| Background:Endocrine therapy is widely used in the treatment of hormone receptor positive breast cancer,because of its efficacy,low toxicity and good tolerance.However,its efficacy is limited to some extent by primary drug resistance,secondary drug resistance caused by long-term drug use,and its role in promoting tumor progression.At present,the mechanism of endocrine drug resistance in breast cancer has not been clarified.As a member of the Translation Initiation Factor family,EIF4H(Eukaryotic Translation Initiation Factor 4H)was first identified to promote translation in rabbit reticulocytes by increasing EIF4A(Eukaryotic Translation Initiation Factor 4A)processing capacity to enhance the activity of EIF4 A helicase,thereby promoting protein synthesis.However,there are few studies on the relationship between EIF4 H and tumorigenesis,and its role in breast cancer remains unclear.We previously screened the differentially expressed proteins in wild-type MCF7 and Tamoxifen-resistant MCF7(MCF7/TAMR)by protein expression microarray,and found that EIF4 H expression was significantly up-regulated in TAM resistant MCF7.Furthermore,overexpression of EIF4 H in breast cancer cells can activate the PI3K-AKT signaling pathway,through protein expression microarray.Therefore,we hypothesized that EIF4 H could affect the sensitivity of endocrine therapy in breast cancer through activation of PI3K-AKT signaling pathway.Purpose:Our aim is to elucidate the role and molecular mechanism of EIF4 H in endocrine therapy sensitivity of breast cancer,which is expected to provide a theoretical basis for the determination of therapeutic targets for breast cancer.Methods:1.The expression of EIF4 H in MCF7/TAMR cells was verified by Western blot.The Expression differences of EIF4 H in breast cancer tissues and normal breast tissues were analyzed by the public database GEO(Gene Expression Omnibus).Immunohistochemistry(IHC)was used to detect the expression of EIF4 H in 20 paired breast cancer tissues and adjacent tissues.The protein expression of EIF4 H in normal breast cancer cells and breast cancer cell lines was detected.2.The expression level of EIF4 H in 226 invasive breast cancer tissue samples was determined by IHC,in order to analyze the relationship between the expression of EIF4 H and the clinicopathological characteristics of breast cancer patients.KaplanMeier was used to analyze the relationship between different EIF4 H expression levels and prognosis of breast cancer patients.Univariate and multivariate analyses were performed to determine whether EIF4 H was an independent prognostic indicator for breast cancer patients.3.According to the expression of EIF4 H in breast cancer cell lines,MCF7 stable cell lines with stable overexpression of EIF4 H were constructed by exogenous transfection of EIF4 H lentivirus expression plasmid,and the expression level of e IF4 H was detected by Western blot.CCK-8(Cell Counting Kit-8),plate clone formation,Ed U and cell cycle assay were used to evaluate the effect of EIF4 H expression level on the proliferation ability of breast cancer cell line MCF7 in vitro.The effect of EIF4 H on the cell cycle progression of breast cancer was determined by cell cycle synchronization experiment.The EIF4H-sh RNA(small hairpin RNA)lentivirus expression plasmid was exogenously transfected into MCF7/TAMR stable cell lines with stable and downregulated EIF4 H expression.The effects of EIF4 H expression on the endocrine sensitivity of MCF7/TAMR cells were verified by in vitro functional experiments and subcutaneous tumor-forming experiments in mice.4.Co-immunoprecipitation(co-IP)assay was used to verify the interaction between EIF4 H and EGFR.The regulation of EIF4 H on PI3K-AKT signaling pathway and downstream molecules was detected by Western blot assay.Endogenous and exogenous ubiquitination experiments were performed to confirm that EIF4 H inhibited EGFR ubiquitination degradation.The effect of EIF4 H on the nucleation of ERα phosphorylation was detected by immunofluorescence assay and nucleoplasmic protein separation assay.5.The effects of E2/ERα on EIF4 H protein and m RNA expression levels in MCF7 cells were detected by Western blot and Nuclear Run-On assay.Chromatin Immunoprecipitation(Ch IP)assay demonstrated that ERα binds to the promoter region of EIF4 H.The proteins which could bind with EIF4 H promoter region containing ERα binding site were screened by Reverse Ch IP.The methylation levels of EIF4 H promoters(-500 ～-200 bp)in breast cancer cell lines MCF7 and MCF7/TAMR were determined by sulfite sequencing.Results:1.Analysis of GEO database GSE65194 showed that EIF4 H m RNA expression was up-regulated in breast cancer tissues(P < 0.001).Immunohistochemistry also showed that EIF4 H expression was significantly up-regulated in matched breast cancer tissues(P < 0.05),and EIF4 H expression was negatively correlated with ER status(P = 0.001).Compared with normal breast cell line MCF10 A,EIF4H expression was upregulated in multiple breast