Role Of BMI1 Research Articles

BMI-1 is a potential therapeutic target in diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG) is a poor-prognosis pediatric brain tumor. No effective curative therapy is currently available and no therapeutic advances have been made in several decades. BMI-1 is a member of the multimeric protein complex Polycomb repressor complex 1. It is highly expressed in a number of diseases and malignancies and has been implicated in self-renewal of normal and cancer cells, and in DNA damage signaling. The role of BMI-1 in DIPG is largely unknown. Here, we show that BMI-1 is highly expressed in tumor tissue samples of DIPG patients and in patient-derived cancer stem-like cells. BMI-1 downregulation leads to the inhibition of DIPG patient-derived neurosphere cell proliferation, cell cycle signaling, self-renewal, telomerase expression and activity, and suppresses DIPG cell migration. Moreover, targeted inhibition of BMI-1 sensitizes DIPG cells to radiomimetic drug-induced DNA damage. Together, our data validate BMI-1 as a potential therapeutic target to treat children with DIPG.

THE EXPRESSION PATTERN OF BMI-1 GENE IN ACUTE MYELOID LEUKEMIA

Objectives: B-cell specific moloney murine leukemia virus integration site-1 (BMI-1) gene is a stem cell gene that modulates stem cell pluripotency and is also implicated in the regulation and accumulation of leukemic stem cells (LSCs). The current study aimed at characterizing BMI-1 gene expression in de novo AML patients before the start of chemotherapy and in those who achieved complete remission (CR). Methods: Real-time polymerase chain reaction was used to assess the gene expression in 54 de novo AML patients: 43 AML and 11 in CR as well as in 21 non malignant bone marrow samples. Results: AML patients showed a higher BMI-1 median fold change in expression (median=0.157) as compared to AML-CR patients (median=0.000) but this difference was not statistically significant. A higher median fold change was observed in patients with intermediate/unfavourable risk groups (2.381) than with favourable risk (0.000). BMI-1 expression levels were not seen to be influenced by clinicopatholoical factors of the disease or to affect response to first induction, overall and disease-free survival. Conclusion: The role of BMI-1 in myeloid leukemogenesis needs further delineation to determine its significance in acute leukemia pathogenesis. Our results point to its possible role in AML risk stratification but further studies are needed.

Silencing BMI1 radiosensitizes human breast cancer cells by inducing DNA damage and autophagy.

Overexpression of BMI1 in human cancer cells, a member of the polycomb group of repressive complexes, correlates with advanced stage of disease, aggressive clinico-pathological behavior, poor prognosis, and resistance to radiation and chemotherapy. Studies have shown that experimental reduction of BMI1 protein level in tumor cells results in inhibition of cell proliferation, induction of apoptosis and/or senescence, and increased susceptibility to cytotoxic agents and radiation therapy. Although a role for BMI1 in cancer progression and its importance as a molecular target for cancer therapy has been established, information on the impact of silencing BMI1 in triple-negative breast cancer (TNBC) and its consequence on radiotherapy have not been well studied. Therefore, in the present study we investigated the potential therapeutic benefit of radiation therapy in BMI1-silenced breast cancer cells and studied the mechanism(s) of radiosensitization. Human MDA-MB-231 and SUM159PT breast cancer cells that were either stably transfected with a lentiviral vector expressing BMI1 shRNA (shBMI1) or control shRNA (shControl) or transient transfection with a BMI1-specific siRNA were used. Silencing of BMI1 resulted in marked reduction in BMI1 both at the mRNA and protein level that was accompanied by a significant reduction in cell migration compared to control cells. Further, BMI1 knockdown produced a marked enhancement of DNA damage as evidenced by Comet Assay and γH2AX foci, resulting in a dose-dependent radiosensitization effect. Molecular studies revealed modulation of protein expression that is associated with the DNA damage response (DDR) and autophagy pathways. Our results demonstrate that BMI1 is an important therapeutic target in breast cancer and suppression of BMI1 produces radiation sensitivity. Further, combining BMI1-targeted therapeutics with radiation might benefit patients diagnosed with TNBC.

Downregulation of Bmi-1 suppresses epithelial‑mesenchymal transition in melanoma.

