Browse
Submit Data
Databases
API
Help

Dataset Information

20 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Transcriptional and metabolic rewiring of colorectal cancer cells expressing the oncogenic KRAS^G13D mutation.

ABSTRACT:

Background

Activating mutations in KRAS frequently occur in colorectal cancer (CRC) patients, leading to resistance to EGFR-targeted therapies.

Methods

To better understand the cellular reprogramming which occurs in mutant KRAS cells, we have undertaken a systems-level analysis of four CRC cell lines which express either wild type (wt) KRAS or the oncogenic KRAS^G13D allele (mtKRAS).

Results

RNAseq revealed that genes involved in ribosome biogenesis, mRNA translation and metabolism were significantly upregulated in mtKRAS cells. Consistent with the transcriptional data, protein synthesis and cell proliferation were significantly higher in the mtKRAS cells. Targeted metabolomics analysis also confirmed the metabolic reprogramming in mtKRAS cells. Interestingly, mtKRAS cells were highly transcriptionally responsive to EGFR activation by TGFα stimulation, which was associated with an unexpected downregulation of genes involved in a range of anabolic processes. While TGFα treatment strongly activated protein synthesis in wtKRAS cells, protein synthesis was not activated above basal levels in the TGFα-treated mtKRAS cells. This was likely due to the defective activation of the mTORC1 and other pathways by TGFα in mtKRAS cells, which was associated with impaired activation of PKB signalling and a transient induction of AMPK signalling.

Conclusions

We have found that mtKRAS cells are substantially rewired at the transcriptional, translational and metabolic levels and that this rewiring may reveal new vulnerabilities in oncogenic KRAS CRC cells that could be exploited in future.

SUBMITTER: Charitou T

PROVIDER: S-EPMC6738113 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Extensive rewiring of the EGFR network in colorectal cancer cells expressing transforming levels of KRASG13D.

Project description:Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRASG13D) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping >6000 PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRASG13D (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes.

| S-EPMC6981206 | biostudies-literature

Clinical significance of the KRAS G13D mutation in anastomotic recurrence of colorectal cancer

Project description:The genetic risk factors for anastomotic recurrence (AR) after curative surgery for colorectal cancer (CRC) are unclear. The present study is a single-center retrospective observational study that aimed to elucidate the association between the KRAS G13D mutation and AR in CRC. The present study included 21 patients with AR and 67 patients with non-anastomotic local recurrence (NALR) following curative surgery for CRC between January 2005 and December 2019. KRAS G13D mutation status was examined by droplet digital polymerase chain reaction. Data of clinicopathological findings and oncological outcomes were analyzed and compared between the AR group and the matched NALR group. The prevalence of the KRAS G13D mutation was significantly higher in the AR group (AR vs. NALR, 33.3 vs. 4.8%; P=0.047). Comparing the KRAS G13D mutation-positive and KRAS G13D mutation-negative patients in the AR group, there was no significant difference in the time from initial surgery to AR or resection rate of AR; however, all patients with KRAS G13D mutation who underwent resection of AR had subsequent recurrence within 2 years after resection, and overall survival was poor (3-year survival rate: Positive vs. negative, 68.6 vs. 90.9%; P=0.02). The prevalence of the KRAS G13D mutation was significantly higher in patients with AR, and KRAS G13D-mutant patients with AR had a poorer prognosis than those that were negative for the KRAS G13D mutation. In conclusion, postoperative surveillance and treatment strategies should be considered with attention to the possibility of AR and subsequent recurrence in KRAS G13D-mutant patients.

| S-EPMC10126653 | biostudies-literature

KRAS G13D Mutation and Sensitivity to Cetuximab or Panitumumab in a Colorectal Cancer Cell Line Model.

Project description:BackgroundThe treatment of metastatic colorectal cancer (mCRC) includes drugs targeting the epidermal growth factor receptor (EGFR). Mutation in codon 12 or 13 in the Kirsten rat sarcoma viral oncogene homolog (KRAS) gene, downstream of the EGFR, evokes constitutive activation of the RAS/RAF/MAPK signaling pathway and correlates with resistance to anti-EGFR monoclonal antibody (mAb) therapies. However, a retrospective study reported that a proportion of patients with the KRAS G13D mutation may respond to cetuximab. A similar analysis for panitumumab was not as conclusive. We sought to determine the sensitivity of CRC cell lines to cetuximab or panitumumab treatment and to investigate the correlation of the KRAS mutational status of the CRC cell lines to the responsiveness to cetuximab or panitumumab.MethodsTo determine the responsiveness of CRC cell lines to cetuximab or panitumumab, cell lines were treated with an optimized concentration of each mAb, and proliferation assays were conducted.ResultsAfter treatment with cetuximab or panitumumab, at the optimum concentration of 8 μg/well, the KRAS G13D mutant cell lines HCT-116, LoVo, and T84 showed intermediate sensitivity to both treatments, between the resistant KRAS G12V mutant cell line SW480 and the sensitive KRAS wild-type cell line LIM1215. One of the G13D cell lines was significantly more sensitive to panitumumab than to cetuximab (P = .02).ConclusionThe specific KRAS mutation determines the responsiveness to anti-EGFR monoclonal antibody treatment, corresponding to reported clinical observations.

| S-EPMC3930148 | biostudies-literature

Oncogenic KRAS and BRAF Drive Metabolic Reprogramming in Colorectal Cancer.

