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Dynamic proteomics reveals unexpected plasticity of cellular proteome: growth-related and drug-induced changes in cancer cells are comparable

ABSTRACT: In proteomics, the changes occurring in cellular proteomes upon drug treatment are used to identify the drug targets and the mechanism of action. However, proteomes of cultured cells undergo also natural alteration associated with changes in the media, attaining a degree of confluence as well as due to cell division, metabolic and circadian cycles. These changes are implicitly assumed to be smaller in magnitude than the drug-induced changes that ultimately lead to cell demise. In this study, we tested this assumption experimentally by comparing the proteome dynamics of cancer cells during the first 48 h after treatment with methotrexate, paclitaxel or vehicle (equivalent to untreated control). Time series revealed considerable growth-related proteome changes in the control group, comparable in magnitude to drug-induced changes. The magnitude of these variations reveals an unexpected plasticity of the cellular proteome and reinforces the need, generally accepted in theory but not always followed in practice, to use a time-matched control when measuring drug-induced proteome changes.

INSTRUMENT(S): Orbitrap Fusion, Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Colon Cancer,Melanoma

SUBMITTER: Pierre Sabatier

LAB HEAD: Roman Zubarev

PROVIDER: PXD007762 | Pride | 2018-11-07

REPOSITORIES: Pride

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Dynamic proteomics reveals unexpected plasticity of cellular proteome

Project description:In proteomics, the changes occurring in cellular proteomes upon drug treatment are used to identify the drug targets and the mechanism of action. However, proteomes of cultured cells undergo also natural alteration associated with changes in the media, attaining a degree of confluence as well as due to cell division, metabolic and circadian cycles. These changes are implicitly assumed to be minimal. In this study, we tested this assumption experimentally by comparing the proteome dynamics of untreated HCT116 and A375 cancer cells during 48 h. Time series revealed considerable growth-related proteome changes. The magnitude of these variations reveals an unexpected plasticity of the cellular proteome and reinforces the need, generally accepted in theory but not always followed in practice, to use a time-matched control when measuring drug-induced proteome changes.

2018-11-07 | PXD010805 | Pride

Quantitative proteomics to systematically assess protein changes after HUWE1 knockout in SW620 and A375 cell line

Project description:Quantitative proteomics to systematically assess protein changes after HUWE1 knockout in SW620 and A375 cell line

2023-03-23 | PXD038892 | Pride

Analysis of Amino Acid Variants in Malignant Melanoma Cells Resistant to BRAF inhibition

Project description:Analysis of patient-specific nucleotide variants is a cornerstone of personalised medicine. Although only 2% of the genomic sequence is protein-coding, mutations occurring in these regions have the potential to influence protein structure and may have severe impact on disease aetiology. Of special importance are variants that affect modifiable amino acid residues, as protein modifications involved in signal transduction networks cannot be analysed by genomics. Proteogenomics enables analysis of proteomes in context of patient- or tissue-specific non-synonymous nucleotide variants. Here, we developed a proteogenomics workflow and applied it to study resistance to serine/threonine-protein kinase B-raf (BRAF) inhibitor (BRAFi) vemurafenib in malignant melanoma cell line A375. This approach resulted in high identification and quantification of non-synonymous nucleotide variants and (phospho)proteins. We integrated multi-omic datasets to reconstruct the perturbed signalling networks associated with BRAFi resistance and to predict drug therapies with the potential to disrupt BRAFi resistance mechanism in A375 cells. Notably, we showed that aurora kinase A (AURKA) inhibition is effective and specific against BRAFi resistant A375 cells. Furthermore, we investigated nucleotide variants that interfere with protein post-translational modification (PTM) status and potentially influence cell signalling. Mass spectrometry (MS) measurements confirmed variant-driven PTM changes in 12 proteins; among them was the runt-related transcription factor 1 (RUNX1) displaying a variant on a known phosphorylation site S(Ph)276L. We confirmed the loss of phosphorylation site by MS and demonstrated the impact of this variant on RUNX1 interactome.

