Investigation Of The Effect Of Epidermal Growth Factor On IRF-1 Expression In Lung Adenocarcinoma And Its Underlying Mechanism

Objective: Lung cancer is the leading cause of cancer death globally.Despite immunotherapy has brought new hope in the treatment of lung cancer,the responding rate in NSCLC patients with EGFR mutations remains at a low level.Few tumorinfiltrating T lymphocytes contribute to the lack of responsiveness to immunotherapy in patients with EGFR mutation.Chemotaxis is one of the important mechanisms affecting immune cells infiltrating tumors.It remains unknown whether EGFR signaling affects immune cell chemotaxis in NSCLC.Methods: The association between IRF-1 and chemotaxis in lung adenocarcinoma was analyzed using the TCGA-LUAD dataset.The expression levels of EGF in LUAD and normal lung tissues were assessed in TCGA and GEO databases.The EGF protein expression in LUAD was assessed by IHC.The expression levels of EGFR in A549,PC9 and H2030 cells was analyzed by flow cytometry.RT-PCR and Western Blot were used to determine the IRF-1 expression level.Overexpression of IRF-1 cell lines was constructed using lentiviral transduction.Cell proliferation was detected by CCK-8.U0126 and LY294002 were used to inhibit MAPK and PI3 K,respectively.Results: In the KEGG pathway analysis of IRF-1 related genes in the TCGA-LUAD dataset,chemotaxis reaction and its related pathways were highly enriched.The expression of EGF in LUAD was significantly higher than that in normal lung tissues in both TCGA-LUAD and GEO dataset.The expression of EGF protein was also detected in lung adenocarcinoma tissue samples.Flow cytometry results showed that EGFR receptor was expressed in A549,PC9 and H2030 cells.IRF-1 expression was significantly downregulated in A549,PC9 and H2030 cells after the treatment with EGF.Furthermore,EGF treatment led to the reduction of CXCL10 expression.IFN-γstimulation resulted in a significantly increased CXCL10 expression.Whereas IFN-γ in combination with EGF significantly inhibited CXCL10 expression.Overexpression of IRF-1,the basal expression of CXCL10 in lung adenocarcinoma cells was upregulated compared to IRF-1 wild type cells.RAS-RAF-MEK-ERK and PI3K-AKT pathways were activated by EGF in A549,PC9,and H2030.The mechanistic analysis showed that inhibition of the RAS-RAF-MEK-ERK pathway and PI3K-AKT pathway by using U0126 and LY294002 respectively,did not affect EGF-induced downregulation of IRF-1.Additionally,we found that EGF promoted the proliferation of A549 and PC9 cells.EGF also upregulated cyclin D1 expression.However,overexpression of IRF-1abrogated EGF induced cyclin D1 expression.Conclusions: EGF downregulated IRF-1 expression thereby inhibiting the production of CXCL10.EGFR signal also reduced the upregulation effect of IFN-γ on IRF-1 and subsequently downregulating CXCL10.Mechanistic analysis revealed that the regulatory effect of EGF on IRF-1 expression was independent of PI3K-AKT and RASRAF-MEK-ERK pathways.Moreover,EGF promoted the proliferation of tumor cells.EGF upregulated the expression of cyclin D1.While IRF-1 overexpression attenuated EGF induced cyclin D1 expression.