Expression Level Of Bmi-1 Research Articles

Abstract 4901: Polycomb group proteins in myelodysplastic syndromes and its progression to acute myeloid leukemia

Abstract Polycomb group BMI-1 and EZH2 in addition to SALL4 and P16 were detected in 89 MDS bone marrow including 49 newly diagnosed MDS and 40 treated MDS by using qRT-PCR. We found the expression level of BMI-1, EZH2, SALL4 and P16 were remarkably higher than that in non-MDS cytopenias, such as iron-deficiency anemia (P<0.05). BMI1 expression level has an increasing tendency from MDS-RA to MDS-RAEB without statistically significant. On the contrary, the EZH2 expression level of MDS-RAEB is lower than that of MDS-RA (P<0.05). Although BMI1 expression level was down-regulated when MDS patients were in morphological remission, EZH2, SALL4 and P16 expression levels still remain abnormally high level (P<0.05). We also found that BMI-1 expression level in MDS CD34+ cells was correlated with International Prognostic Scoring System (IPSS) score (R=0.712, P=0.012). BMI-1 had the highest positive rate in MDS-AML. To explore the function of BMI-1 and its possible role in the pathogenesis of MDS transformation into acute leukemia. We have cloned cDNA of BMI-1 and transfected into U937, K562 leukemic cells mediated by MSCV retroviral vector. The viability In vitro of BMI1 transfected cell was higher than control and BMI1 transfected cell also inhibited As2O3-induced apoptosis. Furthermore, BMI1 repressed the expression of EZH2, PTEN and P16 in U937, and repressed the expression of EZH2 and PTEN in K562. Our results provide evidence suggesting that the clonal disorder of MDS patients remained in molecular aberration even after achieving clinical remission. The In vitro results suggest that BMI-1 may play a key role in MDS progression via P16 pathway or PTEN pathway. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4901.

Bmi‐1 is critical for the proliferation and invasiveness of gastric carcinoma cells

Bmi-1 is a transcriptional repressor belonging to the Polycomb group and is associated with the cell proliferation and carcinogenesis of a variety of human cancers. The level of Bmi-1 expression correlates with the aggressiveness of many cancers, and is considered an important marker for cancer diagnosis. However, its role in gastric carcinoma is unknown. We used lentiviral mediated interfering short hairpin RNA to knockdown Bmi-1 expression in gastric carcinoma human gastric cancer cell line (AGS cells), then tested the cell proliferation by MTT assay, rate of colony formation by colony formation assay, cell cycle distribution by fluorescence-activated cell sorting and cell invasiveness by cell invasion assay. To analyze the expression and localization of Bmi-1 in gastric tumor tissues, we further performed the immunohistochemistry analysis on a gastric cancer tissue array. We found that knocking down Bmi-1 led to slower cell growth, lesser cell invasiveness, decelerated colony formation, and altered cell cycle progression. In addition, a positive relationship between nuclear expression of Bmi-1 and gastric cancer was observed, suggesting that nucleus localization of Bmi-1 in the cells may be a novel marker of gastric cancer. Our study highlights critical roles for Bmi-1 in gastric cancer, and suggests that Bmi-1 nuclear localization could be an important marker for the diagnosis of gastric cancer.

Expression of Proteins P16INK 4a, P53 and Bmi-1 in Hematopoietic Stem Cells of Patients with MDS. the Role of Cellular Senescence.

