The Mechanism Study Of S100A10 In Gastric Cancer Metastasis And Growth

Background:Gastric cancer(GC)is one of the most common cancer worldwide,with a high recurrence rate and poor prognosis.The improvement in public health awareness and the development of endoscopic technology have led to an increased detection rate of early gastric cancer(EGC).EGC patients without LNM can be curatively treated with minimally invasive endoscopic resection.However,once metastases occur in lymph nodes,a surgical resection with lymphadenectomy should be performed.The prognosis for these patients is usually dismal in terms of survival and quality of life.Prerequisite for tumor lymphatic metastasis is that tumor cells proliferate infiltratively and break through the basement membrane,initiate human lymphatic endothelial cells(HLECs)migration and newborn lymphatic vessels formation.Then tumor cells migrated and invade surrounding lymphatic vessels,causing lymph node metastasis and distant metastasis.Therefore,elucidating the mechanism underlying GC lymphatic metastasis and uncovering new therapeutic targets are warranted.Gastric cancer acts as a malignant solid tumor.In order to satisfy high demands for material and energy,tumor cells often undergo a metabolic switch from oxidative phosphorylation to aerobic glycolysis.The intermediate products during aerobic glycolysis can be used as materials for the biosynthesis of macromolecules indispensable for cancer malignant proliferation,like nucleic acids and lipids.Moreover,aerobic glycolysis generates large amounts of lactate,causing an acidification of the tumor microenvironment,and helping immune escape and tumor metastasis.Therefore,gaining insight into the mechanism of by which cancer cells adopt aerobic glycolysis could potentially contribute to targeted anti-cancer therapies.In recent decades,a few genome-wide transcriptomic biomarker discoveries have been performed for screening metastasis in various types of cancer.Therefore,exploring the molecular mechanisms of EGC development and progression as well as establishing the important molecular targets have become the hot topic in translational medicine.S100A10,a member of calcium-binding S100 protein family,has been found to play an important role in regulating various biological and pathological process,such as exocytosis,endocytosis,plasmin generation,signal transduction and cell proliferation.Recent evidences showed that S100A10 was implicated in tumorigenesis and development.S100A10 worked as a driver of pancreatic cancer,ovarian cancer and liver cancer growth and migration.Recent studies have linked S100A10 with accumulation in metastatic lymph nodes of GC.Yet,the significance of S100A10 in GC metastasis and glycolysis is currently unclear.Therefore,this study was divided into three parts to explore the expression of S100A10 in human EGC specimens,and to explore the mechanism of S100A10 on tumor metastasis,and to detect the mechanism of S100A10 on tumor growth via aerobic glycolysis.Part Ⅰ:The expression and clinical significance of S100A10 in early gastric cancerAims:1.To detect the expression of S100A 10 in GC tissues using Oncomine and TCGA database and clinical specimens.2.To explore the expression of S100A 10 in EGC specimens and normal gastric mucosa tissues.3.To analyze the relationship between S100A10 expression and clinicopathological parameters in EGC.Methods:1.Oncomine,TCGA and UALCAN database were used to detect the expression of S100A 10 in GC.Kaplan-Meier plotter was used to determine the prognosis of S100A10 in GC.2.RT-qPCR was used to detect the expression of S100A10 in 17 cases of normal gastric mucosa,23 cases of atrophic gastritis tissues,17 cases of EGC tissues and 24 cases of AGC tissues.3.Immunohistochemistry(1HC)was used to explore the expression of S100A10 in 92 cases of paraffin-sectioned EGC tissues.The clinical and histopathological information were collected to analyze the relationship between S100A10 expression and clinicalpathological parameters.Results:1.B ioinformatic analysis reveals that the expression of S100A10 is higher in GC tissues than normal