Expression Level Of Bmi-1 Research Articles

Potential roles of EZH2, Bmi-1 and miR-203 in cell proliferation and invasion in hepatocellular carcinoma cell line Hep3B

To investigate the potential roles of enhancer of zeste homolog2 (EZH2), Bmi-1 and miR-203 in cell proliferation and invasion in hepatocellular carcinoma (HCC) cell line Hep3B. A total of 73 patients who underwent surgical resection at Fuzong Clinical Medical College of Fujian Medical University were enrolled in this study. Hep3B cells were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37 °C. Vectors that containing cDNA of the EZH2 gene or miR-203 targeted shRNA plasmid were constructed, and then transfected into Hep3B cells. The mRNA expression of miR-203, EZH2, and Bmi-1 was analyzed using quantitative real-time polymerase chain reaction analysis, and the protein levels of EZH2 and Bmi-1 were detected by Western blot analysis. Effect of EZH2 or miR-203 on cell proliferation was observed by methyl thiazolyl tetrazolium assay, and cell apoptosis was assessed using flow cytometry. Besides, effect of EZH2 or miR-203 on tumor cell invasion was detected using Transwell assay. The mRNA levels of EZH2 and Bmi-1 in HCC tissues and in Hep3B cells were significantly higher compared with those in normal samples (P < 0.01), while miR-203 level was significantly lower in HCC tissues (P < 0.01). Hep3B cells transfected with EZH2-shRNA or miR-203-shRNA showed lower expression levels of EZH2 and Bmi-1 (P < 0.05). Compared with controls, Hep3B cells transfected with EZH2-shRNA had relative slow cell proliferation, indicating that low expression of EZH2 and Bmi-1 and overexpression of miR-203 could inhibit Hep3B cell proliferation (P < 0.05). The average apoptosis rate of Hep3B cells transfected with EZH2-shRNA vector was about 18.631%, while that of Hep3B cells transfected with shRNA vector was about 5.33%, suggesting that EZH2 was down-regulated by transfecting with EZH2-shRNA, and the down-regulated EZH2 contributed to the cell apoptosis. Low expression of EZH2 and Bmi-1 and overexpression of miR-203 could reduce Hep3B cell invasion (P < 0.05). Our study suggests that EZH2 and Bmi-1 are up-regulated while miR-203 is down-regulated in Hep3B cells. MiR-203 may contribute to the metastasis and enhance apoptosis of HCC cells by regulating EZH2 and Bmi-1. Our study may provide a theoretical basis for metastasis of HCC and targeted therapy of HCC.

Up-Regulation of MiR-452 Inhibits Metastasis of Non-Small Cell Lung Cancer by Regulating BMI1

Background/Aims: MicroRNAs (miRNAs) have been regarded as a new class of regulators in cellular processes in non-small cell lung cancer (NSCLC). However, the relationship between miR-452 and the development of NSCLC remains unclear. Methods: qRT-PCR was used to detect the expression of miR-452 and its target gene in NSCLC samples (n=60). The transwell assay was used to test the cell invasion capability. The regulation mechanism was confirmed by luciferase reporter assay and western blot assay. Results: In the current study, a relatively lower miR-452 and higher BMI1 expression levels were confirmed to be associated with advanced tumor stage and more extent of lymph nodes metastasis. In vitro, down-regulated miR-452 could enhance cell invasion capability. Furthermore, miR-452 modulated BMI1 expression by binding to its 3ʹ-UTR. The enhancement of cell invasion capability induced by down-regulated miR-452 was eliminated by repression of BMI1. Conclusions: Our results suggest that miR-452 plays a vital role in development of NSCLC, and this miR-452-BMI1 pathway might generate a novel insight into the treatment of NSCLC.

Favorable Clinical Outcome and Unique Characteristics in Association with Twist1 overexpression in De Novo Acute Myeloid Leukemia