Project description:Metabolic reprogramming, in which altered utilization of glucose and glutamine supports rapid growth, is a hallmark of most cancers. Mutations in the oncogenes KRAS and BRAF drive metabolic reprogramming through enhanced glucose uptake, but the broader impact of these mutations on pathways of carbon metabolism is unknown. Global shotgun proteomic analysis of isogenic DLD-1 and RKO colon cancer cell lines expressing mutant and wild type KRAS or BRAF, respectively, failed to identify significant differences (at least 2-fold) in metabolic protein abundance. However, a multiplexed parallel reaction monitoring (PRM) strategy targeting 73 metabolic proteins identified significant protein abundance increases of 1.25-twofold in glycolysis, the nonoxidative pentose phosphate pathway, glutamine metabolism, and the phosphoserine biosynthetic pathway in cells with KRAS G13D mutations or BRAF V600E mutations. These alterations corresponded to mutant KRAS and BRAF-dependent increases in glucose uptake and lactate production. Metabolic reprogramming and glucose conversion to lactate in RKO cells were proportional to levels of BRAF V600E protein. In DLD-1 cells, these effects were independent of the ratio of KRAS G13D to KRAS wild type protein. A study of 8 KRAS wild type and 8 KRAS mutant human colon tumors confirmed the association of increased expression of glycolytic and glutamine metabolic proteins with KRAS mutant status. Metabolic reprogramming is driven largely by modest (<2-fold) alterations in protein expression, which are not readily detected by the global profiling methods most commonly employed in proteomic studies. The results indicate the superiority of more precise, multiplexed, pathway-targeted analyses to study functional proteome systems. Data are available through MassIVE Accession MSV000079486 at ftp://MSV000079486@massive.ucsd.edu.

| S-EPMC5013308 | biostudies-literature

Reduced dynamic complexity allows structure elucidation of an excited state of KRASG13D.

Project description:Localized dynamics of RAS, including regions distal to the nucleotide-binding site, is of high interest for elucidating the mechanisms by which RAS proteins interact with effectors and regulators and for designing inhibitors. Among several oncogenic mutants, methyl relaxation dispersion experiments reveal highly synchronized conformational dynamics in the active (GMPPNP-bound) KRASG13D, which suggests an exchange between two conformational states in solution. Methyl and 31P NMR spectra of active KRASG13D in solution confirm a two-state ensemble interconverting on the millisecond timescale, with a major Pγ atom peak corresponding to the dominant State 1 conformation and a secondary peak indicating an intermediate state different from the known State 2 conformation recognized by RAS effectors. High-resolution crystal structures of active KRASG13D and KRASG13D-RAF1 RBD complex provide snapshots of the State 1 and 2 conformations, respectively. We use residual dipolar couplings to solve and cross-validate the structure of the intermediate state of active KRASG13D, showing a conformation distinct from those of States 1 and 2 outside the known flexible switch regions. The dynamic coupling between the conformational exchange in the effector lobe and the breathing motion in the allosteric lobe is further validated by a secondary mutation in the allosteric lobe, which affects the conformational population equilibrium.

| S-EPMC10238545 | biostudies-literature

SREBP1 regulates mitochondrial metabolism in oncogenic KRAS expressing NSCLC.

Project description:Cancer cells require extensive metabolic reprograming in order to provide the bioenergetics and macromolecular precursors needed to sustain a malignant phenotype. Mutant KRAS is a driver oncogene that is well-known for its ability to regulate the ERK and PI3K signaling pathways. However, it is now appreciated that KRAS can promote the tumor growth via upregulation of anabolic metabolism. We recently reported that oncogenic KRAS promotes a gene expression program of de novo lipogenesis in non-small cell lung cancer (NSCLC). To define the mechanism(s) responsible, we focused on the lipogenic transcription factor SREBP1. We observed that KRAS increases SREBP1 expression and genetic knockdown of SREBP1 significantly inhibited the cell proliferation of mutant KRAS-expressing cells. Unexpectedly, lipogenesis was not significantly altered in cells subject to SREBP1 knockdown. Carbon tracing metabolic studies showed a significant decrease in oxidative phosphorylation and RNA-seq data revealed a significant decrease in mitochondrial encoded subunits of the electron transport chain (ETC). Taken together, these data support a novel role, distinct from lipogenesis, of SREBP1 on mitochondrial function in mutant KRAS NSCLC.

| S-EPMC8335704 | biostudies-literature

Tissue-Specific Oncogenic Activity of KRASA146T.

