Metabolomic profiling is an increasingly important method for identifying potential biomarkers in cancer cells with a view towards improved diagnosis and treatment. Nuclear magnetic resonance (NMR) provides a potentially noninvasive means to accurately characterize differences in the metabolomic profiles of cells. In this work, we use1H NMR to measure the metabolomic profiles of water soluble metabolites extracted from isogenic control and oncogenic HRAS-, KRAS-, and NRAS-transduced BEAS2B lung epithelial cells to determine the robustness of NMR metabolomic profiling in detecting differences between the transformed cells and their untransformed counterparts as well as differences among the RAS-transformed cells. Unique metabolomic signatures between control and RAS-transformed cell lines as well as among the three RAS isoform-transformed lines were found by applying principal component analysis to the NMR data. This study provides a proof of principle demonstration that NMR-based metabolomic profiling can robustly distinguish untransformed and RAS-transformed cells as well as cells transformed with different RAS oncogenic isoforms. Thus, our data may potentially provide new diagnostic signatures for RAS-transformed cells.
Alternative RNA splicing is widely dysregulated in cancers including lung adenocarcinoma, where aberrant splicing events are frequently caused by somatic splice site mutations or somatic mutations of splicing factor genes. However, the majority of mis-splicing in cancers is unexplained by these known mechanisms. We hypothesize that the aberrant Ras signaling characteristic of lung cancers plays a role in promoting the alternative splicing observed in tumors.
We recently performed transcriptome and proteome profiling of human lung epithelial cells ectopically expressing oncogenic KRAS and another cancer-associated Ras GTPase, RIT1. Unbiased analysis of phosphoproteome data identified altered splicing factor phosphorylation in KRAS-mutant cells, so we performed differential alternative splicing analysis using rMATS to identify significantly altered isoforms in lung epithelial cells. To determine whether these isoforms were uniquely regulated by KRAS, we performed a large-scale splicing screen in which we generated over 300 unique RNA sequencing profiles of isogenic A549 lung adenocarcinoma cells ectopically expressing 75 different wild-type or variant alleles across 28 genes implicated in lung cancer.
Mass spectrometry data showed widespread downregulation of splicing factor phosphorylation in lung epithelial cells expressing mutant KRAS compared to cells expressing wild-type KRAS. We observed alternative splicing in the same cells, with 2196 and 2416 skipped exon events in KRASG12Vand KRASQ61Hcells, respectively, 997 of which were shared (
Proteomic and transcriptomic profiling of lung epithelial cells uncovered splicing factor phosphorylation and mRNA splicing events regulated by oncogenic KRAS. These data suggest that in addition to widespread transcriptional changes, the Ras signaling pathway can promote post-transcriptional splicing changes that may contribute to oncogenic processes.
- NSF-PAR ID:
- 10385797
- Publisher / Repository:
- Springer Science + Business Media
- Date Published:
- Journal Name:
- BMC Cancer
- Volume:
- 22
- Issue:
- 1
- ISSN:
- 1471-2407
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract KRAS is the most frequently mutated oncogene in human lung adenocarcinomas (hLUAD), and activating mutations frequently co-occur with loss-of-function mutations in TP53 or STK11/LKB1. However, mutation of all three genes is rarely observed in hLUAD, even though engineered comutation is highly aggressive in mouse lung adenocarcinoma (mLUAD). Here, we provide a mechanistic explanation for this difference by uncovering an evolutionary divergence in the regulation of triosephosphate isomerase (TPI1). In hLUAD, TPI1 activity is regulated via phosphorylation at Ser21 by the salt inducible kinases (SIK) in an LKB1-dependent manner, modulating flux between the completion of glycolysis and production of glycerol lipids. In mice, Ser21 of TPI1 is a Cys residue that can be oxidized to alter TPI1 activity without a need for SIKs or LKB1. Our findings suggest this metabolic flexibility is critical in rapidly growing cells with KRAS and TP53 mutations, explaining why the loss of LKB1 creates a liability in these tumors.
Significance: Utilizing phosphoproteomics and metabolomics in genetically engineered human cell lines and genetically engineered mouse models (GEMM), we uncover an evolutionary divergence in metabolic regulation within a clinically relevant genotype of human LUAD with therapeutic implications. Our data provide a cautionary example of the limits of GEMMs as tools to study human diseases such as cancers.
