Study On Regulation Of GABRP On Stemness Of Triple-Negative Breast Cancer

Objective:Due to the lack of specific therapeutic targets,there are relatively few effective treatment options for Triple negative breast cancer(TNBC).Epidermal growth factor receptor(EGFR)signaling is highly active in TNBC and is associated with poor prognosis.Most EGFR antagonists,which significantly improve outcome in lung and colon cancer,have shown limited clinical effects in breast cancer.Therefore,exploring specific genes that are highly expressed in TNBC and involved in the regulation of EGFR expression and targeting them as therapeutic targets can effectively increase the sensitivity of TNBC to EGFR antagonists.Methods:1)Breast cancer gene-expression minerV4.5(http://begenex.ico.unicancer.fr)was used to screen out gene sets related to EGFR,and the TCGA clinical data set(BRCA.Merged_only_clinical_clin_format.TXT)was used to screen out gene sets highly expressed in TNBC,then the two gene sets were intersected to obtain genes that were both related to EGFR and highly expressed in TNBC;2)GO analysis of gamma-aminobutyric Acid Type A Receptor Pi Subunit(GABRP)related genes was performed using Breast cancer gene-expression Miner V4.5;3)GABRP knockdown stable cell lines were constructed by lentivirus transfection;4)Real-time fluorescence quantitative polymerase chain reaction(Q-PCR)and Western blot were used to detect knockdown efficiency;5)Q-PCR,colony formation assay,and cell flow cytometry were used to verify whether GABRP affected TNBC cell stemness;6)MTS cell number assay,Ki67 immunofluorescence assay,and xenograph assay were used to verify whether GABRP affected the proliferation ability of TNBC cells;7)TCGA database was used to detect the expression abundance of GABRP and GABA-related genes,and MTS assay was used to detect whether GABRP regulated TNBC cells through the same signaling axis of GABA-GABA A receptor.8)Western blot,Co-IP,immunofluorescence,cell flow cytometry,and Q-PCR were used to test the correlation between EGFR and GABRP.And Western blot was used to detect the effect of GABRP silencing on c-Myc;9)UALCAN(http://ualcan.path.uab.edu)database was used to test GABRP abundance expressed in various types of tumors,and MTS assay was used to test whether retigabine influence sensitivity of TNBC to gefitinib.Results:1)GABRP is highly expressed in TNBC,which is closely related to EGFR expression.2)Further analysis of GABRP related genes suggested that GABRP was closely related to stemness maintenance and proliferation of breast cancer.3)GABRP silencing inhibited the stemness and proliferation of TNBC.4)GABRP does not depend on GABA-GABA A receptor pathway to regulate TNBC.5)GABRP interacted with EGFR and maintained the expression of EGFR by inhibiting the protein degradation of EGFR and promoting EGFR transcription.6)GABRP induced resistance to gefitinib in TNBC patients,and retegabine was beneficial to increase the sensitivity of TNBC to gefitinibConclusion:Our findings show that GABRP sustained the stemness of TNBC via modulating EGFR expression,suggesting that GABRP may be a potential therapeutic target that address EGFR inhibitor resistance in TNBC.