| BackgroundWith the rapid development of comprehensive treatment measures such as surgery,radiotherapy,chemotherapy,targeted therapy and immunotherapy,the mortality rate of breast cancer has decreased.However,nearly 90%of breast cancer eventually died of metastasis.At present,the "seed and soil" hypothesis of tumor metastasis model is widely acceptable.The cancer cells metastasis to distant organs requires the invasiveness of tumor cells and the microenvironment supporting their metastasis.There are two classical models of tumor metastasis.One is the linear metastasis model.The model was defined that heterogeneous mutated cells in the primary tumor form new clones,accumulate and survive in the target organs.The other is the parallel metastasis mode.The tumor cells acquire metastatic potential very early.Even if the initial lesion is small or undetectable,tumor cells have been implanted into other distant tissues.These two models suggest that the internal drive of our genes and the changes of microenvironment may be involved in the metastasis of tumor cells.The formation process of metastatic tumor is complex.Firstly,the surrounding tissues were invaded by cancer cells.The cancer cells penetrate the tumor tissues and then inject into blood vessels or lymphatic vessels,survive in the circulation system as circulating tumor cells(CTC),and finally colonize distant organs to form metastatic foci,so as to spread from the primary tumor to distant organs.Breast cancer mainly develops distant metastasis through lymphatic system and blood system.At present,drugs have been developed for targeting hematogenous metastasis,such as bevacizumab,and are applied in clinical practice.However,anti-tumor angiogenesis drugs have not significantly improved the overall survival of breast cancer.Therefore,we should not neglect another important pathway of breast cancer metastasis,lymphatic metastasis.With the discovery of lymphatic markers and lymphangiogenic factors,the study of lymphatic vessels has gradually become a hot spot.At present,most scholars believe that tumor tissue can cause the proliferation of lymphatic endothelial cells and promote the regeneration of tumor lymphatic vessels through autocrine or paracrine lymphangiogenic factors.Lymphangiogenesis is a complex cellular event involving cell proliferation,cell migration,lymphatic sprouting and duct formation.In the past,it was considered that lymphatic vessels only passively provide metastasis channels in tumor metastasis.However,recent clinicopathological studies have shown that lymphatic vessels can actively change and adapt to environmental changes,which is conducive to tumor metastasis.These changes include lymphangiogenesis.Therefore,the study of lymphangiogenesis has an important clinical value for cancer metastasis.NADPH oxidase(Nox)is a membrane-bound enzyme complex.The main function is to produce superoxide anion O2-by transfering electrons from NADPH to oxygen molecules.The mainly action is rapidly converted into hydrogen peroxide(H2O2)by superoxide dismutase(SOD).Nox was first observed in neutrophils and macrophages.In the process of inflammatory reaction,these two cells produce "oxidative explosion"and produce a large number of reactive oxygen species(ROS),which constitutes the body's first line of defense against pathogens.However,Nox has been proved to be closely related to the recurrence and progression of cancer.Because it produces a large number of ROS,oxygen ions and superoxide can destroy cell membrane,DNA transcription and protein oxidation.By regulating the expression of Nox4,reducing lymphangiogenesis and destroying the chance of lymphatic metastasis of breast cancer.It may provide a new idea for the treatment of breast cancer.It is divided into two parts.The first part is to collect the clinical and pathological data of operable breast cancer,and preliminarily determine the value of Nox4 in breast cancer metastasis.Finally,we analyzed the relationship between the different expression levels of Nox4 and the relapse-free survival(RFS)and the overall survival(OS).Further through animal experiments,we used the existing Nox4 inhibitor GKT137831 to treat breast cancer bearing mice,to verify the role of Nox4 in lymphangiogenesis and breast cancer metastasis,and to provide a new plan for breast cancer treatment.Aims1.To analyze the relationship between Nox4 and clinicopathological factors,the correlation between Nox4 and lymph node metastasis and relationship between Nox4 expression and prognosis in breast cancer.2.To confirm that Nox4 promotes proliferation of lymphatic endothelial cells,lymphangiogenesis and distant metastasis of breast cancer in vitro and in vivo animal experiments.Methods1.Nox4 expression of HLECs in breast cancer tissues and normal breast tissuesThe NCBI GEO databases(http://www.ncbi.nlm.nih.gov/geo/)was detected for the transcription profile dataset of HLECs.The accession number was GSE73613.The data derived from the human lymphatic endothelial cells extracted from primary invasive breast tumors(N=2)and human normal mammary glands(N=2).R software(version 3.2.3;https://www.r-project.org/)was used in data mining and statistical analyses.2.Expression of Nox4 in breast cancer and its correlation