The Effect Of TNFAIP3 On FGFR1 Signaling-induced Proliferation And Tumorigenesis Of The Ras-transformed Human Mammary Epithelial Cells

Objective:On the base of breast cancer cell lines expressing inducible FGFR1,the aim of this study is to investigate how FGFR1 activation promotes breast cancer cell growth by using breast cancer cell culture models and mouse models.The specific objectives include the following 3 aspects.(1)To investigate whether TNFAIP3 is regulated by FGFR1.(2)To analyze signal transduction mechanism involved in TNFAIP3 upregulation by FGFR1 activation.(3)To study the effects of FGFR1 activation induced TNFAIP3 upregulation on breast cancer cell proliferation and tumorigenesis.Methods:we generated DCIS-iFGFR1 cells expressing a truncated iFGFR1-fusion protein that was activated by AP20187 and DCIS-Ctrl cells with empty vectors from the mutant HRAS-transformed DCIS.com cells.With these cell lines,the following experiments were conducted:(1)Cells were treated with iFGFR1 agonist AP20187 alone or together with FGFR inhibitor LY284455 or AZD4547,mRNA and protein were extracted and q PCR method and western blot method was employed to examine TNFAIP3 expression.Through the above experiments,we determined whether TNFAIP3 was regulated by FGFR1.(2)Cells were treated with AP20187 alone or together with MEK inhibitor PD0325901 or ERK1/2 inhibitor GDC0994,cellular protein were extracted to analyze change of TNFAIP3.ERK1 and ERK2 gene were knockout respectively in DCIS-iFGFR1 cells by using CRISPR/CAS9 system.ERK knockout cells and control knockout cells were treated with AP20187,and cellular protein were extracted to determine change of TNFAIP3.These experiments were designed to examine which signaling pathway is responsible for FGFR1 induced TNFAIP3 upregulation.(3)Using CRISPR/CAS9 method,the TNFAIP3 was knocked out in DCIS-iFGFR1 cells.With TNFAIP3 knockout cells and control knockout cells,cell growth assays were performed in both 2D cell culture and 3D cell sphere culture.In vivo xenograft nude mice models also were established with these cells lines,and tumor growth were recorded.These experiments were designed to demonstrate the effect of TNFAIP3 in cancer cell growth upon FGFR1 activation.Rusults:(1)Activation of iFGFR1 robustly upregulates TNFAIP3 mRNA and protein in DCIS-iFGFR1 cells.(2)The upregulation of TNFAIP3 is depend on FGFR1 kinase and ERK1/2 activity,and knockout of ERK2 but not ERK1 completely inhibited this upregulation,suggesting that ERK2 mediates FGFR1-upregulated TNFAIP3 expression.(3)TNFAIP3 is required for the FGFR1signaling-induced growth of the Ras-transformed breast epithelial cells(DCIS.COM).Specifically,in both 2D cell culture and 3D mammosphere growth assays,activation of the FGFR1 signaling pathway significantlyincreased the growth of DCIS-i FGFR1 cells,while knockout of TNFAIP3 not only abolished these growths induced by the activated FGFR1 signaling but also further decreased these growths to much lower levels observed in DCIS-iFGFR1 cells without the activated FGFR1 signaling.Conclusions:(1)Activated FGFR1 signaling significantly upregulates TNFAIP3 expression through activating ERK2 MAPK in breast cancer cells;(2)TNFAIP3 is essential for both the FGFR1 signaling driven and the mutant Ras-stimulated breast cancer growth.