2021-11-04 | PXD018305 | Pride

Transcription profiling of QUB HCT116 sensitive and resistant colorectal cancer time series

Project description:HCT116 parental, HCT116 5-FU resistant and HCT116 oxaliplatin resistant cells have been transiently treated with with their respective drug (5-FU or oxaliplatin) for 0, 6 12 or 24h in 3 independent experiments.

2006-01-01 | E-MEXP-390 | biostudies-arrayexpress

Differential patterns of protein expression in monolayer or spheroid melanoma cell models of acquired resistance to BRAF and MEK1/2 inhibitors

Project description:Mitogen activated protein kinase (MAPK) inhibitors are important therapies for treating many cancers. However, the development of acquired resistance to most protein kinase inhibitors limit their ability to provide durable responses. Approximately 50% of malignant melanomas contain activating mutations in the BRAF kinase, which promote cancer cell survival by activating the extracellular signal-regulated kinase-1/2 (ERK1/2) pathway through direct phosphorylation of the MAPK/ERK kinase-1/2 (MEK1/2). Although combination treatment with BRAF and MEK1/2 inhibitors is a recommended approach to treat melanoma, the development of drug resistance remains a barrier to achieving long term patient benefits. Using high-resolution label-free mass spectrometry, the current studies compared relative protein changes in BRAF and MEK1/2 inhibitor resistant A375 melanoma cells grown as monolayers or 3D spheroids. While approximately 66% of proteins identified were common in both monolayer and spheroid cultures, only 6.2% or 3.6% of proteins that significantly increased or decreased, respectively, compared to drug sensitive cells were common between the drug-resistant monolayer and spheroid cells. Major changes in drug-resistant monolayers suggested upregulation of alternative kinase signaling pathways that promote growth and metastasis. In contrast, the major changes in drug-resistant spheroids indicated increased catabolic metabolism to support oxidative phosphorylation and energy requirements.

2022-02-17 | PXD026952 | Pride

A375 melanoma cell line, Expanded database search comparisons

Project description:Thousands of protein post-translational modifications (PTMs) dynamically impact nearly all cellular functions. Mass spectrometry is well suited to PTM identification, but proteome-scale analyses are biased towards PTMs with existing enrichment methods. To measure the full landscape of PTM regulation, software must overcome two fundamental challenges: intractably large search spaces and difficulty distinguishing correct from incorrect identifications. Here, we describe TagGraph, software that overcomes both challenges with a string-based search method orders of magnitude faster than current approaches, and probabilistic validation model optimized for PTM assignments. When applied to a human proteome map, TagGraph tripled confident identifications while revealing thousands of modification types on nearly one million sites spanning the proteome. We expand known sites by orders of magnitude for highly abundant yet understudied PTMs such as proline hydroxylation, and derive tissue-specific insight into these PTMs’ roles. TagGraph expands our ability to survey the full landscape of PTM function and regulation.

2019-03-12 | PXD005912 | Pride

Global, site-resolved analysis of ubiquitylation occupancy and turnover rate reveals systems properties

Project description:Ubiquitylation regulates most proteins and biological processes in a eukaryotic cell. However, the site-specific occupancy (stoichiometry) and turnover rate of ubiquitylation have not been quantified. Here we present an integrated picture of the global ubiquitylation site occupancy and half-life. Ubiquitylation site occupancy spans over four orders of magnitude, but the median ubiquitylation site occupancy is three orders of magnitude lower than that of phosphorylation. The occupancy, turnover rate, and regulation of sites by proteasome inhibitors are strongly interrelated, and these attributes distinguish sites involved in proteasomal degradation and cellular signaling. Sites in structured protein regions exhibit longer half-lives and stronger upregulation by proteasome inhibitors than sites in unstructured regions. Importantly, we discovered a surveillance mechanism that rapidly and site-indiscriminately deubiquitylates all ubiquitin-specific E1 and E2 enzymes, protecting them against accumulation of bystander ubiquitylation. The work provides a systems-scale, quantitative view of ubiquitylation properties and reveals general principles of ubiquitylation-dependent governance.

