Bcl-2-associated Transcription Factor 1 Phosphorylation Mediated DNA Damage Response in Gastric Cancer

Abstract

Background: DNA damage response plays critical roles in tumor pathogenesis and radiotherapy resistance. Various cellular activities including the initiation and major steps DNA damage response are regulated by protein phosphorylation, whereas the molecular mechanism remains largely unknown. Methods: A quick label-free phosphoproteomics using high-resolution mass spectrometry and an open search approach was applied to paired tumor and adjacent tissues from five patients with gastric cancer. The dysregulated phosphoproteins were identified and their associated-pathways analyzed using Gene Set Enrichment Analysis (GSEA). The mostly regulated phosphoproteins and their potential functions were validated by the specific antibodies against the phosphorylation sites. Specific protein phosphorylation was further analyzed by functional and clinical approaches. Results: 832 gastric cancer-associated unique phosphorylated sites were identified, among which 25 were up- and 52 down-regulated. Markedly, the dysregulated phosphoproteins were primarily enriched in DNA-damage-response-associated pathways. Particularly, the phosphorylation of Bcl-2-associated transcription factor 1 (BCLAF1) at Ser290 was significantly upregulated in tumor. The upregulation of BCLAF1 Ser290 phosphorylation (pBCLAF1(Ser290)) in tumor was confirmed by tissue microarray studies and further indicated in association with poor prognosis of gastric cancer patients. Eliminating the phosphorylation of BCLAF1 at Ser290 suppressed gastric cancer (GC) cell proliferation. Upregulation of pBCLAF1(Ser290) was found in association with irradiation-induced γ-H2AX expression in the nucleus, leading to an increased DNA damage repair response, and a marked inhibition of irradiation-induced cancer cell apoptosis. Conclusions: The phosphorylation of BCLAF1 at Ser290 is involved in the regulation of DNA damage response, indicating an important target for the resistance of radiotherapy.