Abstract 5049Expression of Proteins P16INK 4a, P53 and Bmi-1 in Hematopoietic Stem Cells of Patients with MDS. The Role of Cellular Senescence.Irene Constandinidou1, Eleni Kalivioti1,Vasilios Fertakis1, Costas Dallas1, Polyxeni Lampropoulou1, Evangelia Tzouvara1, Panagiotis Zoumboulis2, Nicholas Zoumbos.11Hematology Division, University of Patras, Medical School, Patras, Greece2Orthopaedic Division, University Hospital of Patras, Patras, Greece IntroductionThe myelodysplastic syndromes (MDS) comprise a spectrum of heterogeneous clonal stem/progenitor cell disorders characterized by marrow failure. One potential reason, apart from apoptosis, for hematopoietic stem cells (HSCs) functional failure is cellular senescence, which is believed to have evolved as a tumor suppressor mechanism capable of arresting growth to reduce risk of malignancy. It can be activated by both telomere-dependent (telomere shortening) and telomere-independent pathways (DNA damage, oncogenic or oxidative stress). There are two widely recognized major tumor suppressor pathways p16/pRb and p53-p14/ARF that regulate cellular senescence. Activation of either pathway is profoundly associated with cellular senescence. Bmi-1, on the other hand, is a transcriptional repressor that plays an essential role in the self-renewal of HSCs and leukemic stem cells as well. One of its major targets is the INK/ARF locus, which encodes p16 and p53 independently. AimTo study the role of cellular senescence in the pathogenesis of MDS through the expression of p16, p53, phospho p53 (activated form of p53, phosphorylated at Ser-15) and bmi-1. Patients and methodsWe examined the expression level of p16INK4a, Bmi-1, p53 and phospho p53 by flow cytometry (direct and indirect staining) in CD34+ bone marrow mononuclear cells (BMMNCs). Furthermore, we analyzed the relative telomere length (RTL) in BMMNCs by quantitative fluorescence in situ hybridization assay using flow cytometry (flow-FISH).We verified the presence of p16 by SDS PAGE western blotting. We studied 36 samples of MDS patients (11 RA, 6 RARS, 3 RCMD, 4 5q- and 12 RAEB I-II), 17 samples of age-matched healthy controls after informed consent and 8 cord blood samples. All MDS diagnoses were histologically confirmed by bone marrow examination and categorized into low and high risk according to the international prognostic scoring system (IPSS). ResultsExpression of p16 is significantly increased in low risk MDS (mean± SD=8,7±12) when compared to healthy controls (p=0,02) and high risk patients(p=0,004). Patients with 5q- syndrome express lower level of p16 in comparison with the other low risk group patients. Increased level of bmi-1 expression is noticed in high risk group (mean± SD=30,3 ±33,9) when compared to low risk group (mean± SD=25±24,6) and healthy controls (mean± SD=18 ±16,4), while 5q- syndrome patients appear to express higher level of bmi-1 than any other risk group. Significantly increased expression of p53 (p=0,03) is noticed in high risk group (mean ±SD=64,3±27) when compared to low risk (mean±SD=34,2±28,8). Patients with 5q- syndrome express increased level of p53 (mean± SD=57,4±26,3) similar to that of high risk group. Expression of phospho p53 seams increased (p=0,055) in low risk group(mean± SD=7,1± 13,9) in comparison to high risk group (mean± SD=0,09± 0,18) and healthy controls (mean± SD=0,19± 0,63). Phospho p53 is expressed in lower level in 5q- syndrome patients. Regarding the RTL there is a significant difference (p=0,0018) between MDS (mean± SD=9,7± 2,7) and cord blood samples (mean± SD=13,8± 1,9). ConclusionsIncreased levels of p16 and phospho p53 in low risk MDS suggest that cellular senescence may contribute to the ineffective hematopoiesis of MDS and probably in a telomere independent way. Low expression level of of phospho p53 in high risk MDS raises the question of genetic integrity of p53 in this group of patients and when combined with high levels of bmi-1 and low expression of p16 may play a role in the disease progression to AML. Further investigation of bmi-1, especially in the 5q- group - and other markers of cellular senescence (SA- b galactosidase, SAHF) is needed in order to clarify the impact of cellular senescence in different risk groups of MDS. DisclosuresNo relevant conflicts of interest to declare.

BMI1, the Polycomb-Group Gene, Is Recurrently Targeted by Genomic Rearrangements in Progressive B-Cell Leukemia/Lymphoma.

