| Objective : Breast cancer is one of the most common malignant tumors that affect women’s health.It is predicted that its morbidity and mortality will increase significantly within a few years.Among them,the incidence of breast cancer has increased significantly in women younger than 45 years of age,shows that breast cancer is the leading cause of cancer-related deaths worldwide.Although substantial progress has been made in the early diagnosis and treatment of breast cancer,its refractory and rising relapse rate is still worthy of attention.Therefore,we need to raise the awareness of breast cancer.Exploring the pathogenesis and progression of breast cancer is helpful for screening diagnosis and targeted treatment of breast cancer,and it can also provide new ideas and basis for the prognosis of breast cancer.The mitogen-activated protein kinase kinase(MEK)-extracellular-signal regulated kinase(ERK)pathway is an evolutionarily conserved signal transduction pathway of crucial importance in tumorigenesis.It is often aberrantly activated in malignant tumors,resulting in signal amplification during tumor invasion and metastasis.Abnormal activation leads to loss of differentiation and apoptosis and increased proliferation and invasion,causing tumorigenesis and eventual metastasis.The MEK-ERK pathway is activated at the cell membrane by Ras,which activates Raf,starting a phosphorylation cascade that results in the sequential activation of MEK1/2 and ERK1/2.ERK expression is significantly higher in breast cancer tissue than in benign hyperplastic breast tissue,and ERK phosphorylation is increased in severe atypical hyperplasia and breast cancer tissues compared with that in benign proliferating breast tissue,suggesting that abnormally increased ERK activation plays an important role in the development of atypical hyperplasia into cancer,and can stimulate the proliferation of breast cancer cells.Phosphorylated ERK(p-ERK)enters the nucleus to phosphorylate specific transcription factors such as c-Myc and c-Jun.Decreased p-ERK reduces the expression of MMP1 and MMP9,significantly inhibiting breast cancer invasiveness.In breast cancer cells,ERK inactivation is accompanied by the inactivation of cyclin D1 and BCL2,leading to apoptosis.Family sequence similarity(FAM)genes are families of uncharacterized genes of similar protein sequence.Several of these families have been linked to the development of tumors,including breast cancer,non-small cell lung cancer,lung adenocarcinoma,renal cell carcinoma,prostate cancer,colorectal cancer,and esophageal squamous cell carcinoma,where they are thought to play important roles in proliferation,apoptosis,migration,and invasion.FAM53 is a vertebrate-specific family of proteins that bind to transcriptional regulators of proliferation and neural tube development,encoded by three homologous genes: FAM53 A,FAM53B,and FAM53 C.FAM53A,also known as dorsal neural tube nuclear protein,is thought to play an important role in neuro development by specifying the fate of dorsal cells within the neural tube.Expression quantitative trait loci variants of FAM53 A identified in TP53-based interaction analysis are associated with the use of therapeutic doxorubicin in breast cancer.In the triple-negative TP53-missense mutant breast cancer cell line MDAMB-231,downregulation of FAM53 A increased doxorubicin resistance.However,in the luminal B p53-truncated mutant line MDA-MB-361 and the luminal A p53-wild-type line MCF7,downregulation of FAM53 A resulted in increased sensitivity to doxorubicin.The role of FAM53 A in breast cancer is unclear,and its relevance to the clinical pathology of breast cancer has not been reported.In this study,we examined the expression and localization of FAM53 A in breast cancer tissues and cell lines.We then altered FAM53 A expression in two breast cancer cell lines to explore its effects on the cells and gain mechanistic insight into how FAM53 A affects cancer.Methods:1.Immunofluorescence Breast cancer cells cultured in 24-well plates for 24 h were fixed in 2% paraformaldehyde for 15 min,blocked in 5% BSA for 2 h,and incubated with anti-FAM53 A antibody(1:100)at 4 ? C overnight.Cells were then incubated with a tetramethylrhodamine-labeled secondary antibody at room temperature for 2 h in the dark;the nuclei were counterstained with 4′,6-diamidino-2-phenylindole.Images were captured using an Olympus FV1000 laser scanning confocal microscope.2.Immunohistochemistry We selected primary tumor specimens from 199 patients for clinicopathological correlation