| Part Ⅰ The role and mechanism of Tim-3 in breast cancer cellBackgroundBreast cancer is one of the most common malignancies in women and the leading cause of cancer-related death in women.Dysregulation of immune checkpoints plays an important role in tumor immune evasion by inducing T cell exhaustion.T cell immunoglobulin and mucin-domain containing-3(Tim-3)is also known as Hepatitis A Virus Cellular Receptor 2(HAVCR2),belonging to the Tim gene family.Tim-3 is a type I transmembrane protein including an extracellular domain,a transmembrane domain and a C-terminal cytoplasmic tail.Tim-3 has four ligands,including galectin-9(Gal-9),carcinoembryonic antigen cell adhesion molecule 1(CEACAM-1),high mobility group box B1(HMGB1),and phosphatidylserine(PS).As a negative immune checkpoint,Tim-3 can be expressed on various immune cells such as T cells,regulatory T cells,DC cells,B cells,macrophages,NK cells and mast cells.Gradually increasing preclinical studies have shown that Tim-3 inhibitors have also achieved similar effectiveness to PD-1 inhibitors.In addition,the PD-1 antibody can cause an increase in the expression of Tim-3,suggesting that Tim-3 may be used as a molecular marker of resistance to PD-1 inhibitors.Therefore,Tim-3 is expected to become a promising candidate therapeutic target.Tim-3 induces immune tolerance and is therefore implicated in the pathogenesis of tumors,asthma,food allergy,autoimmune diseases,and chronic viral infections.Tim-3 can inhibit tumor immunity by inducing T cell exhaustion.Tim3+CD8+T cells can downregulate the STAT5 and P38 signaling pathways,and blocking the Tim-3 pathway can enhance antitumor immunity and enhance the release of IFN-r from T cells.Anti-Tim-3 antibody can also induce the release of IFN-r from peripheral NK cells.Recent studies have found that Tim-3 is not only expressed on immune cells,but also expressed on various types of tumor cells.Tim-3 expression in tumor cells was associated with later clinical stage of non-small cell lung cancer,lymphovascular invasion of gastric cancer,lung metastasis of renal clear cell carcinoma and lymphatic metastasis of colon cancer.In vitro studies have found that Tim-3 induces epithelial-mesenchymal transition(EMT)through the Akt/GSK-3/Snail signaling pathway and promotes the metastasis of esophageal squamous cell carcinoma(ESCC).Knockout of Tim-3 significantly inhibited the proliferation,migration and invasion of ESCC cell lines.In addition,interfering with Tim-3 expression can significantly inhibit the proliferation and metastasis of osteosarcoma cells through NF-κB/snail signaling pathway and EMT.Clinically,high expression of Tim-3 in solid tumors significantly shortened overall survival.But there are also contrary results reported,down-regulation of Tim-3 can promote the invasion and metastasis of colorectal cancer cells,and the low expression of Tim-3 in prostate cancer tissue is associated with poor prognosis in patients with metastatic prostate cancer.Different tumor types and different Tim-3 expression localization may lead to different prognostic outcomes.The prognostic significance of Tim-3 in different cancers is still controversial and needs further study.Small-sample studies have shown that Tim-3 is also overexpressed in breast cancer cells and is associated with poor prognosis.However,the exact mechanism of Tim-3 in breast cancer development and invasion has not been elucidated.Furthermore,Tim-3 was resistant to antiangiogenic drugs(sunitinib)and mTOR inhibitor(rapamycin)in renal cell carcinoma cell lines and was able to induce chemotherapy to doxorubicin and carboplatin in ATN 1 cells of lymphoma drug resistance,which means it may play a role in tumor angiogenesis and chemotherapy resistance.Therefore,in this study,we first analyzed the expression of Tim-3 in breast cancer and its effect on prognosis,then explored its effect on breast cancer cell proliferation,migration and invasion and its mechanism,and then evaluated its role in angiogenesis and cellular tight junctions.Methods1.The gene expression level of Tim-3 in breast cancer and normal tissues and its correlation with clinicopathological factors in TCGA-BRCA database were analyzed.2.To explore the correlation between Tim-3 and local recurrence and distant metastasis of breast cancer,and to explore the role and significance of Tim-3 in different molecular types of breast cancer,the KM-plotter breast cancer database was analyzed.3.The triple-negative breast cancer line MDA-MB-231 and Luminal breast cancer cell line MCF7 overexpressing Tim-3 were constructed by lentiviral transfection.In vitro experiments,the effects and mechanisms of Tim-3 on the cell proliferation,adhesion,invasion,migration and tumor-associated angiogenesis of breast cancer cells were explored.Using electric cell substrate Impedance Sensing(ECIS)monitoring the initial attachment and connection of cells,measuring