EML4-ALK enhances programmed cell death-ligand 1 expression in pulmonary adenocarcinoma via hypoxia-inducible factor (HIF)-1 alpha and STAT3

Abstract

Programmed cell death (PD)-1/PD-1 ligand-1 (PD-L1)-targeted therapy has emerged as a promising therapeutic strategy for lung cancer. However, whether EML4-ALK regulates PD-L1 expression in lung cancer remains unknown. A total of 532 pulmonary adenocarcinomas (pADCs), including 58 ALK-translocated tumors, were immunohistochemically evaluated for PD-L1 and PD-1. H23 (EGFR(Wild-type)EML4-ALK(-)PD-L1(Low)) and H2228 (EGFR(Wild-type)EML4-ALK(+)PD-L1(High)) cells were transfected with EML4-ALK or ALK short interfering RNAs and used to investigate the alterations in PD-L1 expression. PD-L1 expression was detected in 81% of ALK-translocated pADCs; this value was significantly higher than those of pADCs with EGFR mutation, KRAS mutation or lacking ALK, EGFR or KRAS mutation (p<0.005 for all). Moreover, ALK-translocated pADC with PD-L1 expression showed significantly higher numbers of tumor-infiltrating PD-1(+) cells. ALK knockdown or inhibition (crizotinib treatment) in H2228 cells downregulated PD-L1 expression. Transfection of H23 cells with EML4-ALK enhanced PD-L1 expression, which was compromised by crizotinib treatment. This ALK-dependent upregulation of PD-L1 expression was mediated by STAT3 and hypoxia-inducible factor (HIF)-1 alpha under normoxia and hypoxia. Furthermore, EML4-ALK enhanced HIF-1 alpha expression through increasing transcription and decreasing ubiquitination of HIF-1 alpha. In ALK-translocated pADC tissues, significant positive correlations between PD-L1 and nuclear HIF-1 alpha (p < 0.05) or pSTAT3 expression levels (p<0.005) were observed. Among patients with ALK-translocated pADC, strong PD-L1 expression was significantly associated with shorter progression-free (p = 0.001) and overall survival (p = 0.002) after crizotinib treatment. Collectively, our findings demonstrate that ALK-derived pADCs increase PD-L1 expression via HIF-1 alpha and/or STAT3, thus providing a rationale for PD-1/PD-L1 pathway-targeted therapy in ALK-translocated lung cancer.