BMI1 Promoter Research Articles

Mechanism of E2F1 in the proliferation, migration, and invasion of endometrial carcinoma cells via the regulation of BMI1 transcription.

Endometrial carcinoma (EC) is the most prevalent gynecological cancer. Transcription factor (TF) regulates a large number of downstream target genes and is a key determinant of all physiological activities, including cell proliferation, differentiation, apoptosis, and cell cycle. The transcription factor E2F1 shows prominent roles in EC. BMI1 is a member of Polycomb suppressor Complex 1 (PRC1) and has been shown to be associated with EC invasiveness. It is currently unclear whether E2F1 can participate in the proliferation, migration, and invasion processes of EC cells by regulating BMI1 transcription. We investigated whether E2F1 could participate in the proliferation, migration, and invasion processes of EC cells by regulating BMI1 transcription, in order to further clarify the pathogenesis and etiology of EC, and provide reference for identifying potential therapeutic targets and developing effective prevention and treatment strategies for this disease. Human endometrial epithelial cells (hEECs) and human EC cell lines were selected. E2F1 expression was assessed by Western blot. E2F1 was silenced in AN3CA or overexpressed in HEC-1 by transfections, or E2F1 was silenced and BMI1 was overexpressed in AN3CA by cotransfection. Cell proliferation, migration, and invasion were detected by MTT, wound healing, and Transwell assays. The binding sites between E2F1 and BMI1 promoters were predicted through JASPAR website, and the targeted binding was verified by dual-luciferase report and ChIP assays. E2F1 was up-regulated in human EC cell lines, with its expression highest in AN3CA, and lowest in HEC-1. AN3CA invasion, migration, and proliferation were repressed by E2F1 knockdown, while those of HEC-1 cells were promoted by E2F1 overexpression. E2F1 overexpression increased the activity of wild type BMI1 reporter vector promoter, while this promotion was weakened after mutation of the predicted binding site in the BMI1 promoter. In the precipitated E2F1, BMI1 promoter site level was higher than that of IgG immunoprecipitant. BMI1 silencing suppressed AN3CA cell growth. BMI1 overexpression partially abrogated E2F1 silencing-inhibited EC cell growth. E2F1 promoted EC cell proliferation, invasion, and migration by promoting the transcription of BMI1.

Nuclear translocation of YAP drives BMI-associated hepatocarcinogenesis in hepatitis B virus infection.

Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) development and progression. The aim of this study was to mechanistically investigate the involvement of Hippo signalling in HBV surface antigen (HBsAg)-dependent neoplastic transformation. Liver tissue and hepatocytes from HBsAg-transgenic mice were examined for the Hippo cascade and proliferative events. Functional experiments in mouse hepatoma cells included knockdown, overexpression, luciferase reporter assays and chromatin immunoprecipitation. Results were validated in HBV-related HCC biopsies. Hepatic expression signatures in HBsAg-transgenic mice correlated with YAP responses, cell cycle control, DNA damage and spindle events. Polyploidy and aneuploidy occurred in HBsAg-transgenic hepatocytes. Suppression and inactivation of MST1/2 led to the loss of YAP phosphorylation and the induction of BMI1 expression in vivo and in vitro. Increased BMI1 directly mediated cell proliferation associated with decreased level of p16INK4a , p19ARF , p53 and Caspase 3 as well as increased Cyclin D1 and γ-H2AX expression. Chromatin immunoprecipitation and the analysis of mutated binding sites in dual-luciferase reporter assays confirmed that the YAP/TEAD4 transcription factor complex bound and activated the Bmi1 promoter. In chronic hepatitis B patients, paired liver biopsies of non-tumour and tumour tissue indicated a correlation between YAP expression and the abundance of BMI1. In a proof-of-concept, treatment of HBsAg-transgenic mice with YAP inhibitor verteporfin directly suppressed the BMI1-related cell cycle. HBV-associated proliferative HCC might be related to the HBsAg-YAP-BMI1 axis and offer a potential target for the development of new therapeutic approaches.

Natural Compounds with BMI1 Promoter Inhibitory Activity from Mammea siamensis and Andrographis paniculata.

A new coumarin derivative (1) and 30 known compounds were isolated from Mammea siamensis and Andrographis paniculata, guided by B cell-specific Moloney murine leukemia virus insertion region 1 (BMI1) promoter inhibitory activity. Among the isolated compounds, 15 compounds showed BMI1 promoter inhibitory activity, and five compounds were found to be cytotoxic. 14-Deoxy-11,12-dehydroandrographolide (18) was highly cytotoxic to DU145 cells with an IC50 value of 25.4 µM. Western blotting analysis of compound 18 in DU145 cells suggested that compound 18 suppresses BMI1 expression.