Epithelial-mesenchymal transition (EMT) is a critical process for inducing stem-like properties of epithelial cancer cells. However, the role of EMT inducers in hematologic malignancies is unknown. Twist1, an EMT inducer necessary for cell migration, has recently been found to have transcriptionally regulatory activity on the expression of Bmi1, and these two are capable of promoting tumorigenesis in a synergized manner. Knowing that Bmi1 expression is essential for maintenance of leukemic stem cells, we speculate that Twist1 might govern the pathogenesis of acute myeloid leukemia (AML) development as well.We used AML cell lines KG1a and THP1 cells, which had low and high expression levels of Twist1, respectively to demonstrate that upregulated Twist1 increased Bmi1 expression in AML. Bmi1 overexpression endued leukemic cells a higher proliferative potential and increased resistance to apoptosis. In primary AML samples, there was strong positive correlation between the expression levels of Twist1 and Bmi1. AML Patients whose leukemic blasts harbored overexpressed Twist1 had a more aggressive clinical phenotype, but they were more likely to have a better clinical outcome after standard therapy. In vitro studies confirmed that Twist1-overexpressing leukemic cells were more susceptible to cytarabine, but not daunorubicin, cytotoxicity.Our findings suggest that, in a subset of AML patients, Twist1 plays a prominent role in the pathogenesis of the disease that leads to unique clinical phenotypes. The result indicates that Twist1 could represent a powerful biomarker that serves as a prognostic as well as therapeutic guide for patients with AML. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2529: Optimization of small molecules targeting BMI1 protein expression. Part 1. Amino-thiazoles: the first-in-class highly potent inhibitors of BMI1 protein

Abstract BMI1 (B-cell specific Moloney Murine Leukemia Virus Insertion 1) is a gene predominantly expressed in stem cells. A deficiency of BMI1 protein leads to a reduction in the adult stem cell population. Decreasing of BMI1 protein levels by siRNA causes apoptosis and/or senescence in tumor cells in vitro and increases susceptibility to cytotoxic agents. In tumors, the level of BMI1 protein is often elevated. Consequently, the expression level of BMI1 is highly prognostic in many types of cancers. Since BMI1 is non-enzymatic, targeting this protein by traditional drug discovery approaches has not met with success. PTC Therapeutics has developed a novel screening technology referred to as GEMSTM (Gene Expression Modulation by Small molecules) to identify small molecules that modulate the levels of a protein of interest by targeting post-transcriptional regulatory processes. Using this screening technology, we have identified a number of compounds that can selectively reduce levels of BMI1 protein. Schematically, the lead scaffold could be represented by four key components: a bicyclic right core, a heterocyclic middle ring (thiazole), a linking group and a left aryl region. Structure-activity relationships associated with this series have been investigated through medicinal chemistry efforts. Incorporation of a small electron-donating para- substituent at the left part of the molecule is highly effective in decreasing levels of BMI1, indicating the strong influence of both steric and electronic effects on activity. An amino (linking group) moiety was found to be essential for activity. Variation at the right-hand moiety appears to be more tolerated, allowing improvements in both pharmacokinetic and activity profiles. These SAR findings have revealed important pharmacophoric and structural features required for the desired biological activity with this series, and provide a foundation for further lead optimization efforts. Citation Format: Nadiya Sydorenko, Ramil Baiazitov, Soongyu Choi, Chang-Sun Lee, Liangxian Cao, Thomas W. Davis, Neil G. Almstead, Young-Choon Moon. Optimization of small molecules targeting BMI1 protein expression. Part 1. Amino-thiazoles: the first-in-class highly potent inhibitors of BMI1 protein. [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 2529. doi:10.1158/1538-7445.AM2014-2529

Clinical implications of BMI-1 in cancer stem cells of laryngeal carcinoma.

The objective of this study is to investigate the chemoresistance of CD133(+) cancer stem cells in Hep-2 cells of laryngeal cancer and detect the expression mRNA and protein levels of BMI-1 in CD133(+) cells and CD133(-) cells. The response of Hep-2 cells to different chemotherapeutic agents was investigated, and the expression of CD133 was studied. Fluorescence-activated cell sorting analysis was used to identify CD133, and the CD133(+) subset of cells was separated and analyzed chemotherapy resistance. Colony formation assays were studied and cells were injected subcutaneously into axillary fossa of node mice to measure the tumor-forming ability. RT-PCR and Western blot analyses were used to detect the expression levels of BMI-1 in the different subpopulation cells. It was concluded that chemotherapy enriched the CD133(+) subpopulation 2-fourfold, relative to the untreated cells. 1.55 ± 0.28% of Hep-2 cells were observed to be CD133(+) cells. Flow cytometric analysis revealed that after the treatment with these chemotherapeutic agents, the expression of CD133 was up to 5.16 ± 0.86%, 4.94 ± 0.58%, 3.66 ± 0.59%. After 5-FU treatment, the expression of CD133 was 6.7 ± 1.6% relative to the untreated mice 2.6 ± 0.96% by nude mice tumor xenograft model. CD133(+) cancer stem cells were more resistant to chemotherapy; the proliferation capability and tumor-forming ability were no difference after chemotherapy. Semi-quantitative RT-PCR and Western blot analyses provided strong evidence that BMI-1 expression in CD133(+) cells is different from CD133(-) cells remarkably. Taken together, it was confirmed that CD133(+) cancer stem cells were chemoresistant and BMI-1 was highly expressed in these CD133(+) cells.