Epithelial-mesenchymal transition (EMT) contributes to the invasion and metastasis of numerous malignant cancers, including melanoma. A significant higher expression of B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1) has been reported in cell lines from metastatic melanoma compared to cell lines from primary melanoma. There are studies that show that knockdown of Bmi-1 could induce E-cadherin expression in melanoma cells. However, the role of Bmi-1 in mediating EMT-like changes in melanoma has not yet been fully studied. In the present study, knockdown of Bmi-1 by shRNA transduction decreased the invasion properties of the cultured human melanoma cells A375 by a Matrigel invasion assay, along with alterations in EMT-related markers E-cadherin, α-catenin, vimentin and N-cadherin. The aforementioned altered expression of EMT markers was verified in BALB/c-nude mouse xenografts. Furthermore, to explore the underlying regulatory mechanism of EMT, we detected the significant downregulation of p-Akt/p‑NF-κB/MMP-2 and the upregulation of PTEN in Bmi-1-silenced A375 cells. The present study demonstrated that knockdown of Bmi-1 significantly inhibited the aggressive behavior of melanoma by reversing EMT-like changes via the PTEN/p-Akt/p‑NF-κB/MMP-2 pathway.

Abstract 2427: Polycomb group protein BMI1 protects neuroblastoma cells from DNA damage-induced apoptotic death in cooperation with L3MBTL2

Abstract [Introduction] BMI1 is a polycomb protein and its overexpression has been correlated with cancer development and aggressiveness. We previously reported that v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN)-induced BMI1 positively regulated neuroblastoma (NB) cell proliferation via the transcriptional suppression of tumor suppressors in NB cells (ONCOGENE, 2010). The role of BMI1 in DNA damage repair pathways has recently been highlighted. The ubiquitination of H2AK119 is also up-regulated locally at DNA damage sites, namely those of double-strand breaks and UV-damaged lesions. BMI1 and other PcG proteins make foci at DNA damage sites and are involved in DNA damage repair pathways; however, the exact role of PcG proteins in DNA damage repair pathways remains to be elucidated. [Results and Discussion] In order to evaluate the potential of BMI1 as a new target for NB therapy, we herein examined the effects of BMI1 reductions on NB cell differentiation and apoptotic NB cell death. BMI1 knockdown (KD) up to 7 days in NB cells significantly induced their differentiation. BMI1 depletion up to 14 days significantly induced apoptotic NB cell death along with the activation of p53, increases in p73, and induction of p53 family downstream molecules. BMI1 depletion in vivo markedly suppressed NB xenograft tumor growth. A pathological analysis using the TUNEL assay indicated the induction of apoptotic cell death. BMI1 reductions activated ATM and increased γ-H2AX in NB cells. The recently identified polycomb protein L3MBTL2 was clearly decreased in BMI1 KD NB cells. BMI1 KD-induced L3MBTL2 reductions occurred at the protein level. BMI1 interacted with Ring1b, but not with L3MBTL2. Furthermore, L3MBTL2 KD by siRNA effectively accelerated apoptosis induced by the BMI1 inhibitor, PTC-209, related to apoptotic cell death accompanied by Jun/EGR-1/p53 pathway activation in NB cells. In neuronal cells, NGF depletion induced JUN pathway activation and apoptotic cell death. Furthermore, JUN modulates myeloma cell apoptosis by interacting with EGR-1, which down-regulates Survivin and triggers caspase signaling; JUN up-regulates EGR-1 expression by binding its promoter, which suggests that EGR-1 is the direct downstream target gene of the JUN transcription factor. In solid tumors, EGR-1 regulates radiation-induced cell death via apoptotic pathways including p53-dependent apoptosis. These results clarified, for the first time, the cooperation between PcG BMI1 and L3MBTL2 in DNA damage responses. Therefore, BMI1 and L3MBTL2 appear to be promising targets in new therapies for NB tumors. Citation Format: Nobuhiro Akita, Hisanori Takenobu, Hiroshi Chikaraishi, Miki Ohira, Takehiko Kamijo. Polycomb group protein BMI1 protects neuroblastoma cells from DNA damage-induced apoptotic death in cooperation with L3MBTL2. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2427.

Abstract 2512: Bmi1 identifies treatment-refractory stem cells in human glioblastoma

