| BackgroundBreast cancer is one of the most prevalent malignancies in women,accounting for more than 24%of new female cancer cases and about 15%of cancer-related death around the world.Despite the development in diagnostic and therapeutic methods,the prognosis of breast cancer patients remains unsatisfactory,particularly concerning metastatic breast cancer.Previous studies reported that 3-10%of breast cancer patients occur distant metastases at the time of primary diagnosis,and 25%of primary non-metastatic cases finally develop metastasis.Moreover,the issues of intrinsic and acquired drug resistance and heterogeneity in breast cancer greatly limited the efficiency of chemotherapy,often attributed to treatment failure and tumor recurrence.Therefore,characterization of the underlying molecular mechanisms of chemoresistance and metastasis would help to develop novel therapeutic strategy to improving the overall survival rate in breast cancer patients.Several previous studies reported that various cancer treatment methods could induce cell-specific programmed cell death,which was known to be closely associated with tumor development and progression.Ferroptosis,a novel form of programmed cell death first identified in 2012,has gained immense attention as a potential therapeutic pathway for cancer treatment.Ferroptosis is characterized by iron-dependent lipid peroxide accumulation,which differs from traditional apoptosis,autophagy,or necrosis in terms of morphology,biochemistry,and genetics.Numerous genes have been previously identified as markers,inducers,or inhibitors of ferroptosis,collectively called ferroptosis-related genes(FRGs),such as GPX4,CISD1,and NRF2.Previous studies identified a pivotal role of ferroptosis in tumor progression and therapies.Although the sensitivity of different types of tumor cells towards ferroptosis are diverse,a combination of erastin and chemotherapeutics could improve curative effects in various cancers,such as ovarian cancer,lung cancer,and breast cancer.Moreover,ferroptosis is associated with the prognosis of various cancers,and some ferroptosis-related prognostic models have been constructed in glioma,Melanoma,and renal cell carcinoma,further indicating the potential value of FRGs as prognostic markers and therapeutic targets in human cancers.Accumulated evidences indicated that ferroptosis played a significant role in the fate of cancer cells and response to various cancer treatments,such as chemotherapy,radiotherapy,and immunotherapy.Many reports also showed that ferroptosis has strong association with breast cancer,indicating that ferroptosis may be expected to become important biomarkers of breast cancer.Therefore,set up ferroptosis related predictive model and further elucidate the molecular mechanism of ferroptosis,would help to predict the prognosis of breast cancer and improve the therapeutic effect,further to improve the prognosis of breast cancer patients.The tumor immune microenvironment usually comprises immune cells and immune-related molecules that are present around the tumor,which shows significant roles in the tumor stroma interaction and the response towards immunotherapy.In recent years,the association of immune cells and immune molecules with iron metabolism has gained immense attention.Various types of immune cells,including Thl cells,natural killer T cells,and macrophages,have been previously shown to be associated with the maintenance of iron homeostasis.Interestingly,ferroptosis in tumor cells makes tumor antigens increase to expose to immune cells,which further promotes the anti-tumor efficacy of immunotherapy.However,the role of ferroptosis in immunotherapy of breast cancer has not been fully elucidated.In this study,a ferroptosis-related prognostic model was constructed based on mRNA expression profiles and clinical data of breast cancer patients obtained from GSE20685 cohort.The model was further validated using the data from METABRIC cohort.Moreover,gene signature characteristics in the tumor microenvironment were also evaluated through ssGSEA and immune infiltration analysis.Based on tissue expression and prognosis analysis,heat shock protein β-1(HSPB1)was selected for further study in this study to comprehensively explore the role and regulatory mechanism of HSPB1 in breast cancer.Functional experiments verified that HSPB1 showed a crucial role in the progression of breast cancer.We further revealed the relevance of HSPB1 in the regulation of chemoresistance of breast cancer for the first time,which was mediated