Abstract 1099Poster Board I-121The 10p11 chromosomal site is recurrently affected by genomic abnormalities in B cell leukemia/lymphoma, but the molecular consequences of these aberrations remain unknown. We have collected three cases (2 female, 1 male; 67-69 years) of chronic lymphocytic leukemia (CLL) with a novel IGH-mediated t(10;14)(p11;q32) (#1 and 2) or its IGL variant t(10;22)(p11;q11) (#3), and one case of blastic mantle cell lymphoma (MCL) with a complex t(11;14)-positive karyotype including add(10)(p12) (#4). Case 1 showed unmutated VH genes and usage of VH3-21/D1-26/JH6. Cases 2 and 3 revealed mutated VH genes and usage of VH3-21/D1-14/JH4 and VH3-11/D4-4/JH6, respectively. In cases 1 and 2 with a monoallelic loss of RB1, the t(10;14)(p11,q32) was found as a secondary change at time of CLL progression/Richter transformation observed 148 and 50 months after diagnosis, respectively. In case 3, the sole t(10;22)(p11;q11) was identified at time of CLL diagnosis. All three patients died from disease progression 37-149 months after diagnosis. Of note, both patients with t(10;14) died from refractory disease 1 and 3 months after CLL progression/transformation and genetic detection f t(10;14). The patient #3 did not require therapy, but died from septic shock 37 months after diagnosis, and autopsy revealed diffuse and massive lymph node and spleen involvement. The patient with MCL (#4), 67 years old man with a previous history of prostatic adenocarcinoma and CLL, developed secondary central nervous system liesions and died 40 months after diagnosis of MCL. Extensive FISH studies of t(10;14) and t(10;22) mapped the 10p11 breakpoint in the region of the BMI1 locus. SNP array analysis of case 4 complemented by FISH identified amplification of the 10p11 region covering BMI1. Immunostaining with anti-BMI1 serum performed in 2 available cases (#2 and 3) showed expression of the BMI1 protein by neoplastic cells. This approach, however, did not allow to estimate whether the level of BMI1 expression in these cases was higher than in control CLL cases. BMI1 (B-cell-specific Moloney murine leukemia virus integration site 1) is a member of the Polycomb group of genes that are important epigenetic chromatin modifiers with an essential role in embryogenesis and the maintenance of adult stem cells. BMI1 was first identified as a protooncogene that cooperates with c-myc in the generation and development of mouse pre B-cell lymphomas. The BMI1 protein is a component of Polycomb repressive complex1 (PRC1), one of two multiprotein PRCs that maintain cell identity by gene suppression. The gene plays an important role in self-renewal of hematopoietic and neural stem cells. Multiple lines of evidence implicate BMI1 and other Polycomb genes in tumorigenesis. Among others, an aberrant expression of BMI1 has been observed in a wide spectrum of cancers and usually correlated with unfavourable clinical outcome. BMI1 is one of the genes marking the ABC-type signature of DLBCL, associated with a poor prognosis. High levels of BMI1 mRNA/protein were also detected in CLL, MCL, MM and HL. In some MCLs, overexpression of BMI1 correlated with genomic amplification of the BMI1 gene detected by aCGH in 12-24% of analysed cases (Jares and Campo, 2008). BMI1 has been shown to contribute to cell cycle regulation and senescence by acting as transcriptional repressor of the INK4a-ARF tumor suppressors. Despite the wealthy information on BMI1 contributing to a variety of tumors, molecular mechanisms underlying deregulation of BMI1 in neoplasms and functional consequences of these events remain elusive.In summary, we show for the first time that BMI1, a postulated lymphoma-related oncogene, is recurrently targeted by IG-mediated chromosomal translocations in CLL and confirm its amplification in MCL. We found that in both cases with t(10;14)/IGH-BMI1, the translocation was acquired during clinical transformation of CLL initially harboring the del(13q)/RB1. The adverse prognostic impact of BMI1 aberrations in all four collected cases is reflected by a constantly aggressive clinical course of disease and short survival of the affected patients (1-50 months). In this regard, further molecular studies of the BMI1 pathway and its downstream partners are necessary to develop novel therapeutic approaches targeting this oncogene in human cancers. DisclosuresNo relevant conflicts of interest to declare.

Polycomb Group Proteins in Myelodysplastic Syndromes and Its Transformation Into Acute Myeloid Leukemia.

Abstract 1756Poster Board I-782The myelodysplastic syndrome (MDS) is a group of clonal blood disorders characterized by refractory cytopenia in peripheral blood, dysplastic cellular morphology in bone marrow, and a high risk of malignant transformation. Recently, expression of BMI1, a polycomb group protein, has been used as an important marker for predicting progression of MDS to acute myeloid leukemia(AML). However the function of BMI1 in MDS is remained unknown. Presently, we have analyzed the expression of SALL4, EZH2, P16 and BMI1 in bone marrow mononuclear cells from 48 newly diagnosed MDS patients and 40 treated MDS patients. Real-time polymerase chain reaction has shown that expression levels of these genes in MDS was frequently higher than that of non-MDS cytopenians (P<0.05). A high level of SALL4 expression in newly diagnosed MDS was always associated with high level of BMI1 expression. When MDS cases were divided into two groups based on their SALL4 mean level, The BMI1 expressed significantly higher in SALL4 higher group than that of SALL4 lower group (P<0.05). The BMI1 expression pattern showed an increasing tendency from MDS-RA moving to MDS-RAEB, without a statistically significance. On the contrary, the EZH2 expression level of MDS-RAEB is lower than that of MDS-RA (P<0.05). Although BMI1 expression level has been down-regulated when MDS patients are in morphological remission, the expression level of SALL4 and EZH2, P16 still remain abnormal (P<0.05). A significantly high level of BMI1 expression could usually be observed in MDS patients in progression, i.e. from RA to RAEB or from MDS to AML. To study whether BMI1 contributes to the MDS progression, we have transfected U937 and K562 cell line with BMI1 full-length cDNA mediated by MSCV retroviral vector and selected subclone cells. The viability of BMI1 transfected cell is much higher than the control cells cultured serum-free medium. Flow cytometry has shown that BMI1 transfected cell also inhibit apoptosis induced by As2O3. By real-time RT-PCR, it was found that BMI1 can repress the expression of EZH2, PTEN and P16 in BMI1 transfected U937 cell line, while only repress the expression of EZH2 in BMI1 transfected K562 cell line. These results suggest that the polycomb group family genes probably play a pivotal role in MDS progression, and BMI1 cooperated with EZH2 may act via an epigenetic mechanism in the pathogenesis of MDS. DisclosuresNo relevant conflicts of interest to declare.