Abstract Glioblastoma (GBM) is an aggressive and fatal primary adult brain tumor. Even with surgery, chemotherapy with temozolomide (TMZ), and radiation, tumor re-growth and patient relapse are inevitable. Brain tumor initiating cells (BTICs), a rare subset of GBM cells with stem cell properties, were shown to be both chemo- and radio-resistant. We hypothesize that these treatment-resistant BTICs cause tumor relapse and a subset of neural stem cell genes regulate BTIC self-renewal, driving GBM recurrence. Using patient-derived primary GBM samples, we designed an in vitro model of tumor recurrence by treating cells with TMZ and radiation. We also adapted the existing treatment protocol for adults with primary GBM for in vivo treatment of immunocompromised mice engrafted with GFP+ GBM cells. Post-chemoradiotherapy, GFP+ cells were recovered from mouse brains and profiled for self-renewal, proliferation and mRNA expression of important stem cell genes. Using in vitro and in vivo gain-of-function/loss-of-function experiments, we investigated the regulatory functions of Bmi1 in primary neural stem & progenitor cells (NSPCs) and GBM tumor formation. To understand the consequences of Bmi1 dysregulation on target gene expression, we performed global RNA-seq profiling on NSPCs and GBMs. GBM cells showed an increase in Bmi1 levels post-chemoradiotherapy, suggesting the presence of a treatment-refractory BTICs. GFP+ cells extracted from chemoradiotherapy treated human tumor xenografts showed increased self-renewal and elevated BTIC marker expression. Although treated mice responded to therapy with decreased tumor size, we observed tumor relapse post-chemoradiotherapy with increased Bmi1 protein expression. Knockdown of Bmi1 diminished self-renewal and proliferation of GBM cells and delayed tumorigenesis in xenografted mice, highlighting a critical role for Bmi1 in tumor initiation and maintenance. Conversely, over-expressing Bmi1 in NSPCs induced stem cell properties in vitro, but failed to initiate tumor formation in vivo. Using high-throughput sequencing data, we generated a map of signaling pathways dysregulated in GBM that may lead to tumor recurrence. Our data confirms the existence of a rare treatment-refractory BTICs population that escapes therapy, and drives tumor relapse and recurrence with enhanced self-renewal capacity. Our human BTIC in vitro assays and human-mouse BTIC xenograft model provide fundamental tools to characterize the functional relevance and of key stem cell self-renewal genes in GBM recurrence. Citation Format: Parvez Vora, Maleeha Qazi, Chitra Venugopal, Minomi Subapanditha, Sujeivan Mahendram, Chirayu Chokshi, Mohini Singh, David Bakhshinyan, Nicole McFarlane, Sheila Singh. Bmi1 identifies treatment-refractory stem cells in human glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2512.

Bmi1 inhibition enhances the sensitivity of pancreatic cancer cells to gemcitabine.

As the standard therapy for pancreatic cancer, gemcitabine shows limited efficacy in pancreatic cancer patients because of chemoresistance. Aberrant expression of Bmi1 has been reported to activate multiple growth-regulatory pathways and confer anti-apoptotic abilities to many cancer cells. However, the role of Bmi1 in response of pancreatic cancer cells towards gemcitabine resistance remains elusive. In this study, we found that certain dose of gemcitabine treatment induced Bmi1 expression in pancreatic cancer cells. Knockdown of Bmi1 enhanced ROS production and promoted the cytotoxic effect of gemcitabine. The increased oxidative stress upon gemcitabine treatment could disrupt mitochondrial membrane and decrease mitochondrial membrane potential, eventually leading to apoptosis. Bmi1 inhibition also suppressed the activation of NF-κB signaling and the expressions of downstream molecules in pancreatic cancer cells treated with gemcitabine. Moreover, we observed Bmi1 inhibition sensitized the pancreatic xenograft tumors to gemcitabine in vivo. Taken together, our study demonstrated that Bmi1 could decrease the sensitivity of pancreatic cancer cells to gemcitabine through increasing oxidative stress and inhibiting NF-κB signaling, thus Bmi1 may serve as a promising target for sensitizing pancreatic cancer cells to chemotherapy.

A product of BMI1 gene, a key component of the Polycomb family, positively regulates adipocyte differentiation of mouse mesenchymal stem cells

Bmi1 is a key component of the Polycomb (PcG) family, which in mammals regulates the basic properties of somatic stem cells (SSC): self-renewal and differentiation. Bmi1 supports SSC via transcriptional suppression of genes associated with cell cycle and differentiation. The best studied targets of Bmi1 are Ink4 locus genes p14Arf and p16Ink4a, an inhibitor of cyclin-dependent kinases. Their suppression due to BMI1 activating mutations results in generation of cancer stem cells (CSC) and carcinomas in various tissues. In contrast, inactivation of BMI1 results in cell cycle arrest and cell senescence. Although clinical phenomena of BMI1 hypo- and hyperactivation are well known, its targets and role in the regulation of tissue specific SSC are largely unknown. The goal of this study was to evaluate the regulatory role of BMI1 in adipocyte differentiation (AD) of mouse mesenchymal stem cells (MSC). AD induction in mouse C3H10T1/2 cell line was accompanied with increased expression of BMI1, pRb family genes (RB, p130) and demethylase UTX, but not methyltransferase EZH2 methylated H3K27. H3K27me3 plays the role of an AD epigenetic switch in human MSC (Hemming et. al., 2014). BMI1 inactivation using specific siRNA slowed and decreased AD but did not abolish it. It was accompanied with total inhibition of expression of adipogenic markers PPARγ2, ADIPOQ and RB, p130 but not UTX. These results provide evidence that epigenetic mechanisms regulating AD differentiation in mouse and human MSC are different.