by chemotherapeutics-induced ferroptosis.Our study highlights HSPB1 as a novel regulator of chemoresistance in breast cancer,providing the potential that targeting HSPB1 might be a novel strategy to prevent breast cancer progression and overcome therapy resistance,which might assist in improving clinical outcomes of breast cancer patients when subjected to personalized treatment.Part ⅠConstruction of ferroptosis-related prognostic model and analysis of immune infiltration in breast cancerObjective1.Classify breast cancer patients based on the expression of ferroptosis-related genes,and analyze the differences in gene expression,clinical characteristics and prognosis among different subgroups of breast cancer patients.2.Construct and verify ferroptosis-related prognosis prediction model for breast cancer patients using GEO and METABRIC databases.3.Evaluate the predictive value of the prognosis model based on ferroptosis-related genes,and construct a nomogram model.4.Analyze the differences in survival,immune infiltration,and immunotherapy response between high-risk group and low-risk group breast cancer patients.5.Evaluate the potential of targeting ferroptosis-related genes in breast cancer therapy.Methods1.Ferroptosis-related genes were obtained based on databases and literature reports,and expression profile of ferroptosis-related genes and clinical information of breast cancer patients were obtained from GSE20685 database.Breast cancer patients were grouped and verified using NMF,t-SNE,and PCA analysis,and the gene expression,clinical characteristics,and prognosis of different group of breast cancer patients were analyzed.2.Treat the GSE20685 database as a training set and the METABRIC database as a validation set.A ferroptosis-related prognostic model in breast cancer patients was established and verified using COX univariate,Lasso and COX multivariate regression analysis.Kaplan-Meier and ROC analysis were used to evaluate the predictive value of the model.3.A nomogram model was established by univariate and multivariate COX regression analysis,and its clinical value was evaluated by calibration curve,ROC,and DCA analysis.4.Based on the ferroptosis-related prognostic model,breast cancer patients were divided into high-risk and low-risk groups.The ESTIMATE,CIBERSORT,and ssGSEA algorithms were used to analyze the immune infiltration of breast cancer patients in high risk and low risk groups,and the difference of immunotherapy response between the two groups was evaluated by SubMap analysis.5.Cellular function assay was used to evaluate the role of ferroptosis-related genes in breast cancer progression.Download the CellMiner database to obtain the drug z score and the corresponding gene expression of NCI-60 cancer cell line,and search for potential therapeutic drugs targeting ferroptosis-related genes.Results1.Ferroptosis-related gene information was obtained from GeneCards database,FerrDb database,and other related literature.After integration,314 ferroptosis-related genes were included.Screened by MAD,a total of 171 genes were used for subsequent subgroup analysis.Breast cancer patients were divided into two subgroups(cluster 1 and cluster 2)by NMF analysis,and the accuracy of this grouping was confirmed by t-SNF and PCA analysis.The survival rate of cluster 2 was significantly lower,and there were significant differences in ferroptosis-related gene expression and clinical characteristics between two groups.2.The RNA expression of ferroptosis-related gene and clinical information of 327 breast cancer patients obtained from the GSE20685 database were regarded as the training set,and the RNA expression and clinical information of 1904 breast cancer patients from METABRIC database were regarded as the verification set.Univariate Cox,Lasso,and multivariate Cox regression analyses were performed using GEO data,and a prognostic model containing 9 ferroptosis-related genes was constructed.Kaplan-Meier survival analysis showed that overall survival was significantly lower in the high-risk group.ROC analysis showed the significant specificity and sensitivity of established prognostic model in predicting overall survival rate of breast cancer patients.Using METABRIC database as a validation set,the prediction efficiency of the proposed prognostic model is verified.3.Univariate and multivariate Cox regression analysis of the 9-gene prognostic model and the clinical characteristics of breast cancer patients showed that TNM stage and risk score were independent predictors,and a nomogram