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Quantitative proteomic and phosphoproteomic comparison of human colon cancer DLD-1 cells differing in ploidy and chromosome stability.

ABSTRACT: Although aneuploidy is poorly tolerated during embryogenesis, aneuploidy and whole chromosomal instability (CIN) are common hallmarks of cancer, raising the question of how cancer cells can thrive in spite of chromosome aberrations. Here we present a comprehensive and quantitative proteomics analysis of isogenic DLD-1 colorectal adenocarcinoma cells lines, aimed at identifying cellular responses to changes in ploidy and/or CIN. Specifically, we compared diploid (2N) and tetraploid (4N) cells with posttetraploid aneuploid (PTA) clones and engineered trisomic clones. Our study provides a comparative data set on the proteomes and phosphoproteomes of the above cell lines, comprising several thousand proteins and phosphopeptides. In comparison to the parental 2N line, we observed changes in proteins associated with stress responses and with interferon signaling. Although we did not detect a conspicuous protein signature associated with CIN, we observed many changes in phosphopeptides that relate to fundamental cellular processes, including mitotic progression and spindle function. Most importantly, we found that most changes detectable in PTA cells were already present in the 4N progenitor line. This suggests that activation of mitotic pathways through hyper-phosphorylation likely constitutes an important response to chromosomal burden. In line with this conclusion, cells with extensive chromosome gains showed differential sensitivity toward a number of inhibitors targeting cell cycle kinases, suggesting that the efficacy of anti-mitotic drugs may depend on the karyotype of cancer cells.

SUBMITTER: Vigano C

PROVIDER: S-EPMC5921571 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Quantitative proteomic and phosphoproteomic comparison of human colon cancer DLD-1 cells differing in ploidy and chromosome stability.

Viganó Cristina C von Schubert Conrad C Ahrné Erik E Schmidt Alexander A Lorber Thomas T Bubendorf Lukas L De Vetter Judith R F JRF Zaman Guido J R GJR Storchova Zuzana Z Nigg Erich A EA

Molecular biology of the cell 20180501 9

Although aneuploidy is poorly tolerated during embryogenesis, aneuploidy and whole chromosomal instability (CIN) are common hallmarks of cancer, raising the question of how cancer cells can thrive in spite of chromosome aberrations. Here we present a comprehensive and quantitative proteomics analysis of isogenic DLD-1 colorectal adenocarcinoma cells lines, aimed at identifying cellular responses to changes in ploidy and/or CIN. Specifically, we compared diploid (2N) and tetraploid (4N) cells wit ...[more]

PMID: 29496963

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Project description:Restenosis, re-narrowing of arterial lumen following intervention for cardiovascular disease, remains a major issue limiting the long-term therapeutic efficacy of treatment. The signaling molecules, TGFβ (transforming growth factor-beta) and Smad3, play important roles in vascular restenosis, but very little is yet known about the down-stream dynamics in global protein expression and phosphorylation. Here, we develop a highly multiplexed quantitative proteomic and phosphoproteomic strategy employing 12-plex N,N-dimethyl leucine (DiLeu) isobaric tags and The DiLeu Tool software to globally assess protein expression and phosphorylation changes in smooth muscle cells (SMCs) treated with TGFβ/Smad3 and/or SDF-1α (stromal cell-derived factor). A total of 4086 proteins were quantified in the combined dataset of proteome and phosphoproteome across 12-plex DiLeu-labeled SMC samples. 2317 localized phosphorylation sites were quantified, corresponding to 1193 phosphoproteins. TGFβ/Smad3 induced up-regulation of 40 phosphosites and down-regulation of 50 phosphosites, and TGFβ/Smad3-specific SDF-1α exclusively facilitated up-regulation of 27 phosphosites and down-regulation of 47 phosphosites. TGFβ/Smad3 inhibited the expression of contractile-associated proteins including smooth muscle myosin heavy chain, calponin, cardiac muscle alpha-actin, and smooth muscle protein 22α. Gene ontology and pathway enrichment analysis revealed that elevated TGFβ/Smad3 activated cell proliferation and TGFβ signaling pathway, sequentially stimulating phosphorylation of CXCR4 (C-X-C chemokine receptor 4). SDF-1α/CXCR4 activated extracellular signal-regulating kinase signaling pathway and facilitated the expression of synthetic marker, osteopontin, which was validated through targeted analysis. These findings provide new insights into the mechanisms of TGFβ regulated SMC dedifferentiation, as well as new avenues for designing effective therapeutics for vascular disease.

Project description:BackgroundColon cancer is one of the most prevalent digestive cancers worldwide. Results of epidemiological, experimental, and clinical studies suggest that aspirin inhibits the development of colon cancer. This study aimed to systematically elucidate the molecular mechanisms by which aspirin prevents colon carcinogenesis.MethodsWe determined the global protein expression profiles of colorectal cancer and aspirin-treated cells using quantitative proteomic analysis. We analyzed the proteomic results using bioinformatics (including differential proteins, protein annotation, Kyoto Encyclopedia of Genes and Genomes [KEGG] pathways, and protein-protein interaction [PPI] network). The viability of the colon cancer cell line and HT29 cells treated with aspirin was determined using the cell counting kit-8 assay. The differentially expressed proteins, such as p53 and cyclin-dependent kinase 1 (CDK1), were quantified using real-time polymerase chain reaction (PCR) and Western blotting. We measured cell cycle distribution and apoptosis in HT29 cells exposed to aspirin using fluorescence-activated cell sorting (FACS).ResultsWe found that 552 proteins were significantly dysregulated, of which 208 and 334 were upregulated and downregulated, respectively, in colon cancer cells exposed to 10 mmol/L of aspirin (95% confidence interval [CI]: -1.269 to -0.106, P < 0.05). Further gene enrichment analysis revealed that cell cycle-related proteins, such as p53 and CDK1, were significantly differentially expressed. Proteomic analysis showed that after 24 h of aspirin exposure, the level of p53 increased by 2.52-fold and CDK1 was downregulated to half that of the controls in HT29 cells (95% CI: -0.619 to -0.364, P < 0.05). Real-time PCR and Western blotting results showed that p53 was upregulated (95%CI: -3.088 to -1.912, P < 0.001) and CDK1 was significantly downregulated after aspirin exposure in colon cancer cells (95% CI: 0.576 to 1.045, P < 0.05). We observed that aspirin promoted G1/S cell cycle arrest in HT29 cells. We confirmed that aspirin induces apoptosis in human HT29 colon cancer cells in a concentration-dependent manner.ConclusionsThese results indicate that aspirin induces G1 arrest and apoptosis in colorectal cancer cells via the p53-CDK1 pathway. Aspirin may be a promising drug candidate for colon cancer prevention.

Dataset Information

Quantitative proteomic and phosphoproteomic comparison of human colon cancer DLD-1 cells differing in ploidy and chromosome stability.

Publications

Quantitative proteomic and phosphoproteomic comparison of human colon cancer DLD-1 cells differing in ploidy and chromosome stability.

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