NR4A1 Regulates Tamoxifen Resistance by Suppressing ERK Signaling in ER-Positive Breast Cancer

Endocrine therapy is used to treat estrogen receptor (ER)-positive breast cancer. Tamoxifen is effective against this cancer subtype. Nonetheless, approximately 30% of patients treated with tamoxifen acquire resistance, resulting in therapeutic challenges. NR4A1 plays key roles in processes associated with carcinogenesis, apoptosis, DNA repair, proliferation, and inflammation. However, the role of NR4A1 in tamoxifen-resistant ER-positive breast cancer has not yet been elucidated. Here, we propose that NR4A1 is a promising target to overcome tamoxifen resistance. NR4A1 gene expression was downregulated in tamoxifen-resistant MCF7 (TamR) cells compared to that in MCF7 cells. Kaplan-Meier plots were used to identify high NR4A1 expression correlated with increased survival rates in patients with ER-positive breast cancer following tamoxifen treatment. Gain and loss of function experiments showed that NR4A1 restores sensitivity to tamoxifen by regulating cell proliferation, migration, invasion, and apoptosis. NR4A1 localized to the cytoplasm enhanced the expression of apoptotic factors. In silico and in vitro analyses revealed that NR4A1 enhanced responsiveness to tamoxifen by suppressing ERK signaling in ER-positive breast cancer, suggesting that the NR4A1/ERK signaling axis modulates tamoxifen resistance. These results indicate that NR4A1 could be a potential therapeutic target to overcome tamoxifen resistance in ER-positive breast cancer.ope

Up-regulation of HOXB cluster genes are epigenetically regulated in tamoxifen-resistant MCF7 breast cancer cells.

Tamoxifen (TAM) is commonly used to treat estrogen receptor (ER)-positive breast cancer. Despite the remarkable benefits, resistance to TAM presents a serious therapeutic challenge. Since several HOX transcription factors have been proposed as strong candidates in the development of resistance to TAM therapy in breast cancer, we generated an in vitro model of acquired TAM resistance using ER-positive MCF7 breast cancer cells (MCF7-TAMR), and analyzed the expression pattern and epigenetic states of HOX genes. HOXB cluster genes were uniquely up-regulated in MCF7-TAMR cells. Survival analysis of in slico data showed the correlation of high expression of HOXB genes with poor response to TAM in ER-positive breast cancer patients treated with TAM. Gain- and loss-of-function experiments showed that the overexpression of multi HOXB genes in MCF7 renders cancer cells more resistant to TAM, whereas the knockdown restores TAM sensitivity. Furthermore, activation of HOXB genes in MCF7-TAMR was associated with histone modifications, particularly the gain of H3K9ac. These findings imply that the activation of HOXB genes mediate the development of TAM resistance, and represent a target for development of new strategies to prevent or reverse TAM resistance.ope

Akt1 Decreases Gcn5 Protein Stability through Regulating The Ubiquitin-Proteasome Pathway in Mouse Embryonic Fibroblasts

General control non-derepressible 5 (Gcn5) is a member of histone acetyltransferase (HAT) that plays key roles during embryogenesis as well as in the development of various human cancers. Gcn5, an epigenetic regulator of Hoxc11, has been reported to be negatively regulated by Akt1 in the mouse embryonic fibroblasts (MEFs). However, the exact mechanism by which Akt1 regulates Gcn5 is not well understood. Using protein stability chase assay, we observed that Gcn5 is negatively regulated by Akt1 at the post-translational level in MEFs. The stability of Gcn5 protein is determined by the competitive binding with the protein partner that interacts with Gcn5. The interaction of Gcn5 and Cul4a-Ddb1 complex predominates and promotes ubiquitination of Gcn5 in the wild-type MEFs. On the other hand, in the Akt1-null MEFs, the interaction of Gcn5 and And-1 inhibits binding of Gcn5 and Cul4a-Dbd1 E3 ubiquitin ligase complex, thereby increasing the stability of the Gcn5 protein. Taken together, our study indicates that Akt1 negatively controls Gcn5 via the proteasomal degradation pathway, suggesting a potential mechanism that regulates the expression of Hox genes.ope

Post-transcriptional Regulation of Gcn5, a Putative Regulator of Hox in Mouse Embryonic Fibroblast Cells

Hox proteins containing DNA-binding homedomain act as transcription factors important for anteroposterior body patterning during vertebrate embryogenesis. However, the precise mechanisms by which signal pathways are transduced to regulate the Hox gene expression are not clear. In the course of an attempt to isolate an upstream regulatory factor(s) controlling Hox genes, protein kinase B alpha (Akt1) has been identified as a putative regulator of Hox genes through in silico analysis (GEO profile). In the Gene Expression Omnibus (GEO) dataset GDS1784 at the NCBI (National Center for Biotechnology Information) site, Hox genes were differentially expressed depending on the presence or absence of Akt1. Since it was not well known how Akt1 regulates the specific Hox genes, whose transcription was reported to be regulated by epigenetic modifications such as histone acetylation, methylation etc., the expression of Gcn5, a histone acetyltransferase (HAT), was analyzed in wild type (WT) as well as in Akt1-/- mouse embryonic fibroblast (MEF) cells. RT-PCR analysis revealed that the amount of Gcn5 mRNA was similar in both WT and Akt1-/- MEFs. However, the protein level of Gcn5 was significantly increased in Akt1-/- MEF cells. The half life of Gcn5 was 1 hour in wild type whereas 8 hours in Akt1-/- MEF. These data all together, indicate that Gcn5 is post-transcriptionally down-regulated and the protein stability is negatively regulated by Akt1 in MEF cells.ope