model is constructed.Calibration curve,ROC and DC A analysis proved the clinical value of the nomogram model.4.The ESTIMATE algorithm showed that the tumor tissue of breast cancer patients in the high-risk group had higher tumor purity and lower immune score.CIBERSORT algorithm found that breast cancer patients in the high-risk group had a higher immunosuppressive microenvironment.ssGSEA analysis showed that the proportion of immune cell infiltration of high-risk breast cancer patients was generally lower.SubMap analysis found that breast cancer patients in the low-risk group may benefit from anti-PD-1 therapy.5.Cellular function experiments showed that ferroptosis inducer can induce ROS production and inhibit the proliferation,activity,migration,and drug resistance of breast cancer cells.Database analysis suggests that targeting ferroptosis-related gene might play an important role in breast cancer therapy.Conclusions1.Breast cancer patients can be divided into two subgroups based on the expression profile of ferroptosis-related genes,and the two groups have different ferroptosis-related gene expression patterns,TNM stages,and prognosis.2.Based on the expression of 9 ferroptosis-related genes,a prognostic model of breast cancer was constructed and verified.3.The 9-ferroptosis-related genes prediction model has significant specificity and sensitivity in predicting the overall survival rate of breast cancer patients.4.According to the constructed prognostic prediction model,breast cancer patients can be divided into high risk and low risk groups,and there are significant differences in prognosis,immune infiltration pattern,and immunotherapy response between two groups.5.Ferroptosis inducers play an important role in inhibiting drug-resistant and metastasis of breast cancer,and targeting ferroptosis-related genes is expected to become a new strategy for breast cancer treatment.Part II Screening and functional study of ferroptosis-related gene HSPB1 in breast cancerObjective1.Screen the key ferroptosis-related gene in breast cancer.2.Analyze the expression of HSPB1 in breast cancer tissues and normal tissues,and evaluate the correlation between HSPB1 expression and prognosis of breast cancer patients.3.Explore the effect of HSPB1 on the proliferation,metastasis,drug resistance,and ferroptosis using in vitro experiments.4.Study the effect of HSPB1 on the drug resistance and metastasis through in vivo experiments.Methods1.According to the hierarchical cluster analysis of TCGA and GEO database,as well as detection of differentially expressed genes between breast cancer and normal tissue samples using immunohistochemical and qRT-PCR assays,breast cancer related gene HSPB1 was selected.Subsequently,Kaplan-Meier analysis,Chi-square test,univariate and multivariate Cox regression analysis were used to explore the correlation between HSPB1 and the prognosis and clinical characteristics of breast cancer.2.The HSPB1 overexpression vector and siRNA were constructed,and the following in vitro experiments were carried out in MDA-MB-231 and MDA-MB-468 cell lines with HSPB1overexpressing or knocking down.Cell proliferation,colony formation,Edu assays were used to explore the effect of HSPB1 on the proliferation of breast cancer cells.Wound healing,migration and invasion assays,and western blot were performed to explore the effect of HSPB1 on the migration and invasion of breast cancer cells.IC50 detection and cytotoxicity assay were used to detect the effect of HSPB 1 on doxorubicin resistance of breast cancer cells.Reactive oxygen species(ROS)analysis and malondialdehyde(MDA)assay were used to detect the effect of HSPB 1 on ferroptosis of breast cancer cells.3.A xenograft transplantation model in nude mice was used to further evaluate the effect of HSPB1 on breast cancer in vivo.MDA-MB-231 cells with stable HSPB1 overexpression or control vectors were implanted subcutaneously into the right flank regions of the BALB/c nude mice.When the subcutaneous tumor reached 50 mm3,each group of nude mice were randomly divided equally into two groups and intravenous injected DOX or the same volume of solvent control.After a certain period of time,the mice were killed and the tumors were harvested for IHC analysis of related gene expression.In addition,lung metastasis model was used to study the effect of HSPB1 on breast cancer metastasis in vivo.H&E staining was used to compare the size and number of pulmonary metastatic nodules.Results1.Hierarchical cluster analysis of TCGA and GEO database and tissue