Downregulation of Bmi-1 is associated with suppressed tumorigenesis and induced apoptosis in CD44⁺ nasopharyngeal carcinoma cancer stem-like cells.

Bmi-1 (B-cell-specific Moloney murine leukemia virus insertion site 1) is a member of the Polycomb group gene (PcG) family, which is involved in the proliferation, migration and tumorigenesis of several types of cancer stem cells (CSCs). However, its precise role and mechanism in CD44+ nasopharyngeal carcinoma (NPC) cancer stem-like cells (CSC-LCs) remain poorly understood. In our previous study, we successfully silenced Bmi-1 by short hairpin RNA (shRNA) in CD44+ NPC CSC-LCs and obtained stable Bmi-1 knockdown (KD) cell lines. In the present study, we tested the cell proliferation by CCK-8 assay and apoptosis by flow cytometry. Scratch wound healing assay, together with Transwell migration and invasion assays were used to measure the migration and invasion capacity. We further evaluated the tumorigenicity of CD44+ NPC CSC-LCs transfected with Bmi-1 shRNA in vivo. Based on our results, knockdown of Bmi-1 by shRNA resulted in the inhibition of tumor proliferation, migration and invasion in vitro, followed by cell apoptosis. In addition, our results preliminarily demonstrated that inhibition of Bmi-1 expression by shRNA increased tumor apoptosis through the p16INK4a-p14ARF-p53 pathway. Bmi-1 silencing in CD44+ NPC CSC-LCs also resulted in the failure to develop tumors in vivo. These results provide important insights into the role of Bmi-1 in the occurrence and development of NPC. Based on our findings, regulation of Bmi-1 in CD44+ NPC CSC-LCs may provide a potential molecular target for the therapy of NPC, and targeted silencing of Bmi-1 by shRNA may have clinical future implications in NPC therapy.

ATPS-92Bmi1 IDENTIFIES TREATMENT-REFRACTORY STEM CELLS IN HUMAN GLIOBLASTOMA

PURPOSE: Glioblastoma (GBM) is a fatal primary adult brain tumor, characterized by extensive cellular heterogeneity. Even with surgery, chemotherapy with temozolomide (TMZ), and radiation, tumor re-growth and patient relapse are inevitable. Brain tumor initiating cells (BTICs), a rare subset of GBM cells with stem cell properties, were shown to be both chemo- and radio-resistant. We hypothesize that these treatment-resistant BTICs cause tumor relapse and a subset of genes regulating BTIC self-renewal drives GBM recurrence. METHODS: Using patient-derived primary GBM samples, we designed an in vitro model of tumor recurrence by treating cells with TMZ and radiation. We then adapted the existing treatment protocol for adults with primary GBM for treatment of immunocompromised mice engrafted with GFP+ human GBM cells. Post-chemoradiotherapy, GFP+ tumor cells were recovered from mouse brains and profiled for self-renewal, proliferation and mRNA expression of important stem cell genes. Using in vitro and in vivo loss-of-function experiments, we investigated the regulatory functions of key BTIC genes in tumor formation. RESULTS: GBM cells showed an increase in Bmi1 levels post-chemoradiotherapy in vitro, suggesting the presence of a treatment-refractory BTIC population. GFP+ cells extracted from chemoradiotherapy treated human tumor xenografts showed increased self-renewal and elevated BTIC marker expression. Although treated mice responded to therapy with decreased tumor size, we observed tumor relapse post-chemoradiotherapy with increased Bmi1 expression. Knockdown of Bmi1 diminished self-renewal and proliferation of GBM cells and delayed tumorigenesis in xenografted mice, highlighting a critical role for Bmi1 in tumor initiation and maintenance. CONCLUSION: Our data confirms the existence of a rare treatment-refractory BTICs population that escapes therapy, and drives tumor relapse and recurrence with enhanced self-renewal capacity. Our human BTIC in vitro assays and human-mouse BTIC xenograft model provide fundamental tools to characterize the functional relevance and of key stem cell self-renewal genes in GBM recurrence.

BMI1, ATM and DDR.

BMI1, ATM and DDR.

