Stanniocalcin 1 Promotes Lung Metastasis Of Breast Cancer Through The PEGFR-pERK-S100A4 Pathway

BackgroudBreast cancer has become the most prevalent cancer worldwide,and lung metastasis from breast cancer is an important cause of poor prognosis and increased mortality of breast cancer,but its molecular mechanisms has not been clearly understood.Stanniocalcin 1(STC1)is a glycoprotein involved in a variety of physiological and pathological processes.In recent years,STC1 has been found to promote breast cancer metastasis,but its mechanism remains unclear.Studying the role and mechanism of STC1 in breast cancer lung metastasis is beneficial to gain insight into the mechanism of breast cancer metastasis and provide potential therapeutic targets for breast cancer treatment.Methods1.KM Plotter database and GEO data set were used to clarified the relationship between STC1 and prognosis of patients with lung metastasis of breast cancer.2.The expression of STC1 in lung metastatic breast cancer cell lines was detected by RT-qPCR and Western blot.3.Lentivirus infection was used to establish breast cancer cell lines with STC1 stable overexpression and downregulation.4.CCK8 proliferation assay,transwell migration and invasion assay and nude mouse model were used to detect the effect of STC1 on proliferation and metastasis of breast cancer cells.5.Immunohistochemistry was used to detect the effect of STC1 on the tumor microenvironment of breast cancer lung metastases.6.The effects of STC1 on HUVECs tubule formation and MRC5 migration were detected by tubule formation assay and transwell migration assay,respectively.7.The effect of STC1 on the expression of inflammatory cytokines in MRC5 cells was detected by RT-qPCR.8.RNA-seq,RT-qPCR,Western blot,ChIP-qPCR,IHC and other methods were used to explore the molecular mechanism of STC1 upstream and downstream.Results1.STC1 is associated with breast cancer lung metastasis.STC1 was highly expressed in lung metastatic breast cancer cell line.The higher expression of STC1 was associated with poorer overall survival in breast cancer patients(HR=1.53,95%CI:1.21～1.94,P=0.00035),distant metastasis-free survival(HR=1.35,95%CI:1.12～1.63,P=0.0016)and lung metastasis-free survival(HR=2.36,95%CI:1.23～4.38,P=0.0039).2.Established breast cancer cells with STC1 stable overexpression and knockdown.Successfully established breast cancer cells with STC1 stable overexpression and knockdown,which were named MDA-MB-231-EV,MDA-MB-231-STC1,LM2-shNC,and LM2-shSTC1,respectively.3.STC1 did not affect the proliferation of breast cancer cells.The recombinant protein STC1 did not affect the proliferation of breast cancer cells in vitro.The proliferation of MDA-MB-231-STC1 cells and MDA-MB-231-EV cells were comparable,and the proliferation of LM2-shSTC1 cells and LM2-shNC cells were not significantly different.In the Balb/c nude mouse model,there was no significant difference in the weight of tumors formed by MDA-MB-231-STC1 cells((0.41± 0.17)g)and those formed by MDA-MB-231-EV cells((0.31±0.14)g)(P>0.05).4.STC1 promoted migration and invasion of breast cancer cells.The recombinant protein STC1 treatment enhanced the migration and invasion of breast cancer cells in vitro(P<0.05).Compared with MDA-MB-231-EV cells,MDA-MB-231-STC1 cells had stronger migration and invasion abilities in vitro(P<0.05).Compared with LM2-shNC cells,LM2-shSTC1 cells had weaker migration and invasion abilities in vitro(P<0.05).5.STC1 promoted lung metastasis of breast cancer.In the nude mouse model of breast cancer lung metastasis,the number of lung tumor nodules formed by MDA-MB-231-STC1 cells(136.8±13.9)was higher than the number of lung tumor nodules formed by MDA-MB-231-EV cells(65.5±49.2)(P<0.05).The number of lung tumor nodules formed by LM2-shSTC1 cells(33.7 ±14.8)was lower than the