gastric tissues.Kaplan-Meier plotter analysis showed that patients with high expression of S100A10 had a worse survival than patients with low expression of S100A10.2.The expression of S100A10 was higher in EGC and AGC tissues than normal gastric tissues and atrophic gastritis tissues,while there was no significance between AGC and EGC tissues.3.IHC results demonstrated that the staining level showed a gradual increasing trend in the throughout normal gastric tissues,EGC without LNM tissues and EGC with LNM tissues,respectively.Correlation study results showed that S100A10 expression was positively correlated with larger tumor size,lymph node metastasis,advanced pTNM stage and vessel invasion.Conclusions:S100A10 was confirmed to exhibit abnormally high expression level in EGC tissues relative to adjacent normal tissues,especially in EGC tissues with LNM.S100A10 expression was positively associated with larger tumor size,lymph node metastasis and vessel invasion.Our findings suggested that S100A10,as a valuable biomarker,might be a promising predictor of EGC lymphatic metastasis.Part Ⅱ:The mechanism study of S100A10 in Gastric CancerMetastasisAims:1.To evaluate the biological functions of S100A10 in GC.2.To explore the upstream regulation mechanisms of S100A10.3.To explore the downstream signaling pathway of S100A10.4,To evaluate the effects of targeting-S 100A10 inhibition on GC growth and metastasis in vivo.Methods:1.The biological functions of S100A10 in GC in vitroGC cells were transfected with pcDNA3.1-S100 A10,si-S 100 A10,sh-S 100A10 or corresponding negative control.Transwell assay was used to detect the effect of S100A10 on cell migration and invasion.Western blot assay was used to evaluate the effect of S100A10 on EMT.CCK8,EdU and colony formation assays were used to explore the effects of S100A10 on cell proliferation.Flow cytometry was used for cell apoptosis detection.Tumor conditioned medium(TCM)from supernatants of GC cells was collected and performed HLECs migration,tube formation assays.Western blot assay and ELISA assay were used to measure the expression of VEGF C secretion.Tumor-endothelium adhesion assay was established to explore the effects of S100A10 on tumor cell-endothelium adhesion ability.2.The upstream regulation of S100A10(1)To identify the core promoter region of S100A10,the putative S100A10 promoter fragments were constructed into pGL3-Basic luciferase reporter plasmid.Dual-luciferase assays were performed to detect the promoter activity of S100A10.The online transcription factor prediction software JASPAR,AnimalTFDB and PROMO databases were used to search for putative transcription factor.Dual-luciferase assay,Western blot assay and ChIP assay were performed to identify that the potential transcription factors binding to the S100A10 promoter.(2)Transwell assay and CCK8 assay were performed to explore the effects of c-Jun on GC cells migration,invasion and proliferation.3.The downstream signaling pathway of S100A10(1)The effects of S100A10 overexpression or knockdown on Src/ANXA2/AKT/mTOR pathway activation were identified by Western blot assay.The effects of S100A10 on VEGF C,E-Cadherin and c-Jun were also investigated.(2)MK-2206,a highly selective allosteric inhibitor of AKT,was added to investigate whether this inhibitor could inverse the effects of S100A10 on GC cells.4.The effects of targeting-S100A10 inhibition on GC growth and metastasis in vivo.(1)GC cells were subcutaneously injected into the right flank of BALB/c nude mice.Then lentiviral packaged LV-sh-S100A10 or LV-NC was injected into the tumor xenografts model every three days accordingly.Record changes in tumor volume every week and tumor weight at the endpoint.(2)The total proteins were extracted from the xenograft tumor tissues.Western blot assay was used to detect the expression of downstream effectors.