with clinicopathological factorsThere are 81 operable breast cancer patients who underwent surgical treatment from May 1,2014 to September 30,2019 in Shandong Cancer Hospital were collected.Immunohistochemical staining was used to detect ER,PR,HER2,Ki-67,Nox4 and LYVE-1.The main aim is to analyze Nox4 and LYVE-1 and their clinicopathological characteristics.3.Analysis of the relationship between Nox4 expression and prognosis of breast cancerKaplan-Meier Plotter online analysis tool was used to analyze the effect of Nox4 expression on RFS and OS of breast cancer.There were 4929 cases with complete RFS data and 1879 cases with complete OS data.According to auto select best cutoff,the effects of Nox4 with different expression levels were analyzed in RFS and OS,and the effect on breast cancer prognosis was discussed.4.To study the effects of Nox4 on proliferation,migration,tube formation and thoracic duct germination of HLECs in vitro(1)The low expression of Nox4 was constructed by lentivirus infection method.The lentivirus infection efficiency was detected by qRT-PCR and western blot.(2)The proliferation ability of HLEC was detected by CCK-8 assay.Different concentrations of GKT137831 were used to treat HLECs and GKT137831 was used to treat HLECs for different time to observe the cell activity.(3)ROS was detected with DCFH-DA fluorescent dye.The treated cells were incubated with DCFH-DA,photographed with laser confocal microscope and detected the fluorescence intensity.(4)Wound healing and transwell cell migration test were performed to detect cell migration ability of HLECs.(5)Mitochondrial membrane potential(?? m)detection and flow cytometry were used to detect the apoptosis of HLECs.HLECs were pretreated with different concentrations of Nox4 inhibitor GKT137831 before the experiment,the mitochondrial membrane potential was tested by mitochondrial membrane potential detection kit(JC-1)according to its instruction,and observed and photographed under laser confocal microscope.HLECs were cultured with different concentrations of GKT137831(0,5,10,15?M),and treated with annexin V-FITC and PI,and then were detected by flow cytometry within 1 hour.(6)Tube formation in vitro:HLECs were treated with Nox4 shRNA lentivirus or GKT137831 respectively.Lymphatic vessels were cultured on matrigel,counted and photographed 12 hours later.(7)Thoracic duct germination in vitro:We cultured thoracic ducts from C57BL/6J mice that were 2-3months of age.Thoracic ducts were dissected by microsurgery to generate fragments and then embedded into matrigel.8?M GKT137831 and/or 20ng/ml recombinant mouse VEGF-C were added into the matrix gel medium.After 7 days of cultivation,the continuous expansion grid of thoracic duct boundary was drawn by binary image and sholl analysis,and analyzed by Image J software.At the end of the test,RNA and protein were extracted,and the expression of Nox4 was detected by RT-PCR and western blot.5.Animal study of Nox4 on breast cancer proliferation and metastasis(1)To investigate the in vivo effects of Nox4 on cancer metastasis,4T1 orthotopic mice were used.(2)The mice were randomly divided into two groups(5 mice in each group)after the tumor volume reached the volum of 80-100mm3,and given orally with GKT137831(50mg/kg)or normal saline containing the same concentration of DMSO every two days for 28 days.The tumor tissue,lymph nodes and lung tissue were removed.The localization of Nox4 in lymphatic vessels was detected by immunofluorescence.Results1.The expression Nox4 on HLECs was higher in breast cancer tissues than that in normal breast tissuesTo identify the differences of Nox4 expression in HLECs of breast cancer tissue and their normal lymphatic endothelial cell counterparts,we obtained data from NCBI GEO datasets and found that Nox4 mRNA expression increased obviously in tumor-associated lymphatic endothelial cells with the calculating criteria of P<0.05 and log2FC=3.14.2.High expression of Nox4 in breast cancer tissue is dedicated to develop breast cancer lymph node metastasis,and Nox4 expression is positively correlated with LYVE-1 expression.(1)The expression levels of Nox4 and LYVE-1 in breast cancer tissues were detected by immunohistochemical staining.The results showed that there are 81 cases of breast cancer accounted for 49.4%(40/81),highly expressed Nox4,and the high expression of LYVE-1 accounted for 54.3%(44/81).Nox4 was highly expressed in breast cancer patients with high histological grade and lymph node positive.LYVE-1 relatively high expression in patients with tumor size>2cm,lymph node positive,ER negative,PR negative,HER2 overexpression and TNBC subtype.(2 Spearman correlation analysis showed that Nox4 and LYVE-1 showed a significant positive correlation in breast cancer tissues.3.Nox4 overexpression in breast cancer has a poor prognosis.The effect of Nox4 on RFS and OS in breast cancer patients were detected by online analysis software which was called Kaplan-Meier Plotter.The results showed that the breast cancer patients with low expression of Nox4 were with a median RFS of 57 months in the 4929 breast cancer patients,the high expression of Nox4 was 37 months.In the 1879 cases of breast cancer,the median OS of the patients with low expression of Nox4 was 106.8 months,the high expression of Nox4 was 66 months,and the difference was statistically significant.4.Nox4 can promote the HLECs proliferation,migration,anti-apoptosis,tube formation and germination of thoracic duct.