NF-κB pathway is involved in inflammation; however, recent data shows its role also in cancer development and progression, including metastasis. To understand the role of NF-κB interactome dynamics in cancer, we study the complexity of breast cancer interactome in luminal A breast cancer model and its rearrangement associated with NF-κB modulation. Liquid chromatography-mass spectrometry measurement of 160 size-exclusion chromatography fractions identifies 5460 protein groups. Seven thousand five hundred sixty eight interactions among these proteins have been reconstructed by PrInCE algorithm, of which 2564 have been validated in independent datasets. NF-κB modulation leads to rearrangement of protein complexes involved in NF-κB signaling and immune response, cell cycle regulation, and DNA replication. Central NF-κB transcription regulator RELA co-elutes with interactors of NF-κB activator PRMT5, and these complexes are confirmed by AlphaPulldown prediction. A complementary immunoprecipitation experiment recapitulates RELA interactions with other NF-κB factors, associating NF-κB inhibition with lower binding of NF-κB activators to RELA. This study describes a network of pro-tumorigenic protein interactions and their rearrangement upon NF-κB inhibition with potential therapeutic implications in tumors with high NF-κB activity.

• Klíčová slova
• AlphaPullDown, NF-κB, RELA, breast cancer, interaction, protein complexes, protein correlation profiling, proteomics,
• MeSH
• karcinogeneze metabolismus   MeSH
• lidé MeSH
• mapování interakce mezi proteiny MeSH
• mapy interakcí proteinů * MeSH
• nádorové buněčné linie MeSH
• nádory prsu * metabolismus   patologie   MeSH
• NF-kappa B * metabolismus   MeSH
• proteinarginin-N-methyltransferasy metabolismus   MeSH
• signální transdukce MeSH
• transkripční faktor RelA * metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• NF-kappa B * MeSH
• proteinarginin-N-methyltransferasy MeSH
• RELA protein, human MeSH Prohlížeč
• transkripční faktor RelA * MeSH

BACKGROUND: Desmocollin-1 (DSC1) is a desmosomal transmembrane glycoprotein that maintains cell-to-cell adhesion. DSC1 was previously associated with lymph node metastasis of luminal A breast tumors and was found to increase migration and invasion of MCF7 cells in vitro. Therefore, we focused on DSC1 role in cellular and molecular mechanisms in luminal A breast cancer and its possible therapeutic modulation. METHODS: Western blotting was used to select potential inhibitor decreasing DSC1 protein level in MCF7 cell line. Using atomic force microscopy we evaluated effect of DSC1 overexpression and modulation on cell morphology. The LC-MS/MS analysis of total proteome on Orbitrap Lumos and RNA-Seq analysis of total transcriptome on Illumina NextSeq 500 were performed to study the molecular mechanisms associated with DSC1. Pull-down analysis with LC-MS/MS detection was carried out to uncover DSC1 protein interactome in MCF7 cells. RESULTS: Analysis of DSC1 protein levels in response to selected inhibitors displays significant DSC1 downregulation (p-value ≤ 0.01) in MCF7 cells treated with NF-κB inhibitor parthenolide. Analysis of mechanic cell properties in response to DSC1 overexpression and parthenolide treatment using atomic force microscopy reveals that DSC1 overexpression reduces height of MCF7 cells and conversely, parthenolide decreases cell stiffness of MCF7 cells overexpressing DSC1. The LC-MS/MS total proteome analysis in data-independent acquisition mode shows a strong connection between DSC1 overexpression and increased levels of proteins LACRT and IGFBP5, increased expression of IGFBP5 is confirmed by RNA-Seq. Pathway analysis of proteomics data uncovers enrichment of proliferative MCM_BIOCARTA pathway including CDK2 and MCM2-7 after DSC1 overexpression. Parthenolide decreases expression of LACRT, IGFBP5 and MCM_BIOCARTA pathway specifically in DSC1 overexpressing cells. Pull-down assay identifies DSC1 interactions with cadherin family proteins including DSG2, CDH1, CDH3 and tyrosine kinase receptors HER2 and HER3; parthenolide modulates DSC1-HER3 interaction. CONCLUSIONS: Our systems biology data indicate that DSC1 is connected to mechanisms of cell cycle regulation in luminal A breast cancer cells, and can be effectively modulated by parthenolide.

• Klíčová slova
• Breast cancer, DIA, DSC1, Metastasis, Proteomics, Pull-down,
• MeSH
• chromatografie kapalinová MeSH
• desmokoliny * metabolismus   MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• nádory * MeSH
• proteom MeSH
• seskviterpeny farmakologie   MeSH
• tandemová hmotnostní spektrometrie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• desmokoliny * MeSH
• DSC1 protein, human MeSH Prohlížeč
• parthenolide MeSH Prohlížeč
• proteom MeSH
• seskviterpeny MeSH

Renal cell carcinoma (RCC) represents about 2-3% of all cancers with over 400,000 new cases per year. Sunitinib, a vascular endothelial growth factor tyrosine kinase receptor inhibitor, has been used mainly for first-line treatment of metastatic clear-cell RCC with good or intermediate prognosis. However, about one-third of metastatic RCC patients do not respond to sunitinib, leading to disease progression. Here, we aim to find and characterize proteins associated with poor sunitinib response in a pilot proteomics study. Sixteen RCC tumors from patients responding (8) vs. non-responding (8) to sunitinib 3 months after treatment initiation were analyzed using data-independent acquisition mass spectrometry, together with their adjacent non-cancerous tissues. Proteomics analysis quantified 1996 protein groups (FDR = 0.01) and revealed 27 proteins deregulated between tumors non-responding vs. responding to sunitinib, representing a pattern of deregulated proteins potentially contributing to sunitinib resistance. Gene set enrichment analysis showed an up-regulation of epithelial-to-mesenchymal transition with transgelin as one of the most significantly abundant proteins. Transgelin expression was silenced by CRISPR/Cas9 and RNA interference, and the cells with reduced transgelin level exhibited significantly slower proliferation. Our data indicate that transgelin is an essential protein supporting RCC cell proliferation, which could contribute to intrinsic sunitinib resistance.

• Klíčová slova
• DIA-MS, mccRCC, resistance, sunitinib, transgelin,
• Publikační typ
• časopisecké články MeSH