BMI1 gene expression in myeloid leukemias and its impact on prognosis.

BMI1 is a polycomb group (PcG) protein and is overexpressed in leukemia. It plays a key role in the self-renewal of stem cells. Leukemic cells lacking BMI1 underwent proliferation arrest and showed signs of differentiation and apoptosis. This study was aimed to investigate the expression and impact of BMI1 in myeloid leukemias. Expression levels of BMI1 in 100 acute myeloid leukemia (AML), 100 chronic myeloid leukemia (CML) patients and 20 healthy controls were measured by real time quantitative polymerase chain reaction (RQ-PCR). The results showed that the expression of BMI1 was significantly higher in AML and CML versus control subjects (p<0.001 for both). The 2-year overall and disease free survival rates were significantly lower in patients expressing higher BMI1. Multivariate analysis showed that BMI1 was independent prognostic factor for OS for AML cases (p=0.015, HR=3.204, 95% CI=1.250-8.212). Accelerated and blastic phases in CML cases expressed higher BMI1 than chronic phase (p<0.001). We concluded that detecting BMI1 is helpful for predicting the survival in AML patients and monitoring the aggressiveness and progression in patients with CML.

BMI1 and TWIST1 downregulated mRNA expression in basal cell carcinoma.

BMI1, TWIST1 and SNAI2/SLUG have been implicated in aggressive behavior of squamous cell carcinoma (SCC) and melanoma and BMI1 expression could identify subtypes of Merkel cell carcinoma (MCC). However, BMI1, TWIST1 and SNAI2 expression levels in basal cell carcinomas (BCCs) have not been elucidated. We hypothesized BCC could be a good model system to decipher mechanisms which inhibit processes that drive tumor metastasis. The aim of this study was to examine the mRNA expression level of BMI1, TWIST1, and SNAI2 in BCCs. Thirty-five fresh non-metastatic BCC tissue samples and seven fresh normal skin tissue samples were evaluated by real-time RT-PCR. BMI1 and TWIST1 demonstrated marked down-regulation (p<0.00l, p=0.00l respectively), but SNAI2 showed no significant change (p=0.12). Previous literature has clearly demonstrated a positive association between BMI1 and TWIST1 expression and metastatic BCC, aggressive SCC and melanoma. Here, we demonstrated a negative association between BMI1 and TWIST1 mRNA expression level and BCC.

Adenovirus assembly is impaired by BMI1-related histone deacetylase activity

Polycomb ring finger oncogene BMI1 (B cell-specific Moloney murine leukemia virus integration site 1) plays a critical role in development of several types of cancers. Here, we report an inverse relationship between levels of BMI1 expression and adenovirus (Ad) progeny production. Enforced BMI1 expression in A549 cells impaired Ad progeny production. In contrast, knocking-down of endogenous BMI1 expression enhanced progeny production of a conditionally replicating Ad and wild-type Ad5 and Ad11p. Ad vectors overexpressing BMI1 were not impaired in the replication of progeny genomes and in the expression of E1A and Ad structural proteins. However, 293 cells infected by Ad vector overexpressing BMI1 contained a large proportion of morphologically irregular Ad particles. This effect was reversed in 293 cells pre-treated with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) in parallel with the production of infectious Ad particles. Our findings suggest an inhibitory role of BMI1 in Ad morphogenesis that can be implied in Ad tropism and Ad-mediated cancer therapy.

