PLSCR1 Activates STAT1 Signaling To Promote The Progression Of Basal-like Breast Cancer

Breast cancer is a kind of malignant tumors that occur in the epithelial tissue of the breast.The global incidence of breast cancer has been on the rise since the late 1970s,and has become a major public health problem in the current society.In China,the incidence of breast cancer is also increasing year by year,with about 200,000 women diagnosed each year.At present,breast cancer has become the first killer of women in China.Breast cancer is a heterogeneous disease,which can be divided into four subtypes according to the different gene expression:luminal epithelial A type,luminal epithelial B type,Her2 overexpression type and basal-like type.Among them,basal-like breast cancer has the characteristics of strong migration,invasion and proliferation ability,is easily resistant to chemotherapy,and lacks the corresponding targeted therapy,so the prognosis is the worst.Therefore,aiming at the characteristics of the occurrence and development of basal-like breast cancer,finding out the key targets and regulatory signals has a high guiding significance for the clinical diagnosis and treatment of breast cancer.Phospholipid scramblase 1?PLSCR1?is a Ca²⁺binding palmitoylated type II membrane protein.Non palmitoylated PLSCR1 can be imported into the nucleus to bind genomic DNA sequence.Initial studies suggested that the main function of PLSCR1 is to promote the turnover rearrangement of membrane phospholipids during cell activation,injury or apoptosis through the activity of phospholipid crawling enzymes.However,more and more studies have showed that the biological function of PLSCR1 is not limited to the transmembrane movement of phospholipid molecules in cell membranes;it has even been reported that it does not have the function of phospholipid transporters and may play other roles in cells.There are data showing that PLSCR1 is closely related to the stage,metastasis and prognosis of breast cancer patients,but its function and mechanism in breast cancer are still unclear.Therefore,the expression,biological function and mechanism of PLSCR1 in breast cancer were studied in this study.We first analyzed gene expression databases and tumor samples from multiple breast cancer patients and found that PLSCR1 expression was significantly increased in basal-like breast cancer compared with other subtypes.The expression of PLSCR1 in breast cancer cell lines was detected by reverse transcription PCR,real-time quantitative PCR and Western Blot,and the proteins were separated from different subtypes of tumor tissues.The results showed that the m RNA and protein expression of PLSCR1 were significantly increased in basal-like breast cancer.To study the biological function of PLSCR1 in breast cancer,we knocked down the expression of PLSCR1 in basal-like breast cancer cells and introduced exogenous wild-type overexpression or mutated PLSCR1 in phospholipid scramblase activity sites,while wild-type or mutant PLSCR1was overexpressed in luminal breast cancer cells.The effects of PLSCR1 on the proliferation,migration and invasion of breast cancer cells were investigated by CCK-8and transwell chamber migration and invasion experiments.It was found that the proliferation,migration and invasion ability of breast cancer cells were positively correlated with the expression of PLSCR1,but not with the activity of its phospholipid scramblase.The expression of PLSCR1 was distributed in cell membrane,cytoplasm and nucleus.However,under normal cell culture conditions,PLSCR1 was detected in a little proportion of the nucleus.PLSCR1 can interact with EGFR when stimulated by EGF.To investigate the effect of EGFR signaling pathway on the subcellular localization of PLSCR1,we used laser confocal analysis to analyze the subcellular localization of PLSCR1 under EGF stimulation,and Western Blot to detect the subcellular localization of PLSCR1 after nucleoplasmic protein isolation.The results showed that EGF stimulation could promote the entry of PLSCR1 into the nucleus.Since EGF can promote the phosphorylation of PLSCR1 tyrosine?Tyr 69/74?,to verify whether this phosphorylation site affects the nuclear translocation of PLSCR1,we mutated these two phosphorylation sites?PLSCR1-Y69,74F?.Knock down the endogenously expressed PLSCR1 in breast cancer cell lines and exogenously overexpress wild-type or phosphorylation site mutated PLSCR1.Through Western Blot and immunofluorescence experiments,we found that phosphorylation of Tyr 69/74 contributed to PLSCR1transport to the nucleus.In cells with knockdown of endogenous PLSCR1 expression,we overexpressed PLSCR1 with mutations in both palmitoylation and phosphorylation sites,and then detected changes in PLSCR1 subcellular localization by immunofluorescence.The results showed that EGF-induced PLSCR1 nuclear translocation required phosphorylation of PLSCR1 Tyr69 and Tyr74.To