(3)Construct stable S100A10 knockdown cell line and inject them through the tail vein,and evaluate the metastatic capability of GC cells by lung metastasis in nude mice.Results:1.S100A10 accelerated malignant biological behaviors of GC cells.S100A10 overexpression promoted GC cells migration,invasion and proliferation,and inhibited cell apoptosis,while inhibition of S100A10 expression reversed this phenomenon.S100A10 overexpression significantly promoted HLEC migration and lymphangiogenesis via increasing the expression and secretion of VEGF C.Tumor-endothelium adhesion assay demonstrated that S100A10-overexpressed GC cells had a significantly higher level of adhesion to HLECs than negative control cells.2.C-Jun activates S100A10 transcription in GC cellsDual-luciferase assay demonstrated that the region-681/-532 bp upstream of the transcription start site(TSS)of S100A10 is its proximal promoter.C-Jun transcriptionally up-regulated S100A10 expression through direct binding its promoter.C-Jun overexpression enhanced GC cell migration,invasion and proliferation.3.S100A10 enhanced GC metastasis through Src/ANXA2/AKT/c-Jun positive feedback loop.(1)High expression of S100A10 may activate Src kinase and promote the phosphorylation of Annexin A2(ANXA2),which in turn can activate the AKT/mTOR signaling pathway.Interestingly,c-Jun acts the downstream targets of AKT signaling pathway,thus forming a positive feedback loop that drives the malignant metastasis of GC.(2)AKT inhibitor,MK-2206,could reverse the effect of S100A10 on GC cells migration and growth.4.S100A10 could be used as a therapy target in suppressing tumor growth and metastasis in vivo(1)Injection of LV-sh-S100A10 recombinant lentivirus was able to diminish GC cells growth and invasion through deactivating the Src/ANXA2/AKT signaling pathway.(2)The incidence of lung metastasis was decreased in S100A10 knockdown nude mice.Conclusions:C-Jun could initiate S100A10 transcription,thereby promoting S100A10 expression in GC.S100A10 overexpression could activate Src/ANXA2/AKT/mTOR signaling pathway and increase c-Jun and VEGF C expression,thus forming a positive feedback loop to accelerate GC metastasis.Part Ⅲ:Part Ⅲ The mechanism study of S100A10 on growth of Gastric Cancer via aerobic glycolysisAims:1.To explore the roles of S100A10 in GC aerobic glycolysis.2.To explore the molecular mechanisms of S100A10 in GC aerobic glycolysis.Methods:1.The effects of S100S10 on GC glycolysis(1)GEPIA database and RT-qPCR were used to analyze the relationship between S100A10 and glycolysis-associated molecules expression.This relationship was verified by RT-qPCR.(2)Measurement of aerobic glycolysis:the measurement of glucose consumption and GLUT1 expression,the measurement of lactate production and LDH activity and expression,the measurement of ATP production,the measurement of ECAR and OCR.2.The mechanism of S100A10 on aerobic glycolysisWestern blot assay was used to detect the changes of mTOR signaling pathway after S100A10 overexpression or knockdown.Western blot assay and glycolytic measurement were performed to detect whether the treatment of rapamycin and GSK-2837808A could abolish S100A10 mediated promotion.3.The effect of S100A10 overexpression and rapamycin treatment on tumor growth by in vivo assayConstruct stable S100A10 overexpressing cell line and inject them subcutaneously,and injected with rapamycin or PBS.Western blot assay was performed to evaluate the downstream effectors changes in xenograft tumor tissues protein.Results:1.GEPIA analysis and RT-qPCR results demonstrated that S100A10 expression was closely associated with glycolysis-related markers.2.S100A10 overexpression favors glucose consumption through GLUT1.S100A10 facilitates lactate production by modulating glycolytic enzymes.S100A10 reduces ATP production by suppressing oxidative phosphorylation,whereas inhibition of S100A10 expression attenuated these events.3.Tumor volume and tumor weight were remarkably larger in S100A10 overexpressing group than the control group.Rapamycin treatment could significantly abolish the promoting effect of S100A10 on tumor growth.Conclusions:S100A10 may function as a positive regulator of glycolysis in GC through activating mTOR signaling pathway,while rapamycin suppresses the activation of the mTOR pathway,thus abolishing the promoting effects of S100A10 and inhibiting tumor growth.