Oncogenic Mutations of KRAS Modulate Its Turnover by the CUL3/LZTR1 E3 ligase Complex
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive HF
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Permanent Cell Line Cell
SUBMITTER: 
Svenja Hester
LAB HEAD: Andreas Damianou
PROVIDER: PXD029725 | Pride | 2024-03-06
REPOSITORIES: Pride
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Project description:KRAS is one of the most frequently mutated genes across all cancer subtypes. Two of the most frequent oncogenic KRAS mutations observed in patients result in glycine to aspartic acid substitution at either codon 12 (G12D) or 13 (G13D). Although the biochemical differences between these two predominant mutations are not fully understood, distinct clinical features of the resulting tumors suggest involvement of disparate signaling mechanisms. When we compared the global and phosphotyrosine proteomic profiles of isogenic colorectal cancer cell lines bearing either G12D or G13D KRAS mutation, we observed both shared as well as unique signaling events induced by the two KRAS mutations.
2021-06-09 | PXD009843 | Pride
Project description:Cancer treatment decisions are increasingly guided by which specific genes are mutated within each patient’s tumor. For example, agents inhibiting the epidermal growth factor receptor (EGFR) benefit many colorectal cancer (CRC) patients, with the general exception of those whose tumor includes a KRAS mutation. However, among the various KRAS mutations, the G13D mutation behaves differently; for unknown reasons, CRC patients with the KRAS G13D mutation (also written KRASG13D) appear to benefit from the EGFR-blocking antibody cetuximab. Controversy surrounds this observation, because it appears to contradict the well-established mechanisms of EGFR signaling and of RAS mutations. Here, we identified a systems-level, mechanistic basis that explains why KRASG13D cancers respond to EGFR inhibition. We first investigated the problem with a computational model of RAS signaling, which unexpectedly revealed that the known biophysical differences between the three most common KRAS mutant proteins are sufficient to generate different sensitivities to inhibition. Computation and experimentation together revealed a non-intuitive, mutant-specific dependency of wild-type RAS activation by EGFR that is determined by the interaction strength between KRAS and the tumor suppressor neurofibromin (NF1). KRAS mutants that strongly interact with NF1 drive wild-type RAS activation in an EGFR independent manner through competitive inhibition of NF1, whereas KRAS G13D cells remain dependent upon EGFR for wild-type Ras activation because they cannot competitively inhibit NF1 due to a weak interaction between these two proteins. Overall, our work demonstrates how systems approaches enable mechanism-based inference in genomic medicine and can help identify patients for selective therapeutic strategies.
2020-05-27 | PXD014755 | Pride
Project description:HCT116 Kras WT and Kras G13D isogenic pair cell lines were cultured in either 25 mM or 1 mM for one week.
2012-07-01 | GSE31084 | GEO
Project description:HCT116 Kras WT and Kras G13D isogenic pair cell lines were cultured in either 25 mM or 1 mM for one week. Two independent replicate samples for each cell line and for each treatment were profiled using Affymetrix HG U133 plus 2.0 arrays
2012-06-30 | E-GEOD-31084 | biostudies-arrayexpress
Project description:The small GTPase KRAS transmits signals from cell surface receptors to various downstream signaling pathways, thereby regulating a multitude of cellular processes. Activating KRAS mutations occur in approximately 30% of human cancers and contribute to malignant transformation through constitutive activation of downstream signaling cascades. These mutations cluster in several hotspots, with codons 12 and 13 being most commonly. It has been suggested that the position and type of amino acid exchange influence the transforming capacity of mutant KRAS proteins. To address this hypothesis, we used MCF10A human mammary epithelial cells to establish isogenic cell lines that express eight different cancer-associated KRAS mutations (G12C, G12D, G12V, G13C, G13D, A18D, Q61H, K117N) at physiological levels, and investigated the biochemical and functional consequences of the different KRAS variants in vitro. In addition, selected effects were evaluated in comparison to cells that overexpress mutant KRAS. The overall effects of mutants expressed at normal levels were moderate compared to overexpressed variants, but allowed delineation of biological functions that were related to specific alleles rather than KRAS expression level. None of the mutations induced morphological changes or migratory abilities. KRAS-G12D, -G12V, -G13D, and -K117N were able to mediate EGF-independent proliferation, whereas anchorage-independent growth was primarily induced by the K117N and Q61H variants. Analysis of known RAS effectors showed that none of the mutations led to increased phosphorylation of ERK, PDK1, or AKT. Both codon 13 mutations were associated with increased EGFR expression, which was not seen with other variants. Finally, comparative gene expression analysis of MCF10A-G13D versus MCF10A-G12D revealed distinct transcriptional changes, such as enhanced cytokine and p53 signaling, upon G13D expression. Together, we describe a useful resource for investigating the function of multiple KRAS mutations and provide insights into the differential effects of these variants in MCF10A cells.