number of lung tumor nodules formed by LM2-shNC cells(70.4±22.5)(P<0.05).6.The effect of STC1 on stromal cells in breast cancer lung metastases.The number and staining intensity of CD31 positive cells in lung metastases formed by breast cancer cells in MDA-MB-231-STC1 group were significantly higher than those in MDA-MB-231-EV group.The number and staining intensity of CD31 positive cells in lung metastases formed by breast cancer cells in the LM2-shSTC1 group were significantly lower than those in the LM2-shNC group.7.The effect of STC1 on angiogenesis.The recombinant protein STC1 did not affect the tube formation ability of HUVECs(P>0.05).Breast cancer cells pretreated with recombinant protein STC1 had stronger ability to promote tube formation of HUVECs(P<0.05).Breast cancer cells overexpressing STC1 had an enhanced ability to promote the tube formation of HUVECs(P<0.05),while breast cancer cells that knocked down STC1 had a reduced ability to promote the tube formation of HUVECs(P<0.05).8.The effect of STC1 on lung fibroblasts migration.The recombinant protein STC1 did not affect the migration of lung fibroblasts MRC5(P>0.05).Breast cancer cells pretreated with recombinant protein STC1 had stronger ability to promote the migration of MRC5 cells(P<0.05).Breast cancer cells overexpressing STC1 had an enhanced ability to promote the migration of MRC5 cells(P<0.05),while breast cancer cells that knocked down STC1 had a reduced ability to promote the migration of MRC5 cells(P<0.05).9.The effect of STC1 on inflammation of lung fibroblasts.The expression levels of pro-inflammatory cytokines IL1B,IL6 and IL8 in MRC5 cells co-cultured with lung metastatic breast cancer cells LM2 were significantly higher than those in MRC5 cells co-cultured with MDA-MB-231 cells(P<0.05).The recombinant protein STC1 did not affect the expression of pro-inflammatory factors IL1B,IL6 and IL8 in MRC5 cells(P>0.05).Breast cancer cells pretreated with recombinant protein STC1 had a stronger ability to promote the expression of pro-inflammatory factors in MRC5 cells(P<0.05).Breast cancer cells overexpressing STC1 had an enhanced ability to promote the expression of pro-inflammatory factors in MRC5 cells(P<0.05),while breast cancer cells that knocked down STC1 had a reduced ability to promote the expression of pro-inflammatory factors in MRC5 cells(P<0.05).10.STC1 promoted the expression of S100A4 in lung metastatic breast cancer.Transcriptome sequencing showed that the expression of S100A4 was higher in MDA-MB-231-STC1 cells than in MDA-MB-231-EV cells.RT-qPCR,Western blot and immunohistochemistry demonstrated that S100A4 was positively regulated by STC1.Mechanistically,STC1 upregulated the expression of S100A4 by promoting the phosphorylation of EGFR and its downstream ERK signaling pathway.11.S100A4 mediated the function of STC1.Knocking down the expression of S100A4 could reverse the effect of STC1 overexpression on promoting breast cancer cell invasion,and could reverse the effect of breast cancer cells overexpressing STC1 on vascular endothelial cells and lung fibroblasts.12.Expression of STC1 activated by JNK signaling pathway in lung metastatic breast cancer.The JNK signaling pathway was activated in lung metastatic breast cancer cells.After inhibition the JNK signaling pathway,the expression of STC1 was down-regulated.The transcription factor c-Jun downstream of the JNK signaling pathway could bind to the promoter region of STC1.Conclusion1.STC1 promoted lung metastasis of breast cancer cells.2.STC1 affected the tumor microenvironment by promoting angiogenesis and fibroblasts inflammation.3.The STC1-pEGFR-pERK-S100A4 signaling axis existed in lung metastatic breast cancer cells.4.Activated JNK signaling pathway in lung metastatic breast cancer cells upregulated the expression of STC1.