Abstract PO1-25-03: Programmed Death-Ligand 1 and Receptor Tyrosine Kinases in Breast Cancer

Abstract

Abstract Programmed death-ligand 1 (PD-L1) is an immune checkpoint expressed in a wide range of malignancies leading to immune tolerance and cancer cell immune evasion. Receptor tyrosine kinases (RTKs) are key regulators of cancer cell proliferation, survival, and invasion. The Hepatocyte Growth Factor (HGF) receptor, MET, and the Epidermal Growth Factor Receptor (EGFR) are RTKs known for their tumorigenic potential in a variety of human malignancies. This study aimed to evaluate the effect of the small-molecule tyrosine kinase inhibitors (TKIs) on the expression of PD-L1 in breast cancer cells and the effect of their combination with inflammatory cytokines. The study also assessed the association of the expression of the tumoral PD-L1 mRNA with each of MET and EGFR mRNA expression and the tumor features and treatment outcomes in breast cancer patients using the METABRIC dataset publicly available from cBioPortal for Cancer Genomics. The TKIs crizotinib [a MET inhibitor] and gefitinib [an EGFR inhibitor] were used as a single treatment and in combination with the cytokines; tumor necrosis factor-α (TNF-α) or interferon-γ (INF-γ) in MCF7 and MDA-MB-231 breast cancer cells in vitro. The cells were also treated with the mitogens HGF and EGF. The viability of cells after the different treatments was determined using the MTT viability assay. The effects of the combination treatments were analyzed using combination index (CI) analysis. The expression level of PD-L1 in cancer cells was assessed using Western blotting. The combination treatment of crizotinib with TNF-α and INF-γ produced a synergistic growth inhibition of MCF7 and MDA-MB-231 cells with CI values of 0.31 and 0.55, respectively. Alternatively, combined crizotinib and TNF-α produced an antagonist effect on the viability of MCF7 cells (CI=2.49). The combination of gefitinib with TNF-α produced a synergistic growth inhibition in both MCF7 and MDA-MB-231 cells with CI values of 0.39 and 0.78, respectively. Alternatively, gefitinib resulted in an additive effect when combined with INF-γ (CI=0.99) and an antagonistic effect when combined with TNF-α (CI=2.68) in MCF7 and MDA-MB-231 cells, respectively. Treatment with HGF (50 and 100 ng/ml) increased the protein levels of PD-L1 while EGF (50 and 100 ng/ml) reduced its levels in MDA-MB-231 cells. Treatment with the TKIs crizotinib (0.1-4 µM) and gefitinib (1-40 µM) significantly reduced PD-L1 levels in MDA-MB-231 cells compared to vehicle-treated cells. The analysis of the METABRIC dataset revealed that the mRNA expression of PD-L1 was positively correlated with the mRNA expression of the EGFR gene (r= 0.081, p< 0.001) but not the MET gene. A double-high PD-L1/MET expression was significantly associated with younger age at diagnosis, high-grade carcinoma, greater tumor size, hormone receptor-negative status, HER2-positivity, and non-luminal disease compared to patients with a double-low PD-L1/MET expression. Similar findings were reported for patients with a double-high PD-L1/EGFR expression compared to patients with a double-low PD-L1/EGFR expression. Nevertheless, the mRNA expression of the PD-L1, MET, and EGFR genes as well as the co-expression of PD-L1/MET or PD-L1/EGFR genes did not affect the overall survival of breast cancer patients. Collectively, MET and EGFR TKIs could modulate the effects of inflammatory cytokines differently based on the type of cytokine and the molecular subtype of breast cancer cells. The expression of PD-L1 in breast cancer cells was upregulated by HGF while TKIs reduced its expression. The co-expression of the PD-L1 gene with MET and EGFR genes in breast cancer was associated with advanced disease presentation and worse prognosticators in patients. Together, TKIs that target MET or EGFR could be an appealing therapeutic target in breast cancer, particularly in younger patients with the non-luminal disease. Citation Format: Nehad Ayoub, Muhsen Al-Diabat, Moath Al-Shorman, Laith Al-Eitan. Programmed Death-Ligand 1 and Receptor Tyrosine Kinases in Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-25-03.