Smad1 And P38 MAPK-c-Myc Axis Participating In Regulating Estrogen To Promote Breast Cancer Cell Proliferation

BackgroundBreast cancer is the most prevalent cancer malignancy and the leading cause of cancer-related mortality in women in developed countries.A survey,in 2014,in the United States,estimated 232,670 women would be diagnosed with invasive breast cancer,and 40,000 might die from it.Breast cancer,a clinically and biologically heterogeneous disease,is induced by dysregulation of multiple cellular pathways,which may contribute to the research of tumorgenesis.A report recently released from the Ministry of Health shows that breast cancer is the most common female cancer in Shanghai,Beijing,Tianjin and other major cities,and the incidence is rising year by year.Because of its different sensitivities to treatment,a wide range of survival durations for patients has shown.The ER,encoded by the ESR1 gene,belonging to the nuclear receptor superfamily,and sharing the typical structural configuration and functional aspects associated with its family members,is predominantly a nuclear protein---a ligand-dependent transcription factor.The ER-ligand complex binds directly to a specific DNA sequence called the estrogen response element(ERE),and interacts with co-activator or co-repressor proteins to regulate the transcription of estrogen-responsive genes that are important in various physiological and pathological processes,including tumorigenesis and tumor progression,of which the mechanism is not entirely clear.Thus the research on how the estrogen works in breast cancer is very important.BMP-Smadl pathway involves in embryonic development and tissue homeostasis,and is related to the DNA damage response and tumorigenesis.When genotoxic irritation occurs,BMP-activated Smadl is phosphorylated by ATM on serine 239 in the nucleus,destroying the interaction between Smadl protein phosphatase PPM1A so that Smadl activation upregulation is enhanced.Furthermore,Smadl interacts with p53 to inhibit Mdm2-mediated p53 ubiquitination and degradation.Eventually,cell proliferation and survival was adjusted.Therefore,further study of the role of BMP-Smadl pathway in tumors,particularly in the cycle and apoptosis of breast cancer cells will have important biological significance.Purpose1)To study whether estrogen promoted development and progression of breast cancer cells is dependent on the BMP-Smad1 pathway,and explore other important collaborated pathway in the process of estrogen action in breast cancer.2)To clarify the mechanism of BMP-Smad pathway working in estrogen promoted development and progression of breast cancer.3)To assess the possibility of Smadl and other target of this pathway as diagnostic and prognostic markers of breast cancer,and explore their feasibility as breast cancer treatment targets.Methods1)Different concentrations of estrogen was added in breast cancer cell line MCF-7 and MDA-MB-231 cell lines and primary mouse embryonic fibroblast MEFs,RNA and protein was then extracted,use real-time PCR and WB to testify that estrogen can activate BMP-Smadl pathway.2)Clarify that estrogen promotes cell proliferation by BMP-Smadl pathway.(A)Estrogen was added in breast cancer cell line MDA-MB-231 and MCF-7 Smad1 knockout cells,then tested by WST-1.(B)Use Annexin V-FITC/PI kit on flow cytometry to conduct apoptosis detection of cell line MCF-7-ShSmadl/ShCTRL,MDA-MB-231-ShSmadl/ShCTRL.(C)Use cell line MDA-MB-231-ShSmadl/ShCTRL,MCF7-ShSmadl/ShCTR to conduct nude mice experiment,tumor volume was observed in nude mice.3)CHIP experiment was performed after estrogen was added in MDA-MB-231-ShSmadl and MDA-MB-231-CTRL cell lines.4)Knock down Smadl and p38 to study the activation of the downstream transcription factor,observe the cell proliferation and cell cycle in the presence of estrogen.5)Collect and analysis of clinical tumor samples and normal samples,using tissue chip to stain Estrogen receptor,p-Smad1,Smad1,and give correlation analysis.Results1.Estrogen activating BMP/Smadl pathway.We find estrogen could activate BMP-Smadl pathways in MEFs and breast cancer cell lines MCF7 and MDA-MB-231.Smadl,a key gene in BMP-Smadl pathway,could increase transcription and translation in the stimulation of estrogen.p-Smadl is activated after four hour activation of estrogen and then reach maximum at twelve hour later.When knocking down Smadl,estrogen could not affect Smadl expression.2.BMP/Smad1 is activated in breast cancer,and relates positively to estrogen receptor.Smadl enhance expression and that p-Smad1 activation is inhibited probably because of BMP receptor mutant,which indicates BMP-Smad1 pathway playing complex controlling role in the process of cell occurrence and development.Meanwhile,by using WB and IHC,we find Smadl having positive relationship with ERa levels.3.Estrogen promotes breast cancer cell proliferation and migration depend partially on Smadl.In Smad1 knocked down breast cancer cell,estrogen impacts less on progression of proliferation and migration which indicates estrogen manipulation in cell depending partially on Smadl high expression.In nude mice tumor formation experiment,after tumor formatted by cell knocked down by Smad1,that tumor size is less than control group may be influenced by,when estrogen stimulation,cell cycle lagging in G1 after Smadl knocked down in breast cancer cell.4.p38 MAPK-c-Myc pathway Manipulates estrogen induced breast cancer cell proliferationWe find,by meticulously filtering that the c-Myc protein levels is enhanced under p38 MAPK downstream.c-Myc expression is inhibited partially when Smad1 was knocked down with estrogen treatment.We use CHIP experiment to find Smadl binding to the promoter of c-Myc and its combination ability enhanced when added stimulation of estrogen.5.Estrogen promotes c-Myc expression by Smad1 and p38 MAPK pathway,thereby improving cell proliferation,regulating cell cycleBy studying cell proliferation,when Smadl is knocked down,estrogen promoting proliferation is weakened to over-express c-Myc and then to rescue this phenomenon.Meanwhile cell circle experiment confirmed that G1 checkpoint was reversed.ConclusionSmadl and p38 MAPK-c-Myc axis participating in regulating estrogen to promote breast cancer cell proliferation...