The Function And Mechanism Of Transcriptional Coregulator NUPR1 In Tamoxifen Resistant Breast Cancer Cells

Objects: The drug resistance of breast cancer is a major cause of recurrence and metastasis.Currently,numerous studies reported that autophagy is closely connected to drug resistance,but the precise molecular mechanisms of this drug resistance remain unclear.The biological function of Nuclear protein 1(NUPR1,also known as p8 or candidate of metastasis 1,Com-1)in tumors has drawn much attention from researchers,but as a transcriptional regulator,NUPR1’s function in drug resistant breast cancer cells has not been reported.The object of our research was to unravel the biological function and molecular mechanism of NUPR1 during the development of tamoxifen resistance and validated it in clinical samples.Meanwhile,,we would explore the feasibility of reversing the drug resistant breast cancer cells based on the biological effects of NUPR1,which has vital clinical significance.This project will help us to understand the regulatory pathway of autophagy in breast cancer and expand the understanding of molecular mechanisms in drug resistant breast cancer cells.Methods: The NUPR1’s expression in tissue microarray of human breast cancers and changes of these cells in the development of drug resistance at the autophagy level.Evaluating the expression and distribution of NUPR1 in tissue samples from 133 breast cancer patients by immunohistochemical staining.The relationship between NUPR1 and the survival rate in patients was further analyzed.RT-PCR and Western blot were used to detect the expression of NUPR1 in breast epithelial cells,various breast cancer cells and tamoxifen resistant breast cancer cells.The results of autophagy markers(like SQSTM1 and LC3B)showed changed autophagy flux and autolysosomal efflux.Next,interfering autophagic process by bafilomycin A1(Baf A1)and chloroquine(CQ),then the cellular proliferation was detected by Brd U assay.The biological function of NUPR1 in mediating drug resistance of breast cancer.The morphological changes and ultrastructure of NUPR1 depleted cells were observed by optical microscope and transmission electron microscope,respectively.The m Cherry-GFP-LC3 assay and Lyso-tracker Red and mono dansylcadaverine(MDC)staining were applied to detect changes in autophagy flow.To explore NUPR1’s function during development of resistance to tamoxifen,cell cycle analysis,Brd U assay,SA-β-gal Staining,clonogenic and invasion assays,cells subcutaneous injection were employed.The molecular mechanism of NUPR1 mediated tamoxifen resistance through autophagic survival process.Affinity purification and mass spectrometry were used to identify the associated proteins with NUPR1.Next,assessing the transcriptional profile of the involved genes upon NUPR1 depletion using RNA sequencing,then comparing the different expression genes.The downstream genes directly regulated by NUPR1 were verified by Ch IP-re-Ch IP,Luciferase and EMSA assays.Results: NUPR1 was strongly detected in the nucleus of breast cancers cells,and its high expression level correlates with low overall survival rate in breast cancer patients.Further analysis showed that the expression of NUPR1 was closely related to clinical stage and age(P <0.005).Immunoblotting data showed that NUPR1 expression was induced by tamoxifen treatment.Moreover,induced NUPR1 expression by tamoxifen treatment at a consistent level in a timedependent manner.With induced NUPR1 protein level,autophagy flux and autolysosomal efflux increased.Either we inhibit the autophagic flux or autolysosomal efflux,the cellular proliferations were all reduced by Brd U assay.NUPR1-depleted cells showed a typical phenotype of premature senescence and increased cell death compared to control.Lyso-tracker Red and mono dansylcadaverine(MDC)staining suggested the intracellular acidic environment.Our data showed that autolysosomal process was impaired.Likewise,the ultrastructural analysis indicated a significant increased lysosomes and autolysosomes in NUPR1-depleted cells compared with an irrelevant fire fly luciferase sh RNA cells(hereafter referred to as control)under transmission electron microscopy.Additionally,NUPR1 depletion in cells resulted in reduced malignancy in vitro and in vivo.The capacity of proliferation,invasion and colony formation were all reduced.A NUPR1-regulated gene signature is both prognostic and predictive for drug resistance.We depleted endogenous NUPR1 in cells and then assessed the transcriptional profile of the involved genes by RNA-sequencing.The KEGG pathway analysis revealed that lysosomal process,cell cycle and estrogen signaling pathways were enriched(BECN1,LCN2,RAB31 and NEDD9,etc).Ch IP-re-Ch IP assay verified that NUPR1 and ESR1 were obviously enriched in the promoter of BECN1 gene.Luciferase reporter assay showed that knockdown NUPR1 increased BECN1 promoter activity.However,deletion of ESR1 binding site impaired the activating effect on BECN1 promoter activity.Further EMSA experiments in vitro suggested that NUPR1 bound to the promoter region of BECN1 gene.