Disulfiram/Copper Suppresses Cancer Stem Cell Activity in Differentiated Thyroid Cancer Cells by Inhibiting BMI1 Expression.

Differentiated thyroid carcinomas (DTCs), which have papillary and follicular types, are common endocrine malignancies worldwide. Cancer stem cells (CSCs) are a particular type of cancer cells within bulk tumors involved in cancer initiation, drug resistance, and metastasis. Cells with high intracellular aldehyde hydrogenase (ALDH) activity are a population of CSCs in DTCs. Disulfiram (DSF), an ALDH inhibitor used for the treatment of alcoholism, reportedly targets CSCs in various cancers when combined with copper. This study reported for the first time that DSF/copper can inhibit the proliferation of papillary and follicular DTC lines. DSF/copper suppressed thyrosphere formation, indicating the inhibition of CSC activity. Molecular mechanisms of DSF/copper involved downregulating the expression of B lymphoma Mo-MLV insertion region 1 homolog (BMI1) and cell cycle-related proteins, including cyclin B2, cyclin-dependent kinase (CDK) 2, and CDK4, in a dose-dependent manner. BMI1 overexpression diminished the inhibitory effect of DSF/copper in the thyrosphere formation of DTC cells. BMI1 knockdown by RNA interference in DTC cells also suppressed the self-renewal capability. DSF/copper could inhibit the nuclear localization and transcriptional activity of c-Myc and the binding of E2F1 to the BMI1 promoter. Overexpression of c-Myc or E2F1 further abolished the inhibitory effect of DSF/copper on BMI1 expression, suggesting that the suppression of c-Myc and E2F1 by DSF/copper was involved in the downregulation of BMI1 expression. In conclusion, DSF/copper targets CSCs in DTCs by inhibiting c-Myc- or E2F1-mediated BMI1 expression. Therefore, DSF is a potential therapeutic agent for future therapy in DTCs.

Correlation between the expression of cancer stem cell marker BMI1 and glioma prognosis

B-cell-specific Moloney murine leukemia virus integration site 1 (BMI1) appears to be essential for promoting certain types of cancer, and its inhibition effectively reduced the stemness of cancer cells. Therefore, this study aimed to investigate the potential role of BMI1 in glioma. To this end, we first investigated BMI1 expression in brain tumors using microarray datasets in ONCOMINE, which indicated that BMI1 levels were not commonly increased in clinical brain tumors. Moreover, survival plots in PROGgeneV2 also showed that BMI1 expression was not significantly associated with reduced survival in glioma patients. Interestingly, stressful serum deprivation and anchorage independence growth conditions led to an increased BMI1 expression in glioma cells. A stress-responsive pathway, HDAC/Sp1, was further identified to regulate BMI1 expression. The HDAC inhibitor vorinostat (SAHA) prevented Sp1 binding to the BMI1 promoter, leading to a decreased expression of BMI1 and attenuating tumor growth of TMZ-resistant glioma xenografts. Importantly, we further performed survival analysis using PROGgeneV2 and found that an elevated expression of HDAC1,3/Sp1/BMI1 but not BMI1 alone showed an increased risk of death in both high- and low-grade glioma patients. Thus, HDAC-mediated Sp1 deacetylation is critical for BMI1 regulation to attenuate stress- and therapy-induced death in glioma cells, and the HDAC/Sp1 axis is more important than BMI1 and appears as a therapeutic target to prevent recurrence of malignant glioma cells persisting after primary therapy.

