ArfGAP with the SH3 Domain, Ankyrin Repeat and PH Domain 1 Inversely Regulates Programmed Death-Ligand 1 Through Negative Feedback of Phosphorylated Epithelial Growth Factor Receptor and Activation of Nuclear Factor-Kappa B in Non-Small Cell Lung Cancer.

Background: Signaling pathways centered on the G-protein ADP-ribosylation factor 6 (Arf6) and its downstream effector ArfGAP with the SH3 Domain, Ankyrin Repeat and PH Domain 1 (AMAP1) drive cancer invasion, metastasis, and therapy resistance. The Arf6-AMAP1 pathway has been reported to promote receptor recycling leading to programmed cell death-ligand 1 (PD-L1) overexpression in pancreatic ductal carcinoma. Moreover, AMAP1 regulates of nuclear factor-kappa B (NF-κB), which is an important molecule in inflammation and immune activation, including tumor immune interaction through PD-L1 regulation. In this study, we investigated the function of AMAP1 on PD-L1 expression using lung cancer cells.

Methods: We used two non-small cell lung cancer cell lines. Protein expression was evaluated by Western blotting. AMAP1 and NF-kB expression were reduced by conventional siRNA methods, and osimertinib was used as an epithelial growth factor receptor (EGFR) inhibitor. Multiple analysis of receptor tyrosine kinases (RTKs) was conducted using a semi-comprehensive RTKs assay.

Results: We found that AMAP1 inversely regulated PD-L1 expression. Based on these results, we examined the activation levels of RTKs associated with both AMAP1 and PD-L1. Following a semi-comprehensive phosphorylated RTK assay, we observed the upregulation of phosphorylated EGFR (pEGFR) led by the downregulation of AMAP1. The inhibition of pEGFR by osimertinib downregulates PD-L1 expression. We investigated the relationships between AMAP1, NF-κB, and PD-L1 expression. AMAP1 knockdown upregulated the expression of both NF-κB and PD-L1. Subsequently, NF-κB knockdown downregulated PD-L1 levels, while double knockdown of AMAP1 and NF-κB, restored PD-L1 expression.

Conclusion: AMAP1 may inversely regulate PD-L1 through negative feedback of pEGFR and activation of NF-κB in NSCLC cell lines.