Abstract 4647: Bioactive tanshinones and tea polyphenols inhibit prostate cancer stem cells by targeting bmi1

Abstract Prostate cancer (CaP) is the most common invasive malignancy and the second leading cause of cancer-related death in U.S. men. Androgen-independent/hormone-refractory CaP remains the primary cause of CaP death. The search for effective therapeutic and preventive strategies, particularly the ways to intervene in the progression of androgen-independent tumors remains the top priority of CaP research. Prostate cancer stem cells (CSCs) are suggested to be responsible for initiation, progression, drug resistance, recurrence and metastasis of CaP. Strategies that target prostate CSCs may significantly prevent and/or delay cancer progression and metastasis. The objective of this study was to identify the potent natural bioactive compounds for targeting prostate CSCs. The sphere-forming efficiencies in LNCaP, DUCaP, PC3 and DU145 prostate cancer cell lines were 2.8%, 15.7%, 1%, and 5.6%, respectively. The expression of several CSC related biomarkers, such as c-met, c-kit, bmi1 and CD44 were increased, whereas the expression of androgen receptor was decreased in prostate spheres. Natural compounds, especially tanshinones (tanshinone I (T1), tanshinone IIA (T2A), cryptotanshinone) and tea compounds (epigallocatechin gallate (EGCG) and theaflavines), inhibited the sphere-forming efficiency of CaP cells with T1 and T2A the most potent. Furthermore, T1 and EGCG significantly downregulated Bmi1 gene expression and protein level. Since Bmi1 has been reported to be a crucial regulator of stem cell self-renewal and malignant transformation, we further studied the functional role of bmi1 in self-renewal of prostate CSCs. Silencing of bmi1 dramatically inhibited the growth and decreased the sphere-forming ability of CaP cells. Animal studies showed that T1 and EGCG inhibited the growth and metastasis of prostate tumors associated with downregulation of bmi1 expression. The results derived from our preliminary studies suggest that bmi1 may play an important role in self-renewal ability of prostate CSCs, and that some natural bioactive agents, such as T1 and EGCG, may inhibit self-renewal of prostate CSCs in part via downregulation of bmi1 gene expression and function. Supported in part by NCI/NIH R21 CA153355. Note: This abstract was not presented at the meeting. Citation Format: Jin-Rong Zhou, Yi Gong, Hamid Abdolmaleky. Bioactive tanshinones and tea polyphenols inhibit prostate cancer stem cells by targeting bmi1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4647. doi:10.1158/1538-7445.AM2015-4647

Role of Bmi-1 in regulation of ionizing irradiation-induced epithelial-mesenchymal transition and migration of breast cancer cells.

Radiotherapy is a widely used treatment for cancer. However, recent studies suggest that ionizing radiation (IR) can promote tumor invasion and metastasis. Bmi-1, a member of the polycomb group protein family, has been observed as a regulator of oxidative stress and promotes metastasis in some tumors. But, its potential role in the metastasis induced by IR of breast cancer has not been explored. In our study, we found that increased levels of Bmi-1 were correlated to EMT of breast cancer cells. Through analyzing the EMT state and metastasis of breast cancer induced by IR, we found the metastatic potential of breast cancer cells can either be inhibited or accelerated by IR following a time-dependent pattern. Silencing Bmi-1 completely abolished the ability of the IR to alter, reduce or increase, the migration of breast cancer cells. Also, when Bmi-1 was knocked down, the effect of inhibition of PI3K/AKT signaling on EMT affected by IR was blocked. These results suggest that Bmi-1 is a key gene in regulation of EMT and migration of breast cancer cells induced by IR through activation of PI3K/AKT signaling; therefore, Bmi-1 could be a new target for inhibiting metastasis caused by IR.

Abstract 224: The role of Bmi-1 in benzyl isothiocyanate-mediated suppression of breast cancer stem cell self-renewal

Abstract We have shown previously that benzyl isothiocyanate (BITC), a nontoxic small-molecule constituent of edible cruciferous vegetables (eg, garden cress) inhibits breast cancer stem cells (bCSC) in vitro and in vivo but the underlying mechanisms are not fully understood. Bmi-1 is a member of the polycomb group family of proteins that function in the epigenetic silencing of genes governing self-renewal, differentiation, and proliferation. The present study was undertaken to investigate the role of Bmi-1 in BITC-mediated inhibition of bCSC. BITC treatment downregulated protein level of Bmi-1 in cultured human breast cancer cells (MCF-7, SUM159 and MDA-MB-231), and in MDA-MB-231 xenografts in vivo. Ectopic expression of Bmi-1 alone was sufficient to drive stemness in MCF-7 cells. In addition, Bmi-1 overexpression in MCF-7 cells conferred marked protection against BITC-mediated inhibition of bCSC as evidenced by mammosphere frequency and aldehyde dehydrogenase 1 (ALDH1) activity. Consistent with these observations, silencing of Bmi-1 in SUM159 cells remarkably reduced the portion of bCSC. Apoptosis induced by BITC was also partially attenuated by overexpression of Bmi-1 in MCF-7 cells but its silencing had no effect on BITC-induced apoptosis in SUM159 cells. Previous studies have shown that miRNA128 inhibits cell proliferation and self-renewal by direct regulation of Bmi-1. BITC treatment modestly but statistically significantly increased the expression of miRNA128 in both MCF-7 and SUM159 cells. Inhibition of miRNA128 increased the mammosphere formation in MCF-7 and SUM159 cells and partially attenuated BITC-mediated inhibition of mammosphere formation. These observations led us to conclude that Bmi-1 is a novel target of BITC-mediated inhibition of bCSC. This study was supported by the grant RO1 CA129347 awarded by the National Cancer Institute. Citation Format: Su-Hyeong Kim, Shivendra V. Singh. The role of Bmi-1 in benzyl isothiocyanate-mediated suppression of breast cancer stem cell self-renewal. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 224. doi:10.1158/1538-7445.AM2014-224

