Novel Role Of STYK1-SPINT2 Signaling Pathway In Regulating Tumor Growth And Metastasis Of Non-Small Cell Lung Cancer

Background:Lung cancer is the most frequently diagnosed cancer with the highest morbidity and mortality in China and worldwide.Non-small cell lung cancer(NSCLC)is the main pathological type of lung cancer accounting for about 87%,and the 5-year survival rate of Chinese lung cancer patients is just 16.1%.Therefore,NSCLC has become the most serious malignancy in China,and it is important to explore the molecular mechanism of NSCLC progression and to find the potential therapeutic target for NSCLC.Serine threonine tyrosine kinase 1(STYK1)was found as a oncoprotein in 2003 and identified as a new member of the receptor protein tyrosine kinase(RTK)-like protein family.RTKs are involved in the progression of NSCLC tumors,and a variety of RTKs targeted inhibitors were applied in the clinical treatment of NSCLC.Previous studies reported that STYK1 overexpression enhanced the abilities of proliferation and metastasis in the mouse Ba F3 cells.Moreover,several studies found STYK1 was elevated in the tissues of human colon cancer and liver cancer,and high STYK1 expression was correlated with poor prognosis of these cancers.However,the function and mechanisms of STYK1 in mediating NSCLC progression remains unclear.Thus,NSCLC cell lines and nude bearing mice models will be used to investigate the underlining mechanisms of STYK1 in regulating NSCLC progression both in vivo and in vitro.In the meantime,we will comprehensively analyze the correlation between these signaling and clinical prognosis using NSCLC patient’s tissues.Above all,this study will provide new anti-cancer method and theoretical basis for targeting STYK1 during NSCLC treatment.Objectives:1.To investigate the expression of STYK1 in NSCLC tissues and the correlation of STYK1 expression and the survival prognosis of NSCLC patients.2.To investigate the role of STYK1 in mediating the abilities of proliferation,metastasis and epithelial-mesenchymal transition(EMT)in NSCLC cells.3.To further explore the downstream signaling of STYK1,and to investigate whether SPINT2 is involved in the progression of NSCLC.4.To investigate the expression of SPINT2 in NSCLC tissues and the correlation of SPINT2 expression and the survival prognosis of NSCLC patients.Moreover,to further evaluate the NSCLC prognosis via combining the expression STYK1 and SPINT2.Methods:1.The public cancer bioinformatics databases were used to analyze the expression of STYK1 gene in NSCLC tissues and its role on the prognosis of NSCLC patients.Immunohistochemistry(IHC)was applied to detect the STYK1 expression in 347 pairs of NSCLC tissues and corresponding adjacent noncancerous tissues.Later,we further analyzed the correlation of STYK1 expression and clinicopathological parameters or the survival prognosis of NSCLC patients.2.We established the stable STYK1 overexpressed or STYK1 knockdown NSCLC cell lines through using the STYK1 or sh STYK1 lentivirus.The CCK-8 cell viability assay,colony formation assay,the 5-Ethynyl-2′-deoxyuridine(Ed U)incorporation assay and in vivo tumor xenograft assays were used to detect the ability of proliferation in NSCLC cells.The TUNEL assay was used to evaluate the apoptotic level in NSCLC cells,and the wound healing assay and transwell migration and invasion assays were used to detect the ability of metastasis of NSCLC cells.Moreover,western blot was applied to examine the expression of proliferation markers,apoptosis markers and EMT markers.3.RNA-seq based transcriptome analysis was applied to estimate the transcriptome changes in H1299 cells among the NC and STYK1 OE groups,and the changes were further validated by q PCR and western blot analyses.The co-immunoprecipitation was used to detect whether STYK1 binds with SPINT2.Moreover,we further upregulated the SPINT2 expression by infecting SPINT2 lentivirus on the NSCLC cells overexpressing STYK1,and to further analyze whether SPINT2 is involved in the progression of NSCLC via using the methods in part 2.4.IHC was applied to detect the SPINT2 expression in 347 pairs of NSCLC tissues and corresponding adjacent noncancerous tissues.Later,we further analyzed the correlation of SPINT2 expression and clinicopathological parameters or the survival prognosis of NSCLC patients.Moreover,further analyses were