Down Regulation of miR200c Promotes Radiation‐Induced Thymic Lymphoma by Targeting BMI1

The miR-200c has recently been implicated in the epithelial to mesenchymal transition (EMT) process by directly target the EMT related transcriptional factors ZEB1 and ZEB2. The expression of this miRNA is inversely correlated with tumorgenecity and invasiveness in several human cancers. However, little is known about the expression and targets of the miR-200c in radiation carcinogenesis. Here in this study, using a split radiation induced thymic lymphoma (RITL) model in BALB/c mice, we found that miR-200c is down-regulated in RITL samples. Cell death and apoptosis in lymphoma cells was induced by miR-200c mimic while decreased by miR-200c inhibitor. Computational analysis found a putative target site of miR-200c in the 3'UTR of one of the polycomb group (PcG) protein BMI1 mRNA, which was verified by a luciferase reporter assay. Forced over-expression of miR-200c decreased the level of BMI1 protein and moreover, over-expression of BMI1 rescued the biological effects of miR-200c, indicating BMI1 is a direct mediator of miR-200c functions. Furthermore, the BMI1 expression level was up-regulated and inversely correlated with miR-200c in RITL samples. Finally, our data also indicates that Adenovirus over-expression of pre-miR-200c reduced tumorgenesis in vivo. Taken together, we conclude that down-regulated expression of miR-200c and up-regulation of its direct target BMI1 in radiation-induced thymic lymphoma, which may indicate a novel therapeutic method for RITL through induction of miR-200c or inhibition of BMI1.

MiR-203 Inhibits the Proliferation and Self-Renewal of Esophageal Cancer Stem-Like Cells by Suppressing Stem Renewal Factor Bmi-1

Cancer stem-like cells exist in many malignancies and several stem cell-related genes and microRNAs, such as Bmi-1 and miR-203, have been identified as cancer stem-like cell regulators using gene microarray or sequencing analysis. Previously, we used side population (SP) sorting to enrich cancer stem-like cells from esophageal squamous cell carcinoma (ESCC) cell line EC9706. Our results demonstrated that EC9706 SP cells shared common features of cancer stem-like cells. In this study, we examined the expression of Bmi-1 and miR-203 in ESCC SP and non-SP (NSP) cells. Our results showed that, when compared with NSP cells, Bmi-1 was up-regulated and miR-203 was down-regulated in SP cells. During the differentiation from SP to NSP cells, the expression levels of Bmi-1 were gradually decreased. Overexpression of miR-203 resulted in a significant reduction of endogenous Bmi-1 protein level in EC9706 cells. SP and NSP analyses revealed that the SP cell fraction was markedly decreased in miR-203 overexpressed cells. miR-203 overexpressed cells also showed a significant reduction in colony formation, which was resistant to chemotherapeutic drug treatment and tumorigenicity in nude mice. Rescue experiments demonstrated that ectopic expression of Bmi-1 in miR-203 overexpressed cells increased the SP fraction and restored cell proliferation. Taken together, these results indicated that stem renewal factor Bmi-1 was a direct target of miR-203. The regulation of Bmi-1 by miR-203 may play an important role in controlling cell proliferation and self-renewal of esophageal cancer stem-like cells. It may also promote the development of new therapeutic strategies and efficient drugs that target ESCC stem-like cells.

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.

Proto-oncogene Bmi-1 and head and neck cancer

Bmi-1 is a core member of the polycomb group genes.As a proto-oncogene,Bmi-1 plays an important role in cell self-renewal,proliferation and apoptosis.Several studies have shown that Bmi-1 is highly expressed in some head and neck malignant tumors,such as nasopharyngeal cancer and laryngeal carcinoma.Furthermore,the expression level of Bmi-1 is closely related to the occurrence,development,incursion and prognosis of tumor.Bmi-1 is expected to become a novel tumor molecular marker,and provides a new direction for the treatment of the head and neck malignant tumor. Key words: Genes; Head and neck neoplasms; Bmi-1

In aggressive variants of non-Hodgkin lymphomas, Ezh2 is strongly expressed and polycomb repressive complex PRC1.4 dominates over PRC1.2