cancer cell lines,and was lower in ER-positive breast cancer cell lines,and higher in ER-negative breast cancer cell lines and MCF7/TAMR.2.Among 226 cases of invasive breast cancer,109 cases(109/226,48.2%)were high expression of EIF4 H,and 117 cases(117/226,51.8%)were low expression of EIF4 H.The expression of EIF4 H was significantly correlated with ER status(P=0.001),PR status(P = 0.004),the number of lymph node metastases(P = 0.007)and age(P =0.038).Patients with high EIF4 H expression had worse Disease-free Survival(P < 0.0001)and Overall Survival(P = 0.010)than those with low EIF4 H expression.In ER-positive breast cancer cases,EIF4 H expression was correlated with DFS and OS.However,there was no correlation between EIF4 H expression and DFS and OS in ER-negative breast cancer patients.Univariate and multivariate analyses showed that EIF4 H was an independent prognostic indicator of DFS and OS in patients with breast cancer.KM-Plotter prognosis prediction showed poor prognosis in breast cancer patients with high EIF4 H expression after systematic treatment,and subgroup analysis also showed that EIF4 H expression level was associated with prognosis in breast cancer patients with endocrine therapy.3.In vitro functional experiments showed that EIF4 H promoted the proliferation of MCF7 cells.Furthermore,it was found that EIF4 H regulates the G1/S phase of breast cancer cell cycle by promoting the expression of Cyclin D1 through cell cycle synchronization assay.4.In vitro functional experiments confirmed that silencing EIF4 H could significantly improve the sensitivity of Tamoxifen to MCF7/TAMR cells.Subcutaneous tumorigenesis assay and oral administration of Tamoxifen showed that the tumor volume in knockdown of EIF4 H group was significantly lower than that in the control group.Ki-67,Cyclin D1 and c-Myc were detected by immunohistochemistry,which were significantly lower in the knockdown of EIF4 H group than in the control group.5.EIF4 H overexpression could significantly increase the expression of EGFR in PI3K-AKT signaling pathway by protein expression microarray combined with bioinformatic analysis.Western blot analysis showed that EIF4 H could significantly up-regulate the phosphorylation levels of ERK,m TOR,AKT and EGFR of PI3K-AKT signaling pathway,as well as the phosphorylation levels of downstream molecules ERα and the expression levels of Cyclin D1 and c-Myc.Through endogenous and exogenous ubiquitination degradation experiments,we found that EIF4 H overexpression significantly inhibited EGFR ubiquitination degradation.The interaction between EIF4 H and EGFR was found by CoImmunoprecipitation.Cell immunofluorescence assay and cytoplasmic protein isolation assay showed that EIF4 H could promote the transition of ERα-phosphorylation into the nucleus.6.The protein expression level of EIF4 H was significantly down-regulated after the treatment of MCF7 with estrogen(E2),while the protein expression level of EIF4 H was significantly up-regulated after the treatment of MCF7 with E2 and Tamoxifen compared with E2 alone.E2 promoted the expression of EIF4 H m RNA in a short period of time but inhibited the expression of EIF4 H in a long period of time.It was found that ERα could bind to the promoter region of EIF4 H by Ch IP assay.The protein HDAC1 and DNMT3 b,which are involved in epigenetic regulation,can bind to the ERα binding region by reverse Ch IP technique.Further,through the methylation prediction website,we found that the EIF4 H promoter region exists Cp G island.The methylation level of EIF4 H promoter region in MCF7/TAMR cells was significantly lower than that in MCF7 cells by bisulfite sequencing.Conclusions:The expression of EIF4 H is high in breast cancer and negatively correlated with ER expression.The prognosis of ER-positive breast cancer patients with high EIF4 H expression is significantly lower than that of patients with low EIF4 H expression.EIF4 H promoted the proliferation of ER-positive breast cancer cells and promoted the transition from G1 phase to S phase in the process of breast cancer cell cycle by upregulating Cyclin D1.EIF4 H is highly expressed in Tamoxifen-resistant MCF7/TAMR cells,and the knockdown of EIF4 H reverses Tamoxifen resistance in MCF7/TAMR cells.EIF4 H can lead to activation of PI3K-AKT signaling pathway and promote the nucleation of ERα phosphorylation via inhibiting ubiquitination degradation of EGFR.E2/ERα transcriptionally inhibits EIF4 H expression by DNA methylation of the EIF4 H promoter.Long-term use of endocrine therapy drugs results in the inability of ER and co-repressors to bind to EIF4 H promoter,leading to the demethylation of EIF4 H promoter DNA and upregulation of EIF4 H expression.When EIF4 H accumulates to a certain extent,the binding of EIF4 H to EGFR inhibits EGFR ubiquitination and degradation,thus stabilizing EGFR expression and activating PI3 KAKT signaling pathway,leading to endocrine therapy resistance of breast cancer. |
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