CSIG-12. HDACs-Sp1-BMI1 PATHWAY IS INVOLVED IN THE ATTENUATION OF STRESS- AND THERAPY-INDUCED DEATH IN GLIOMA

Abstract BMI-1 is a transcriptional repressor and overexpresses in various human cancer tissues, which is also known to involve in the maintenance of “stem cell” features in a cell sub-population within the tumor. Here, we investigate the roles of BMI1 oncogene in brain tumors, notably involving drug-resistant metabolism. Interestingly, microarray datasets in OMCOMINE indicated that BMI1 is not commonly increased in clinical brain tumors, and survival plots in PROGgeneV2 also showed that BMI1 expression is not statistically associated with glioma patients. However, stressful serum starvation and anchorage independence growth conditions enabled to induce upregulation of BMI1. Further investigation, we identified HDAC-Sp1 is a stress-responsive pathway, critically promoting BMI1 expression. HDAC inhibition via vorinostat treatment prevented the binding of Sp1 to BMI1 promoter, leading to decreased BMI1 expression and attenuated tumor growth of TMZ-resistant glioma xenografts. Importantly, increased expression of the HDAC-Sp1-BMI1 axis but not BMI1 alone showed an increased risk of death in both high- and low-grade glioma. Thus, the HDAC-Sp1-BMI1 axis is critical to attenuate stress- and therapy-induced death in glioma cells and the axis inhibition may prevent the recurrence of malignant cells persisting after primary therapy.

Nonactic Acid Derivatives Isolated From Streptomyces werraensis IFM12104 in a Screening Program for BMI1 Promoter Inhibitory Activity

Upon screening for compounds with B cell-specific Moloney murine leukemia virus region 1 (BMI1) promoter inhibitory activity, 3 nonactic acid derivatives (1-3) were isolated from Streptomyces werraensis IFM12104. Although spectral data of compound 1 were identical with those reported in literature for 4α-(3, 5-dihydroxy-heptyl)-3α-methyl-2-oxetanone (4), compound 1 was revealed to be homononactic acid by X-ray analysis. Compounds 2 and 3 were identified as bishomononactic acid and nonactic acid using spectral data. The 3 compounds 1 to 3 inhibited BMI1 promoter activities with IC50 values of 240, 160, and 180 μM, respectively.

Identification of BMI1 promoter inhibitors from Streptomyces sp. IFM-11958.

B-cell-specific Moloney murine leukemia virus region 1 (BMI1) is a central component of polycomb repressive complex 1 (PRC1), which maintains epigenetic repression of genes expression via chromatin condensation. BMI1 overexpression downregulates the expression of tumor suppressor genes, such as p16Ink4a and PTEN. BMI1 expression is upregulated in cancer stem cells (CSCs). Therefore, inhibitors of BMI1 expression have potential as therapeutic agents for cancer. This study aimed to identify BMI1 promoter inhibitors from actinomycetes. Using a recently constructed BMI1 promoter assay, we isolated three known compounds, elaiophylin (1), 2-methylelaiophylin (2), and nocardamin (3), from Streptomyces sp. IFM-11958 that inhibited BMI1 promoter activity with IC50 values of 30 nM, 447 nM, 22 µM, respectively. Elaiophylin (1) was the most potent. Western blot and PCR analyses revealed that elaiophylin (1) inhibited BMI1 expression at the mRNA level in human prostate cancer cells (DU145). Elaiophylin (1) also inhibited the sphere-forming activity of human hepatocellular carcinoma cells (Huh7), indicating that elaiophylin (1) suppresses the self-renewal capacity of CSCs. Elaiophylin (1) is the first BMI1 promoter inhibitor isolated from actinomycete metabolites.

Abstract 3999: Intracellular redox status determines self-renewal activity of breast cancer stem-like cells through activation of Nrf2-mediated FoxO3a-Bmi-1 signaling

