The Research Of B7-H4 Expression Regulation

Emerging anti-cancer immunotherapies blocking immune checkpoints such as PD-1,PD-L1 and CTLA-4 have shown promising responses in a variety of malignancies.B7-H4 is another less known immune checkpoint that widely expressed on tumor cells,infiltrating myeloid cells and vessels.The apparently T cell-inhibitory function,coupled with the demonstration of enhanced expression on human tumors,indicates B7-H4 has the potential to be another useful target in tumor immunotherapy.However,the stimuli that induce B7-H4 expression are not well characterized.Up to now it is known that IL-6 and IL-10 take part in the B7-H4 expression stimulation.Our study showed that IGF-1 and EGF significantly enhanced B7-H4 protein level in various cell lines,including A549,SPC-A-1,MCF-7 and B7-H4 transgene cells.As Akt and ERK are the common molecules downstream of IGF-1 and EGF pathways,we investigated which molecule mediated IGF-1 and EGF-induced B7-H4 expression.The specific inhibitors targeting signal molecules were used to treat cells.The result showed that LY294002 and Wortmannin,the specific AKT pathway inhibitors,did not alter B7-H4 expression in A549 and SPC-A-1 cell.However,SCH772984 which is the specific inhibitor of ERK,downregulated B7-H4 protein level in A549 and SPC-A-1 cells.To further verify the involvement of MAPK/ERK pathway in B7-H4 expression modulation,rapamycin(the specific inhibitor of mTOR)and BI-D1870(the specific inhibitor of p90RSK)are used to treat cells.The result showed that both rapamycin and BI-D1870 inhibited B7-H4 expression in A549 and SPC-A-1 cells.Taken together,our study verified that B7-H4 expression was modulated by MAPK/ERK pathway.The paper includes the following two parts:Part ?Studies on the relationship between the negative costimulatory molecular B7-H4 and two singal pathways that are PI3K/Akt and MAPK/ERKObjective: Whether PI3K/Akt signaling pathway or MAPK/ERK signaling pathway regulate the expression of B7-H4.Methods: After 24 hours of IGF-1 stimulation in A549,SPC-A-1,MCF-7,H08910,hela and B7-H4 transgenic cells,extract total proteins then detect B7-H4 protein by western blot.Using real-time PCR detect B7-H4 mRNA after 24 hours of IGF-1 stimulation in A549 and MCF-7 cells.After 24 hours of IGF-1 and EGF stimulation in A549 and MCF-7 respectively,extract total proteins then detect B7-H4 protein,molecules participating in PI3K/Akt and MAPK/ERK signaling pathways by western blot.Results: IGF-1 can up-regulate the expression of B7-H4 protein in A549,SPC-A-1,MCF-7,H08910,hela and B7-H4 transgenic cells.B7-H4 mRNA is no change after IGF-1 stimulation in A549 and MCF-7 cells.IGF-1 and EGF can up-regulate the expression of p-Akt and p-ERK in MCF7 and A549 cells.Conclusion: PI3K/Akt signaling pathway or MAPK/ERK signaling pathway may regulate the expression of B7-H4 protein.Part? MAPK/ERK signal pathway regulates the expression of B7-H4 protein in lung cancer cellsObjective: To confirm which signaling pathway regulates the expression of B7-H4 protein.Methods: After 24 hours of LY294002 and Wortmannin that are specific inhibitors of Akt signaling pathway stimulation,extract total proteins then detect B7-H4 protein by western blot in A549 and SPC-A-1 cells.SCH772984 that is the specific inhibitor of ERK signaling pathway,rapamycin that is the mTOR specific inhibitor and BI-D1870 that is the p90 RSK specific inhibitor do the same.After the 24 hours of EGF or PMA stimulation in SPC-A-1 cells,extract the total proteins then detect the expression of B7-H4,p-ERK and p-p90 RSK by western blot.After 24 hours of inhibitors of ERK signal pathway before 1 hour of EGF stimulation in SPC-A-1 cells,extract the total proteins then detect the expression of B7-H4,p-ERK and p-p90 RSK by western blot,and detect the membrane B7-H4 by flow cytometry.Results: B7-H4 protein is increase after the stimulation of LY294002 and Wortmannin in A549 and SPC-A-1 cells.SCH772984,rapamycin and BI-D1870 down regulate the expression of B7-H4 protein in A549 and SPC-A-1 cells.EGF or PMA can up-regulate the expression of B7-H4,but EGF together with inhibitors of ERK signal pathway down regulate.The mebrane B7-H4 is increase after EGF stimulation in SPC-A-1 cells,EGF together with SCH772984 is opposite.Conclusion: MAPK/ERK signaling pathway can regulate the expression of B7-H4 protein in lung cancer cells.