Project description:KRAS is the most frequently mutated oncogene. The incidence of specific KRAS alleles varies between cancers from different sites, but it is unclear whether allelic selection results from biological selection for specific mutant KRAS proteins. We used a cross-disciplinary approach to compare KRASG12D, a common mutant form, and KRASA146T, a mutant that occurs only in selected cancers. Biochemical and structural studies demonstrated that KRASA146T exhibits a marked extension of switch 1 away from the protein body and nucleotide binding site, which activates KRAS by promoting a high rate of intrinsic and guanine nucleotide exchange factor-induced nucleotide exchange. Using mice genetically engineered to express either allele, we found that KRASG12D and KRASA146T exhibit distinct tissue-specific effects on homeostasis that mirror mutational frequencies in human cancers. These tissue-specific phenotypes result from allele-specific signaling properties, demonstrating that context-dependent variations in signaling downstream of different KRAS mutants drive the KRAS mutational pattern seen in cancer. SIGNIFICANCE: Although epidemiologic and clinical studies have suggested allele-specific behaviors for KRAS, experimental evidence for allele-specific biological properties is limited. We combined structural biology, mass spectrometry, and mouse modeling to demonstrate that the selection for specific KRAS mutants in human cancers from different tissues is due to their distinct signaling properties.See related commentary by Hobbs and Der, p. 696.This article is highlighted in the In This Issue feature, p. 681.

| S-EPMC6548671 | biostudies-literature

KRAS G13D sensitivity to neurofibromin-mediated GTP hydrolysis.

Project description:KRAS mutations occur in ?35% of colorectal cancers and promote tumor growth by constitutively activating the mitogen-activated protein kinase (MAPK) pathway. KRAS mutations at codons 12, 13, or 61 are thought to prevent GAP protein-stimulated GTP hydrolysis and render KRAS-mutated colorectal cancers unresponsive to epidermal growth factor receptor (EGFR) inhibitors. We report here that KRAS G13-mutated cancer cells are frequently comutated with NF1 GAP but NF1 is rarely mutated in cancers with KRAS codon 12 or 61 mutations. Neurofibromin protein (encoded by the NF1 gene) hydrolyzes GTP directly in complex with KRAS G13D, and KRAS G13D-mutated cells can respond to EGFR inhibitors in a neurofibromin-dependent manner. Structures of the wild type and G13D mutant of KRAS in complex with neurofibromin (RasGAP domain) provide the structural basis for neurofibromin-mediated GTP hydrolysis. These results reveal that KRAS G13D is responsive to neurofibromin-stimulated hydrolysis and suggest that a subset of KRAS G13-mutated colorectal cancers that are neurofibromin-competent may respond to EGFR therapies.

| S-EPMC6825300 | biostudies-literature

Structural dataset for the fast-exchanging KRAS G13D.

Project description:Cancers bearing the KRAS G13D mutation are notable for their distinct clinical behavior relative to other oncogenic KRAS mutations. We hypothesized that primary biochemical or biophysical properties of KRAS G13D might contribute to these clinical observations and as part of our study undertook structural studies using x-ray crystallography. In this data article we discuss several x-ray diffraction datasets that yielded structures of oncogenic KRAS mutants including a high resolution (1.13 Å) structure of KRAS G13D. The datasets are typical for high resolution x-ray diffraction data and allow the construction of atomic resolution, three dimensional structural models with high confidence. This data can be correlated with biochemical information such as defects in substrate binding kinetics, GTPase activities and interactions with the RAS effector RAF kinase.

| S-EPMC4773362 | biostudies-literature

MEK1/2 inhibitor withdrawal reverses acquired resistance driven by BRAFV600E amplification whereas KRASG13D amplification promotes EMT-chemoresistance.

Project description:Acquired resistance to MEK1/2 inhibitors (MEKi) arises through amplification of BRAFV600E or KRASG13D to reinstate ERK1/2 signalling. Here we show that BRAFV600E amplification and MEKi resistance are reversible following drug withdrawal. Cells with BRAFV600E amplification are addicted to MEKi to maintain a precise level of ERK1/2 signalling that is optimal for cell proliferation and survival, and tumour growth in vivo. Robust ERK1/2 activation following MEKi withdrawal drives a p57KIP2-dependent G1 cell cycle arrest and senescence or expression of NOXA and cell death, selecting against those cells with amplified BRAFV600E. p57KIP2 expression is required for loss of BRAFV600E amplification and reversal of MEKi resistance. Thus, BRAFV600E amplification confers a selective disadvantage during drug withdrawal, validating intermittent dosing to forestall resistance. In contrast, resistance driven by KRASG13D amplification is not reversible; rather ERK1/2 hyperactivation drives ZEB1-dependent epithelial-to-mesenchymal transition and chemoresistance, arguing strongly against the use of drug holidays in cases of KRASG13D amplification.

| S-EPMC6497655 | biostudies-literature