Abstract A subpopulation of cancer cells, termed cancer stem cells (CSCs), has a self-renewal property, which drives cancer recurrence and tumor resistance. It has been reported that CSCs possess enhanced capabilities to protect against oxidative stress caused by reactive oxygen species (ROS) compared with non-stem-like cancer cells. In the present study, we found that the redox-sensitive transcription factor NF-E2-related factor 2 (Nrf2) plays a role in self-renewal activity of human breast cancer cells, and that this was mediated by Bmi-1. The manifestation of stemness in breast CSC-like cells was associated with an elevated ratio of reduced to oxidized glutathione (GSH) and consequently, lowered ROS levels compared with parent breast cancer (MCF-7) cells. We observed that there was a concurrent increase in the expression of CD44, glutamate cysteine ligase (GCLC) and Bmi-1 in CD24low/CD44high breast cancer stem-like cells sorted from MCF-7 mammospheres. Treatment with buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis, led to a decrease in the number and the size of mammospheres, which was accompanied by the reduction of Bmi-1 expression. Conversely, treatment with a membrane permeable GSH ethyl ester (GSH-MEE) enhanced the size and the number of mammospheres. In addition, phosphorylation of AMPK (Thr 172) and FoxO3a (Ser 7) as well as expression of Bmi-1 was increased in mammosphere cells treated with GSH-MEE. When GSH-MEE-treated mammospheres were challenged with hydrogen peroxide, the phosphorylation of AMPK was abolished. Notably, FoxO3a directly bound to the Bmi-1 promoter as assessed by the chromatin immunoprecipitation assay. Moreover, elevated nuclear accumulation of Nrf2 protein was evident in cells growing under sphere-forming conditions. Under physiologic conditions, Nrf2 is sequestered in the cytoplasm as an inactive complex with Keap1. Sphere-forming breast cancer cells exhibited an enhanced interaction between Keap1 and CD44, which may account for an increased translocation of Nrf2 in nucleus. We found that expression of GCLC, FoxO3a and Bmi-1 was inhibited following introduction of Nrf2 siRNA into MCF-7 mammsophere cells. Knockdown of Nrf2 suppressed the xenograft growth of orthotopically injected breast cancer stem-like cells in athymic nude mice, and this was accompanied by decreased expression of GCLC, FoxO3a and Bmi-1. In conclusion, CD44 promotes Nrf2 accumulation in breast cancer stem-like cells, which, in turn, upregulates GCLC expression and consequently enhances GSH biosynthesis with concomitant reduction in intracellular ROS levels. The consequent upregulation of nuclear FoxO3a and its binding to the Bmi-1 promoter may stimulate self-renewal and growth of breast cancer stem-like cells. Citation Format: Do-Hee Kim, Jeong-Hoon Jang, Young-Joon Surh. Intracellular redox status determines self-renewal activity of breast cancer stem-like cells through activation of Nrf2-mediated FoxO3a-Bmi-1 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3999.

Hsp90α Mediates BMI1 Expression in Breast Cancer Stem/Progenitor Cells through Facilitating Nuclear Translocation of c-Myc and EZH2.

Heat shock protein 90 (Hsp90) is a molecular chaperone that facilitates the correct folding and functionality of its client protein. Numerous Hsp90-client proteins are involved in cancer development. Thus, Hsp90 inhibitors have potential applications as anti-cancer drugs. We previously discovered that Hsp90α expression increased in breast cancer stem cells (BCSCs), which can initiate tumorigenesis and metastasis and resist treatment. In the present study, we further demonstrated that 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), an inhibitor of Hsp90, could suppress the self-renewal of BCSCs by downregulating B lymphoma Mo-MLV insertion region 1 homolog (BMI1), a polycomb family member with oncogenic activity in breast cancer. Through immunoprecipitation analysis, we found that BMI1 did not interact with Hsp90α and that the downregulation of BMI1 by 17-DMAG was mediated by the inhibition of c-Myc and enhancement of zeste homolog 2 (EZH2) expression. The transcriptional and BMI1 promoter-binding activities of c-Myc in BCSCs were inhibited by 17-DMAG treatment. The overexpression of EZH2 attenuated the inhibitory effect of 17-DMAG on BMI1 and c-Myc expression. Furthermore, Hsp90α could be co-immunoprecipitated with c-Myc and EZH2 and bind to the BMI1 promoter. Treatment with 17-DMAG decreased the nuclear expression of EZH2 and c-Myc but not that of Hsp90α. In conclusion, our data suggested that Hsp90α could positively regulate the self-renewal of BCSCs by facilitating the nuclear translocation of c-Myc and EZH2 to maintain BMI1 expression.

Targeting MUC1-C suppresses polycomb repressive complex 1 in multiple myeloma

The polycomb repressive complex 1 (PRC1) includes the BMI1, RING1 and RING2 proteins. BMI1 is required for survival of multiple myeloma (MM) cells. The MUC1-C oncoprotein is aberrantly expressed by MM cells, activates MYC and is also necessary for MM cell survival. The present studies show that targeting MUC1-C with (i) stable and inducible silencing and CRISPR/Cas9 editing and (ii) the pharmacologic inhibitor GO-203, which blocks MUC1-C function, downregulates BMI1, RING1 and RING2 expression. The results demonstrate that MUC1-C drives BMI1 transcription by a MYC-dependent mechanism. MUC1-C thus promotes MYC occupancy on the BMI1 promoter and thereby activates BMI1 expression. We also show that the MUC1-C→MYC pathway induces RING2 expression. Moreover, in contrast to BMI1 and RING2, we found that MUC1-C drives RING1 by an NF-κB p65-dependent mechanism. Targeting MUC1-C and thereby the suppression of these key PRC1 proteins was associated with downregulation of the PRC1 E3 ligase activity as evidenced by decreases in ubiquitylation of histone H2A. Targeting MUC1-C also resulted in activation of the PRC1-repressed tumor suppressor genes, PTEN, CDNK2A and BIM. These findings identify a heretofore unrecognized role for MUC1-C in the epigenetic regulation of MM cells.