transepithelial resistance(Transepithelial Resistance,TER)and cell permeability(Paracellular permeability,PCP)experiments to explore the function and mechanism of Tim-3 in tight junction of cells.Results1.The pooled analysis of the TCGA database showed that the expression level of Tim-3 gene was significantly increased in breast cancer tissues compared with normal tissues(P<0.001).There were no significant differences in the expression level of Tim-3 between different molecular subtypes of breast cancer,breast cancer patients with different clinical stages and different axillary lymph node stages.2.Analysis of the KM-plotter breast cancer database showed that the recurrence-free survival(RFS)of patients with high Tim-3 expression decreased significantly(P=0.004),and the overall survival(OS)also had a similar trend,but the difference was not statistically significant(P=0.099),suggesting that high expression of Tim-3 is associated with poor prognosis of breast cancer in the overall population.High expression of Tim-3 was associated with poorer RFS in Luminal A(P<0.001)and Luminal B(P=0.039)subtypes.In basal-like breast cancer,high expression of Tim-3 was associated with better RFS(P<0.001).For OS,high expression of Tim-3 was associated with poor prognosis in luminal A subtype(P=0.019),while in basal subtype,patients with high expression of Tim-3 had a better prognosis(P<0.001),suggesting that Tim-3 may play different roles in different molecular subtypes of breast cancer.3.Breast cancer cells overexpressing Tim-3 promoted cell invasion,proliferation,migration and tumor-associated angiogenesis.Mechanistically,Tim-3 exert its function by activating NF-κB/STAT3 signaling pathway and regulating downstream gene expression[upregulation of cyclin D1(CCND1),C-Myc,matrix metalloproteinase-1(MMP1),TWIST,vascular endothelial growth factor(VEGF),accompanied by downregulation of E-cadherin].In addition,Tim-3 downregulates tight junction-associated molecules zone occluder(ZO)-2,ZO-1,and Occludin to reduce tight junctions between cells,which further promotes tumor progression.Conclusions1.Bioinformatics analysis showed that the expression level of Tim-3 gene in breast cancer tissue was significantly higher than that in normal tissue.Breast cancer patients with high expression of Tim-3 are associated with poor recurrence-free survival in the overall population.2.Overexpression of Tim-3 promoted the proliferation,migration,invasion and tumor-related angiogenesis of breast cancer cells,while reducing the function of tight junctions between cells.Mechanistically,Tim-3 exert its function by activating NF-κB/STAT3 signaling pathway and regulating downstream gene expression[upregulation of cyclin D1(CCND1),C-Myc,matrix metalloproteinase-1(MMP1),TWIST,vascular endothelial growth factor(VEGF),accompanied by downregulation of E-cadherin].Tim-3 further promotes tumor progression by downregulating tight junction-related molecules ZO-2,ZO-1,and Occludin.Part Ⅱ The role and mechanism of Tim-3 in vascular endothelial cellsBackgroundMolecules of the T-cell immunoglobulin and mucin family have attracted increasing attention for their association with a variety of diseases including allergy,autoimmunity,and cancer.Tim-3 is an important negative immune checkpoint that plays a key role in regulating immune cell activity and is considered a promising target for cancer immunotherapy.Tim-3 is also expressed on endothelial cells and plays a role in melanoma,lymphoma,rickettsial infection,and atherosclerosis.Melanoma endothelial cells expressing Tim-3 increase the metastatic potential of tumor cells by promoting cellular intravasation and extravasation.Tim-3 expression in lymphoma-derived endothelial cells promotes lymphoma growth and dissemination by inhibiting CD4+T cell activation by interacting with circulating T cells.Clinically,it has also been observed that the expression level of endothelial Tim-3 in B-cell lymphoma is associated with dissemination and poor prognosis.Furthermore,Tim-3 was shown to be a negative regulator of atherosclerosis,accompanied by increased levels of circulating monocytes and diseased macrophages and decreased levels of regulatory T cells and regulatory B cells.Tim-3 also protects human umbilical vein endothelial cells(HUVEC)from bovine low-density lipoprotein(LDL)-induced apoptosis through the c-Jun N-terminal kinase(JNK)pathway and reverses the effect on migration inhibition.In summary,Tim-3 plays an important role in human endothelial-related diseases.Distant metastasis of tumor is one of the main causes of cancer-related death.Bidirectional interactions between tumor cells and their microenvironment,including tumor stroma,are important for tumor progression and metastasis.Tumor angiogenesis includes the degradation of vascular basement membrane,the activation,proliferation and migration of vascular endothelial cells,and the reconstruction