2014-12-31 | GSE60695 | GEO
Project description:The small GTPase KRAS transmits signals from cell surface receptors to various downstream signaling pathways, thereby regulating a multitude of cellular processes. Activating KRAS mutations occur in approximately 30% of human cancers and contribute to malignant transformation through constitutive activation of downstream signaling cascades. These mutations cluster in several hotspots, with codons 12 and 13 being most commonly. It has been suggested that the position and type of amino acid exchange influence the transforming capacity of mutant KRAS proteins. To address this hypothesis, we used MCF10A human mammary epithelial cells to establish isogenic cell lines that express eight different cancer-associated KRAS mutations (G12C, G12D, G12V, G13C, G13D, A18D, Q61H, K117N) at physiological levels, and investigated the biochemical and functional consequences of the different KRAS variants in vitro. In addition, selected effects were evaluated in comparison to cells that overexpress mutant KRAS. The overall effects of mutants expressed at normal levels were moderate compared to overexpressed variants, but allowed delineation of biological functions that were related to specific alleles rather than KRAS expression level. None of the mutations induced morphological changes or migratory abilities. KRAS-G12D, -G12V, -G13D, and -K117N were able to mediate EGF-independent proliferation, whereas anchorage-independent growth was primarily induced by the K117N and Q61H variants. Analysis of known RAS effectors showed that none of the mutations led to increased phosphorylation of ERK, PDK1, or AKT. Both codon 13 mutations were associated with increased EGFR expression, which was not seen with other variants. Finally, comparative gene expression analysis of MCF10A-G13D versus MCF10A-G12D revealed distinct transcriptional changes, such as enhanced cytokine and p53 signaling, upon G13D expression. Together, we describe a useful resource for investigating the function of multiple KRAS mutations and provide insights into the differential effects of these variants in MCF10A cells.
2014-12-31 | GSE60695 | GEO
Project description:Purpose: The uncommonness of gallbladder cancer in the developed world has contributed to the generally poor understanding of the disease. The development of new and effective treatment has been and continues to be a major public health imperative. Methods: We report mutational and copy number analysis of 44 predominantly early-staged gallbladder tumors and 5-gallbladder cancer cell lines by a combination of directed and whole exome sequencing at an average coverage of 100X and above. Using gallbladder cancer cell lines and xenograft mouse models we performed phospho-proteome array profiling, followed by an in-depth functional characterization. Results: We describe recurrent activating ERBB2 somatic mutation in 6 of 44 gallbladder primary tumors with an overall mutation frequency of 13%, along with KRAS activating mutations in 3 of 44 samples. Consistent with whole exome findings, a phospho-proteomic array profile of 49-tyrosine kinase revealed constitutive phosphorylation of ERBB2 and EGFR that were found to heterodimerize. We demonstrate that treatment with ERBB2-specific, EGFR-specific shRNA or with covalent EGFR family inhibitor BIBW-2992 inhibits transformation, survival, migration, invasion, and tumor forming characteristics of gallbladder cancer cells harboring wild type or KRAS (G13D) but not KRAS (G12V) mutation. Furthermore, we show in vivo reduction in tumor size is paralleled by a reduction in the amounts of phospho-ERK in KRAS (G13D) but not in KRAS (G12V) xenografts, validating the in vitro findings Conclusion: Findings from this study implicate ERBB2 as an important therapeutic target in early stage gallbladder cancer. We also present the first evidence that the presence of KRAS (G12V), but not KRAS (G13D) mutation, may preclude gallbladder cancer patients to respond to anti-EGFR treatment, similar to the clinical algorithm commonly practiced to opt for anti-EGFR treatment in colorectal cancer.