The Polycomb protein, Bmi1, regulates insulin sensitivity.

ObjectiveThe Polycomb Repressive Complexes (PRC) 1 and 2 function to epigenetically repress target genes. The PRC1 component, Bmi1, plays a crucial role in maintenance of glucose homeostasis and beta cell mass through repression of the Ink4a/Arf locus. Here we have explored the role of Bmi1 in regulating glucose homeostasis in the adult animal, which had not been previously reported due to poor postnatal survival of Bmi1−/− mice.MethodsThe metabolic phenotype of Bmi1+/− mice was characterized, both in vivo and ex vivo. Glucose and insulin tolerance tests and hyperinsulinemic-euglycemic clamps were performed. The insulin signaling pathway was assessed at the protein and transcript level.ResultsHere we report a negative correlation between Bmi1 levels and insulin sensitivity in two models of insulin resistance, aging and liver-specific insulin receptor deficiency. Further, heterozygous loss of Bmi1 results in increased insulin sensitivity in adult mice, with no impact on body weight or composition. Hyperinsulinemic-euglycemic clamp reveals increased suppression of hepatic glucose production and increased glucose disposal rate, indicating elevated glucose uptake to peripheral tissues, in Bmi1+/− mice. Enhancement of insulin signaling, specifically an increase in Akt phosphorylation, in liver and, to a lesser extent, in muscle appears to contribute to this phenotype.ConclusionsTogether, these data define a new role for Bmi1 in regulating insulin sensitivity via enhancement of Akt phosphorylation.

ShRNA-mediated Bmi-1 silencing sensitizes multiple myeloma cells to bortezomib.

The introduction of bortezomib has resulted in a paradigm shift in the treatment of multiple myeloma (MM) and has contributed to the improved survival of patients with MM. Inevitably, resistance to therapy develops, and thus the clinical efficacy of bortezomib is hampered by drug resistance. The oncogene B-cell‑specific Moloney murine leukemia virus insertion site‑1 (Bmi-1) has been implicated in the pathogenesis of various human malignancies. Furthermore, RNA interference (RNAi)‑mediated Bmi-1 silencing has been shown to sensitize tumor cells to chemotherapy and radiation. The role of Bmi-1 in influencing the response to bortezomib therapy has not been investigated to date. In the present study, Bmi-1 was silenced in two MM cell lines (U266 and RPMI8226) using short hairpin RNA (shRNA) targeting Bmi-1 (shBmi-1). A cell counting kit-8 (CCK-8) assay was performed to analyze cell proliferation and evaluate the 50% inhibitory concentration (IC50) values of bortezomib. Cell cycle progression and apoptosis were analyzed by flow cytometry (FCM), and the mRNA and protein expression of associated genes (Bmi-1, p14, p21, Bcl-2 and Bax) was quantified by RT-qPCR and western blot analysis, respectively. The IC50 values significantly decreased in the cells transfected with shBmi-1 (p<0.05). The depletion of Bmi-1 sensitized the MM cells to bortezomib, which increased the G1 phase duration and enhanced bortezomib‑induced apoptosis (p<0.05). The expression of p21 and Bax (apoptosis inducer) was upregulated, whereas that of the anti-apoptotic protein, Bcl-2, was downregulated in the Bmi-1‑silenced cells (p<0.05). The depletion of Bmi-1 enhanced the sensitivity of MM cells to bortezomib by inhibiting cell proliferation and inducing cell cycle arrest and apoptosis. Our data suggest that Bmi-1 may serve as an important novel therapeutic target in MM.

