Cancer Res Treat.  2023 Oct;55(4):1134-1143. 10.4143/crt.2023.311.

Targeting CD73 to Overcomes Resistance to First-Generation EGFR Tyrosine Kinase Inhibitors in Non–Small Cell Lung Cancer

• Kim M
• Kim S
• Yim J
• Keam B
• Kim TM
• Jeon YK
• Kim DW
• Heo DS

• KMID: 2547788
• DOI: http://doi.org/10.4143/crt.2023.311

Abstract

Purpose
In patients with epidermal growth factor receptor (EGFR)-mutant non–small cell lung cancer (NSCLC), EGFR tyrosine kinase inhibitors (TKIs) improve response rate and survival. However, most patients eventually develop resistance. This study aimed to identify the role of CD73 in EGFR-mutant NSCLC and explore whether CD73 inhibition may serve as a therapeutic strategy in NSCLC patients with acquired resistance to EGFR-TKIs.
Materials and Methods
We evaluated the prognostic role of CD73 expression in EGFR-mutant NSCLC using tumor samples from a single institution. We silenced CD73 in EGFR-TKI–resistant cell lines using short hairpin RNA (shRNA) targeting CD73 and also transfected a vector alone as a negative control. Using these cell lines, cell proliferation and viability assays, immunoblot assays, cell cycle analysis, colony-forming assays, flow cytometry, and apoptosis analysis were performed.
Results
High expression of CD73 was associated with shorter survival in patients with metastatic EGFR-mutant NSCLC treated with first-generation EGFR-TKI. CD73 inhibition synergistically inhibited cell viability with first-generation EGFR-TKI treatment compared with the negative control. When CD73 inhibition and EGFR-TKI treatment were combined, G0/G1 cell cycle arrest was induced through the regulation of p21 and cyclin D1. In addition, the apoptosis rate was increased in CD73 shRNA-transfected cells treated with EGFR-TKI.
Conclusion
High expression of CD73 adversely affects the survival of patients with EGFR-mutant NSCLC. The study demonstrated that inhibiting CD73 in EGFR-TKI–resistant cell lines resulted in increased apoptosis and cell cycle arrest, which overcame the acquired resistance to first-generation EGFR-TKIs. Further research is needed to determine whether blocking CD73 plays a therapeutic role in EGFR-TKI–resistant patients with EGFR-mutant NSCLC.

Keyword

Non-small cell lung carcinoma; ErbB receptors; CD73; Tyrosine kinase inhibitor; Resistance

Figure

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  Fig. 1 Kaplan-Meier curve for overall survival of progression-free survival (A) and overall survival (B) of patients with metastatic epidermal growth factor receptor (EGFR)–mutant non–small cell lung cancer treated with EGFR tyrosine kinase inhibitor.

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  Fig. 2 Representative images of CD73 immunohistochemistry in non–small cell lung cancer patients. (A) Changes of CD73 expression before and after resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) (n=26). (B) Representative immunohistochemistry staining (×200) of CD73 protein expression before and after EGFR-TKI in the same patient.

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  Fig. 3 The shCD73 transduced lung cancer cell lines are more sensitive to epidermal growth factor receptor (EGFR) inhibitors than parental lung cancer cell lines. (A) Cell viability assay with various erlotinib and gefitinib concentrations in the shCD73 group and negative control (NC) group of PC9ER and PC9GR cell line. (B) Long-term colony-forming assay. ShCD73 group and NC group cells treated with control (DMSO) and gefitinib or erlotinib were stained by Coomassie brilliant Blue-R250 on day 14. (C) Cells were treated with EGFR-TKI for 6 hours and harvested cell lysates were analyzed by immunoblot assay for proteins involved in the mitogen-activated protein kinase pathway. All experiments were performed in three independent replicates and IC50 values represent the mean±standard deviation. *p < 0.001. E, erlotinib; G, gefitinib; NT, no treatment; 0.1, 100 nmol/L; 1, 1 μmol/L.

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  Fig. 4 CD73 can synergistic inhibition with epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) to lung cancer cell lines. (A) Cell cycle progression of the CD73 group and negative control (NC) group in PC9–EGFR-TKI–resistant cell lines were analyzed after drug treatment by flow cytometry. (B) The sub-G1 fractions were analyzed by flow cytometry. The graphs shown represent results from three independent experiments. (C) Cells were treated with EGFR-TKI or vehicle for 24 hours and harvested cell lysates were analyzed by immunoblot assay for cell cycle–related proteins. (D) Representative dot plots of Annexin V–propidium iodide (PI) staining. E, erlotinib; G, gefitinib; NT, no treatment; 0.1, 100 nmol/L; 1, 1 μmol/L.

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