Identification of BMI1 Promoter Inhibitors from Beaumontia murtonii and Eugenia operculata.

B-Cell-specific Moloney murine leukemia virus insertion region 1 (BMI1) is a core component of the polycomb repressive complex 1 (PRC1). Abnormal expression of BMI1 is associated with a number of human malignances and cancer stem cells (CSCs), which cause chemotherapy resistance. Therefore, small molecules that inhibit BMI1 expression are potential candidates for cancer therapy. In this study, a cell-based reporter gene assay was developed that allowed BMI1 promoter activity to be measured in 293T human embryonic kidney cells based on luciferase expression levels. Using this screening assay, the methanol-soluble extracts of Beaumontia murtonii and Eugenia operculata were selected as leads. Bioassay-guided fractionation of the extracts led to the isolation of three known cardenolides (1-3) and one new compound (4) from B.murtonii and two known triterpenoids (5 and 6) and one new compound (7) from E.operculata. These seven compounds inhibited BMI1 promoter activity (IC50 range 0.093-23.0 μM), and the most active compound, wallichoside (1), was further evaluated. Western blot analysis revealed that wallichoside (1) decreases BMI1 protein levels in HCT116 human colon carcinoma cells, and flow cytometry analysis showed that it significantly reduced levels of the CSC biomarker epithelial cell adhesion molecule. Wallichoside (1) also inhibited sphere formation of Huh7 human hepatocellular carcinoma cells, indicating that it diminished the self-renewal capability of CSCs.

Antitumor activity and inhibitory effects on cancer stem cell-like properties of Adeno-associated virus (AAV) -mediated Bmi-1 interference driven by Bmi-1 promoter for gastric cancer.

Bmi-1 is aberrantly activated in various cancers and plays a vital role in maintaining the self-renewal of stem cells. Our previous research revealed that Bmi-1 was overexpressed in gastric cancer (GC) and it's overexpression was an independent negative prognostic factor, suggesting it can be a therapeutic target. The main purpose of this investigation was to explore the antitumor activity of Bmi-1 interference driven by its own promoter (Ad-Bmi-1i) for GC. In this study, we used adenoviral vector to deliver Bmi-1 shRNA driven by its own promoter to treat GC. Our results revealed that Ad-Bmi-1i could selectively silence Bmi-1 in GC cells which overexpress Bmi-1 and suppress the malignant phenotypes and stem-like properties of GC cells in vitro and in vivo. Moreover, direct injection of Ad-Bmi-1i into xenografts suppressed tumor growth and destroyed cancer cells in vivo. Ad-Bmi-1i inhibited the proliferation of GC cells mainly via inducing senescence in vitro, but it suppressed tumor through inducing senescence and apoptosis, and inhibiting angiogenesis in vivo. Bmi-1 knockdown by Ad-Bmi-1i downregulated VEGF via inhibiting AKT activity. These results suggest that Ad-Bmi-1i not only inhibits tumor growth and stem cell-like phenotype by inducing cellular senescence directly, but also has an indirect anti-tumor activity by anti-angiogenesis effects via regulating PTEN/AKT/VEGF pathway. Transfer of gene interference guided by its own promoter by an adeno-associated virus (AAV) vector might be a potent antitumor approach for cancer therapy.

Bmi1 drives stem-like properties and is associated with migration, invasion, and poor prognosis in tongue squamous cell carcinoma.

