R-Ras subfamily proteins elicit distinct physiologic effects and phosphoproteome alterations in neurofibromin-null MPNST cells.
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ABSTRACT:
SUBMITTER: Weber SM
PROVIDER: S-EPMC8447793 | biostudies-literature | 2021 Sep
REPOSITORIES: biostudies-literature
Publications
R-Ras subfamily proteins elicit distinct physiologic effects and phosphoproteome alterations in neurofibromin-null MPNST cells.
Weber Shannon M SM Brossier Nicole M NM Prechtl Amanda A Barnes Stephen S Wilson Landon S LS Brosius Stephanie N SN Longo Jody Fromm JF Carroll Steven L SL
Cell communication and signaling : CCS 20210916 1
<h4>Background</h4>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-R ...[more]
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Project description:Neurofibromin, the tumor suppressor encoded by the neurofibromatosis type 1 (NF1) gene, potentially suppresses the activation of H-Ras, N-Ras, and K-Ras. However, it is not known whether these classic Ras proteins are hyperactivated in NF1-null nerve sheath tumors, how they contribute to tumorigenesis, and what signaling pathways mediate their effects. Here we show that H-Ras, N-Ras, and K-Ras are coexpressed with their activators (guanine nucleotide exchange factors) in neurofibromin-null malignant peripheral nerve sheath tumor (MPNST) cells, and that all 3 Ras proteins are activated. Dominant negative (DN) H-Ras, a pan-inhibitor of the classic Ras family, inhibited MPNST proliferation and survival, but not migration. However, NF1-null MPNST cells were variably dependent on individual Ras proteins. In some lines, ablation of H-Ras, N-Ras, and/or K-Ras inhibited mitogenesis. In others, ablation of a single Ras protein had no effect on proliferation; in these lines, ablation of a single Ras protein resulted in compensatory increases in the activation and/or expression of other Ras proteins. Using mass spectrometry-based phosphoproteomics, we identified 7 signaling networks affecting morphology, proliferation, and survival that are regulated by DN H-Ras. Thus, neurofibromin loss activates multiple classic Ras proteins that promote proliferation and survival by regulating several distinct signaling cascades.
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Project description:BackgroundGrowth factors induce a characteristically short-lived Ras activation in cells emerging from quiescence. Extensive work has shown that transient as opposed to sustained Ras activation is critical for the induction of mitogenic programs. Mitogen-induced accumulation of active Ras-GTP results from increased nucleotide exchange driven by the nucleotide exchange factor Sos. In contrast, the mechanism accounting for signal termination and prompt restoration of basal Ras-GTP levels is unclear, but has been inferred to involve feedback inhibition of Sos. Remarkably, how GTP-hydrolase activating proteins (GAPs) participate in controlling the rise and fall of Ras-GTP levels is unknown.ResultsMonitoring nucleotide exchange of Ras in permeabilized cells we find, unexpectedly, that the decline of growth factor-induced Ras-GTP levels proceeds in the presence of unabated high nucleotide exchange, pointing to GAP activation as a major mechanism of signal termination. Experiments with non-hydrolysable GTP analogues and mathematical modeling confirmed and rationalized the presence of high GAP activity as Ras-GTP levels decline in a background of high nucleotide exchange. Using pharmacological and genetic approaches we document a raised activity of the neurofibromatosis type I tumor suppressor Ras-GAP neurofibromin and an involvement of Rsk1 and Rsk2 in the down-regulation of Ras-GTP levels.ConclusionsOur findings show that, in addition to feedback inhibition of Sos, feedback stimulation of the RasGAP neurofibromin enforces termination of the Ras signal in the context of growth-factor signaling. These findings ascribe a precise role to neurofibromin in growth factor-dependent control of Ras activity and illustrate how, by engaging Ras-GAP activity, mitogen-challenged cells play safe to ensure a timely termination of the Ras signal irrespectively of the reigning rate of nucleotide exchange.
