Journal Article

Epithelial-to-mesenchymal transition and NF-kB pathways are promoted by a mutant form of DDB2, unable to bind PCNA, in UV-damaged human cells

Bassi, Elisabetta, Tricarico, Anna, Stivala, Lucia Anna, Cazzalini, Ornella, Perucca, Paola, Prosperi, Ennio, Vetro, Martina

2024

Overview

Background DNA-Damaged Binding protein 2 (DDB2) is a protein involved in the early step of Nucleotide Excision Repair. Recently, it has been reported that DDB2 is involved in epithelial-to-mesenchymal transition (EMT), key process in tumour invasiveness and metastasis formation. However, its role is not completely known. Methods Boyden chamber and cell adhesion assays, and ICELLigence analysis were performed to detect HEK293 adhesion and invasion. Western blotting and gelatine zymography techniques were employed to assess the EMT protein levels and MMP enzymatic activity. Immunofluorescence analysis and pull-down assays facilitated the detection of NF-kB sub-cellular localization and interaction. Results We have previously demonstrated that the loss of DDB2-PCNA binding favours genome instability, and increases cell proliferation and motility. Here, we have investigated the phenotypic and molecular EMT-like changes after UV DNA damage, in HEK293 clones stably expressing DDB2 Wt protein or a mutant form unable to interact with PCNA (DDB2 PCNA− ), as well as in HeLa cells transiently expressing the same DDB2 constructs. Cells expressing DDB2 PCNA− showed morphological modifications along with a reduced expression of E-cadherin, an increased activity of MMP-9 and an improved ability to migrate, in concomitance with a significant upregulation of EMT-associated Transcription Factors (TFs), whose expression has been reported to favour tumour invasion. We observed a higher expression of c-Myc oncogene, NF-kB, both regulating cell proliferation and metastatic process, as well as ZEB1, a TF significantly associated with tumorigenic potential and cell migratory ability. Interestingly, a novel interaction of DDB2 with NF-kB was detected and found to be increased in cells expressing the DDB2 PCNA− , suggesting a direct modulation of NF-kB by DDB2. Conclusion These results highlight the role of DDB2-PCNA interaction in counteracting EMT since DDB2 PCNA− protein induces in HEK293 transformed cells a gain of function contributing to the acquisition of a more aggressive phenotype.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Analysis / Antibodies / Biomedical and Life Sciences / Biomedicine / Biotechnology / Boyden chamber / Breast cancer / c-Myc protein / Cancer Research / Cell Adhesion / Cell Movement / Cell Proliferation / Cells / Cloning / DNA binding proteins / DNA Damage / DNA repair / DNA-Binding Proteins - genetics / DNA-Binding Proteins - metabolism / DNA-Damaged binding protein 2 / E-cadherin / Emt protein / EMT-TFs / Enzymatic activity / Enzymes / Epithelial-Mesenchymal Transition / Gelatinase B / Genomic instability / Health aspects / Health Promotion and Disease Prevention / HEK293 Cells / HeLa Cells / Humans / Immunofluorescence / Invasion / Invasiveness / Localization / Medicine/Public Health / Metastases / Metastasis / Morphology / Motility / Mutants / Mutation / Myc protein / NF-kappa B - metabolism / NF-κB protein / Nucleotide excision repair / Oncology / p-EMT / Penicillin / Phenotypes / Proliferating cell nuclear antigen / Proliferating Cell Nuclear Antigen - metabolism / Protein Binding / Proteins / Scientific equipment and supplies industry / Signal Transduction / Stem cells / Surgical Oncology / Transcription factors / Transformed cells / Tumors / Ultraviolet Rays - adverse effects / Western blotting