SPP1 Promotes Radiation Resistance Through JAK2/STAT3 Pathway In Esophageal Carcinoma

BackgroundEsophageal carcinoma(ESCA)is a common clinical malignant tumor type of digestive tract,which is the seventh most common cancer and the sixth leading cause of cancer-related deaths worldwide.As the fourth leading cause of death due to malignant tumor in China,it is a very important cancer type in clinical practice and scientific research.Despite the recent development in comprehensive treatment regimens,the prognosis of ESCA remains poor.This is mainly because most ESCA patients have been locally advanced when diagnosed,and local invasion and metastasis of tumor have occurred.Therefore,it is of great importance to study the potential mechanisms of esophageal cancer growth and metastasis and to find new biomarkers for early diagnosis and prognosis of ESC A.In addition,radiotherapy is one of the standard treatments for ESCA,which can efficiently kill cancer cells,alleviate symptoms and prolong patients’ survival.However,ESCA tissue frequently develops radiation resistance during radical,perioperative,or palliative radiotherapy treatments,which ultimately results in treatment failure and has become one of the major obstacles to ESCA treatment in clinical practices.Therefore,it is of great importance to explore the specific molecular mechanism of ESCA radiation resistance,and then identify the related molecular targets and signaling pathways of radiation resistance,so as to develop new combined therapies,so as to improve the local control rate and patient survival of ESCA.Secreted phosphoprotein 1(SPP1),also known as osteopontin,belongs to a family of secreted acidic proteins.SPP1 is commonly expressed in cardiac fibroblasts,osteocytes,macrophages,smooth muscle and endothelial cells,regulates many biological processes such as biomineralization,bone remodeling,chemotaxis,cell activation and apoptosis.Under pathological conditions,SPP1 was found to highly express in a variety type of cancers,it has been reported that the level of SPP1 in blood has a certain correlation with the diagnosis and prognosis of esophageal cancer through bioinformatics methods.However,the SPP1 level in ESCA tumor microenvironment(TME)can more fully prove the relationship between SPP1 and the diagnosis and prognosis of ESCA.Therefore,the diagnostic and prognostic value of SPP1 still needs to be further verified by clinical tissue samples of ESCA.And SPP1 was considered oncogenic by contributing to tumor proliferation and invasion in breast,colon and prostate cancer,which all facilitate tumorigenesis and progression,however,its role in ESCA is less studied,and more importantly,there’s no study on the relationship between SPP1 and radio-sensitivity of ESCA.Therefore,it is of great significance and innovation to study the specific roles of SPP1 in the occurrence and development of ESCA.The JAK(Janus kinase)family belongs to the family of non-receptor tyrosine kinases,currently consisting of four members:JAK1,JAK2,JAK3 and TYK2.The N-terminal domain of JAK can bind to receptors,and the C-terminal domain is the kinase domain.Each member of the JAK family binds to specific cytokine receptors to activate downstream signaling pathways.The substrate of JAK is signal and activator of transcription(STAT).There are seven members of the STAT family:STAT1 to STAT7.When phosphorylated by JAK,STAT is dimerized and enters the nucleus to regulate the expression of related genes,thereby affecting cell function.Jak-stat signaling pathway is closely related to cell proliferation,differentiation,survival and other basic cell biological behaviors,and plays an important role in the occurrence,progression,metastasis and treatment resistance of a variety of tumors.It has been reported that JAK-STAT is the downstream signaling pathway of SPP1 in breast cancer and is involved in the biological behavior of tumor malignancy mediated by SPP1.However,the relationship between JAK-STAT pathway and SPP1 in ESCA is still unclear,and the role of JAK-STAT pathway in radiation resistance of ESCA has not been studied.Objectives1.To study the correlation between SPP1 and prognosis of ESCA.2.To study the role of SPP1 in the malignant biological behavior of ESCA and explore the effect of SPP1-targeted therapy combined with radiotherapy on the malignant biological behavior of ESCA.3.To study the effect of radiation on SPP1 expression level.4.To study the relationship between SPP1 and ESCA in radiation resistance and explore the specific molecular mechanism.Materials and methods1.Database search:The protein level of SPP1 in ESCA,the relationship between SPP1 expression and ESCA clinical features,and the survival curve of ESCA associated with SPP1 were obtained by TCGA database.2.Bioinformatics analysis:mRNA level of SPP1 was obtained from GEO database and the volcano map was made by R language.Gene enrichment analysis of SPP1 and JAK-STAT pathway was conducted by R software and figure was drawn.3.Cell line construction:Eca-109 and Kyse-150 cell lines were transfected with SPP1-knockdown and SPP1-overexpression lentivirus.4.Cell proliferation detection:(1)EdU incorporation assay:The cells were planted in 24-well plates with a density of 1×105 cells/well,and incubated at 37℃ for 24 h.EdU kit was used for detection and cell fluorescence images were collected.