HOXA5 confers tamoxifen resistance via the PI3K/AKT signaling pathway in ER-positive breast cancer

Tamoxifen is a commonly used drug to treat estrogen receptor-positive patients with breast cancer. Despite the outstanding efficacy of tamoxifen, approximately one-third of patients develop resistance toward it, thereby presenting a therapeutic challenge. HOX genes may be involved in the acquisition of tamoxifen resistance. In this study, we identified HOXA5, a member of the HOX gene family, as a marker of tamoxifen resistance. Using ChIP assay, we found that HOXA5 expression was significantly overexpressed in tamoxifen-resistant MCF7 (TAMR) breast cancer cells because of reduced H3K27me3 binding. HOXA5 upregulation resulted in activation of the PI3K/AKT signaling cascade, which in turn, led to p53 and p21 reduction, ultimately making the TAMR cells less apoptotic. Furthermore, elevated HOXA5 expression resulted in breast cancer cells acquiring more mesenchymal-like and stem cell traits associated with aggressive breast cancer phenotypes. In conclusion, our results delineate a mechanism by which HOXA5 promotes tumorigenesis, cancer progression, and tamoxifen resistance in breast cancer cells.ope

The mitotic checkpoint regulator RAE1 induces aggressive breast cancer cell phenotypes by mediating epithelial-mesenchymal transition

The gene RAE1 encodes ribonucleic acid export 1 (RAE1), which is involved in mRNA export and is known to serve as a mitotic checkpoint regulator. In addition, RAE1 haplo-insufficiency leads to chromosome missegregation and early aging-associated phenotypes. In humans, a positive correlation has been found between RAE1 copy number abnormalities and gene amplification in breast cancer cells. However, the precise functional role of RAE1 in breast cancer remains to be determined. An in silico analysis of data retrieved from GENT and cBio-Portal identified RAE1 upregulation in breast cancer tissues relative to normal breast cells. Functional studies of various cell lines showed that RAE1 induced invasive and migratory abilities by regulating epithelial-mesenchymal transition signals. A tissue microarray was constructed to demonstrate the interrelationship between clinicopathological features and RAE1 expression. Immunohistochemistry revealed a positive correlation between RAE1 expression and a high histologic grade. Furthermore, RAE1 overexpression was associated with considerably poorer disease-free survival and distant metastasis-free survival, especially in patients with oestrogen receptor-positive tumours. In summary, RAE1 may be a prognostic marker and therapeutic intervention target in malignant breast cancers.ope

Direct Interaction Between Akt1 and Gcn5 and its Plausible Function on Hox Gene Expression in Mouse Embryonic Fibroblast Cells

Hox genes encode transcription factors important for anterior‐posterior body patterning at early stages of embryonic development. However, the precise mechanisms by which signal pathways are stimulated to regulate Hox gene expression are not clear. In the previous study, protein kinase B alpha (Akt1) has been identified as a putative upstream regulator of Hox genes, and Akt1 has shown to regulate Gcn5, a prototypical histone acetyltransferase (HAT), in a negative way in mouse embryonic fibroblast (MEF) cells. Since the activity of HAT such as the CBP/p300, and PCAF (a Gcn5 homolog), was down‐regulated by Akt through a phosphorylation at the Akt consensus substrate motif (RXRXXS/T), the amino acid sequence of Gcn5 protein was analyzed. Mouse Gcn5 contains an Akt consensus substrate motif as RQRSQS sequence while human Gcn5 does not have it. In order to see whether Akt1 directly binds to Gcn5, immunoprecipitation with anti‐Akt1 antibody was carried out in wild‐type (WT) mouse embryonic fibroblast (MEF) cells, and then western blot analysis was performed with anti‐Akt1 and anti‐Gcn5 antibodies. Gcn5 protein was detected in the Akt1 immunoprecipitated samples of MEFs. This result demonstrates that Akt1 directly binds to Gcn5, which might have contributed the down regulation of the 5’ Hoxc gene expressions in wild type MEF cells.ope

RAE1 mediated ZEB1 expression promotes epithelial-mesenchymal transition in breast cancer

Breast cancer metastasis accounts for most of the deaths from breast cancer. Since epithelial-mesenchymal transition (EMT) plays an important role in promoting metastasis of cancer, many mechanisms regarding EMT have been studied. We previously showed that Ribonucleic acid export 1 (RAE1) is dysregulated in breast cancer and its overexpression leads to aggressive breast cancer phenotypes by inducing EMT. Here, we evaluated the functional capacity of RAE1 in breast cancer metastasis by using a three-dimensional (3D) culture system and xenograft models. Furthermore, to investigate the mechanisms of RAE1-driven EMT, in vitro studies were carried out. The induction of EMT with RAE1-overexpression was confirmed under the 3D culture system and in vivo system. Importantly, RAE1 mediates upregulation of an EMT marker ZEB1, by binding to the promoter region of ZEB1. Knockdown of ZEB1 in RAE1-overexpressing cells suppressed invasive and migratory behaviors, accompanied by an increase in epithelial and a decrease in mesenchymal markers. Taken together, these data demonstrate that RAE1 contributes to breast cancer metastasis by regulating a key EMT-inducing factor ZEB1 expression, suggesting its potential as a therapeutic target.ope

RAE1의 상피-간충직 이행을 통한 유방암 진행 조절

Department of Medical Scienceprohibition박