EML4-ALK enhances programmed cell death-ligand 1 expression in pulmonary adenocarcinoma via hypoxia-inducible factor (HIF)-1α 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 <i>ALK</i>-translocated tumors, were immunohistochemically evaluated for PD-L1 and PD-1. H23 (<i>EGFR</i>^Wild-type<i>EML4-ALK</i>^-PD-L1^Low) and H2228 (<i>EGFR</i>^Wild-type<i>EML4-ALK</i>⁺PD-L1^High) cells were transfected with <i>EML4-ALK</i> or ALK short interfering RNAs and used to investigate the alterations in PD-L1 expression. PD-L1 expression was detected in 81% of <i>ALK</i>-translocated pADCs; this value was significantly higher than those of pADCs with <i>EGFR</i> mutation, <i>KRAS</i> mutation or lacking <i>ALK, EGFR</i> or <i>KRAS</i> mutation (<i>p</i> <0.005 for all). Moreover, <i>ALK</i>-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 <i>EML4-ALK</i> 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)-1α under normoxia and hypoxia. Furthermore, EML4-ALK enhanced HIF-1α expression through increasing transcription and decreasing ubiquitination of HIF-1α. In <i>ALK</i>-translocated pADC tissues, significant positive correlations between PD-L1 and nuclear HIF-1α (<i>p</i> < 0.05) or pSTAT3 expression levels (<i>p</i><0.005) were observed. Among patients with <i>ALK</i>-translocated pADC, strong PD-L1 expression was significantly associated with shorter progression-free (<i>p</i> = 0.001) and overall survival (<i>p</i> = 0.002) after crizotinib treatment. Collectively, our findings demonstrate that <i>ALK-</i>derived pADCs increase PD-L1 expression via HIF-1α and/or STAT3, thus providing a rationale for PD-1/PD-L1 pathway-targeted therapy in <i>ALK</i>-translocated lung cancer.