BAIAP2L1 Promotes Breast Cancer Progression And Chemo-resistance Via PI3K-AKT Signaling Pathway By Interacting With RPL3

Background: Breast Cancer(BC)is a phenomenon in which breast epithelial cells proliferate out of control under the action of a variety of carcinogens.The early stage of the disease often manifests as breast lumps,nipple discharge,axillary lymphadenopathy and other symptoms.In the late stage,cancer cells may metastasize to a distance,and multiple organ diseases may appear,which directly threatens the life of the patient.According to the latest data from the International Agency for Research on Cancer(IARC)in 2018,the incidence of breast cancer in female cancers worldwide is 24.2%,ranking first among female cancers.In my country,the incidence of breast cancer is increasing year by year,and more than 300,000 women are diagnosed with breast cancer every year.In the eastern coastal areas and economically developed large cities,the incidence of breast cancer has increased significantly.From the age of onset,the incidence of breast cancer in my country has gradually increased after the age of 20,reaching a high value at the age of 45-50.With the popularization of new treatment strategies and methods,the global mortality rate of breast cancer is gradually decreasing.However,in China,especially in the vast rural areas,the decline in breast cancer mortality is not significant.Therefore,exploring the complex molecular mechanisms in the development of breast cancer,discovering efficient molecular markers for early diagnosis and prognostic evaluation of breast cancer,and finding sites for targeted therapy have important clinical significance.Brain-specific angiogenesis inhibitor 1(BAI1)related protein 2-like protein(BAIAP2L1),also known as insulin receptor tyrosine kinase substrate(Insulin Receptor Tyrosine Kinase Substrate,IRTKS),the gene encoding this protein is 2000 A new gene was electronically cloned by the National Human Genome Southern Research Center in the process of large-scale EST sequencing of the human pituitary-hypothalamus-adrenal neuroendocrine system.BAIAP2L1 can inhibit TP53-mediated apoptosis and transcriptional activity.Studies have shown that BAIAP2L1 can bind to MDM2 and promote MDM2-mediated ubiquitination of TP53.In a variety of tumor cells,especially gastrointestinal tumors,the expression of BAIAP2L1 increases,which promotes the formation of a large number of complexes of BAIAP2L1,TP53 and MDM2,Which increases the ubiquitination and degradation of TP53 and promotes cell proliferation.However,the biological functions and mechanisms in breast cancer are still unclear.Therefore,we explored its complex molecular mechanisms in the occurrence and development of breast cancer to provide new ideas and targets for the treatment of breast cancer.Methods: We analyzed the difference in m RNA expression of BAIAP2L1 in cancerous and paracancerous non-cancerous tissues in the breast by TCGA data and in breast cancer tissue specimens from our unit.The protein expression of BAIAP2L1 was further examined in breast cancer tissues and breast cancer cell lines(MDA-MB-231,MDA-MB-453,MDA-MB-468,BT-549),and non-triple negative breast cancer cells(MCF-7,SKBR-3)using Western blot.Meanwhile,we examined the protein expression of BAIAP2L1 at the cellular and tissue levels using cellular immunofluorescence chemical staining and immunohistochemical staining.Finally,we applied chi-square test and Kaplan meier survival to analyze the relationship between BAIAP2L1 expression and clinicopathological data and prognosis.Further,we constructed BAIAP2L1 stable knockdown and stable overexpression breast cancer cells,and applied MTT cell proliferation assay,colony formation assay,tumor stem cell sphere-forming assay,and Transwell assay to detect the effects of BAIAP2L1 on breast cancer cell proliferation,stem cell characteristics,invasion and chemotherapy resistance phenotypes.We also applied nude mice to construct a subcutaneous tumorigenesis model and a lung metastasis model to verify the biological functions of BAIAP2L1 in vivo.In terms of mechanism exploration,we first used bioinformatics and database to predict the signaling pathways that BAIAP2L1 may be involved in regulating and their corresponding downstream target genes,and combined GO functional enrichment and KEGG pathway analysis to screen out the enriched signaling pathways.Meanwhile,we performed RNA sequencing analysis on BAIAP2L1 stable knockdown breast cancer cells and compared the sequencing results with the predicted signaling pathways in the database to identify the same signaling pathway,and then further searched