The role of Bmi-1 gene in submandibular gland of mice

To determine the role of Bmi-1 in the submandibular gland (SMG) of mice. SMG of 4-week wild-type (WT) and Bmi-1 null (Bmi-1(-/-)) mice was analyzed on the weight, salivary flow rate, hematoxylin-eosin staining morphological differences and the changes in proliferation and aging by histology, immunohistochemistry and Western blotting. Compared with WT mice, the average static salivary flow rate [WT:(0.21 ± 0.02) µg/min,Bmi-1(-/-): (0.10 ± 0.02) µg/min] (P = 0.001) and the submandibular gland weight [WT: (1.89 ± 0.15) µg], Bmi-1(-/-): [(1.34 ± 0.07)µg] (P = 0.003) of the male Bmi-1(-/-) mice were significantly decreased, the number of gland duct increased, and the granular convoluted duct showed reduced diameter and branches. More senescence-associated β-galactosidase positive cells existed in SMG of Bmi-1(-/-)mice (WT:0.00, Bmi-1(-/-): 0.18 ± 0.02), and Ki-67 immunopositive cells decreased in SMG of Bmi-1(-/-) mice (WT:0.40 ∼ 0.47, Bmi-1(-/-): 0.18 ∼ 0.20) (P = 0.000). The expression of p16 (WT:1.00 ± 0.12, Bmi-1(-/-): 0.00 ± 0.00) (P = 0.003) and p19 (WT:0.97 ± 0.09, Bmi-1(-/-): 5.09 ± 0.21) (P = 0.004) were up-regulated dramatically in SMG of the Bmi-1(-/-) mice. Bmi-1 gene deficiency causes abnormal function of SMG by inducing senescence phenotype of SMG.

Mitogen-activated Protein Kinase Signaling Mediates Phosphorylation of Polycomb Ortholog Cbx7

Cbx7 is one of five mammalian orthologs of the Drosophila Polycomb. Cbx7 recognizes methylated lysine residues on the histone H3 tail and contributes to gene silencing in the context of the Polycomb repressive complex 1 (PRC1). However, our knowledge of Cbx7 post-translational modifications remains limited. Through combined biochemical and mass spectrometry approaches, we report a novel phosphorylation site on mouse Cbx7 at residue Thr-118 (Cbx7T118ph), near the highly conserved Polycomb box. The generation of a site-specific antibody to Cbx7T118ph demonstrates that Cbx7 is phosphorylated via MAPK signaling. Furthermore, we find Cbx7T118 phosphorylation in murine mammary carcinoma cells, which can be blocked by MEK inhibitors. Upon EGF stimulation, Cbx7 interacts robustly with other members of PRC1. To test the role of Cbx7T118 phosphorylation in gene silencing, we employed a RAS-induced senescence model system. We demonstrate that Cbx7T118 phosphorylation moderately enhances repression of its target gene p16. In summary, we have identified and characterized a novel MAPK-mediated phosphorylation site on Cbx7 and propose that mitogen signaling to the chromatin template regulates PRC1 function.