explore the potential molecular mechanism of PLSCR1 in breast cancer,we analyzed the co-expression of PLSCR1 with other genes in the gene expression dataset and found that the expression of PLSCR1 was positively correlated with STAT1.Western Blot assay was used to detect the expression of PLSCR1 and STAT1 in basal-like breast cancer cell lines and luminal breast cancer cell lines.We found that the expression of PLSCR1 and STAT1 increased in basal-like cell lines and decreased in luminal cell lines.To explore the relationship between PLSCR1 and STAT1,we overexpressed or knocked down PLSCR1 in breast cancer cell lines,and the results showed that STAT1 expression was also increased or decreased accordingly.These data suggest that PLSCR1 can regulate STAT1 expression.To explore the mechanism of the close association between STAT1 and PLSCR1 in basal-like breast cancer,we searched for the proteins interacting with PLSCR1 by immunoprecipitation and mass spectrometry?IP-MS?,combined with the existing reports and bioinformatics analysis to identify several key proteins,and then further verified by co-immunoprecipitation?Co-IP?experiments.We found that PLSCR1could interact with STAT3,and the phosphorylation of PLSCR1 Tyr 69/74 affected the binding of the two proteins.The expression of STAT1 was up-regulated when STAT3 was highly expressed in breast cancer cells,and down-regulated when STAT3 was knocked down.Since phosphorylation modification affects nuclear translocation of PLSCR1 and binding to STAT3,we highly expressed wild-type or phosphorylation site mutated PLSCR1 in breast cancer cell lines,respectively,and found that phosphorylation of PLSCR1 Tyr69 and Tyr74 affected STAT1 expression.Through chromatin immunoprecipitation?Ch IP?technology and semi-quantitative PCR and real-time quantitative PCR analysis,we found that Tyr69 and Tyr74 phosphorylated PLSCR1 interacted with STAT3 in the nucleus,and combined into the promoter region of STAT1 to regulate STAT1 expression.In order to elucidate the mechanism of PLSCR1 in breast cancer cells,we knocked down PLSCR1or expressed wild-type PLSCR1 or PLSCR1 with enzymatic activity mutation in breast cancer cell lines for tumorsphere formation assay,and detected the change of CD44^high/CD24^lowpopulation in these cell lines by flow cytometry.The results showed that PLSCR1 was positively correlated with the number of CD44^high/CD24^low cells.Cancer stem cells?CSCs?possess highly tumorigenic and metastatic properties.We first performed soft agar clonogenic experiments with corresponding breast cancer cells and found that the clonogenic ability of cell lines knocking down the expression of PLSCR1 decreased,while that of cell lines overexpressing wild-type or mutated sites of phospholipid scramblase activity increased.Furthermore,we used breast cancer cell lines with knockdown PLSCR1 expression to carry out in situ tumorigenesis experiments in mice,and found that the tumour growth ability of knockdown PLSCR1 expression was significantly reduced.To explore the clinical significance of PLSCR1 expression on breast cancer progression,we first evaluated the correlation between PLSCR1 expression and tumor size and grade in multiple databases,and found that the higher the PLSCR1expression,the larger the tumor volume and grade.In order to detect the correlation between PLSCR1 and breast cancer metastasis in vivo,we constructed a lung metastasis model by tail vein injection of breast cancer cell lines that knocked down PLSCR1expression,and found that inhibiting PLSCR1 expression could significantly reduce the lung metastasis ability of breast cancer cells.In addition,we analyzed the clinical sample database of breast cancer patients and found that the higher the expression of PLSCR1,the stronger the ability of distal metastasis of tumors.We also analyzed the relationship between PLSCR1 expression and chemosensitivity through the breast cancer patient database and found that PLSCR1 expression was higher in tumors with chemoresistance.Finally,we evaluated the relationship between PLSCR1 expression and patient survival by Kaplan-Meier survival analysis of multiple data sets,and found that patients with high PLSCR1 expression had shorter distant metastasis-free survival?DMFS?,relapse-free survival?RFS?,and overall survival?OS?.According to the above results,we draw the following conclusions:1.High expression of PLSCR1 in basal-like breast cancer is positively correlated with poor prognosis.2.The phosphorylation of PLSCR1 contributes to its nuclear translocation and binding with STAT3.3.Nuclear PLSCR1 can activate STAT1 signal transduction and contributes to the maintenance of BLBC stem cell characteristics to enhance tumorigenicity and metastasis.In conclusion,this study reveals the key mechanism of PLSCR1 promoting the progress of blbc,and proposes the potential prognostic indicators and treatment targets for this challenging disease.