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Project description:Sporadic adrenocortical carcinomas (ACC) are rare endocrine neoplasms with a dismal prognosis. By contrast, benign tumors of the adrenal cortex are common in the general population. Whether benign tumors represent a separate entity or are in fact part of a process of tumor progression ultimately leading to an ACC is still an unresolved issue. To this end, we have developed a mouse model of tumor progression by successively transducing genes altered in adrenocortical tumors into normal adrenocortical cells. The introduction in different orders of the oncogenic allele of Ras (H-Ras(G12V)) and the mutant p53(DD) that disrupts the p53 pathway yielded tumors displaying major differences in histological features, tumorigenicity, and metastatic behavior. Whereas the successive expression of Ras(G12V) and p53(DD) led to highly malignant tumors with metastatic behavior, reminiscent of those formed after the simultaneous introduction of p53(DD) and Ras(G12V), the reverse sequence gave rise only to benign tumors. Microarray profiling revealed that 157 genes related to cancer development and progression were differentially expressed. Of these genes, 40 were up-regulated and 117 were down-regulated in malignant cell populations as compared with benign cell populations. This is the first evidence-based observation that ACC development follows a multistage progression and that the tumor phenotype is directly influenced by the order of acquisition of genetic alterations.
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Project description:Neurofibromatosis type 1 (NF1) is a common genetic disorder affecting 1 in 3500 individuals. Patients with NF1 are predisposed to debilitating skeletal manifestations, including osteopenia/osteoporosis and long bone pseudarthrosis (nonunion fracture). Hyperactivation of the Ras/mitogen-activated protein kinase (MAPK) pathway in NF1 is known to underlie aberrant proliferation and differentiation in cell lineages, including osteoclast progenitors and mesenchymal stem cells (MSCs) also known as osteoblast progenitors (pro-OBLs). Our current study demonstrates the hyper Ras/MAPK as a critical pathway underlying the pathogenesis of NF1-associated fracture repair deficits. Nf1-deficient pro-OBLs exhibit Ras/MAPK hyperactivation. Introduction of the NF1 GTPase activating-related domain (NF1 GAP-related domain) in vitro is sufficient to rescue hyper Ras activity and enhance osteoblast (OBL) differentiation in Nf1(-/-) pro-OBLs and NF1 human (h) MSCs cultured from NF1 patients with skeletal abnormalities, including pseudarthrosis or scoliosis. Pharmacologic inhibition of mitogen-activated protein kinase kinase (MEK) signaling with PD98059 partially rescues aberrant Erk activation while enhancing OBL differentiation and expression of OBL markers, osterix and osteocalcin, in Nf1-deficient murine pro-OBLs. Similarly, MEK inhibition enhances OBL differentiation of hMSCs. In addition, PD98059 rescues aberrant osteoclast maturation in Nf1 haploinsufficient bone marrow mononuclear cells (BMMNCs). Importantly, MEK inhibitor significantly improves fracture healing in an NF1 murine model, Col2.3Cre;Nf1(flox/-). Collectively, these data indicate the Ras/MAPK cascade as a critical pathway in the pathogenesis of bone loss and pseudarthrosis related to NF1 mutations. These studies provide evidence for targeting the MAPK pathway to improve bone mass and treat pseudarthrosis in NF1.
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Project description:In the development of multicellularity, signaling proteins has played a very important role. Among them, RAS family is one of the most widely studied protein family. However, evolutionary analysis has been carried out mainly on super family level leaving sub family information in scanty. Thus, a subfamily evolutionary study on RAS evolutionary expansion is imperative as it will aid in better drug designing against dreadful diseases like Cancer and other developmental diseases. The present study was aimed to understand RAS evolution on both holistic as well as reductive level. All human RAS family genes and protein were subjected to BLAST tools to find orthologs and paralogs with different parameters followed by phylogenetic tree generation. Our results clearly showed that H-RAS is the most primitive RAS in higher eukaryotes and then diverged into other RAS family members due to different gene modification events. Furthermore, a site specific selection pressure analysis was carried out using SELECTON server which showed that H-RAS, M-RAS and N-RAS are evolving faster than K-RAS and R-RAS. Thus, the results ascertain a new ground to cancer biologists to exploit negatively selected K-RAS and R-RAS as potent drug targets in cancer therapeutics.
