Study On The Role And Mechanism Of ESM1 And Ferritin In The Malignant Progression Of Triple Negative Breast Cancer

Breast cancer is the most common cancer worldwide and the leading cause of cancerrelated deaths among women.With the development of first-line endocrine therapy,breast cancer treatment has made great progress.However,69,000 women still died of the disease in China in 2015.The main cause of death was the spread to distant tissues of breast tumors,that is,tumor metastasis.Radiochemotherapy and targeted therapy can achieve good therapeutic effects on breast cancer in situ,but metastatic tumor foci located in the bones,lungs,and brain often show insensitivity to treatment and easily develop treatment resistance.Triple negative breast cancer(TNBC)is more prone to relapse,drug resistance and metastasis than other types of breast cancer,and lacks endocrine therapy targets.Tumor repopulating cell(TRC)is a subpopulation of cancer cell found in most solid cancers including breast cancer.It is characterized by stem-like properties and the potential for tumor formation.TRC proliferation,migration and invasion are considered to be the main causes of tumor recurrence and metastasis.On the other hand,chemotherapeutic drugs often fail to completely kill these cells and induce drug resistance at the same time.Therefore,systematic study of the molecular characteristics of triple negative breast cancer repopulating cells(TNBC-TRC)can provide potential therapeutic targets as well as new ideas and strategies for the diagnosis and treatment of breast cancer.Recent studies have shown that the soft threedimensional fibrin matrix culture method can efficiently and reliably isolate tumor repopulating cells,and has been well applied in the research of melanoma,colon cancer and lung cancer.Endothelial cell-specific molecule 1(ESM1),also known as endocan,is overexpressed in various cancers including breast cancer and may play an important role in cancer progression.In this study,on the one hand,we studied the effect of ESM1 on the proliferation of TNBC cells and its regulatory mechanism.On the other hand,the triple-negative breast cancer cell line MDA-MB-231 was used to obtain TNBC-TRC by the abovementioned soft three-dimensional fibrin matrix culture,and the integrative analysis of proteome and transcriptome were carried out,which were compared with TNBC cells from regular rigid culture;Based on omics analysis,we selected ferritin FTH1 and FTL as key candidate molecules to study their role and mechanism in TNBC-TRC proliferation and drug resistance;Furthermore,exosomes of TNBC-TRC and regularly cultured TNBC were collected for qualitative analysis.Our research results are summarized as follows:1.We found that ESM1 is overexpressed in TNBC patients as well as TNBC cell lines and correlated to poor prognosis.Our results demonstrate that overexpression of ESM1 increases in vitro proliferation,migration and invasion of TNBC cells.Our mechanistic study further discloses that ESM1 promotes the proliferation of TNBC cells through activating an Akt-dependent NF-?B/Cyclin D1 pathway.2.TNBC-TRC was obtained by culturing MDA-MB-231 cells with soft threedimensional fibrin matrix.Compared with the conventionally cultured MDA-MB-231 cells,TNBC-TRC has significantly increased migration ability,plate cloning ability and tumor forming ability.3.To systematically study the molecular characteristics of TNBC-TRC,using the triple negative breast cancer cell line MDA-MB-231,TNBC-TRC and regularly cultured TNBC were examined using transcriptome and proteome detection and analysis.The results showed that multiple molecular pathways related to tumorigenesis and tumor progress and metastasis of are enriched in TNBC-TRC.4.Transcriptome and proteome results showed that the NRF2-mediated oxidative stress pathway was significantly enriched in TNBC-TRC,and the expression of both ferritin FTH1 and FTL in this pathway was significantly up-regulated.5.The application of ferric ammonium citrate,which increases the intracellular iron ion content,can significantly promote sphere formation of TNBC cells;while the application of deferoxamine to reduce the intracellular iron ion content significantly inhibits sphere formation of TNBC cells.6.We screened to obtain the best si RNAs to knockdown FTH1 and FTL respectively.Knockdown of the ferritin expression can significantly increase the sensitivity of TNBC cells to doxorubicin and reduce its ability to form spheres.7.Through exploring the molecular mechanism of upstream and downstream regulation of ferritin,we found that ferritin can down-regulate the expression of KIF18 B and BEST1,and knocking down KIF18 B and BEST1 can significantly increase the sensitivity of TNBC cells to doxorubicin and reduce its ability to form spheres.8.The construction and analysis of protein-protein interaction(PPI)network screened out 13 key molecules,such as f2rl2,CSF2,TLR6,clic5,nsun4,app,VEGFA,EGF,CXCL8,FOS,Cdc42,EGR1 and AR,which play important roles in the development of TNBC-TRC and may serve as candidate targets.9.Through gel electrophoresis staining and analysis of exosomal proteins,we found that in the exosomes of MDA-MB-231-TRC cells,there were two more protein bands compared with those of MDA-MB-231 cells.The results of qualitative detection by mass spectrometry showed that these two protein bands are mainly enriched in keratin family,Rab protein family and ribosomal protein.Our results indicate that multiple molecular pathways related to tumorigenesis,progression,and metastasis are enriched in TNBC-TRC,and the expression of ferritin FTH1 and FTL in oxidative stress pathway is significantly upregulated in TNBC-TRC.Ferritin plays an important role in the sensitivity to chemotherapeutic drugs as well as in proliferation,migration and invasion of TNBC-RTC.Ferritin and the molecules in its upstream and downstream pathways may be effective targets for the treatment of TNBC.Our mechanistic study further discloses that ESM1 promotes the proliferation of TNBC cells through activating an AKT-dependent NF-?B/Cyclin D1 pathway.This study deepens the understanding of the malignant progression of triple-negative breast cancer and its related mechanisms,and provids potential new targets for the treatment of TNBC,while laying the theoretical foundation for the diagnosis,efficacy monitoring as well as prognostic judgment of TNBC.More mechanisms of ferritin and the molecules in its upstream and downstream pathways to regulate the malignant progression of TNBC,as well as the role and mechanisms of other molecules and pathways discovered by our multi-omics analysis in the malignant progression of TNBC need to be further studied.