Bmi1 (B-cell-specific Moloney murine leukemia virus insertion site 1) had been found to involve in self -renewal of stem cells and tumorigenesis in various malignancies. The purpose of this study is to evaluate the role of Bmi1 in the development of tongue squamous cell carcinoma (TSCC) and its functional effect on the migration and invasion of TSCC. Initially, immunohistochemistry revealed that Bmi1 overexpression was a common event in premalignant dysplasia, primary TSCC, and lymph node metastases and was associated with a poor prognosis. A significant correlation between Bmi1 and SOD2 (manganese superoxide dismutase) expression was observed. Side population (SP) cells were used as cancer stem-like cells and further assessed by sphere and colony formation assays, and the expression of stem cell markers. TSCC cells with higher migration and invasion ability (UM1 cell lines) showed a higher proportion of SP cells and Bmi1 expression than TSCC cells with lower migration and invasion ability (UM2 cell lines). Knockdown of Bmi1 in UM1 or SP cells inhibited migration and invasion and decreased the sphere and colony formation, and the expression of stem cell markers and SOD2. Direct binding of C-myc to the Bmi1 promoter was demonstrated by chromatin immunoprecipitation and luciferase assays. Moreover, C-myc knockdown in SP cells inhibited their migration and invasion and decreased the expression of Bmi1 and SOD2. Our results indicate that the deregulation of Bmi1 expression is a frequent event during the progression of TSCC and may have a prognostic value for patients with this disease. The Bmi1-mediated migration and invasion of TSCC is related to cancer stem-like cells and involves the C-myc-Bmi1-SOD2 pathway.

YB-1 Transforms Human Mammary Epithelial Cells Through Chromatin Remodeling Leading to the Development of Basal-Like Breast Cancer

There is growing evidence that cancer-initiation could result from epigenetic changes. Y-box binding protein-1 (YB-1) is a transcription/translation factor that promotes the formation of tumors in transgenic mice; however, the underlying molecular events are not understood. To explore this in a human model system, YB-1 was expressed in mammary epithelial cells under the control of a tetracycline-inducible promoter. The induction of YB-1 promoted phenotypes associated with malignancy in three-dimensional breast acini cultures. This was attributed to YB-1 enhancing the expression and activity of the histone acetyltransferase p300 leading to chromatin remodeling. Specifically, this relaxation of chromatin allowed YB-1 to bind to the BMI1 promoter. The induction of BMI1 engaged the Polycomb complex resulting in histone H2A ubiquitylation and repression of the CDKN2A locus. These events manifested functionally as enhanced self-renewal capacity that occurred in a BMI1-dependent manner. Conversely, p300 inhibition with anacardic acid prevented YB-1 from binding to the BMI1 promoter and thereby subverted self-renewal. Despite these early changes, full malignant transformation was not achieved until RSK2 became overexpressed concomitant with elevated human telomerase reverse transcriptase (hTERT) activity. The YB-1/RSK2/hTERT expressing cells formed tumors in mice that were molecularly subtyped as basal-like breast cancer. We conclude that YB-1 cooperates with p300 to allow BMI1 to over-ride p16(INK4a) -mediated cell cycle arrest enabling self-renewal and the development of aggressive breast tumors.

Estrogen receptor α-coupled Bmi1 regulation pathway in breast cancer and its clinical implications

BackgroundBmi1 has been identified as an important regulator in breast cancer, but its relationship with other signaling molecules such as ERα and HER2 is undetermined.MethodsThe expression of Bmi1 and its correlation with ERα, PR, Ki-67, HER2, p16INK4a, cyclin D1 and pRB was evaluated by immunohistochemistry in a collection of 92 cases of breast cancer and statistically analyzed. Stimulation of Bmi1 expression by ERα or 17β-estradiol (E2) was analyzed in cell lines including MCF-7, MDA-MB-231, ERα-restored MDA-MB-231 and ERα-knockdown MCF-7 cells. Luciferase reporter and chromatin immunoprecipitation assays were also performed.ResultsImmunostaining revealed strong correlation of Bmi1 and ERα expression status in breast cancer. Expression of Bmi1 was stimulated by 17β-estradiol in ERα-positive MCF-7 cells but not in ERα-negative MDA-MB-231 cells, while the expression of Bmi1 did not alter expression of ERα. As expected, stimulation of Bmi1 expression could also be achieved in ERα-restored MDA-MB-231 cells, and at the same time depletion of ERα decreased expression of Bmi1. The proximal promoter region of Bmi1 was transcriptionally activated with co-transfection of ERα in luciferase assays, and the interaction of the Bmi1 promoter with ERα was confirmed by chromatin immunoprecipitation. Moreover, in breast cancer tissues activation of the ERα-coupled Bmi1 pathway generally correlated with high levels of cyclin D1, while loss of its activity resulted in aberrant expression of p16INK4a and a high Ki-67 index, which implied a more aggressive phenotype of breast cancer.ConclusionsExpression of Bmi1 is influenced by ERα, and the activity of the ERα-coupled Bmi1 signature impacts p16INK4a and cyclin D1 status and thus correlates with the tumor molecular subtype and biologic behavior. This demonstrates the important role which is played by ERα-coupled Bmi1 in human breast cancer.

