Functional characterisation of p53 mutants identified in breast cancers with suboptimal responses to anthracyclines or mitomycin

• Published in: Biochimica et biophysica acta Vol. 1830; no. 3; pp. 2790 - 2797
• Main Authors: Berge, Elisabet O., Huun, Johanna, Lillehaug, Johan R., Lønning, Per E., Knappskog, Stian
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2013
• Subjects: anthracyclines; Anthracyclines - pharmacology; Antibiotics, Antineoplastic - pharmacology; apoptosis; Apoptosis - drug effects; beta-galactosidase; binding capacity; Biopsy; Breast; breast neoplasms; Breast Neoplasms - drug therapy; Breast Neoplasms - genetics; Breast Neoplasms - metabolism; Breast Neoplasms - pathology; Cancer; Cell Line, Tumor; Cell Proliferation - drug effects; Cell Survival - drug effects; Drug Resistance, Neoplasm - drug effects; Drug Resistance, Neoplasm - genetics; Female; fluorescent antibody technique; Gene Expression Regulation, Neoplastic - drug effects; genes; Humans; metastasis; mitomycin; Mitomycin - pharmacology; mutants; Mutation; p53; patients; Plasmids; precipitin tests; Protein Binding; Protein Multimerization; Protein Structure, Tertiary; proteins; quantitative polymerase chain reaction; Resistance; Structure-Activity Relationship; Transcription, Genetic - drug effects; Transfection; Tumor Suppressor Protein p53 - genetics; Tumor Suppressor Protein p53 - metabolism; variants; PR; HRP; DEPC; WST-1; p53; Resistance; BSA; SD; PD; NLS; FBS; Breast; Mutation; SDS-PAG; Cancer
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2012.12.004

Approximately 4300 different TP53 mutations have been reported in human cancers. TP53 mutations, in particular those affecting the L2/L3 domains, are associated with resistance to anthracycline or mitomycin treatment in breast cancer patients. While many mutations have been characterised functionally, novel TP53 mutations are continuously reported. Here, we characterise 10 p53 protein variants encoded by mutated TP53 (5 within and 5 outside L2/L3) detected in locally advanced or metastatic breast cancers. Each tumour was previously characterised for response to therapy, allowing comparison between in vivo and in vitro findings. Mutated p53 variants were analysed for their ability to oligomerise with the wild-type protein and their subcellular localisation by immunoprecipitation and immunofluorescence, respectively. Their ability to induce transcription of target genes was determined by qPCR. Cellular growth rate, apoptosis and senescence were monitored by WST-1, TUNEL and beta-galactosidase assays, respectively. Immunoprecipitation assays revealed each mutant protein to retain binding capacity for wild-type p53, thus potentially acting in a dominant negative manner. Even though each p53 variant located predominantly in the nucleus, the percentage of cells with only nuclear p53 localisation varied between 60% and 90%. None of the p53 variants were able to induce target genes to levels similar to wild-type p53, nor where they able to reduce cellular growth rate, induce apoptosis or senescence similar to wild-type p53 after anthracycline treatment in vitro. All the 10 variants studied displayed inferior p53 functionality compared to the wild-type protein. Our data add further information characterising the effects of somatic TP53 mutations on p53 protein function and anthracycline resistance in breast cancer. ► 10 p53 variants encoded by mutated TP53 in breast cancers were characterised. ► Each tumour was characterised for response to anthracycline or mitomycin therapy. ► All mutants bound wild-type p53, potentially acting in a dominant negative manner. ► All mutants showed reduced induction of apoptosis/senescence ► P53 mutations both within and outside the L2/L3 domains may cause treatment failure.