NDRG2 Promotes Adriamycin Sensitivity Through A Bad/p53 Complex At The Mitochondria In Breast Cancer

Breast cancer is the most common cancer among women with the rapidly increasing and much younger incidence worldwide.It is also the leading cause of cancer-related mortality among females worldwide,which is a serious threat to women's physical and mental health.Comprehensive therapy including surgical intervention,radiotherapy,endocrine therapy,chemotherapy and the molecular targeted therapy has become the basic principles and the mainstream patterns of treatment.Chemotherapy is an important part of the comprehensive treatment of breast cancer and it has been widely appreciated.With the advantages of long-term effects,fewer adverse effects and the higher life quality improvement,chemotherapy can be used as a salvage therapy after metastasis.It can reduce the cell mutations and resistant cells,reduce breast cancer primary lesions,increase the chance of breast-conserving surgery,prolong the long-term survival of patients and improve the quality of survival.Chemo-resistance is the chief culprit of restrictions on chemotherapy drug application,meanwhile,it also leads to clinical treatment failure,and even the deaths of patients.To date,various mechanisms of drug resistance have been proposed,including mi RNAs,tumor microenvironment,DNA damage repair,and tumor suppressor genes such as Rb and p53.Among them,the function of p53 in drug sensitivity of breast cancer is widely studied.N-Myc downstream-regulated gene 2(NDRG2),which belongs to the NDRG family,was firstly cloned by our laboratory in 1999 from a normal human brain c DNA library(Gene Bank,Accession No.AF 159092).The NDRG2 gene located at chromosome 14q11.2,and it can be delineated into 16 exons and 15 introns;the entire m RNA of NDRG2 is 2024 bp in length and it encodes a 357 amino acids protein with a calculated molecular mass of 41 k Da.In recent years,a lot of studies about the NDRG2 gene function found that NDRG2 is a candidate tumor suppressor gene.Our previous study showed that ADR,one kind of chemotherapy drug could induce the expression of NDRG2,moreover,DNA damage related genes such as ATM and ATR may regulate NDRG2 expression,suggesting that NDRG2 is involved in the DNA damage.What's more,we found that NDRG2 can be transcriptionally activated by p53 and involved in p53-mediated apoptosis pathway.However,the molecular mechanism of NDRG2 in p53-mediated apoptosis,especially in breast cancer,remains unclear.?Objectives?1.To investigate whether there is a correlation between NDRG2 and adriamycin resistance in breast cancer.2.NDRG2 can sensitize breast cancer cells to ADR in a p53-dependent manner.3.To analyze the underlying mechanisms of NDRG2 promoting apopotosis of breast cancer.4.To explore the underlying mechanisms of NDRG2 promoting ADR sensitivity of breast cancer depending on p53.?Methods and Results?1.NDRG2 expression is suppressed in ADR-Resistant Breast CancersqRT-PCR and Western Blot analyses were performed to examine the endogenous NDRG2 expression in MCF-7/ADR and MCF-7 cells,and results showed that both NDRG2 m RNA and protein levels were decreased in MCF-7/ADR cells compared with MCF-7 cells,indicating that NDRG2 might regulate the ADR resistance of breast cancer.We further established the stable cell lines of NDRG2 overexpression in MCF-7/ADR cells(MCF-7/ADR-NDRG2).When cells were treated with increasing amounts of ADR,overexpression of NDRG2 in MCF-7/ADR cells could obviously increase the cytotoxic effect of ADR and apoptosis compared with MCF-7/ADR cells.Taken together,our data implicated that NDRG2 might increase ADR sensitivity in breast cancer MCF-7 cells.2.NDRG2 promotes ADR sensitivity in breast cancer in a p53-dependent mannerWe overexpressed NDRG2 in two mutant p53 cell lines and one wildtype p53 cell line.MTT assay and Annexin V/PI assay showed that NDRG2 could significantly inhibit the proliferation and promote cell apoptosis of MCF-7 cells under ADR treatment.However,NDRG2 could not alter the drug sensitivity and cell apoptosis in two mutant p53 cells.Moreover,NDRG2 knockdown in MCF-7 cells inhibits