(2)Cell Counting Kit-8(CCK-8)assay:The cells were planted in 96-well plates with a density of 3×103 cells/well,incubated at 37℃,and CCK-8 reagent was added at 24,48,72,96,120 h to measure the OD value at 450 nm.(3)Clone formation assay:The cells were planted in 6-well plates at a density of 1000 cells/well and incubated at 37℃ for about 10 days.Cell colonies were fixed with methanol and stained with crystal violet.The number of clones was counted.5.Cell migration detection:(1)Wound healing assay:Cells were planted into 6-well plates and when cell density reached 70%,a straight line was scratched.Cells were cultured in serum-free medium at 37℃ for 72 h.The wound photographs were collected at 0,48,and 72 h.(2)Transwell assay:Cells were planted in the upper chamber of the Transwell system at a density of 1×105 cells/well,and cell medium containing 20%FBS was added into the lower chamber as a chemotactic agent.After 24 hours,cells were fixed with methanol and stained with crystal violet to count the number of cells.6.Apoptosis detection:(1)Apoptosis chip:The expression of multiple apoptotic proteins was detected simultaneously.(2)Flow cytometry:Cells were planted into 6-well plate at a density of 3×105 cells/well.And cells were exposed to 6 Gy of radiation.Then cells were collected after 12 h and stained with an Annexin-V FITC/PI Staining Kit.Flowjo software was used for data analysis.7.Immunofluorescence:The cells were fixed with methanol,blocked with 5%goat serum,and incubated with 0.5%Triton X-100 PBS for 1 h.Then incubated with primary antibody overnight and incubated with secondary antibody for 2 h.8.Radio-sensitivity detection of ESCA cells:Cells were planted into 6-well plates at a density of 1000 cells/well and given X-ray irradiation at different doses.The number of surviving cell clones under different irradiation doses was recorded.9.Construction of tumor xenograft model in nude mice:BALB/c male mice aged 4 weeks were selected for experiments.Mice were randomly divided and received subcutaneous injection of ESCA cells,with the number of 2×107 cells.The tumors’volume was measured and recorded once every three days when tumors were visible,and the calculation formula was V=X2 × Y × 0.5.About 28 days later,the nude mice were sacrificed to collect tumor tissue and conduct follow-up experiments.10.Acquisition of clinical specimens:Tissue samples of ESCA patients were obtained from fresh ESCA and para-cancer tissues of thoracic surgery department of QILU Hospital,as well as experimental tissue chips purchased,and the required follow-up information was obtained.11.Immunohistochemistry:The tumor tissue was fixed with formalin,embedded in paraffin,sectioned,and stained with immunohistochemical detection kit,then images were collected.Results1.SPP1 was negatively correlated with ESCA prognosis:(1)Information analysis of GEO database showed that the mRNA level of SPP1 in ESCA tissue was higher than normal tissue in multiple datasets.(2)Information analysis of TCGA database showed that the protein level of SPP1 was higher in ESCA tissue than normal tissue;The expression of SPP1 was positively correlated with ESCA tumor stage.Asian patients had the highest expression of SPP1 compared with other races.Overweight or obese ESCA patients have lower SPP1 expression.(3)Information analysis of TCGA database showed that SPP1 expression was negatively correlated with disease-free survival of ESCA.Study of our ESCA cohort showed that SPP1 expression was negatively correlated with overall survival of ESCA.2.Western blotting and cell immunofluorescence results showed that the expression of SPP1 increased after radiation in ESCA cells.3.SPP1 promoted cell proliferation in ESCA:(1)The results of colony formation assay,EdU incorporation assay and CCK-8 assay showed that the proliferation ability of ESCA cells was significantly inhibited after SPP1-knockdown,which was more obvious after radiation.(2)The results of colony formation assay,EdU incorporation assay and CCK-8 assay showed that the proliferation ability of ESCA cells was significantly enhanced after SPP1-overexpression,while the increase of proliferation was inhibited after radiation.(3)Tumor xenograft model with ESCA cells in nude mice further confirmed the results of the above cell experiments.4.SPP1 promoted cell migration in ESCA:(1)Both Transwell assay and wound healing assay results showed that the migration ability of ESCA cells was significantly inhibited after SPP1-knockdown,which was more obvious after radiation.