conducted to evaluate the NSCLC prognosis via combining the expression STYK1 and SPINT2.Results:1.Oncomine database showed that,the STYK1 m RNA levels were much higher in all the NSCLC subtypes(LCC,LUAD and LUSC)compared with the normal lung tissues;Both the Kaplan Meier plotter database and The Human Protein Atlas database analyses showed the positive correlation between high STYK1 expression and poor prognosis of NSCLC patients.IHC results showed that 74.45%(258/347)NSCLC sections and 47.84%(166/347)corresponding adjacent non-cancerous tissue sections were classified as STYK1 positive;the STYK1 expression in NSCLC was significantly higher than that in the adjacent noncancerous samples.The elevated STYK1 expression was positively correlated to tumor size,tumor invasion,distant metastasis,differentiation,and clinical TNM stage.Moreover,STYK1 was found to be an independent prognostic factor for NSCLC patients via Cox survival analysis,and NSCLC,LUAD or LUSC patients with high STYK1 expression were all associated with worse overall survival.2.STYK1 could promote NSCLC cell proliferation determined by CCK-8 cell viability assay,colony formation assay and Ed U incorporation assay;STYK1 also could enhance the growth of H1299 cell xenograft tumors in athymic nude mice.Interestingly,TUNEL assay showed that STYK1 had no effect on the basal apoptotic rate of NSCLC cells under normal cell culture.Moreover,the migratory and invasive potentials were also enhanced by STYK1 in NSCLC cells via wound healing assay and transwell migration and invasion assays.STYK1 could upregulate the c-Myc,Cyclin D and CDK4 expression thus promoting NSCLC cell proliferation;and STYK1 could decrease epithelial biomarker E-cadherin expression and increased pro-EMT Snail levels,thus enhancing EMT in NSCLC cells.3.RNA-seq based transcriptome analysis showed that there were 20 genes upregulated and 114 genes downregulated in the H1299 cells after STYK1 overexpression;decrease of SPINT2 m RNA and protein levels was further validated by q PCR and western blot analyses after STYK1 overexpression.Moreover,co-IP results suggested that STYK1 did not bind to SPINT2 at the protein level.SPINT2 overexpression partially inhibited the STYK1 enhanced proliferative ability,xenograft tumor growth,and metastasis and invasion in NSCLC cells.Additionally,SPINT2 overexpression could partially inhibit STYK1-induced c-Myc,Cyclin D,CDK4 and snail upregulation,and E-cadherin downregulation,thus reversing STYK1 enhanced NSCLC cell proliferation,metastasis and EMT.4.IHC results suggested that the SPINT2 expression in NSCLC was much lower than that in the adjacent non-cancerous samples;the high SPINT2 expression was negatively correlated to tumor invasion,distant metastasis,differentiation,and clinical TNM stage.Furthermore,Cox survival analysis indicated that SPINT2 expression was positively correlated with NSCLC overall survival,and Kaplan Meier survival curves indicated that NSCLC,LUAD and LUSC patients with high SPINT2 expression were all associated with better prognosis.Next,we set out to detect whether prediction of NSCLC prognosis was more accurate according to combined STYK1 and SPINT2 expression than themselves alone.After analyzing the total NSCLC group and the stratified LUAD and LUSC groups,Kaplan Meier survival curves indicated that patients with low STYK1/high SPINT2 had the best prognosis,and patients with high STYK1/low SPINT2 had the worst prognosis.In both high and low STYK1 level subgroups,patients with high SPINT2 level showed better prognosis than the low one.Conclusions:The present study verified that receptor protein tyrosine kinase STYK1 functioned as a tumor promoting factor through downregulating SPINT2 thus enhancing NSCLC progression.STYK1 could significantly inhibit SPINT2 expression thus enhancing NSCLC cell proliferation,metastasis and EMT via c-Myc,Cyclin D,CDK4 and snail upregulation,and E-cadherin downregulation.Further survival analyses showed that NSCLC patients with low STYK1 level and high SPINT2 level had the best prognosis and survival,whereas patients with high STYK1 level and low SPINT2 level had the worst prognosis and survival.Taken together,our studies illustrated the action of STYK1-SPINT2 pathway during facilitating NSLCC proliferation and metastasis.Therefore,targeting STYK1 and SPINT2 may be a novel therapeutic strategy for NSCLC.