Phosphoproteomics Reveals HMGA1, a CK2 Substrate, as a Drug-Resistant Target in Non-Small Cell Lung Cancer

Wang, Yi Ting; Pan, Szu-Hua; Tsai, Chia Feng; Kuo, Ting-Yu; Hsu, Yuan Ling; Yen, Hsin Yung; Choong, Wai-Kok; Wu, Hung Tsung; Liao, Yi-Ying; Hong, Tse-Ming; Sung, Ting Yi; Chen, Yu‐Ju

doi:10.1038/srep44021

Cited by 33 publications

(32 citation statements)

References 60 publications

(64 reference statements)

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“…We identified HMGA1 S102 as a hyperphosphorylated site in cells expressing ectopic SRMS (Additional file 2 : Table S1). HMGA1 S102 is a target site for phosphorylation by CK2 [ 49 , 50 ] and studies have shown that the hyperphosphorylation of HMGA1 S102 impairs the DNA-binding ability of the protein [ 51 , 52 ]. The dissociation of HMGA1 from the DNA in turn promotes efficient DNA repair, presumably by allowing various DNA repair factors to be recruited to the sites of DNA lesions, as reported previously [ 53 , 54 ].…”

Section: Discussionmentioning

confidence: 99%

Global phosphoproteomic analysis identifies SRMS-regulated secondary signaling intermediates

et al. 2018

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BackgroundThe non-receptor tyrosine kinase, SRMS (Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites) is a member of the BRK family kinases (BFKs) which represents an evolutionarily conserved relative of the Src family kinases (SFKs). Tyrosine kinases are known to regulate a number of cellular processes and pathways via phosphorylating substrate proteins directly and/or by partaking in signaling cross-talks leading to the indirect modulation of various signaling intermediates. In a previous study, we profiled the tyrosine-phosphoproteome of SRMS and identified multiple candidate substrates of the kinase. The broader cellular signaling intermediates of SRMS are unknown.MethodsIn order to uncover the broader SRMS-regulated phosphoproteome and identify the SRMS-regulated indirect signaling intermediates, we performed label-free global phosphoproteomics analysis on cells expressing wild-type SRMS. Using computational database searching and bioinformatics analyses we characterized the dataset.ResultsOur analyses identified 60 hyperphosphorylated (phosphoserine/phosphothreonine) proteins mapped from 140 hyperphosphorylated peptides. Bioinfomatics analyses identified a number of significantly enriched biological and cellular processes among which DNA repair pathways were found to be upregulated while apoptotic pathways were found to be downregulated. Analyses of motifs derived from the upregulated phosphosites identified Casein kinase 2 alpha (CK2α) as one of the major potential kinases contributing to the SRMS-dependent indirect regulation of signaling intermediates.ConclusionsOverall, our phosphoproteomics analyses identified serine/threonine phosphorylation dynamics as important secondary events of the SRMS-regulated phosphoproteome with implications in the regulation of cellular and biological processes.Electronic supplementary materialThe online version of this article (10.1186/s12953-018-0143-7) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Global phosphoproteomic analysis identifies SRMS-regulated secondary signaling intermediates

et al. 2018

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“…These techniques have been successful for a large number of resistance mechanisms (11,42–45), but can fail to identify epigenetic/non-genomic mechanisms of resistance. As an additional strategy, several groups have used MS-based phosphoproteomics or RTK-arrays to identify signaling based mechanisms of resistance (16,36,46–48). MS-based phosphoproteomics has typically been applied to in vitro cell culture following long-term treatment and development of resistance, and has been successful in identifying some common mechanisms of resistance, many of which have been validated in vivo (in cell line xenografts or patient derived xenografts).…”

Section: Discussionmentioning

confidence: 99%

Characterization of In Vivo Resistance to Osimertinib and JNJ-61186372, an EGFR/Met Bispecific Antibody, Reveals Unique and Consensus Mechanisms of Resistance

Emdal

Dittmann

Reddy

et al. 2017

Molecular Cancer Therapeutics

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Approximately 10% of non-small cell lung cancer (NSCLC) patients in the U.S. and 40% of NSCLC patients in Asia have activating EGFR mutations and are eligible to receive targeted anti-EGFR therapy. Despite an extension of life expectancy associated with this treatment, resistance to EGFR tyrosine kinase inhibitors and anti-EGFR antibodies is almost inevitable. To identify additional signaling routes that can be co-targeted to overcome resistance, we quantified tumor-specific molecular changes that govern resistant cancer cell growth and survival. Mass spectrometry-based quantitative proteomics was used to profile in vivo signaling changes in 41 therapy resistant tumors from four xenograft NSCLC models. We identified unique and tumor-specific tyrosine phosphorylation rewiring in tumors resistant to treatment with the irreversible third generation EGFR-inhibitor, osimertinib, or the novel dual-targeting EGFR/Met antibody, JNJ-61186372. Tumor-specific increases in tyrosine-phosphorylated peptides from EGFR family members, Shc1 and Gab1 or Src family kinase substrates were observed, underscoring a differential ability of tumors to uniquely escape EGFR inhibition. Although most resistant tumors within each treatment group displayed a marked inhibition of EGFR as well as Src family kinase (SFK) signaling, the combination of EGFR inhibition (osimertinib) and SFK inhibition (saracatinib or dasatinib) led to further decrease in cell growth in vitro. This result suggests that residual SFK signaling mediates therapeutic resistance and that elimination of this signal through combination therapy may delay onset of resistance. Overall, analysis of individual resistant tumors captured unique in vivo signaling rewiring that would have been masked by analysis of in vitro cell population averages.