analysis.All of the above patient samples were taken from female breast cancer patients,aged between 36 and 87 years,were diagnosed as invasive ductal cancer,and performed surgical resection at the First Affiliated Hospital of China Medical University between 2011 and 2013.The 199 breast cancer patients who participated in the pathological correlation analysis did not receive any adjuvant therapy such as chemoradiotherapy or endocrine before surgery.Regarding tumor size classification,we chose five centimeters as the dividing line.There were 92 cases with a tumor diameter of 5 cm or less,and 107 cases with a diameter greater than 5 cm.According to the 2009 AJCC Breast Cancer Staging Standard for TNM staging and patient pathology data,a total of 109 patients in stage I and II and 90 cases in stage III were shown.There were 119 cases of distant lymph node metastasis and 80 cases of distant lymph node metastasis.In all cases,115 cases were negative for p53 staining,and84 cases were positive for p53 staining.There were 23 cases of ER,PR,HER-2 triple negative breast cancer and 176 cases of non-negative breast cancer.Surgically removed tumor specimens were fixed in 10% neutral formalin,embedded in paraffin,and continuously cut into 4-μm thick sections.FAM53 A expression levels were evaluated based on the percentage of positive cells(PP)and the staining intensity(SI)within the whole tissue section.FAM53 A staining intensity was evaluated semi-quantitatively using the immune response score(IRS)and calculated as follows: IRS = PP × SI;where PP:0,no dye;1,1–25%;2,26–50%;3,51–75%;and 4,76–100%;and SI: 0,no staining;1,weak staining;2,medium staining;and 3,strong staining.The scores for each tumor sample were multiplied to give a final score of 0–9;tumor samples with scores >3 were classified as having high FAM53 A expression,while samples with scores ≤ 3 were classified as having low FAM53 A expression.By using SPSS 16.0 software for analysis,a one-sample paired t-test method was selected to analyze the statistical relationship between the expression level of FAM53 A and various clinical pathological factors.A P value of less than 0.05 was defined as statistically significant.3.Cell Culture and Treatment MCF-10 A,MCF-7,T47 D,MDA-MB-231,BT-474,and BT-549 cell lines were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences(Shanghai,China)and identified by short tandem repeat(STR)DNA analysis.The cells were cultured and frozen,and experiments were performed after 10 passages.MCF-10 A cells were cultured in 1:1 Dulbecco ’ s modified Eagle ’ s medium(DMEM)/F12(Gibco,Waltham,MA,USA)supplemented with 5% horse serum,10μg/m L insulin(Sigma-Aldrich Co,St.Louis,MO,USA),and 20 ng/m L epidermal growth factor(EGF).MDA-MB-231 cells were cultured in L15(Gibco)supplemented with 10% fetal bovine serum(FBS).MCF7 and T47 D cell lines were cultured in DMEM(Gibco)supplemented with 10% FBS.BT-474 and BT549 cells were cultured in Roswell Park Memorial Institute(RPMI)-1640(Gibco)supplemented with 10% FBS.All cells were cultured in sterile culture flasks,trypsinized with 0.25% trypsin.Cells were cultured in sterile culture flasks in a 5% CO2 incubator at 37?C and subcultured every 2 days by trypsinization.4.Plasmid Transfection,si RNA Interference,and Inhibitor Treatments Transfection was performed using Xfect Transfection Reagent according to the manufacturer’s instructions.The plasmids p CMV6-ddk-myc and p CMV6-ddk-myc-FAM53 A were purchased from Origene(Rockville,MD,USA).One day before transfection,cells were plated in 2 m L of complete growth medium,aiming for 50–70% confluency at the time of transfection.FAM53A(sc-88998)and non-targeted control(NC;sc-37007)si RNAs werepurchased from Santa Cruz Biotechnology,and transfected into cells using Hi Per Fect Transfection Reagent according to the manufacturers,protocols.Cells were plated in 2 m L of complete growth medium,aiming for 30–50% confluence at the time of si RNA transfection.By up-regulating and down-regulating the protein expression of FAM53 A,it provides the basis for subsequent cell functional experiments and finding related pathways of FAM53 A in breast cancer.5.Cell Proliferation and Colony Formation Assays The cells were cultured in a sterile six-well culture plate,transfected or interfered with FAM53 A to change its protein expression level,and the effect of FAM53 A on the proliferation ability of breast cancer cells was detected using MTT test.After MTT treatment,the absorbance at 490 nm was measured and the growth curve was drawn.For colony formation