2024-04-15 | PXD037009 | Pride

RNAseq of HCT116 p53KO cells transfected with either siRNA silencing ZNF84 or with negative control siRNA, in 2 variants of experiment a) with doxorubicin treatment (doxorubicin added 48h post-transfection, for the next 48h before harvesting) b) untreated, cultured 72h post-transfection

Project description:The aim of this experiments was to characterise the transcriptomic changes occurring in cancer cells, in which ZNF84 gene is silenced, in control conditions and when cells undergo stress by being treated with cytostatic doses of doxorubicin. We anticipated that ZNF84 regulates the level of p21 cell cycle inhibitor and therefore contributes to senescence induction in response to stress. We wanted to know whether attenuation of senescence in cells with downregulated ZNF84 is accompanied by alterations in other biological processes and signalling pathways. Cells were transfected with 30 nM siRNA (against ZNF84 or negative control siRNA) and then either a) doxorubicin was added (48 hours after transfection and cells were cultured in the presence of the drug for another 48 hours) b) no drug was added (cells were cultured in drug-free medium for 72 hours post- transfection) Variants a) and b) were separate RNA sequencing experiments.

2021-05-05 | E-MTAB-9848 | biostudies-arrayexpress

Comprehensive chemical proteomics for target deconvolution of the redox active drug auranofin

Project description:Chemical proteomics encompasses novel drug target deconvolution methods in which compound modification is not required. Herein we use Thermal Proteome Profiling, Functional Identification of Target by Expression Proteomics and multiplexed redox proteomics for deconvolution of auranofin targets to aid elucidation of its mechanisms of action. Auranofin (Ridaura®) was approved for treatment of rheumatoid arthritis in 1985. Because several clinical trials are currently ongoing to repurpose auranofin for cancer therapy, comprehensive characterization of its targets and effects in cancer cells is important. Together, our chemical proteomics tools confirmed thioredoxin reductase 1 (TXNRD1) as a main auranofin target, with perturbation of oxidoreductase pathways as the top mechanism of drug action. Additional indirect targets included NFKB2 and CHORDC1. Our comprehensive data can be used as a proteomic signature resource for further analyses of the effects of auranofin. Here we also assessed the orthogonality and complementarity of different chemical proteomics methods that can furnish invaluable mechanistic information and thus the approach can facilitate drug discovery efforts in general.

2020-03-19 | PXD016776 | Pride

A Quantitative Chemical Proteomics Approach to Profile the Specific Cellular Targets of Andrographolide, a Promising Anticancer Agent that Suppresses Tumor Metastasis

Project description:Drug target identification is a critical step towards the understanding of the mechanism of action of a drug, which will help to improve the current therapeutic regime and to expand the drug’s therapeutic potential. However, current in vitro affinity chromatography-based and in vivo activity- based protein profiling (ABPP) approaches generally face difficulties discriminating specific drug targets from non-specific ones. Here we describe a novel approach combining isobaric tag for relative and absolute quantitation (iTRAQ) with Clickable ABPP, named ICABPP, to specifically and comprehensively identify the protein targets of andrographolide (Andro), a natural product with known anti-inflammation and anti-cancer effects, in live cancer cells. We identified a spectrum of specific targets of Andro, which furthered our understanding of the mechanism of action of the drug. We found that Andro has a potential novel application as the tumor metastasis inhibitor, which was validated through cell migration and invasion assays. Moreover, we have unveiled the target binding mechanism of Andro with a combination of drug analogue synthesis, protein engineering and mass spectrometry-based approaches and determined the drug-binding sites of two protein targets, NF-kappaB and actin.

2019-11-28 | PXD000588 | Pride

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