RESUMO
Perturbation of protein phosphorylation represents an attractive approach to cancer treatment. Besides kinase inhibitors, protein phosphatase inhibitors have been shown to have anti-cancer activity. A prime example is the small molecule LB-100, an inhibitor of protein phosphatases 2A/5 (PP2A/PP5), enzymes that affect cellular physiology. LB-100 has proven effective in pre-clinical models in combination with immunotherapy, but the molecular underpinnings of this synergy remain understood poorly. We report here a sensitivity of the mRNA splicing machinery to phosphorylation changes in response to LB-100 in colorectal adenocarcinoma. We observe enrichment for differentially phosphorylated sites within cancer-critical splicing nodes of U2 snRNP, SRSF and hnRNP proteins. Altered phosphorylation endows LB-100-treated colorectal adenocarcinoma cells with differential splicing patterns. In PP2A-inhibited cells, over 1000 events of exon skipping and intron retention affect regulators of genomic integrity. Finally, we show that LB-100-evoked alternative splicing leads to neoantigens that are presented by MHC class 1 at the cell surface. Our findings provide a potential explanation for the pre-clinical and clinical observations that LB-100 sensitizes cancer cells to immune checkpoint blockade.
Assuntos
Neoplasias do Colo , Splicing de RNA , Humanos , Processamento Alternativo/efeitos dos fármacos , Antígenos de Neoplasias/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/imunologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Inibidores Enzimáticos/farmacologia , Fosforilação , Proteína Fosfatase 2/metabolismo , Splicing de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Piperazinas/farmacologiaRESUMO
Deficiencies in the BRCA1 tumor suppressor gene are the main cause of hereditary breast and ovarian cancer. BRCA1 is involved in the Homologous Recombination DNA repair pathway and, together with BARD1, forms a heterodimer with ubiquitin E3 activity. The relevance of the BRCA1/BARD1 ubiquitin E3 activity for tumor suppression and DNA repair remains controversial. Here, we observe that the BRCA1/BARD1 ubiquitin E3 activity is not required for Homologous Recombination or resistance to Olaparib. Using TULIP2 methodology, which enables the direct identification of E3-specific ubiquitination substrates, we identify substrates for BRCA1/BARD1. We find that PCNA is ubiquitinated by BRCA1/BARD1 in unperturbed conditions independently of RAD18. PCNA ubiquitination by BRCA1/BARD1 avoids the formation of ssDNA gaps during DNA replication and promotes continuous DNA synthesis. These results provide additional insight about the importance of BRCA1/BARD1 E3 activity in Homologous Recombination.