of new blood vessels and vascular network,which are necessary conditions for tumor progression.Recent studies have highlighted the important role of ’angiosecretory factors’ that are released from tumor endothelial cells and contribute to tumor progression.During endocytosis,tumor cells physically contact endothelial cells and interact with them through proximal and paracrine signals,thereby stimulating tumor cell metastasis.Therefore,vascular endothelial cells also play an important role in tumor progression and metastasis.Previous studies have shown that,as a member of the Tim-3 family,overexpression of Tim-1(HAVCR 1)leads to a decrease in the formation of tight junctions in human vascular endothelial cells,which is related to the closure zone protein-1(ZO-1)and closure zone protein-2(ZO-2)related.Therefore,in this part of the study,we investigated the effects of Tim-3 on the proliferation,migration,invasion and angiogenesis of vascular endothelial cells in human lung microvascular endothelial cells(HMVECs)and HUVECs,and subsequently the potential role and mechanism of Tim-3 in tight junctions of vascular endothelial cells were analyzed.Methods1.The expression of Tim-3 in HMVEC and HUVEC was detected by RT-qPCR,and the expression of Tim-3 protein in vascular endothelial cells in breast cancer tissue was evaluated by immunohistochemistry.2.Human Tim-3 overexpression(Tim-3 OE)plasmid or blank control vector[Scramble(Scr)]lentivirus was stably transfected into HMVEC and HUVEC cells,and the expression level of Tim-3 in the two cell lines was detected by RT-qPCR and Western blot to construct a vascular endothelial cell line that overexpressed Tim-3.3.To explore the changes and mechanisms of HMVEC and HUVEC cell proliferation,adhesion,invasion,migration,and angiogenesis before and after overexpression of Tim-3 through cell proliferation experiments,cell matrix adhesion experiments,matrix glue invasion experiments,cell scratch experiments,and angiogenesis experiments.4.ECIS was used to monitor the initial adhesion and connection of endothelial cells,and TER and PCP experiments were conducted to explore the changes in cell tight junction function before and after overexpression of Tim-3.5.Breast cancer cell MDA-MB-231OE/Scr and vascular endothelial cell HUVEC wereadded into the lower and upper chambers of Transwell chamber for coculture,and the difference of TER of the single endothelial cell layer was detected.Results1.Both HUVEC and HMVEC cells expressed Tim-3,but the expression levels were lower.Tim-3 is highly expressed in vascular endothelial cells of breast cancer tissue,which suggests that Tim-3 maybe involved in the formation of microvessels in breast cancer.2.Through lentiviral transfection,a vascular endothelial cell line overexpressing Tim-3 was successfully constructed.After transfection of Tim-3 plasmid,RT-qPCR and Western blot confirmed that Tim-3 was significantly increased at the gene and protein levels.3.Tim-3 promotes cell proliferation,migration,invasion and angiogenesis by activating cyclin D1(CCND1),Ras homologous gene family member A(Rho A)and vascular endothelial growth factor(VEGF)receptor 2(VEGFR2).4.Tim-3 reduces tight junction(TJ)formation and transepithelial resistance(TER)in endothelial cells by reducing the expression levels of TJ protein 2(ZO2),Occludin and claudin 1(CLND1).5.The coculture of breast cancer cells with high expression of Tim-3 and vascular endothelial cells can reduce the TER of single endothelial cell layer.Conclusions1.Tim-3 was expressed in vascular endothelial cell line and breast cancer tissue.2.Tim-3 promotes the proliferation,adhesion,migration,and invasion of vascular endothelial cells by activating CCND1 and RhoA.3.Tim-3 promotes angiogenesis by promoting the expression of VEGFR2 protein.4.Tim-3 reduces intercellular tight junctions and transepithelial resistance by reducing the expression of ZO-2,Occludin,and CLDN1.5.The coculture of breast cancer cells with high expression of Tim-3 and vascular endothelial cells can reduce the TER of the single endothelial cell layer,suggesting that the connection between vascular endothelial cells is looser,which is conducive to tumor cells invading blood vessels and metastasis.Part Ⅲ The role and mechanism of Tim-3 in breast cancer chemoresistanceBackgroundChemotherapy is widely used to improve the survival of breast cancer patients.Although the prognosis of breast cancer patients has gradually improved,many patients still show primary or secondary drug resistance,leading to tumor recurrence and/or metastasis.At present,drug resistance is the main reason for poor prognosis and reduced survival rate of breast cancer patients.In order to better treat these breast cancer patients,solving the problem of drug resistance has been a huge challenge.Many mechanisms related to drug resistance have been found in the treatment of breast cancer,including