for the target genes with the same trend in this signaling pathway.We detected pathway-related downstream proteins associated with BAIAP2L1 protein by rapid high-throughput phosphorylation antibody microarray.We further applied immunoprecipitation combined with mass spectrometry analysis to search for proteins that may interact with BAIAP2L1.And from the mass spectrometry analysis results,we searched for protein molecules that might regulate the pathway and verified them using immunoprecipitation.Finally,we further applied multiple replies experiments to verify that BAIAP2L1 does affect the progression of breast cancer through the above mechanism.Results:TCGA analysis showed that BAIAP2L1 was significantly highly expressed in breast cancer tissues(p<0.0001)and the expression of BAIAP2L1 m RNA was significantly higher in triple-negative breast cancer than in non-triple-negative breast cancer tissues(p<0.0001).Further analysis also showed that BAIAP2L1 m RNA expression was significantly and positively correlated with Nottingham prognostic index(NPI,p<0.0001)and Scarff-Bloom-Richardson grading of breast cancer(p<0.0001).Immunohistochemical results also showed that BAIAP2L1 protein was significantly highly expressed in breast cancer tissues,mainly in breast cancer parenchyma.western blot results also showed that BAIAP2L1 protein was significantly more highly expressed in breast cancer tissues compared to the paracancerous non-cancerous group(p<0.001).Correlation analysis of clinicopathological data showed that high BAIAP2L1 expression was positively correlated with higher TNM(stage)(p=0.001),lymph node metastasis(p=0.001)and triple negative breast cancer(p=0.013),but not with age(p=0.864).survival analysis by Kaplan-Meier method showed that as BAIAP2L1 protein MTT proliferation and colony formation assays examined the proliferative capacity of BAIAP2L1 for breast cancer in vitro and showed that overexpression of BAIAP2L1 significantly promoted the proliferation of breast cancer cells and knockdown of BAIAP2L1 expression significantly inhibited proliferation.Further western blot showed that BAIAP2L1 significantly promoted the expression of Cyclin D1,a cell cycle-related protein,while cell cycle assays suggested that BAIAP2L1 promoted cell cycle progression.Western blot showed that overexpression of BAIAP2L1 significantly promoted the expression of the mesenchymal phenotype-associated protein Snail and inhibited the expression of the epithelium-associated marker E-cadherin.For breast cancer cell stemness assay,western blot results showed that BAIAP2L1 overexpression could significantly promote breast cancer cells to express ALDH1 expression,and the results of tumor sphere formation assay showed that BAIAP2L1 could significantly enhance the growth ability of breast cancer stem cells.Finally,the results of animal experiments showed that BAIAP2L1 could promote breast cancer tumor growth(p<0.01)and lung metastasis(p<0.01)in vivo.We further applied RNA sequencing and phosphokinase array to analyze the possible signaling pathways regulated by BAIAP2L1,and confirmed by western blot that BAIAP2L1 could activate PI3K-AKT signaling pathway.Application of PI3K-AKT pathway inhibitor for reversion assay confirmed that BAIAP2L1 promotes proliferation and invasive phenotype of breast cancer cells through activation of PI3K-AKT signaling pathway.Mechanistically,we confirmed that BAIAP2L1 stabilizes PIK3 CA through the interaction of the SH3 structural domain with the structure consisting of amino acids202-288 of RPL3,thereby promoting AKT phosphorylation.We further promoted BAIAP2L1 expression in cells with knockdown RPL3 and examined the phenotypic changes in breast cancer,confirming that BAIAP2L1 indeed regulates AKT signaling activation through RPL3 and thus affects breast cancer proliferation and invasion.Finally,we further clarified the positive correlation of BAIAP2L1 with p-AKT in breast cancer tissue specimens(p<0.001).Conclusion: 1.BAIAP2L1 is aberrantly highly expressed in triple-negative breast cancer and positively correlates with poor prognosis.2.BAIAP2L1 promotes breast cancer cell proliferation,invasion,EMT progression and tumor cell stemness.3.BAIAP2L1 promotes breast cancer cell proliferation and metastasis at the ex vivo level.4.BAIAP2L1 promotes the proliferation,invasion and drug-resistant phenotype of triple-negative breast cancer cells by activating the PI3K-AKT signaling pathway.5.BAIAP2L1 can interact with RPL3 to promote PI3K-AKT signaling pathway and breast cancer progression.6.BAIAP2L1 can form a ternary complex with RPL3 and PIK3 CA to activate PI3K-AKT signaling pathway.