experiments,the cells were cultured in a sterile six-well culture plate,the protein expression level of FAM53 A was changed,and then the cells were counted.The cells were seeded at a density of 1000 cells / dish in a 40 mm culture dish and incubated for 10-15 days.The corresponding medium containing 10% fetal calf serum was changed every 3 days.The culture dish was collected,and the number and size of cell colony formation were counted through steps such as fixation and staining to measure the effect of FAM53 A on the proliferation of breast cancer cells.At least 3 independent experiments were performed under the same conditions.6.Cell Migration and Invasion Assays Cell migration and invasion experiments were performed using 24-well Transwell chambers with a pore size of 8μm.For the invasion assay,the upper chamber of the Transwell chamber was coated with 100 μL Matrigel;for cell migration assays,no Matrigel was added.Approximately 24 h after transfection,the cells were trypsinized,and 1 × 105 cells in 100 μL of medium supplemented with2% FBS were transferred to the upper chamber.Medium supplemented with 10% FBS was added to the lower chamber as a chemoattractant.After 18 h of incubation,the chamber was removed and stained with hematoxylin,and cells that migrated through the chamber membrane were counted using an Olympus FV1000 laser-scanning confocal microscope.At least three independent experimental replicates were performed.7.Western Blot Analysis Cells and tumor tissues were lysed in lysis buffer,and total protein was quantified using the Bradford method.Proteins were separated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes.Membranes were incubated overnight at 4?C with the appropriate primary antibody and for 2 h at room temperature with horseradish peroxidase-labeled secondary antibody,then visualized with ECL on a Bio Imaging System.Protein levels were analyzed using the Image J software.Western blot detection was used to detect and analyze proteins related to cell proliferation,migration,and invasion to find out the relevant pathways of FAM53 A affecting breast cancer.8.By analyzing the results of Western blot analysis,we found that FAM53 A may affect breast cancer through the MEK / ERK signaling pathway.In order to confirm that FAM53 A does indeed function through the MEK / ERK signaling pathway,based on the existing effects on this signaling pathway,PD98059,a specific MEK / ERK signaling pathway inhibitor,was selected to suppress the effect of this pathway on downstream functions,thereby reversing our conclusions.Results: 1.FAM53 A is localized in the cytoplasm and nucleus,and is lowly expressed in p53 wild-type breast cancer cells MCF-7,and highly expressed in p53 mutant breast cancer cells MDA-MB-231.In the clinicopathological correlation analysis,FAM53 A level was negatively correlated with wild-type p53.2.In p53 wild-type breast cancer cell MCF-7,when FAM53 A is over-expressed,the cell proliferation,migration and invasion ability are significantly reduced,and when FAM53A expression is down-regulated,MCF-7 proliferation,migration and invasion are enhanced.FAM53 A can regulate MCF-7 cell proliferation,migration,and invasion related functional proteins.3.In p53 mutant breast cancer cells MDA-MB-231,FAM53 A overexpression promoted cell proliferation,migration and invasion,while down-regulating FAM53 A inhibited MDA-MB-231 proliferation,migration and invasion.FAM53 A can regulate the proliferation,migration,and invasion of functional proteins related to MDA-MB-231 cells.4.In p53 wild type breast cancer cell MCF-7,FAM53 A negatively regulates EMT-related proteins,while in p53 mutant breast cancer cell MDA-MB-231,FAM53 A promotes EMT.5.FAM53 A affects breast cancer cell proliferation,migration,and invasion through the MEK / ERK pathway,and regulates EMT.6.By changing the p53 status of MCF-7 and MDA-MB-231 cells,the original effects on cell proliferation,migration and invasion,and EMT also changed.Conclusion: 1.FAM53 A Expression in Breast Cancer Cells Is Associated With p532.FAM53 A Inhibits Proliferation,Migration,and Invasion in the p53-Wild-Type Breast Cancer Cell Line MCF-73.FAM53 A Promotes Proliferation,Migration,and Invasion in the p53-Mutant Breast Cancer Cell Line MDA-MB-2314.FAM53 A Differentially Affects the Expression of Epithelial-Mesenchymal Transition(EMT)-Related Proteins5.FAM53 A Regulates Breast Cancer Cells Through the MEK/ERK Signaling Pathway6.The opposite regulatory effect of FAM53 A on MCF-7 and MDA-MB-231 cells depends on their p53 status... |