samples of patients showed that the expression of HSPB1 in breast cancer tissues was higher than that in normal tissues.In addition,the expression of HSPB1 in breast cancer cell lines was higher than that in normal cells.Kaplan-Meier analysis showed that the overall survival and disease-free survival of breast cancer patients with high expression of HSPB1 were worse.Chi-square test showed that the high expression of HSPB1 was significantly correlated with distant metastasis of breast cancer(P<0.001).Univariate and multivariate Cox regression analysis showed that the high expression of HSPB1 predicted the poor prognosis of breast cancer patients.2.A series of in vitro experiments using MDA-MB-231 and MDA-MB-468 cells with overexpressing or knocking down HSPB1 have shown that overexpression of HSPB1 promotes the proliferation,migration and invasion of breast cancer cells.Overexpression of HSPB1 leads to the decrease of the expression of epithelial markers(E-cadherin)and the increase of interstitial markers(Fibronectin,N-cadherin,Vimentin),confirming that HSPB1 plays a significant role in regulating EMT of breast cancer cells.In doxorubicin-resistant breast cancer cells,the expression levels of mRNA and protein of HSPB1 were significantly higher than those of parental cells.Doxorubicin treatment increased the expression of HSPB1 in a dose-and time-dependent manner,and HSPB1 can promote doxorubicin resistance of breast cancer cells.Erastin(the inducer of ferroptosis)increased the expression of HSPB1 in a dose-dependent manner.The overexpression of HSPB1 decreased the concentration of reactive oxygen species induced by erastin in breast cancer cells,while the treatment of ferrostatin-1(Fer-1,the inhibitor of ferroptosis)had a synergistic effect with the overexpression of HSPB1.The ROS and MDA analyses indicated that HSPB1 could inhibit the ferroptosis of breast cancer cells.3.A xenograft model in nude mice found that overexpression of HSPB1 led to an increase in tumor volume and tumor weight.The tumor of mice injected with doxorubicin was smaller and lighter than that of mice treated with PBS,which indicated that doxorubicin could inhibit the growth of breast cancer cells in vivo.IHC analysis showed that HSPB1 could increase the resistance to doxorubicin of breast cancer in vivo.In addition,the lung metastasis model showed that the overexpression of HSPB1 significantly increased the size and number of pulmonary metastatic nodules.In conclusion,the results of in vivo experiments showed that HSPB1 promoted the growth,doxorubicin resistance,and in vivo metastasis of breast cancer cells.Conclusions1.HSPB1 was preliminarily screened as a key ferroptosis-related gene in breast cancer.2.The expression of HSPB1 in breast cancer tissues is significantly higher than that in normal tissues,and high expression of HSPB1 is associated with poor prognosis of breast cancer patients.3.In vitro experiments demonstrated that HSPB1,acting as a tumor-promoting gene,can promote the proliferation,migration and invasion of breast cancer cells,and enhance the doxorubicin resistance of breast cancer cells by inhibiting ferroptosis.4.In vivo experiments have proved that HSPB1 can promote the progression and drug resistance of breast cancer.Part Ⅲ The mechanical study that HSPB1 promotes drug resistance and metastasis of breast cancer through regulating NF-κB pathwayObjective1.Explore the effect of HSPB1 on NF-κB signaling pathway.2.Evaluate the role of NF-κB signaling pathway in HSPB1-mediated breast cancer drug resistance and metastasis.3.Analyze the effect of HSPB1 on macrophage infiltration in breast cancer and explore the related mechanism.4.Elucidate the underlying mechanism of HSPB1 promoting the resistance of breast cancer cells to doxorubicin-induced ferroptosis.Methods1.The effect of HSPB1 on the expression of molecules related to NF-κB signal pathway were detected by Western blot.The effect of HSPB1 overexpression on the activation of NF-κB pathway were investigated by NF-κB report assay,western blot,and qRT-PCR.2.The expression of NF-κB was detected by nuclear and cytoplasmic protein isolation and immunofluorescence assays,and the effect of HSPB1 expression on nuclear translocation of NF-κB was investigated.The effect of HSPB1 expression on the binding of Ikβ-α and NF-κB was investigated by co-IP assay.Breast cancer cells were treated with proteasome inhibitors and lysosome inhibitors to detect the role of HSPB1 in regulating the protein stability of Iβ-α,and explore whether Ikβ-α can be degraded