Activation Of CXCL14/FGFR3 Promotes Small Cell Lung Cancer Invasion Via Up-regulating MMP2/9

Background:Small cell lung carcinoma(SCLC)is a highly malignant neuroendocrine neoplasm with poor prognosis.It accounts for about 15% of lung cancers,metastasis often occurs at the early stage or after chemotherapyin most patients.However,the precise molecular mechanism regulating SCLC invasion and metastasis is still unclear.C-X-C motif chemokine ligand-14(CXCL14),also known as BRAK,belongs to the CXC chemokine family and its receptor is still unknown.Previous studies have shown that CXCL14 plays a key role in regulating the infiltration of white blood cells into inflammatory tissues,as well as other biological processes related to immune surveillance and cancer progression.CXCL14 showed different expression trends in different tumors,showing a background dependent pro-tumor or anti-tumor effect.Fibroblast growth factor receptor(FGFRs)belongs to receptor tyrosine kinases.In tumors,FGFR gene amplification,fusion or exon missense mutations can cause FGFR protein overexpression and over-activation of signaling pathways.In our clinicopathological work,both CXCL14 and FGFR3 are highly expressed in small cell lung carcinoma and CXCL14 protein expression is closely related to the poor prognosis of SCLC patients.Therefore,we intend to study whether CXCL14/FGFR3 affects the invasion and migration of SCLC tumors.Objective:The purpose of this study was to investigate the ligand-receptor relationship of CXCL14/FGFR3 signal axis and its mechanism for regulating the development and invasion of SCLC,especially the mechanism by which this signal axis regulates the expression of MMPs family proteins,so as to provide a basis for further improving the potential therapeutic targets of small cell lung carcinoma.Methods:1.Immunohistochemistry was used to detect the expression of CXCL14,FGFRs,MMP2/9in tumor tissues of SCLC patients.2.The possible interaction sites between CXCL14 and FGFR3 were revealed by molecular simulation of protein interactions in The Molecular Operating Environment(MOE),and the Surface Plasmon Resonance(SPR)was used to determine the interaction and affinity of CXCL14 and FGFR3.3.Cell migration and invasion were evaluated by wound healing and transwell test.4.Western blot was used to detect the changes of various indicators of the protein level after CXCL14 overexpression and knockdown.And to detect the changes of various indicators of protein levels after the addition of recombinant CXCL14 protein to knockdown FGFR3 and the addition of recombinant CXCL14 to knockdown FGFR3.Results:1.Immunohistochemistry of 36 SCLC patients showed that CXCL14 was highly expressed in SCLC tumor tissues(29/36,81%).The survival time of CXCL14 positive patients was shorter than that of CXCL14 negative patients(P = 0.0360).In 36 SCLC specimens,FGFR3 was significantly over-expressed(24/36,67%)and the positive rate was much higher than FGFR1,FGFR 2,or FGFR4(25%,44%,8%).MMP2/9 immunohistochemical positive staining in tumor tissues of SCLC patients was higher.2.MOE’s protein-protein module was used to detect the binding site of CXCL14 to FGFR3 in the region of 477-590,and the affinity constant(KD)was 9.96e-8 by SPR count.3.Transwell and wound healing test results showed that CXCL14 silencing inhibited the migration and invasion of SCLC cells and CXCL14 overexpression promoted the migration and invasion of SCLC cells.In addition,exogenous addition of recombinant CXCL14 protein promoted SCLC cell invasion and migration and FGFR3 knockdown inhibited SCLC cell migration and invasion,while FGFR3 knockdown and recombinant CXCL14 protein also inhibited SCLC cell migration and invasion.4.Western blot assay showed that MMP2/9 of SCLC cells was down-regulated and TIMP1 was up-regulated after CXCL14 was knockdown.Overexpression of CXCL14 and recombinant CXCL14 up-regulated MM2/9 in SCLC cells and down-regulated TIMP1.FGFR3 knockdown down-regulated MMP2/9 in SCLC cells and up-regulated TIMP1,FGFR3 knockdown and recombinant CXCL14 down-regulated MMP2/9 in SCLC cells and up-regulated TIMP1.Conclusion:1.CXCL14 is highly expressed in tumors of SCLC patients and is associated with poor prognosis.2.In small cell lung carcinoma,FGFR3 is a receptor of CXCL4 and activation of the CXCL14/FGFR3 signaling axis plays a key role in the regulation of MMP2/9 expression in small cell lung carcinoma.3.Overactivation of the CXCL14/FGFR3 signal axis plays a key role in the invasion and migration of SCLC and other malignant biological behaviors,which may be a potential target for tumor therapy.