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ArfGAP with the SH3 Domain, Ankyrin Repeat and PH Domain 1 Inversely Regulates Programmed Death-Ligand 1 Through

Naohisa Chiba¹, Toshi Menju¹, Yumeta Shimazu¹

¹Department of Thoracic Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

Cancer Management and Research

|January 27, 2025

Abstract

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.

Insights

ADP-ribosylation factor 6 (Arf6)-associated protein 1 (AMAP1) inversely regulates programmed cell death-ligand 1 (PD-L1) in lung cancer. AMAP1 downregulation increases PD-L1 via EGFR and NF-κB signaling.

Area of Science:

Oncology
Molecular Biology
Cancer Signaling

Background:

The Arf6-AMAP1 pathway is implicated in cancer progression, influencing invasion, metastasis, and therapy resistance.
AMAP1 promotes PD-L1 overexpression in pancreatic cancer via receptor recycling and regulates NF-κB, impacting tumor immunity.
This study investigates the role of AMAP1 in regulating PD-L1 expression within lung cancer models.

Purpose of the Study:

To elucidate the functional relationship between AMAP1 and PD-L1 expression in non-small cell lung cancer (NSCLC).
To explore the underlying molecular mechanisms involving receptor tyrosine kinases (RTKs) and NF-κB signaling.

Main Methods:

Utilized two NSCLC cell lines for experimental analysis.
Employed siRNA to knockdown AMAP1 and NF-κB expression.
Investigated the effects of osimertinib, an EGFR inhibitor, on PD-L1 levels.
Assessed RTK activation using a semi-comprehensive RTKs assay and protein expression via Western blotting.

Main Results:

AMAP1 knockdown led to increased PD-L1 expression, suggesting an inverse regulatory relationship.
Downregulation of AMAP1 resulted in increased phosphorylated EGFR (pEGFR) levels.
Osimertinib treatment inhibited pEGFR and consequently reduced PD-L1 expression.
AMAP1 knockdown upregulated both NF-κB and PD-L1; NF-κB knockdown decreased PD-L1, while combined knockdown restored PD-L1 levels.

Conclusions:

AMAP1 inversely regulates PD-L1 expression in NSCLC cell lines.
This regulation appears to involve a negative feedback loop through pEGFR and activation of NF-κB signaling.

Read full analysis

Keywords:

AMAP1 NF-κB PD-L1 pEGFR

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