ST88-14 MPNST cells. c RRAS2, but not RRAS, knockdown inhibits the proliferation of ST88-14 cells. d Both RRAS and RRAS2 knockdown inhibit the phosphorylation of MYPT1 and ROCK1. Quantification indicated below the blots represents the ratio of the normalized phosphorylated to total protein levels
Fig. 8
Fig. 8
Signaling pathways potentially affected by R-Ras subfamily signaling as implicated by changes in the phosphoproteome triggered by DN R-Ras expression. Proteins rimmed in green have increased phosphorylation following activation of R-Ras and/or R-Ras2, while proteins rimmed in red show reduced phosphorylation following activation of R-Ras and/or R-Ras2
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