Polycomb group (PcG) proteins are important for the regulation of hematopoiesis by regulating chromatin compaction and silencing genes related to differentiation and cell cycle. Overexpression of enhancer of zeste homologue 2 (Ezh2) and Bmi-1/PCGF4 has been implicated in solid organ cancers, while Mel-18/PCGF2 has been reported as a tumor suppressor. Detailed expression profiles of PcG proteins and their diagnostic significance in malignant lymphomas are still unknown. In this study, we analyzed the expression levels of Ezh2, Bmi-1, Mel-18, and Ki67 in 197 Hodgkin's and non-Hodgkin's lymphoma patient samples and in lymphoma cell lines using immunohistochemistry, fluorescent immunocytochemistry, and Western blotting. Immunohistochemical staining showed that Ezh2 expression was significantly increased in aggressive compared to indolent subtypes of B cell neoplasms (P = 0.000-0.030), while no significant differences in Bmi-1 expression were found between these subtypes. Compared to the normal counterpart, T cell lymphomas showed significant overexpression of Bmi-1 (P = 0.011) and Ezh2 (P = 0.000). The Ki67 labeling index showed a positive correlation with Ezh2 expression in B cell lymphomas (correlation coefficient (Co) = 0.983, P = 0.000) and T/NK cell lymphomas (Co = 0.629, P = 0.000). Fluorescent immunohistochemical staining showed coexpression of Ezh2 and Ki67 in the same tumor cells, indicating that Ezh2 expression correlates with cell proliferation. Both B and T/NK cell neoplasms showed low expression of Mel-18 and high expression of both Bmi-1 and Ezh2. In conclusion, in aggressive lymphoma variants, Ezh2 is strongly expressed and polycomb repressive complex PRC1.4 dominates over PRC1.2. Coexpression of Bmi-1 and Ezh2 is a characteristic of aggressive lymphomas. Ezh2 correlates with the proliferation and aggressive nature of non-Hodgkin's lymphomas.

Nrf2 is required to maintain the self-renewal of glioma stem cells

BackgroundGlioblastomas are deadly cancers that display a functional cellular hierarchy maintained by self-renewing glioma stem cells (GSCs). Self-renewal is a complex biological process necessary for maintaining the glioma stem cells. Nuclear factor rythroid 2-related factor 2(Nrf2) plays a significant role in protecting cells from endogenous and exogenous stresses. Nrf2 is a key nuclear transcription factor that regulates antioxidant response element (ARE)-containing genes. Previous studies have demonstrated the significant role of Nrf2 in the proliferation of glioblastoma, and in their resistance to radioactive therapies. We examined the effect of knocking down Nrf2 in GSCs.MethodsNrf2 expression was down-regulated by shRNA transinfected with lentivirus. Expression levels of Nestin, Nrf2, BMI-1, Sox2 and Cyclin E were assessed by western blotting, quantitative polymerase chain reaction (qPCR) and immunohistochemistry analysis. The capacity for self-renewal in vitro was assessed by genesis of colonies. The capacity for self-renewal in vivo was analyzed by tumor genesis of xenografts in nude mice.ResultsKnockdown of Nrf2 inhibited the proliferation of GSCs, and significantly reduced the expression of BMI-1, Sox2 and CyclinE. Knocking down of Nrf2 changed the cell cycle distribution of GSCs by causing an uncharacteristic increase in the proportion of cells in the G2 phase and a decrease in the proportion of cells in the S phase of the cell cycle.ConclusionsNrf2 is required to maintain the self-renewal of GSCs, and its down-regulation can attenuate the self-renewal of GSCs significantly.

Mo1805 Ablation of DCLK1 in Intestinal Epithelium Exacerbates Colonic Epithelial Barrier Damage in Response to DSS Treatment

Background: During normal homeostasis the intestinal epithelium maintains a dynamic architecture with rapid renewal every 3-5 days. Cytotoxic and genotoxic insults cause excessive epithelial damage and crypt injury. Intestinal stem cells (ISCs) are responsible for homeostasis and crypt regeneration. It is believed that the quiescent ISCs may represent a population that can be called upon to rescue the crypt following severe injury. Doublecortinlike kinase 1 (Dclk1) is a putative quiescent ISC and tuft cell marker. It is predominantly expressed in relatively quiescent cells near the traditional +4 location within the intestinal crypt. Dclk1+ cells sorted from murine small intestine form spheroids in suspension culture, and when transplanted into nude mice develop into nodules demonstrating early intestinal epithelial secretory lineage commitment. These data suggest that Dclk1 is a putative ISC marker. The role of Dclk1 in epithelium regeneration in response to injury is not clear. In this study, we investigate the role of Dclk1 in colonic epithelial restitution following injury. Aim: To determine whether Dclk1 regulates colonic epithelial barrier function following DSS-induced colitis. Methods: Age and gender matched control (Dclk1flox/flox) and Dclk1 KO (Villin-Cre;Dclk1flox/flox) mice were fed 3% Dextran Sulfate Sodium (DSS) in drinking water for 7 days, followed by water for 2 days. Body weight and occult blood were monitored daily. To assess epithelial barrier permeability, both Dclk1 KO and control mice were gavaged with FITC-dextran. Blood was collected 4 hours later and the serum concentration of FITC-dextran was measured. Corresponding colon tissues were subjected to histological examination, gene expression, and protein expression analysis. Results: Dclk1 KO mice lost significantly more weight than control mice following DSS treatment. The serum concentration of FITC-dextran in Dclk1 KO mice was approximately 2 fold higher than in control mice. The gene expression levels of ISC markers, Dclk1, Lgr5, Bmi1, mTert, and Msi-1, were significantly downregulated in the colon of Dclk1 KO mice compared to control mice following DSS treatment. Protein levels of Ecadherin, Claudin-1, and Claudin-7 were similar in untreated control and Dclk1 KO mice, but decreased dramatically in Dclk1 KO mice following DSS treatment. H&E staining of colon from DSS-treated mice indicated increased inflammation in Dclk1 KO mice relative to controls. Conclusions: In summary, these results show that loss of Dclk1 in colonic epithelial cells results in exacerbated damage to barrier function following DSS treatment. These data also demonstrate a role for Dclk1 in the regulation of other stem cell markers following DSS-induced injury. Taken together, this study suggests that Dclk1 plays an important role in colonic epithelial restitution in response to severe injury.