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mTOR-regulated U2af1 tandem exon splicing specifies transcriptome features for translational controlAbstract U2 auxiliary factor 1 (U2AF1) functions in 3′-splice site selection during pre-mRNA processing. Alternative usage of duplicated tandem exons in U2AF1 produces two isoforms, U2AF1a and U2AF1b, but their functional differences are unappreciated due to their homology. Through integrative approaches of genome editing, customized-transcriptome profiling and crosslinking-mediated interactome analyses, we discovered that the expression of U2AF1 isoforms is controlled by mTOR and they exhibit a distinctive molecular profile for the splice site and protein interactomes. Mechanistic dissection of mutually exclusive alternative splicing events revealed that U2AF1 isoforms’ inherent differential preferences of nucleotide sequences and their stoichiometry determine the 3′-splice site. Importantly, U2AF1a-driven transcriptomes feature alternative splicing events in the 5′-untranslated region (5′-UTR) that are favorable for translation. These findings unveil distinct roles of duplicated tandem exon-derived U2AF1 isoforms in the regulation of the transcriptome and suggest U2AF1a-driven 5′-UTR alternative splicing as a molecular mechanism of mTOR-regulated translational control.more » « less
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Abstract Background Huntington disease (HD) is a neurodegenerative disease where a genetic mutation leads to excessive polyglutamine (Q) repeats in the huntingtin protein. The polyglutamine repeats create toxic plaques when the protein is cleaved, leading to neuron death. The glycolipid GM1 ganglioside (GM1) has been shown to be neuroprotective in HD models, as it prevents the cleavage of the mutant huntingtin protein by phosphorylation of serine 13 and 16. Previous studies have tested GM1 in both adult-onset and juvenile-onset HD models, but this study set out to investigate whether GM1 mediated cytoprotection is influenced by the length of polyglutamine repeats.
Method and result This study utilized cell culture to analyze the effect of GM1 on cell viability, directly comparing the response between cells with adult-onset HD and juvenile-onset HD. HEK293 cells expressing either wild-type huntingtin (Htt) (19Q) exon 1, adult-onset HD mutant Htt exon 1 (55Q), or Juvenile HD mutant Htt exon 1 (94Q) were assessed for cell viability using the WST-1 assay. Our results suggested moderate doses of GM1 increased cell viability for all cell lines when compared to untreated cells. When comparing HEK293 55Q and 94Q cells, there was no difference in cell viability within each dose of GM1.
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Abstract BACKGROUND Despite widespread interest in next-generation sequencing (NGS), the adoption of personalized clinical genomics and mutation profiling of cancer specimens is lagging, in part because of technical limitations. Tumors are genetically heterogeneous and often contain normal/stromal cells, features that lead to low-abundance somatic mutations that generate ambiguous results or reside below NGS detection limits, thus hindering the clinical sensitivity/specificity standards of mutation calling. We applied COLD-PCR (coamplification at lower denaturation temperature PCR), a PCR methodology that selectively enriches variants, to improve the detection of unknown mutations before NGS-based amplicon resequencing.
METHODS We used both COLD-PCR and conventional PCR (for comparison) to amplify serially diluted mutation-containing cell-line DNA diluted into wild-type DNA, as well as DNA from lung adenocarcinoma and colorectal cancer samples. After amplification of TP53 (tumor protein p53), KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog), IDH1 [isocitrate dehydrogenase 1 (NADP+), soluble], and EGFR (epidermal growth factor receptor) gene regions, PCR products were pooled for library preparation, bar-coded, and sequenced on the Illumina HiSeq 2000.
RESULTS In agreement with recent findings, sequencing errors by conventional targeted-amplicon approaches dictated a mutation-detection limit of approximately 1%–2%. Conversely, COLD-PCR amplicons enriched mutations above the error-related noise, enabling reliable identification of mutation abundances of approximately 0.04%. Sequencing depth was not a large factor in the identification of COLD-PCR–enriched mutations. For the clinical samples, several missense mutations were not called with conventional amplicons, yet they were clearly detectable with COLD-PCR amplicons. Tumor heterogeneity for the TP53 gene was apparent.
CONCLUSIONS As cancer care shifts toward personalized intervention based on each patient's unique genetic abnormalities and tumor genome, we anticipate that COLD-PCR combined with NGS will elucidate the role of mutations in tumor progression, enabling NGS-based analysis of diverse clinical specimens within clinical practice.