Functional Dissection and Targeting Of Bmi1 Independence Of MN1 Leukemia

While PcG protein, Bmi1, plays a critical role in development of leukemic stem cells (LSCs), we have recently shown a differential Bmi1 dependency for LSCs initiated by different oncogenic transcription factors associated with distinct prognostic outcomes. PML-RARA and AML1-ETO leukemias associated with good prognosis are dependent on Bmi1, whereas poor prognostic leukemia induced by MLL fusion that is capable of activating multiple Hox genes is Bmi1 independent. However the role of Bmi1 and the mechanisms of over-coming Bmi1 dependency in other acute myeloid leukemia (AML) subtypes are still largely unknown. Aberrations of the MN1 gene mostly as an over-expression or rarely as a fusion partner of TEL in patients carrying the translocation t(12;22)(p13;q11) are frequently found in AML and myelodysplastic syndrome (MDS). Forced expression of MN1 induces aggressive AML with a 100% penetrance in mouse models within a few weeks, a latency significantly shorter than myeloid malignancy induced by MN1-TEL. Consistently, MN1 over-expression correlates with reduced drug response and poor prognosis of AML patients as well as high level of Bmi1 expression. In spite of these striking experimental and clinical features, the molecular mechanisms underlying MN1 leukemia are still largely unknown, and little progress has been made in targeting this leukemia. In the current study, we sought out to investigate the role of Bmi1 for MN1 mediated leukemic transformation and the mechanisms underlying this aggressive leukemia. In contrast to MN1-TEL that is dependent on Bmi1 for transformation of murine primary hematopoietic cells, MN1 over-expression could transform Bmi1-/- hematopoietic progenitor cells (HPCs) and induced serially transplantable AML in mice with the same latency as MN1 transformed wild type (wt) HPCs. Further molecular analyses revealed a significant up-regulation of p16Ink4a and cellular senescence in MN1-TEL Bmi1-/- HPCs, which was absent in MN1 transformed cells. Senescence in MN1-TEL Bmi1-/- cells could be rescued by re-expression of Bmi1 or suppression of p16 respectively, consistently suggesting an inherent functional difference between MN1 and MN1-TEL reflected by contrasting Bmi1 dependences. To identify the functional domain critical for the Bmi1 independency, structure/function analysis revealed that the removal of DNA binding domain (DBD) in the TEL moiety conferred Bmi1 independent transformation to MN1-TEL (MN1-TELΔDBD). MN1-TELΔDBD was able to induce transplantable leukemia in both the wild type and Bmi1 deficient cells. Conversely, fusion of TEL DBD to full length MN1 abolished its Bmi1 independence, and failed to transform Bmi1 deficient cells. These results strongly suggest that MN1 regulated molecules/pathways critical for Bmi1 independent transformation are misguided by the TEL DBD in MN1-TEL leukemia, providing a unique platform to dissect the pathways for Bmi1 independent transformation in AML. By performing global gene expression analyses on MN1, MN1-TEL and MN1-TELΔDBD transformed cells, we identified 1727 genes differentially expressed in MN1 transformed cells compared with MN1-TEL transformed cells; whereas only 44 genes were differentially expressed in MN1-TEL versus MN1-TELΔDBD transformed cells. When we overlapped these two gene sets together, we generated a unique gene set containing 34 genes associated with Bmi1 independence, including metabolic enzymes, signaling molecules and transcription factors such as Hoxa gene that has previously implicated in Bmi1 independent leukemic transformation. To assess the functional significance and the potential of targeting the candidates in this gene list in overcoming Bmi1 independent transformation, we performed functional analyses using shRNA approaches with a focus on those pharmacologically tractable candidates to suppress Bmi1 independent leukemic transformation. As a result, we were able to identify and demonstrate two different classes of enzymes with rigid catalytic domains that are required for Bmi1 independent transformation by MN1. Together, we dissect the mechanisms underlying Bmi1-independent leukemic transformation, and provide promising novel targets for MN1 leukemia. Disclosures: No relevant conflicts of interest to declare.

Abstract C11: Molecular mechanism driving BMI1-induced melanoma metastasis

Abstract Malignant melanoma is one of the most aggressive human cancers, with a high potential for lethal metastasis and therapeutic resistance. The epigenetic chromatin regulator Bmi1 acts as a key component of the Polycomb Repressor Complex 1 (PRC1) and is a known oncogene. Various studies shown that BMI1 is highly expressed in many human hematopoietic malignancies and in different solid tumor types including prostate, breast, ovarian, colon, brain and lung cancer. Importantly, high Bmi1 expression is an excellent predictor of both metastatic progression and poor therapeutic response. Bmi1's role in tumor onset has been assessed using germline mutant mice showing that Bmi1 ablation efficiently suppress tumor development. This impaired tumorigenicity correlates with an impairment of the self-renewal and proliferative properties of tumor initiating cells, mainly due to Bmi1's repressive activity on the Ink4a/Arf tumor suppressor locus. Nevertheless, this function does not explain why Bmi1 is frequently upregulated in tumors nor Bmi1's role in tumor progression. To evaluate Bmi1's contribution to melanoma progression, we upregulated Bmi1 in two different melanoma cell lines, a murine metastatic cell line and a poorly metastatic human cell line. We found that Bmi1 overexpression activates or increases invasive behavior of both cell lines, without enhancing their proliferative capacity. This finding allowed us to dissect Bmi1's oncogenic activity at later stages of tumorigenesis, in the absence of Bmi1's deregulation of cell proliferation. Our data show that Bmi1 significantly enhances in vitro cell movement and modulates adhesion. Bmi1 also protects cells from apoptosis induced by different stimuli and increases lung metastasis formation in vivo. To characterize Bmi1-induced changes in gene expression, we have performed comparative deep sequencing of total RNA libraries generated from Bmi1 or GFP overexpressing cells. Gene ontology analysis revealed enrichment in cancer-relevant pathways that seem to be controlled by Bmi1, including cell movement, cell death and survival, tissue development and cell morphology. In particular, Bmi1 expression modulates the gene regulatory programs associated with EMT, TGFβ and the non-canonical Wnt pathways. We have identified a new Bmi1 target gene, poorly characterized, that seems to be responsible for Bmi1-induced melanoma progression. These data determine a causal role for Bmi1 in melanoma cell migration and establishment of distant melanoma metastasis. Citation Format: Roberta Ferretti, Jacqueline A. Lees. Molecular mechanism driving BMI1-induced melanoma metastasis. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C11.