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Project description:The severe defects in growth plate development caused by chondrocyte extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) gain or loss-of-function suggest that tight spatial and temporal regulation of mitogen-activated protein kinase signaling is necessary to achieve harmonious growth plate elongation and structure. We provide here evidence that neurofibromin, via its Ras guanosine triphosphatase -activating activity, controls ERK1/2-dependent fibroblast growth factor receptor (FGFR) signaling in chondrocytes. We show first that neurofibromin is expressed in FGFR-positive prehypertrophic and hypertrophic chondrocytes during growth plate endochondral ossification. Using mice lacking neurofibromin 1 (Nf1) in type II collagen-expressing cells, (Nf1col2(-/-) mutant mice), we then show that lack of neurofibromin in post-mitotic chondrocytes triggers a number of phenotypes reminiscent of the ones observed in mice characterized by FGFR gain-of-function mutations. Those include dwarfism, constitutive ERK1/2 activation, strongly reduced Ihh expression and decreased chondrocyte proliferation and maturation, increased chondrocytic expression of Rankl, matrix metalloproteinase 9 (Mmp9) and Mmp13 and enhanced growth plate osteoclastogenesis, as well as increased sensitivity to caspase-9 mediated apoptosis. Using wildtype (WT) and Nf1(-/-) chondrocyte cultures in vitro, we show that FGF2 pulse-stimulation triggers rapid ERK1/2 phosphorylation in both genotypes, but that return to the basal level is delayed in Nf1(-/-) chondrocytes. Importantly, in vivo ERK1/2 inhibition by daily injection of a recombinant form of C-type natriuretic peptide to post-natal pups for 18 days was able to correct the short stature of Nf1col2(-/-) mice. Together, these results underscore the requirement of neurofibromin and ERK1/2 for normal endochondral bone formation and support the notion that neurofibromin, by restraining RAS-ERK1/2 signaling, is a negative regulator of FGFR signaling in differentiating chondrocytes.
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Project description:Low oxygen or hypoxia is a feature of all solid tumors and has been associated with aggressive disease. Here, we describe a novel mechanism for the hypoxia-dependent degradation of the Ras-GTPase-activating protein neurofibromin, by hypoxia-associated factor (HAF). We have previously characterized HAF as an oxygen-independent ubiquitin ligase for HIF-1α. Here, we show that HAF promotes neurofibromin ubiquitination and degradation independently of oxygen and pVHL, resulting in Ras-ERK pathway activation. Hypoxia enhanced HAF:neurofibromin binding independently of HAF-SUMOylation, whereas HAF knockdown increased neurofibromin levels primarily in hypoxia, supporting the role of HAF as a hypoxia-specific neurofibromin regulator. HAF overexpression increased p-ERK levels and promoted resistance of clear cell kidney cancer (ccRCC) cells to sorafenib and sunitinib in both normoxia and hypoxia. However, a greater-fold increase in sorafenib/sunitinib resistance was observed during hypoxia, particularly in pVHL-deficient cells. Intriguingly, HAF-mediated resistance was HIF-2α-dependent in normoxia, but HIF-2α-independent in hypoxia indicating two potential mechanisms of HAF-mediated resistance: a HIF-2α-dependent pathway dominant in normoxia, and the direct activation of the Ras-ERK pathway through neurofibromin degradation dominant in hypoxia. Patients with ccRCC with high HAF transcript or protein levels showed significantly decreased overall survival compared with those with low HAF. Thus, we establish a novel, nonmutational pathway of neurofibromin inactivation through hypoxia-induced HAF-mediated degradation, leading to Ras-ERK activation and poor prognosis in ccRCC. IMPLICATIONS: We describe a novel mechanism of neurofibromin degradation induced by hypoxia that leads to activation of the prooncogenic Ras-ERK pathway and resistance to therapy.
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