(1)shRNA-Nox4 lentivirus and GKT137831 were used to treat HLECs for CCK-8 experiment.The results showed that both Nox4 shRNA and GKT137831 could inhibit the proliferation of HLECs in a concentration dependent manner.The IC50 of GKT 137831 inhibiting HLECs was 8.5±0.7 ?M?(2)Nox is an important source of ROS.Different concentrations(0,1,10,15?M)of GKT137831 were treated HLECs for 24 hours.DCFH-DA was used to detect the ROS.The results showed that ROS expression decreased with the increase of GKT137831 concentration.(3)20MOI Nox4 shRNA lentivirus and 8?M GkT137831were used to detect wound-healing.The results showed that HLECs with low levels of Nox4 whatever shRNA or inhibitor was easy to be wound healing delay and decline VEGF-C-induced HLECs migration.Transwell cell migration experiment showed that the cells are hard to pass through the membrane in shRNA-Nox4 than that in GKT137831 treatment group.Previous studies have confirmed that VEGF-C can promote the proliferation of lymphatic endothelial cells.Even increasing VEGF-C treating cells can not reverse the migration inhibition ability of shRNA-Nox4 and GKT137831 on HLEC.(4)The cells were treated with GKT137831(0,1,10,15 ?M)and then treated with VEGF-C(20ng/ml)and DMSO for 24 hours.The results of JC-1 showed that in the early stage,Nox4 could reduce mitochondrial membrane potential.The apoptosis of HLEC cells increased after GKT137831 treated.(5)HLECs were cultured in matrigel and treated with 8?M GKT137831 or 20 MOI shRNA-Nox4 lentivirus,respectively.The results showed that the tube forming ability of HLECs decreased significantly in shRNA-Nox4 or GKT137831 treated group.Even adding VEGF-C could not reverse the inhibitory effect on tube formation.(6)C57BL/6J mice aged 2-3 months were selected.The thoracic duct was cut into fragments and placed in Matrigel.8 ?M GKT 137831 and/or 20ng/ml of recombinant mouse VEGF-C treatment were adding to the matrigel.The results showed that the germination of thoracic duct increased significantly in VEGF-C group alone,but decreased significantly in GKT137831 group.Even combined with VEGF-C,it could not increase the germination of thoracic duct which There was nowas pretreated with Nox4 inhibition.The results showed that the expression of Nox4 in thoracic duct decreased after GKT 137831 treated.5.Nox4 increases CCL21 expression through ERK pathway and promotes lymphangiogenesis.In vitro experiments have confirmed that Nox4 can promote HLECs proliferation,migration and anti-apoptosis.In the follow-up study,we explored the mechanism.Western blot indicated that with GKT 137831 concentration increasing,the expression of related protein including p-ERK,p-Akt,p-JNK,MMP2 and CCL21 decreased,and cleaved caspase-3 expression increased.After that the treatment of HLECs with ERK,Akt,JNK inhibitors(U0126,Akt inhibitor ? and SP600125)and ROS inhibitor(NAC)showed that the tube forming ability of HLECs was inhibited in NAC treatment group and U0126 treatment group,but there was no significant change in Akt inhibitor VIII and SP600125 treatment group.HLECs were cultured and infected with 20MOI lentivirus to overexpress Nox4.After 24 hours,different concentrations of U0126(5,10,15?M)were given.Western blot showed that the expression of p-ERK decreased accompanied with the decrease of CCL21 protein expression.6.Nox4 promotes lymphangiogenesis and metastasis of orthotopic breast transplantation tumor in mice.The mouse orthotopic transplanted tumor model was established by highly invasive 4T1 cells.The mice were treated with GKT137831(50mg/kg)and DMSO,respectively.No significant differences in tumor size and tumor weight of transplanted mice were observed.The results showed that Nox4 expression on HLECs and regulating the expression of Nox4 may affect the regeneration of lymphatic vessels.In GKT137831 treated mice,there was no significant difference in the number of blood vessels inside and around the tumor,but the lymphatic vessels around the tumor were inhibited,and there was no significant change in the lymphatic vessels inside the tumor.Through HE staining of lymph nodes and lung tissues,we found that lymph node metastasis and lung metastasis decreased significantly after GKT137831 treated in BALB/c mice.Previous studies found that Nox4 could promote the expression of CCL21 in HLECs.Previous studies also have confirmed that the ligand of CCL21 is CCR7.Immunofluorescence detection showed that CCR7 was highly expressed in transplanted tumor tissues,and CCR7 was also highly expressed in lymph nodes and lung metastatic tissues.Conclusion1.Nox4 could increase lymphangiogenesis in breast cancer and vassociated with cancer metastasis.Patient with high expression of Nox4 in breast cancer has a poor prognosis.2.Nox4 promotes tube formation and preexisting lymphatic vessel sprouting by increasing ERK/CCL21 expression in HLECs,which can attract breast cancer cells with CCR7-positive draining to lymph nodes and spread to distant organs. |
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