Abstract 3756: MiR-203 inhibits the proliferation and self-renewals of esophageal cancer stem-like cells by suppression stem renewal factor Bmi-1.

Abstract Recent reports have shown that cancer stem-like cells exist in many malignancies and several stem cell-related gene products and miRNAs, such as Bmi-1 and miR-203, were identified as cancer stem-like cell regulators using gene microarray or sequencing analysis. Bmi-1 is essential for the self-renewal capacity of several types of normal stem cells and cancer stem-like cells while miR-203 is involved in stem cell self-renewal regulation. Previously, we used side population (SP) sorting to enrich cancer stem-like cells from esophageal squamous cell carcinoma (ESCC) cell line EC9706. Our results demonstrated that EC9706 SP cells have the same common features of cancer stem-like cells, such as the ability of self renewal, highly proliferation and tumorigenesis. In this study, we examined expression regulation of Bmi-1 and miR-203 in ESCC SP and none-SP (NSP) cells. Our results showed that, when compared with NSP cells, Bmi-1 was up-regulated and miR-203 was down-regulated in SP cells. During the differentiation from SP to NSP cells, the expression levels of Bmi-1 were gradually decreased, suggesting that Bmi-1 might be required for maintaining the ESCC SP cells. As sequence analysis revealed that the mRNA 3’UTR of Bmi-1 has a highly conserved binding site of miR-203, we overexpressed miR-203 in EC9706 cells using lentivirus mediated gene transduction. Overexpression of miR-203 (lenti-miR-203) resulted in a significant reduction of endogenous Bmi-1 protein levels in EC9706 cells. SP and NSP analyses revealed that the SP cell fraction markedly decreased in lenti-miR-203 cells. Lenti-miR-203 cells also showed significantly reductions of the colony formation, resistant to chemotherapeutic drug treatment and tumorigenicity in nude mice. Rescue experiments demonstrated that ectopic expression of Bmi-1 in lenti-miR-203 cells increased the SP fraction and restored cell proliferation. Taken together, these results indicated that stem renewal factor Bmi-1 is a direct target of miR-203. Regulation of Bmi-1 by miR-203 may play an important role in controlling cell proliferation and self-renewals of esophageal cancer stem-like cells. Citation Format: Xiying Yu, Xingran Jiang, Hongxia Li, Liping Guo, Wei Jiang, Shih-Hsin Lu. MiR-203 inhibits the proliferation and self-renewals of esophageal cancer stem-like cells by suppression stem renewal factor Bmi-1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3756. doi:10.1158/1538-7445.AM2013-3756

RUNX1/AML1 Mutants Collaborate with BMI1 in the Development of Myelodysplastic Syndromes (MDS) / Acute Myeloid Leukemia (AML) in a Mouse BMT Model.