Abstract P1-07-11: ERa regulates epithelial-mesenchymal transition in breast cancers through suppressing Bmi1 expression

Abstract Background: Bmi1, a polycomb-group protein, maintains stem cell self-renewal and is frequently overexpressed in cancers. Upregulation of Bmi1 by Twist1 can promote tumor-initiating capability and Epithelial-mesenchymal transition (EMT) by repressing E-cadherin expression. It has been demonstrated that Post-EMT breast cancer cells express cancer stem cell (CSC) markers including Bmi1, but display decreased ERα expression. ERα is a ligand-activated nuclear hormone receptor that regulates the transcription of E2-responsive genes in diverse target cells. It's reported that ligand-activated ERα could repress slug transcription by binding to the half-site ERE element in its promoter, and then regulate E-cadherin and EMT. Herein, we hypothesized that ERα signaling might regulate E-cadherin and EMT through Bmi1. Methods: First, we determined Bmi1 and ERα expression at both mRNA and protein levels by quantitative RT-PCR and Western blot in various breast cancer cell lines. Further, we quantitatively analyzed mRNA and protein levels of Bmi1 as well as its down-stream genes including E-cadherin after silencing ERα in T47D cells with siRNA, or after overexpressing ERα in BT549 cells. The chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) were also performed to investigate whether ERα binds directly to Bmi1 promoter region. Luciferase assay with Bmi1 reporter was performed to ERα mediated regulation. To identify whether ERα regulates EMT, transwell and matrigel invasion, flow cytometry, mammosphere assay and wound healing assay were performed to invasion, metastases and characterize stem-cell properties of EMT. Finally, we analyzed the expression of ERα, Bmi1 and E-cadherin in human breast cancer specimens with immunohistochemistry. Results: The study showed that Bmi1 inversely correlated with ERα expression in different subtypes of breast cancer cells. We further confirmed that Bmi1 was upregulated at both mRNA and protein levels and E-cadherin was downregulated with siRNA against ERα in T47D cells, and Bmi1 was downregulated and E-cadherin was upregulated when overexpressing ERα in BT549 cells with qRT-PCR and western blot analyses. The present study also demonstrated that ERα could directly binds to the half-ERE region of the Bmi1 gene and repressing Bmi1 expression transcriptionally with CHIP/EMSA and reporter analyses. Overexpression of ERα in BT549 cell significantly decreased levels of migration of transwell and wounding healing, invasiveness, CD44high/CD24low population, and the capabilities of mammosphere formation. Conclusions: Our results demonstrated that ERα can suppress EMT through transcriptionally down-regulating Bmi-1 and its down-stream genes in human breast cancer cells. The inverse relationship between ERα and Bmi1 expression further supports the epithelial phenotype of ERα positive tumors or mesenchymal phenotype of ERα negative tumors. Our findings provide a novel mechanistic insight into how ERα regulates EMT, and is of valuable for developing biomarkers to predict prognosis and targeted therapies in breast cancers in the future. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-07-11.

Abstract A037: YB-1 transforms mammary epithelial cells through chromatin remodeling leading to the development of basal-like breast cancer

Abstract There is growing evidence that cancer-initiation could result from epigenetic changes. Y-box binding protein-1 (YB-1) is a transcription/translation factor that promotes the formation of tumors in transgenic mice; however, the early molecular events leading up to this are not understood. To address this in a human model system, YB-1 was expressed in mammary epithelial cells under a tetracycline-inducible promoter. The induction of YB-1 drove the development of ductal carcinoma in situ (DCIS) in vitro. Underlying these events, YB-1 induced p300, promoted its nuclear localization, and heightened histone acetylation leading to chromatin relaxation. Subsequently, chromatin relaxation allowed YB-1 to bind to the BMI1 promoter based on chromatin immunoprecipitation. The induction of BMI1 engaged the PRC1 complex as histone H2A was ubiquitinated and p16INK4a was silenced. These events manifested functionally in sustained self-renewal that occurred in a BMI1 dependent manner. Conversely, p300 inhibition with anacardic acid prevented YB-1 from binding to the BMI1 promoter and thereby subverted self-renewal. Accordingly, transient expression of YB-1 in the normal mammary 184hTERT and MCF10A cells had the same effect on chromatin remodeling and self-renewal. Despite these early changes, full malignant transformation was not achieved until RSK2 was expressed leading to elevated hTERT activity. The YB-1/RSK2/hTERT expressing cells formed DCIS and invasive ductal carcinoma in mice that were molecularly subtyped as basal-like breast cancer (BLBC). We conclude that YB-1 cooperates with p300 to allow BMI1 to over-ride p16INK4a enabling self-renewal and the formation of tumors. Citation Format: Alastair H. Davies, Kristen M. Reipas, Natalie Firmino, Anna L. Stratford, Rachel Berns, Abbas Fotovati, Mary Pambid, Kaiji Hu, Chris Maxwell, Gordon B. Mills, Sandra E. Dunn. YB-1 transforms mammary epithelial cells through chromatin remodeling leading to the development of basal-like breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A037.