by proteasome or lysosome.The cells were treated with protein synthesis inhibitor to detect the half-life of Ikβ-α.The ubiquitination of Ikβ-α was detected by Western blot.3.NF-κB reporter assay,western blot,and qRT-PCR were used to confirm that knockdown Ikβ-α can activate NF-κB signaling pathway.MTT,colony formation assay,Edu,transwell,wound healing,and ROS assay were used to evaluate whether activation of NF-κB signaling pathway can reduce the inhibited effect induced by HSPB1 knockdown.4.ELISA was used to investigate whether the secretion of IL6 was affected by HSPB1.The effect of IL6 on malignant behaviors of breast cancer cells was examined by cell migration assay and HUVEC tube formation assay.CIBERSORT,xCell,and quanTIseq algorithms were used to evaluate the infiltration level of immune cells in the tumor microenvironment.The supernatant of culture medium from HSPB1 knockdown or overexpression cells was used to evaluate whether HSPB1 could regulate the infiltration of macrophages in breast cancer.Anti-IL6 neutralizing antibodies were added to the culture medium to verify that the promoted malignant behavior of macrophages by HSPB1 was mediated by IL6.Results1.Overexpression of HSPB1 can increase the protein levels of p-Ikβ-α,NF-κB,and its target genes,while the protein expression of Ikβ-α was decrease.qRT-PCR and immunofluorescence detection also showed that Survivin and IL6 were increased after HSPB1 overexpression.In addition,HSPB1 knockdown can inhibit the activation of NF-κB signaling pathway.These data indicate that HSPB1 is involved in the activation of NF-κB signaling pathway.2.Overexpression of HSPB1 can promote the nuclear translocation of NF-κB in breast cancer cells,and the nuclear translocation of NF-κB is inhibited after HSPB1 knockdown.Co-IP experiment confirmed that HSPB1 can bind to Ikβ-α.Proteasome inhibitors can lead to up-regulation of Ikβ-α expression,and HSPB1 overexpression significantly reduces the half-life of Ikβ-α,suggesting that HSPB1 can reduce the protein stability of Ikβ-α.HSPB1 regulates the ubiquitination of Ikβ-α in breast cancer cells,and knockdown of HSPB1 reduces the ubiquitination level of Ikβ-α.These findings suggest that HSPB1 can enhance the ubiquitination mediated degradation of Ikβ-α by binding to Ikβ-α,leading to the release of NF-κB from the cytoplasmic NF-κB/Ikβ-α complex,and promote the nuclear translocation of NF-κB,thereby enhancing the activity of NF-κB.3.The NF-κB report experiment and detection of NF-κB target gene expression confirmed that the increase of Ikβ-α expression and decrease of NF-κB activity induced by HSPB1 knockdown could be recovered by knockdown Ikβ-α,indicating that knockdown Ikβ-αcan activate NF-κB signaling pathway.Ikβ-α knockdown can weaken the inhibitory effect caused by HSPB1 knockdown on cell proliferation,migration and invasion.In HSPB1 knockdown cells,doxorubicin or erastin induced inhibition of cell viability and enhancement of reactive oxygen species production can be restored by simultaneous knockdown of Ikβ-α.These results suggest that HSPB1 regulates the biological behavior of breast cancer cells and doxorubicin-induced ferroptosis through modulating the activity of NF-κB,and restoration of NF-κB activity can reduce the inhibitory effect caused by HSPB1 knockdown in breast cancer cells.4.ELISA assay showed that knockdown of HSPB1 resulted in decreased secretion of IL6,while overexpression of HSPB1 promoted secretion of IL6.The supernatant from HSPB1 overexpressing cells led to increased breast cancer cell migration and HUVEC tube formation,and supplementation with IL6 neutralizing antibodies partially attenuated this stimulative effect.The expression level of HSPB1 was positively correlated with the infiltration level of M2 macrophages and the ratio of M2 to M1,but negatively correlated with the infiltration level of M1 macrophages.These results suggest that HSPB1 promotes malignant behavior of breast cancer cells by regulating the expression of IL6.Conclusions1.HSPB1 can promote the activation of NF-κB signal pathway in breast cancer cells.2.Activation of NF-κB signaling pathway can restore the inhibitory effect caused by HSPB1 knockdown in breast cancer cells.3.HSPB1 inhibits doxorubicin-induced ferroptosis by promoting ubiquitination degradation of Ikβ-α,leading to increased nuclear transmutation of NF-κB in breast cancer cells.4.HSPB1 can promote the infiltration of M2 macrophages by regulating the expression and secretion of IL6. |
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