Abstract 2820RUNX1/AML1 mutations have been frequently detected in patients with myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML). RUNX1 mutations are rarely detected in lower-risk MDS, whereas approximately 20% patients with higher-risk MDS (H-MDS) have the mutations. The mutations were distributed throughout the RUNX1 protein, and replacement of D171 amino acid in runt homology domain was the most frequent target of mutations in the RUNX1 gene. The D171N mutant showed a loss of normal RUNX1 trans-activation potential and dominant-negative suppression. In mouse transplantation systems D171N-transduced mice exhibited AML with multilineage dysplasia in collaboration with Evi1 overexpression. However, EVI1 overexpression was very rare in patients. Instead, most of H-MDS patients with RUNX1 mutations displayed a high expression of BMI1.RUNX1 D171N mutant showed an increased self-renewal capacity, differentiation block, dysplasia in all 3 lineages, slightly increased immature cells and no proliferation ability using enforced expression in human CD34+ cells, and the D171N-transduced cells showed low expression level of BMI1. Both D171N and BMI1 transduced cells displayed long-term proliferation ability. When BMI1 transduced later into D171N cells, the cells expanded with a retained CD34+ cell fraction, suggesting that BMI1 have a potential to boost the D171N cells to H-MDS.To confirm the collaboration of BMI1 overexpression with D171N mutant in vivo, we performed mouse BMT using BM cells transduced with both D171N and BMI1. Ly-5.1 murine BM mononuclear cells infected with retrovirus harboring D171N/BMI1 or control vectors were transplanted into sublethally irradiated syngeneic Ly-5.2 mice.Mice that received transplants of BMI1-transduced cells remained healthy over the observation period (n=12/12), as well as those that were transplanted with empty vectors-transduced cells (n=4/4). Most of the mice that received transplants of D171N -transduced cells developed MDS/AML mainly 6–8 months after transplantation (n=6/11, P<0.0001), as observed in the previous report. Of note, mice that received transplants of BM cells expressing D171N/BMI1 developed MDS/AML with significantly shorter latencies (mainly 3–5 months) compared with the D171N group (n=12/12, P=0.001). Morbid mice with D171N or D171N/BMI1 exhibited similar phenotypes, characterized by leukocytosis, anemia, and marked splenomegaly, while the mice with BMI1 or empty vectors, sacrificed 8 months after BMT, showed none of these phenotypes. In the leukemic mice with D171N or D171N/BMI1, BM and spleen were occupied by immature myeloid cells including myeloid blasts. More myeloblasts in BM were observed in D171N/BMI1 mice than in D171N ones. The leukemic cells displayed similar morphological abnormalities and surface markers: leukemic cells were CD11blow to high, Gr-1low, B220low and c-kitlow to high. The normal structure of the spleen was completely destroyed with massive blast and immature myeloid cell infiltration, and these cells also invaded into the hepatic portal areas in the liver. Meanwhile, BMI1-transduced BM cells did not become dominant in vivo and myeloid cells showed normal differentiation. Collectively, BMI1 overexpression has a strong potential to induce MDS/AML in concert with D171N in a mouse BMT model, although BMI1 overexpression by itself does not result in maturation block or leukemogenesis.BMI1 is well-known to be essential for self-renewal of HSCs, in part via repression of genes involved in senescence, and self-renewal of HSCs is enhanced by BMI1 expression. To address the mechanisms by which BMI1 would contribute to MDS/AML development, we analyzed gene expressions involved in BMI1 downstream signaling pathways. It is suggested that BMI1 overexpression may act as one of the partner abnormalities collaborating with master gene mutations for MDS-genesis.RUNX1 D171N mutant showed no proliferation ability using enforced expression in human CD34+ cells, and D171N-transduced mice exhibited MDS/AML in collaboration with Evi1 overexpression. In addition, co-transduction of D171N and BMI1 into BM cells resulted in faster induction of MDS/AML in BMT mice. Taken together, the RUNX1 mutant may require collaborating genes such as EVI1 and BMI1 to develop MDS/AML. We confirmed that BMI1 have a potential to boost the D171N cells to MDS/AML in vivo. Disclosures:No relevant conflicts of interest to declare.

Clinicopathologic characteristics of high expression of Bmi-1 in esophageal adenocarcinoma and squamous cell carcinoma

