Expression Of MLF1IP In Luminal Breast Cancer And Its Effect On Tamoxifen Sensitivity

Objective:Breast cancer is a highly heterogeneous disease with hormone receptor-positive(or Luminal)tumors representing approximately 60%-70%of all cases.Despite endocrine therapy is highly effective and appropriate for nearly all women with Luminal tumors,both primary and secondary resistance to endocrine treatments still occur,resulting in tumor progression.The myelodysplasia/myeloid leukemia factor 1-interacting protein(MLF1IP)gene encodes a 47-kDa protein and maps to chromosome 4q35.1.MLF1IP has been shown to be involved in tumorigenesis in several types of cancer.However,the potential correlation between MLF1IP and clinical outcome in patients with Luminal breast cancer remains unknown.In this study,We aim to explore the expression of MLF1IP in Luminal breast cancer and its effect on tamoxifen sensitivity,in order to find a method to predict endocrine resistance and to find potential therapeutic targets.Methods:1.The expression of MLF1IP mRNA was detected in 15 paired breast cancer tissues and adjacent non-cancerous tissues by quantitative real-time PCR,and GAPDH was taken as an internal control.In order to verify the experimental results,immumohistochemical staining method was used to detect the expression of MLF1IP protein in Luminal breast cancer tissues.In addition,in order to investigate whether MLF1IP was associated with the development and progression of Luminal breast cancer,we investigated its relationship with clinical features and analyzed follow-up data of TCGA(The Cancer Genome Atlas)cohort.2.After successful construction of MLF1IP gene silencing lentiviral vector,the MCF-7 cell line was infected with corresponding lentiviral vector.And then,quantitative real-time PCR and Western Blot were used to verify its MLF1IP silencing effect.In order to evaluate the impact of knocking down MLF1IP on the biological function of breast cancer cell line.CCK-8 was used to detect the ability of cell proliferation.Flow cytometry was performed to measure the distribution of cells in the cell cycle phases and to assess cellular apoptosis.And Western Blot was used to detect the expression levels of a variety of cell cycle related proteins including Cyclin D1,Cyclin E,CDK2,CDK4 and CDK6.3.Quantitative real-time PCR was used to detect the expression level of MLF1IP mRNA in MCF-7 cells after 4-Hydroxytamoxifen(TAM)treatment.We used CCK-8 to measure the inhibitory rate of MCF7-shRNA and MCF7-NC cells after treated with different concentrations of TAM(8μm,16μm and 32μm).And CCK-8 was also used to detect the cell viability in the presence or absence of TAM stimulation.In addition,flow cytometry analysis was performed to evaluate the apoptosis of MCF7-shRNA and MCF7-NC cells after treated by TAM.To further demonstrate the mechansm of tamoxifem resistance induced by MLF1IP,we evaluated the protein expressions of Bcl-2,Bax,Caspase3,Caspase7,Caspase9,PTEN and p-AKT by Western Blot.Results:1.The expression of MLF1IP mRNA was significantly increased in breast cancer tissues compared with the adjacent normal tissues.To investigate whether the conclusion is valid in Luminal breast cancer patient population,RNA sequencing data of breast cancer was acquired from TCGA database which contained 63 paired breast cancer tissues and nontumorous tissues.Consistently,the expression of MLF1IP mRNA was significantly upregulated in Luminal breast cancer tissues.In TCGA cohort,the clinical information of total 745 Luminal breast cancer patients was available to be used for further analysis.Patients were divided into low and high MLF1IP expression groups according to the median value.High MLF1IP expression group presented more often lymph node metastasis and negative progesterone receptor expression compared with low MLF1IP expression group.Survival analysis showed that patients with high MLF1IP expression had significantly lower overall survival.As respect to the prognostic value of MLF1IP,Cox regression analysis was conducted adjusting for clinical features.It was indicated that the high MLF1IP expression was an independent high risk factor as well as old age(>60)and distant metastasis.2.MLF1IP knockdown model was established in MCF-7,and the mRNA expression level and the protein expression level were both significantly down-regulated compared to the control.The growth curves for MLF1IP knockdown cells were significantly lower than those for control cells,indicating cell proliferation was significantly inhibited in MCF-shRNA compared to MCF-NC.The effects of MLF1IP knockdown on cell cycle distribution were also assessed,and the inhibition of MLF1IP expression in MCF-7 cells by RNAi arrested cells in G1 phase.Moreover,Western Blot analysis showed that the expression level of Cyclin D1 protein in MCF7-shRNA cells was significantly lower than MCF7-NC cells.However,there was no significant difference in the expression level of Cyclin E,CDK2,CDK4,CDK6 protein between the two groups.In apoptosis assay,the MLF1IP-shRNA cells showed significantly higher proportion of Annexin V+7-AAD+ cells compared to the control.No differences were revealed in the proportion of Annexin V+7-AAD-cells between the two groups.3.TAM could induce MLF1IP expression in MCF-7 cells.CCK-8 experiment showed that the inhibitory rate of MCF7-shRNA was significantly higher than that of MCF7-NC cells after treated with different concentrations of TAM(8μm,16μm and 32μm).And the proliferation of MCF7-shRNA cells was weaker than that of MCF7-NC cells under the condition of no TAM.Moreover,the gap between the two groups was greater under the condition of TAM stimuLation.Flow cytometry analysis showed that proportion of early-stage apoptotic cell population and late-stage apoptotic(or dead)cell population of MCF7-shRNA cells were both significantly higher than that of MCF7-NC cells after TAM treatment.Western Blot experiment indicated that the protein expression levels of Bax,Caspase3,Caspase7 and Caspase9 in MCF7-shRNA cells were all significantly higher than that of MCF7-NC cells under the condition of TAM treatment,and Bcl-2 protein expression level was significantly lower than that of MCF7-NC cells.In addition,the protein expression level of PTEN in MCF7-shRNA cells was significantly higher than that of MCF7-NC cells,and p-AKT protein expression level was significantly lower than that of MCF7-NC cells.Conclusion:1.We found that MLF1IP was significantly upregulated in a large cohort of human Luminal breast cancer tissues.And that MLF1IP levels significantly correlated with the clinical characteristics of breast cancer,including lymph node metastasis and progesterone receptor expression.Moreover,survival analysis showed that patients with high MLF1IP expression showed significantly lower overall survival compared with patients with low MLFIIP expression,suggesting that MLFIIP is a potential predictor for survival of patients with Luminal breast cancer.2.Knockdown of MLF1IP expression inhibited the malignant behavior of Luminal breast cancer cells.MLF1IP promotes the proliferation of MCF-7 cells by upregulating Cyclin D1.3.Down-regulation of MLF1IP mRNA expression in MCF-7 cells significantly increased its sensitivity to TAM and significantly increased the apoptosis level of MCF-7 cells under TAM stimuLation,suggesting that MLF1IP-related apoptotic resistance may play an important role in endocrine resistance.MLF1IP can be used as a predictor of tamoxifen sensitivity in Luminal breast cancer.Moreover,MLF1IP may induce tamoxifen resistance by influencing the activity of PI3K-PTEN/AKT signaling pathway.