A2AR-phospho-STAT1 (Y701)-HLA-E axis as a potential immune modulatory pathway in radiotherapy-resistant triple negative breast cancer

Triple-negative breast cancer (TNBC) patients have lower survival rates and higher recurrence risks than non-TNBC patients. Moreover, radiotherapy-resistant TNBC (RT-R-TNBC) exhibits enhanced chemotherapy resistance and invasiveness. Therefore, there is a critical need for innovative treatments for RT-R-TNBC and TNBC patients. Our previous study indicated that NK cells exhibit reduced cytotoxicity against RT-R-TNBCs due to human leukocyte antigen class I histocompatibility antigen, alpha chain E (HLA-E) upregulation. Thus, this study aimed to identify the mechanism responsible for the upregulation of HLA-E and suggest potential therapeutic targets for overcoming the RT-resistance of TNBC. We found that HLA-E expression was significantly higher in TNBC tumor tissues than in normal epithelial tissues and non-TNBC tissues, correlating with A2AR levels. In addition, MDA-MB-231 (TNBC) and RT-R-MDA-MB-231 (RT-R-TNBC) showed an A2AR-dependent HLA-E overexpression. NK cell-mediated cytotoxicity against MDA-MB-231 and RT-R-MDA-MB-231 was reduced and restored by A2AR or STAT1 knockdown. Interestingly, STAT1 phosphorylation (Y701) by adenosine (ADO) aligned with the HLA-E expression pattern by ADO, and fludarabine, a STAT1 inhibitor, effectively reduced phospho-STAT1 (Y701) levels but not phospho-STAT1 (S727) levels. Fludarabine also inhibited ADO-induced HLA-E expression in MDA-MB-231 and RT-R-MDA-MB-231, including basal HLA-E expression in RT-R-MDA-MB-231. Additionally, fludarabine reduced tumor progression, lung metastasis, HLA-E expression, and phospho-STAT1 (Y701) in RT-R-MDA-MB-231-injected mice. Moreover, monalizumab, an NKG2A monoclonal antibody, significantly reduced tumor progression and lung metastasis with increased population of cytotoxic NK cells (CD25 + NK1.1+ and CD69 + NK1.1+) in the inguinal lymph nodes of RT-R-MDA-MB-231-injected mice. This study suggests that the A2AR-phospho-STAT1 (Y701)-HLA-E axis may serve as an alternative target for overcoming RT-resistance in TNBC.