BackgroundHigh expression of Bmi-1, a key regulatory component of the polycomb repressive complex-1, has been associated with many solid and hematologic malignancies including esophageal squamous cell carcinoma. However, little is known about the role of Bmi-1 in esophageal adenocarcinoma. The aim of this study is to investigate the amplification and high expression of Bmi-1 and the associated clinicopathologic characteristics in esophageal adenocarcinoma and squamous cell carcinoma.MethodsThe protein expression level of Bmi-1 was detected by immunohistochemistry (IHC) from tissue microarrays (TMA) constructed at the University of Rochester from using tissues accrued between 1997 and 2005. Types of tissues included adenocarcinoma, squamous cell carcinoma and precancerous lesions. Patients’ survival data, demographics, histologic diagnoses and tumor staging data were collected. The intensity (0–3) and percentage of Bmi-1 expression on TMA slides were scored by two pathologists. Genomic DNA from 116 esophageal adenocarcinoma was analyzed for copy number aberrations using Affymetrix SNP 6.0 arrays. Fisher exact tests and Kaplan-Meier methods were used to analyze data.ResultsBy IHC, Bmi-1 was focally expressed in the basal layers of almost all esophageal squamous mucosa, which was similar to previous reports in other organs related to stem cells. High Bmi-1 expression significantly increased from squamous epithelium (7%), columnar cell metaplasia (22%), Barrett’s esophagus (22%), to low- (45%) and high-grade dysplasia (43%) and adenocarcinoma (37%). The expression level of Bmi-1 was significantly associated with esophageal adenocarcinoma differentiation. In esophageal adenocarcinoma, Bmi-1 amplification was detected by DNA microarray in a low percentage (3%). However, high Bmi-1 expression did not show an association with overall survival in both esophageal adenocarcinoma and squamous cell carcinoma.ConclusionsThis study demonstrates that high expression Bmi-1 is associated with esophageal adenocarcinoma and precancerous lesions, which implies that Bmi-1 plays an important role in early carcinogenesis in esophageal adenocarcinoma.

The differential expression pattern of the BMI-1, SALL4 and ABCA3 genes in myeloid leukemia

Background and methodsIn order to characterize the expression pattern of SALL4, BMI-1 and ABCA3 genes in patients with myeloid leukemia and those who achieved complete remission (CR) after chemotherapy. Real-time PCR was used to determine the expression level of these genes in peripheral blood mononuclear cells from 24 patients with AML, eight patients with AML-CR, 13 patients with CML in the chronic phase (CML-CP), 12 patients with CML in blast crisis (CML-BC), 13 patients with CML-CR and 11 healthy individuals (HI).ResultsOverexpression of the BMI-1 gene was found in the AML, CML-CP and CML-BC groups as compared with HI group, while the BMI-1 expression level was lower in patients who achieved CR. In contrast, significantly increased SALL4 expression was only found in AML group, additionally, SALL4 expression was lower in the CML-CP and CML-CR groups compared with the HI group, while the SALL4 expression level in the CML-BC group was higher and significantly greater than that in the CML-CP and CML-CR groups. Moreover, a positive correlation between the expression of SALL4 and BMI-1 genes was found in samples from most groups. There was no significant difference of ABCA3 expression level in AML and CML-BC group in comparison with HI group. Interestingly, the ABCA3 expression level was significantly decreased in the CML-CP, AML-CR and CML-CR in comparison with the HI group. Moreover, the ABCA3 expression level in all of the CR groups was lower than that in their corresponding groups.ConclusionsThese results describe the altered SALL4, ABCA3 and BMI-1 expression pattern in different phases of myeloid leukemia, which may relate to the development and progression to different diseases. SALL4 expression was strongly correlated with BMI-1 in most of the myeloid leukemia patient groups, providing a potential link between SALL4 and BMI-1 in leukemogenesis.

Bmi-1 confers adaptive radioresistance to KYSE-150R esophageal carcinoma cells

Radiotherapy (RT) is a major modality of cancer treatment. However, tumors often acquire radioresistance, which causes RT to fail. The exact mechanisms by which tumor cells subjected to fractionated irradiation (FIR) develop an adaptive radioresistance are largely unknown. Using the radioresistant KYSE-150R esophageal squamous cell carcinoma (ESCC) model, which was derived from KYSE-150 parental cells using FIR, the role of Bmi-1 in mediating the radioadaptive response of ESCC cells to RT was investigated. The results showed that the level of Bmi-1 expression was significantly higher in KYSE-150R cells than in the KYSE-150 parental cells. Bmi-1 depletion sensitized the KYSE-150R cells to RT mainly through the induction of apoptosis, partly through the induction of senescence. A clonogenic cell survival assay showed that Bmi-1 depletion significantly decreased the radiation survival fraction in KYSE-150R cells. Furthermore, Bmi-1 depletion increased the generation of reactive oxygen species (ROS) and the expression of oxidase genes (Lpo, Noxo1 and Alox15) in KYSE-150R cells exposed to irradiation. DNA repair capacities assessed by γ-H2AX foci formation were also impaired in the Bmi-1 down-regulated KYSE-150R cells. These results suggest that Bmi-1 plays an important role in tumor radioadaptive resistance under FIR and may be a potent molecular target for enhancing the efficacy of fractionated RT.