somatic mutations or epigenetic changes within drug targets,cancer cell heterogeneity,cancer stem cells,cancer related macrophages and immune cell regulation,metabolic reprogramming,and so on.There is increasing evidence that targeted immune checkpoint modulators such as PD-1/PD-L1 and CTLA-4 have a bright future in the treatment of cancer.At the same time,these molecules also play a key role in the chemoresistance of cancer cells.Immune checkpoints affect cancer chemoresistance in a drug and cell type dependent manner.High PD-L1 expression is a poor predictor of local recurrence and distant metastasis of breast cancer,and also an important predictor of chemotherapy resistance of breast cancer.B7 family checkpoint can also cause breast cancer cells to become resistant to chemotherapy drugs,and targeting these immune checkpoint molecules is considered to be an effective way to overcome drug resistance.Recent studies have shown that inhibiting or blocking Tim-3 can enhance the chemotherapy effect of breast cancer,so it has therapeutic value for breast cancer.The CD103+DCs in the tumor overexpress Tim-3,and the Tim-3 antibody promotes the expression of CXCL9[Chemokine(C-X-C motif)ligand 9]in these DCs,thereby enhancing the function of CD8+T cells and improving the therapeutic effect of paclitaxel in breast cancer.Tim-3 can also promote the resistance of lymphoma ATN-1 cells to doxorubicin and carboplatin.In head and neck squamous cell carcinoma(HNSCC),the expression of Tim-3 in HNSCC after radiotherapy and chemotherapy is significantly higher than that in primary HNSCC,suggesting that Tim-3 is involved in the resistance of HNSCC to radiotherapy and chemotherapy.So far,the role of Tim-3 in response to chemotherapy drugs in breast cancer is still unclear.Therefore,this study also explored the role and mechanism of Tim-3 in chemoresistance of breast cancer.Methods1.Twenty-two breast cancer patients who received neoadjuvant chemotherapy and surgery were selected.All patients received AC×4(erubicin and cyclophosphamide)followed by T×4(paclitaxel or docetaxel)regimen for neoadjuvant chemotherapy.The expression of Tim-3 in breast tumor tissues before and after neoadjuvant chemotherapy was detected by immunohistochemistry.2.By constructing MDA-MB-231 and MCF7 cells overexpressing Tim-3,the sensitivity of the cells to paclitaxel and cisplatin chemotherapy drugs was detected,and the mechanism of action was explored by Western blot and qPCR.Results1.Tim-3 was expressed in breast cancer tissues of different molecular types before and after neoadjuvant chemotherapy,but there was no significant difference in the expression level.Compared with before neoadjuvant chemotherapy,the expression level of Tim-3 in tumor tissue was significantly increased after chemotherapy,suggesting that Tim-3 may be involved in the chemotherapy resistance of breast cancer cells.2.Paclitaxel drug sensitivity testMDA-MB-231 Tim-3 OE cells exhibited higher tolerance to paclitaxel than control Scr cells at concentrations of 10,20,and 40 nmol/L.Compared with Scr cells,MCF7 Tim-3 OE cells were also more resistant to paclitaxel at concentrations of 2.5 and 5 nmol/L.After treating MDA-MB-231 and MCF7 cells with a single dose of paclitaxel(10 nmol/L for MDA-MB-231 and 5 nmol/L for MCF7)for 6 and 24 hours,the results of Western blot showed that compared with Scr control cells,the total NF-κB protein level of Tim-3 OE cells was significantly increased after 6 hours of culture,and p-NF-κB was significantly increased in Tim-3 OE cells after 24 hours of culture in the two cell lines.After paclitaxel treatment for 24 hours,the protein level of STAT3 in Tim-3 OE cells was significantly higher than that in MDA-MB-231 and MCF7 Scr cells.CCND1 was upregulated in MCF7 Tim-3 OE cells after 6 hours compared with Scr controls.When NF-κB inhibitor SC75741 or STAT3 inhibitor Stattic were added for cytotoxicity assay,Tim-3-induced paclitaxel resistance was eliminated,further confirming the involvement of NF-κB and STAT3 in Tim-3-mediated paclitaxel resistance.3.Cisplatin drug sensitivity testMDA-MB-231 Tim-3 OE cells were more resistant to cisplatin than Scr cells when cisplatin was added at a concentration of 8μmol/L and 16μmol/L.Compared with Scr,MCF7 Tim-3 OE cells were also more resistant to cisplatin at concentrations of 8 μmol/L,16μmol/L and 32μmol/L.Conclusions1.Compared with before neoadjuvant chemotherapy,the expression level of Tim-3 in tumor tissue after chemotherapy was significantly increased,suggesting that Tim-3 may be involved in the chemotherapy resistance of breast cancer cells.2.Breast cancer cells with high expression of Tim-3 increased their drug resistance to paclitaxel and cisplatin,and the drug resistance to paclitaxel was achieved by activating the NF-κB/STAT3 signaling pathway and changing the expression of CCND1. |
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