ADR sensitivity.To further verify whether the effect of NDRG2 on promoting drug sensitivity depends on p53,we used lentivirus-mediated sh RNA to further knockdown p53 in MCF-7/NDRG2 cells and then measured cell viability and apoptosis.The results suggested that knockdown p53 in MCF-7 cells compromised the pro-apoptotic function of NDRG2.Our data provided strong evidences that NDRG2 promotes ADR sensitivity dependent on p53.3.NDRG2 aggravates DNA damage response in p53 wt but not in p53 mut cellsFollowed by ADR treatment,the DNA damage response functions,which will result in DNA damage repair.We first examined the protein expression of ?-H2 AX and RAD51 by Western Blot and found that only in MCF-7 cells,NDRG2 overexpression significantly promoted ?-H2 AX expression and inhibited RAD51 expression.What's more,RAD51 foci formation showed the same results consistent with Western Blot.We further examine the role of NDRG2 on the homologous recombination(HR)repair mechanism using pdr-GFP,I-Sce I system and found that NDRG2 overexpression caused decreased HR efficiency in MCF-7 cells.However,NDRG2 overexpression did not inhibit the HR efficiency after p53 abrogation.Collectively,these data showed that NDRG2 expression could promote DNA damage response induced by ADR,and thus sensitized breast cancer cells to chemotherapeutic agents in a p53-dependent manner.4.NDRG2 exerts its pro-apoptotic effects by improving Bad expressionWe tested apoptosis related Bcl-2 family proteins by Western Blot and found that NDRG2 induced the expression of the pro-apoptotic protein Bad in MCF-7 cells with or without ADR.Knockdown of Bad decreased the percentage of apoptosis induced by NDRG2,and blocked the ADR sensitivity in NDRG2-overexpressed cells.We further elucidated the underlying mechanisms of how NDRG2 regulates Bad expression.RT-q PCR results showed that NDRG2 did not change the m RNA level of Bad.MG132 and CHX analyses showed that NDRG2 counteracted Bad degradation and prolonged the half-life of Bad.Taken together,we have identified that NDRG2 could increase Bad stability to promote apoptosis of breast cancer cells.5.NDRG2 overexpression attenuates p53 nuclear translocationTo further verify the mechanism of NDRG2 in promoting drug sensitivity in a p53-dependent manner,we evaluated how NDRG2 regulates p53 function.RT-q PCR and Western Blot suggested NDRG2 overexpression had no effect on p53 m RNA and protein level in the presence and absence of ADR.Subsequently,nuclear and cytosolic extracts were prepared for Western Blot analysis and results showed that NDRG2 overexpression promoted p53 location in the cytoplasm in MCF-7 cells.Numerous p53-targeting genes m RNAs remain unchanged with NDRG2 overexpression,which is consistent with the cytosolic localization of p53.Moreover,NDRG2 overexpression made an increasing accumulation of p53 in the mitochondrial fraction,with a corresponding decrease in their cytosolic levels.Furthermore,co-immunoprecipitation experiments showed that NDRG2 could enhance the interaction between Bad and p53 under ADR treatment.These studies for the first time demonstrated that NDRG2 could promote the accumulation of p53 on the mitochondria by Bad/p53 complex and trigger apoptosis to sensitize breast cancer to ADR.?Conclusion?In this study,we uncovered the novel function of NDRG2 in response to chemotherapy drugs with a focus on ADR.Our experiments demonstrated for the first time that tumor suppressor NDRG2 was significantly decreased in MCF-7/ADR cells,and NDRG2 overexpression could improve breast cancer cells sensitivity to ADR.Under ADR treatment,NDRG2 could promote apoptosis and DNA damage response as well as inhibit cell proliferation.But all those functions of NDRG2 was dependent on wild type p53 states.In addition,NDRG2 could inhibit Bad degradation by increasing its protein stability.Moreover,excessive Bad can interact with p53 at the mitochondria,which induces apoptosis through transcription-independent mechanism of p53.Simultaneously,cytosolic p53 is detained by the excessive Bad,thus preventing p53 from entering the nucleus for further transcription of DNA damage repair genes.