Zoledronic Acid Reverses the Epithelial–Mesenchymal Transition and Inhibits Self-Renewal of Breast Cancer Cells through Inactivation of NF-κB

Zoledronic acid, a third-generation bisphosphonate, has been shown to reduce cell migration, invasion, and metastasis. However, the effects of zoledronic acid on the epithelial-mesenchymal transition (EMT), a cellular process essential to the metastatic cascade, remain unclear. Therefore, the effects of zoledronic acid on EMT, using triple-negative breast cancer (TNBC) cells as a model system, were examined in more detail. Zoledronic acid treatment decreased the expression of mesenchymal markers, N-cadherin, Twist, and Snail, and subsequently upregulated expression of E-cadherin. Zoledronic acid also inhibited cell viability, induced cell-cycle arrest, and decreased the proliferative capacity of TNBC, suggesting that zoledronic acid inhibits viability through reduction of cell proliferation. As EMT has been linked to acquisition of a self-renewal phenotype, the effects of zoledronic acid on self-renewal in TNBC were also studied. Treatment with zoledronic acid decreased expression of self-renewal proteins, BMI-1 and Oct-4, and both prevented and eliminated mammosphere formation. To understand the mechanism of these results, the effect of zoledronic acid on established EMT regulator NF-κB was investigated. Zoledronic acid inhibited phosphorylation of RelA, the active subunit of NF-κB, at serine 536 and modulated RelA subcellular localization. Treatment with zoledronic acid reduced RelA binding to the Twist promoter, providing a direct link between inactivation of NF-κB signaling and loss of EMT transcription factor gene expression. Binding of Twist to the BMI-1 promoter was also decreased, correlating modulation of EMT to decreased self-renewal. On the basis of these results, it is proposed that through inactivation of NF-κB, zoledronic acid reverses EMT, which leads to a decrease in self-renewal.

Phosphorylation of Nanog is essential to regulate Bmi1 and promote tumorigenesis

Emerging evidence indicates that Nanog is intimately involved in tumorigenesis in part through regulation of the cancer initiating cell population. However, the regulation and role of Nanog in tumorigenesis are still poorly understood. In this study, human Nanog was identified to be phosphorylated by human PKCε at multiple residues including T200 and T280. Our work indicated that phosphorylation at T200 and T280 modulates Nanog function through several regulatory mechanisms. Results with phosphorylation-insensitive and phosphorylation-mimetic mutant Nanog revealed that phosphorylation at T200 and T280 enhance Nanog protein stability. Moreover, phosphorylation-insensitive T200A and T280A mutant Nanog had a dominant-negative function to inhibit endogenous Nanog transcriptional activity. Inactivation of Nanog was due to impaired homodimerization, DNA binding, promoter occupancy, and p300, a transcriptional co-activator, recruitment resulting in a defect in target gene promoter activation. Ectopic expression of phosphorylation-insensitive T200A or T280A mutant Nanog reduced cell proliferation, colony formation, invasion, migration, and the cancer initiating cell population in head and neck squamous cell carcinoma (HNSCC) cells. The in vivo cancer initiating ability was severely compromised in HNSCC cells expressing phosphorylation-insensitive T200A or T280A mutant Nanog; 87.5% (14/16), 12.5% (1/8), and 0% (0/8) for control, T200A, and T280A, respectively. Nanog occupied the Bmi1 promoter to directly transactivate and regulate Bmi1. Genetic ablation and rescue experiments demonstrated that Bmi1 is a critical downstream signaling node for the pleiotropic, pro-oncogenic effects of Nanog. Taken together, our study revealed, for the first time, that post-translational phosphorylation of Nanog is essential to regulate Bmi1 and promote tumorigenesis.