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“…This category contains the two HSP90 isoforms (HSP90AA1 and HSP90AB1), which harbor PTM sites involved in the regulation of the chaperone activity (Mollapour and Neckers, 2012). These isoforms are known to be phosphorylated at Ser263 and Ser255, respectively (Hu et al, 2015; Wang et al, 2017), within the CK2 phosphorylation motif (S-X-X-E) that is crucial for protein activity. In particular, phosphorylation at Ser255 is required for the activation of MAPK/ERK pathway.…”

Section: Resultsmentioning

confidence: 99%

Comprehensive quantification of the modified proteome reveals oxidative heart damage in mitochondrial heteroplasmy

Bagwan

Bonzón-Kulichenko

Lechuga‐Vieco

et al. 2018

Preprint

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17Post-translational modifications hugely increase the functional diversity of 18 proteomes. Recent algorithms based on ultratolerant database searching are 19 forging a path to unbiased analysis of peptide modifications by shotgun mass 20 spectrometry. However, these approaches identify only half of the modified forms 21 potentially detectable and do not map the modified residue. Moreover, tools for the 22 quantitative analysis of peptide modifications are currently lacking. Here, we 23 present a suite of algorithms that allow comprehensive identification of detectable 24 modifications, pinpoint the modified residues, and enable their quantitative 25 analysis through an integrated statistical model. These developments were used to 26 characterize the impact of mitochondrial heteroplasmy on the proteome and on the 27 modified peptidome in several tissues from 12-week old mice. Our results reveal 28 that heteroplasmy mainly affects cardiac tissue, inducing oxidative damage to 29 proteins of the oxidative phosphorylation system, and provide a molecular 30 mechanism that explains the structural and functional alterations produced in 31 heart mitochondria. 32 33 36 Highlights: 37• Identifies all protein modifications detectable by mass spectrometry 38• Locates the modified site with 85% accuracy 39• Integrates quantitative analysis of the proteome and the modified peptidome 40 5 peptide maps can be obtained including practically all the modifications potentially 77 detectable by MS and closed database searches. Our approach also allows accurate 78 location of the modified residues and quantitative analysis of PTMs in the context of 79 proteome-wide studies. We demonstrate the performance of the new tools by 80 performing a comprehensive, tissue-specific characterization of PTMs that are induced 81 by mitochondrial heteroplasmy in a mouse model. Heteroplasmy is a situation that has 82 recently attracted the attention of the biomedical community because it may be 83 produced during mitochondrial replacement therapies aimed to prevent transmission of 84 pathogenic mutations in mitochondrial DNA (Craven et al., 2010) or to treat infertility 85 (Wolf et al., 2015). Mitochondrial heteroplasmy in mice can be genetically unstable and 86 produce adverse physiological effects (Sharpley et al., 2012). The exact molecular 87 mechanisms that produce the pathological effects are unclear and the potential health 88 risk produced by heteroplasmy in the offspring is still a matter under debate. Our results 89show that heteroplasmy between non-pathological mitochondrial DNA variants induces 90 an array of oxidative modifications in heart that are concentrated in proteins belonging 91 to the oxidative phosphorylation system. The new tools thus improve our ability to 92 interpret the totality of information present in MS/MS datasets and provide new 93 proteome-wide perspectives for systems biology analysis in high throughput 94 proteomics. 95 96 RESULTS 97 Comet-PTM enables comprehensive identification of peptide modifications 98We dev...

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Phosphoproteomics Reveals HMGA1, a CK2 Substrate, as a Drug-Resistant Target in Non-Small Cell Lung Cancer

Cited by 33 publications

References 60 publications

Global phosphoproteomic analysis identifies SRMS-regulated secondary signaling intermediates

Global phosphoproteomic analysis identifies SRMS-regulated secondary signaling intermediates

Characterization of In Vivo Resistance to Osimertinib and JNJ-61186372, an EGFR/Met Bispecific Antibody, Reveals Unique and Consensus Mechanisms of Resistance

Comprehensive quantification of the modified proteome reveals oxidative heart damage in mitochondrial heteroplasmy

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