2018-12-29 | E-MTAB-6619 | biostudies-arrayexpress
Project description:Mutations in LZTR1, an adaptor for cullin 3 (CUL3) ubiquitin ligase complex, are associated with glioma, hepatocarcinoma, paediatric cancers, Schwannomatosis, and Noonan syndrome (NS). NS is a poorly understood and complex disease. The variety of NS phenotypes makes it challenging to elucidate the molecular mechanisms by which mutations in LZTR1 drive human disease. Using an Lztr1 knockout model, we assessed the effect of Lztr1 loss on endothelial function. Moreover, the molecular alterations mediated by Lztr1 loss were evaluated by mass-spectrometry (MS)-based ubiquitome and proteome analysis. These analyses demonstrated that Lztr1 loss affects vesicular trafficking. We identified the ESCRT III member CHMP1B as a substrate for the LZTR1/CUL3 ubiquitin complex and could show that disease-related LZTR1 mutations abolish the interaction with CHMP1B. LZTR1-mediated ubiquitination of CHMP1B facilitates its interaction with IST1 that affects the dynamics of fusion and fission of endosomal vesicles. Dysregulation of vesicular trafficking triggered by Lztr1 loss leads to up-regulation of VEGF signaling due to endosomal accumulation and activation of VEGFR2. This novel molecular mechanism provides a fundamental explanation for the role of LZTR1 in endothelial function and justify the use of anti-VEGF therapies for the treatment of NS patients.
2020-10-14 | PXD011926 | Pride
Project description:Mutations in LZTR1, a substrate adaptor for cullin 3 (CUL3) ubiquitin ligase complexes, have been recently associated with Noonan syndrome and familial Schwannomatosis. Concordantly, we found that Lztr1+/- mice showed craniofacial abnormalities, cardio defects, and premature ageing; whereas loss of LZTR1 in Schwann cells drives their dedifferentiation into proliferating, pro-myelinating cells. In the present dataset we screened for changes in the ubiquitin landscape induced by LZTR1 loss of function by mass spectrometry-based proteomics and identified RAS proteins as substrates of the LZTR1/CUL3 complex. In follow-up experiments we showed that LZTR1/CUL3 inhibits the RAS pathway by ubiquitinating RAS at K170 and triggering its dissociation from the membrane. Disease-associated LZTR1 mutations affect either the LZTR1/CUL3 complex formation, or the interaction with RAS proteins. Together, our results position LZTR1 as a key node in the RAS pathway, loss-of-function of which leads to human disease.
2018-11-07 | PXD007035 | Pride
Project description:K-ras mutations are observed in around 40% human colorectal adenomas and carcinomas and contribute to the pathogenesis of human and rodent colorectal tumour formation. Previously, we developed and characterised a strain of transgenic mice with inducible intestinal epithelial expression of K-rasVal12 via a Cre/LoxP system. To evaluate the influence of mutant K-ras on carcinogen-induced colorectal tumourigenesis, we induced neoplastic alterations in the large intestines of wild-type and K-rasVal12 mice using the colon-selective carcinogen 1, 2-dimethylhydrazine (DMH), which has been widely used to study chemically induced colorectal tumours that are histopathologically similar to those observed in humans. K-rasVal12 expression significantly promoted DMH-induced colorectal tumourigenesis: the average life span of the mice decreased from 38.52±1.97 weeks for 40 wild-type mice to 32.42±2.17 weeks for 26 K-rasVal12 mice (mean+SEM, P<0.05) and the large intestinal tumours increased from 2.27±0.15 per wild-type mouse to 3.85±0.20 in K-rasVal12 mice (mean+SEM, P<0.01). Adenomas from DMH-treated K-rasVal12 mice showed significantly higher levels (10.9±1.69%) of Ki-67-positive proliferating cells compared with those from DMH-treated wild-type mice (7.77±1.15%) (mean+SD, P<0.01) and a mild increase in apoptotic nuclei staining for cleaved caspase-3 (1.94+0.51% compared with 1.15±0.34%, mean+SD, P<0.01). In the adenomas from DMH-treated K-rasVal12 mice, K-rasVal12 transgene recombination and expression were confirmed and shown to promote strong Erk and mild Akt activation compared with adenomas from DMH-treated wild-type mice. Microarray hybridization and cluster analysis demonstrated different expression profiles in adenomas from DMH-treated wild-type and DMH-treated K-rasVal12 mice, indicating involvement of different molecular mechanisms, but array-comparative genomic hybridisation analysis showed chromosome stability in both, with very few chromosome alterations observed in adenomas from either of the two groups. Taken together, these data show that mutant K-ras promotes DMH-induced colorectal tumourigenesis, conferring a proliferative effect, but does not alter chromosome stability in the tumours. This study has 7 samples analysed, 4 Kras mutants and 3 controls, on the Illumina Mouse-6 Beadchip array.
2010-10-02 | E-GEOD-24010 | biostudies-arrayexpress