(2)Both Transwell assay and wound healing assay results showed that the migration ability of ESCA cells was significantly enhanced after SPP1-overexpression,while the increase of migration was inhibited after radiation.5.SPP1 inhibited cell apoptosis in ESCA:(1)Flow cytometry results showed that the apoptosis ratio of ESCA cells increased after SPP1-knockdown,which was more obvious after radiation.(2)Apoptosis protein array results showed that the expressions of pro-apoptotic proteins such as Bax,Cleaved-caspase 3 and FADD increased,and the expressions of anti-apoptotic proteins such as Livin and XIAP decreased after SPP1-knockdown.6.SPP1 up-regulation promoted radiation resistance in ESCA,while SPP1 down-regulation increased radio-sensitivity of ESCA cells:(1)Cell immunofluorescence results showed that SPP1 knockdown increased the signal of γ-H2AX within 12 h compared with negative control,indicating that SPP1-knockdown ESCA cells had reduced DNA repair ability after DNA double-strand break(DSB).(2)Western blotting results showed that after knocking down SPP1,the expression levels of pATM,TP53,P21 and P27 decreased post-radiation compared with the negative control,indicating that SPP1 knockdown might reduce the ability of ESCA cells to stop the cell cycle for DNA repair after DNA damage.(3)The analysis of the cell radiation dose-survival curve after radiation by single-hit multi-targets model showed that SPP1-knockdown increased radio-sensitivity of ESCA cells.(4)Cell immunofluorescence results showed that SPP1 overexpression decreased the signal of γ-H2AX within 12 h compared with negative control,indicating that SPP1-overexpression ESCA cells had increased DNA repair ability after DNA double strand damage.(5)Western blotting results showed that after overexpression of SPP1,the expression levels of pATM,TP53,P21 and P27 increased post-radiation compared with the negative control,indicating that SPP1 overexpression might enhance the ability of ESCA cells to stop the cell cycle for DNA repair after DNA damage.(6)The analysis of the cell radiation dose-survival curve after radiation by single-hit multi-targets model showed that SPP1-overexpression contributed to radiation resistance of ESCA cells and reduced radio-sensitivity.7.SPP1 promoted radiation resistance through JAK2-STAT3 signaling pathway:(1)Gene enrichment analysis showed that the JAK-STAT pathway was significantly enriched with the high expression phenotype of SPP1.(2)Western blotting results showed that the expression level of SPP1 significantly increased after radiation in ESCA cells,and the total protein levels of JAK2,STAT1 and STAT3 were not significantly changed,while their phosphorylation level significantly increased.(3)Cell immunofluorescence results showed that STAT1 and STAT3 were phosphorylated and partially transferred from cytoplasm into nucleus after radiation,indicating that they were activated after radiation.(4)Western blotting and cell immunofluorescence results showed that after SPP1 knockdown,the activation of JAK2 and STAT3 post-radiation in ESCA cells was inhibited,while STAT1 did not change significantly,suggesting that SPP1 specifically activated the JAK2-STAT3 signaling pathway.(5)Western blotting results showed that STAT3 was directly activated by JAK2 after radiation in ESCA cells.(6)Western blotting results showed that adding exogenous recombinant SPP1 into SPP1 knockdown ESCA cells could activate JAK2-STAT3 signaling pathway within 5 min,and this activation could be maintained for at least 72 h.8.Both SPP1 downregulation and inhibition of the JAK2-STAT3 signaling pathway could increase radio-sensitivity in ESCA cells:(1)The results of immunofluorescence,colony formation assay and transwell assay showed that inhibition of STAT3 activation could almost eliminate the enhancement of DNA repair,cell proliferation and cell migration ability induced by SPP1 up-regulation in ESCA cells.(2)The results of immunofluorescence,colony formation assay and transwell assay showed that both SPP1 down-regulation or STAT3 inhibition could weaken the DNA repair,cell proliferation and cell migration ability of ESCA cells,and enhance the radio-sensitivity.(3)Tumor xenograft model in nude mice further confirmed the results of the above cell experiments.Conclusions1.The expression level of SPP1 was negatively correlated with ESCA prognosis.2.SPP1 expression level increased after radiotherapy.3.SPP1 promoted ESCA progression,and the SPP1-targeted therapy can effectively inhibit the progression of ESCA,while the effect is more obvious when combined with radiotherapy.4.SPP1 promoted radiation resistance through JAK2-STAT3 signaling pathway in ESCA cells.