Polycomb group protein Bmi1 is overexpressed and essential in anchorage-independent colony formation, cell proliferation and repression of cellular senescence in cholangiocarcinoma: Tissue and culture studies

Polycomb-group proteins Bmi1 is regarded as a "stemness" gene involved in the maintenance of stem cells, malignant transformation, and biologic aggressiveness of several human carcinomas. We examined the significance of the Bmi1 expression in intrahepatic cholangiocarcinoma. The expression of Bmi1 was examined in intrahepatic cholangiocarcinoma (n = 30; 9 bile ductular carcinoma, 8 intrahepatic cholangiocarcinoma of peripheral type, and 13 of hilar type) by using immunohistochemistry and real-time polymerase chain reaction. The expression level of Bmi1 was assessed in 7 cholangiocarcinoma cell lines. The effect of Bmi1 knockdown was examined in cultured cholangiocarcinoma cells (HuCCT1 and TFK-1) using small interfering RNA. Bmi1 was consistently expressed in nonneoplastic biliary epithelial cells and in all intrahepatic cholangiocarcinoma, irrespective of the location and histological degree of differentiation. The level of mRNA expression was significantly higher in 13 (81.3%) of 16 intrahepatic cholangiocarcinoma compared with the corresponding nonneoplastic tissues. All 7 cultured cholangiocarcinoma cells overexpressed Bmi1 to various degrees. The knockdown of Bmi1 resulted in decreased colony formation, decreased cell proliferation activities, and increased cellular senescence. The overexpression of polycomb-group protein Bmi1 is essential for colony formation and cell proliferation, probably by the repression of cellular senescence in intrahepatic cholangiocarcinoma.

Biological characteristics of highly tumorigenic CD44+CD133+ subpopulation of laryngeal carcinoma cells

To separate the cell subpopulation with high tumorigenic ability and study the biological characteristics of this subpopulation in laryngeal carcinoma cells. Human laryngeal carcinoma cells were obtained by primary tissue culture technique. CD44 and CD133 molecules were used as markers to isolate the CD44(+), CD133(+), CD44(+)CD133(+) and CD44(+)CD133(-) cell subpopulations from the laryngeal carcinoma cells by flow cytometry. A nude mouse tumor xenograft model was developed for the study of the tumorigenic effects of the different cell populations. 1 x 10(6), 1 x 10(5), 1 x 10(4) and 1 x 10(3) cells were injected into the left flank of the mice, respectively. The mice were observed for palpable tumor formation and were sacrificed at 4 weeks later to assess the tumor formation rate, tumor volume and tumor weight. Boyden chamber migration assay was used to determine the migration ability and immunochemistry was used to detect the expression of stem cell antigen SCA-1 and beta1-integrin. Semi-quantities RT-PCR and Western blot analysis were performed to detect the expression level of Bmi-1 in the different cell subpopulations. The growth of subcutaneous tumors in nude mice showed that a tumor can be generated with 1 x 10(3) CD44(+)CD133(+) cells. When the same dose of 1 x 10(6) CD44(+)CD133(+) cells was injected into the mice, both the average weight and volume of the tumors were significantly higher than those generated from other cell subpopulations. Boyden chamber migration assay showed that the invasion ability of CD44(+)CD133(+) cells was significantly higher than that of other cell subsets. The results of immunochemical analysis showed an abundant expression of stem cell antigen SCA-1 and beta1-integrin in the CD44(+)CD133(+) cells. Semi-quantitative RT-PCR and Western blot analysis provided strong evidence that the Bmi-1 expression in CD44(+)CD133(+) and CD133(+) cells was very significantly higher than that in CD44(+), CD44(+)CD133(-) and control cells (P < 0.01). Our findings demonstrate that CD44(+)CD133(+) subset cells in laryngeal carcinoma posses some biological characteristics of tumor stem cells, which may be the original cells of laryngeal carcinoma and may become a new target of tumor therapy.

Association between Bmi1 and clinicopathological status of esophageal squamous cell carcinoma

To investigate the clinicopathological roles of Bmi1 in esophageal squamous cell carcinoma (ESCC). Quantitative real-time polymerase chain reaction and immunohistochemical staining for Bmi1 were performed in cancerous and adjacent non-cancerous paraffin-embedded esophageal specimens. The Bmi1 expression level was unaffected by gender and age. The level of Bmi1 mRNA in ESCC was significantly higher than that in the adjacent non-cancerous tissues (2.181 +/- 2.158 vs 0.931 +/- 0.894, P = 0.0152), and its over-expression was aggressively associated with lymph node metastasis (3.580 +/- 2.487 vs 1.703 +/- 0.758, P = 0.0003), poorer cell differentiation (P = 0.0000) and advanced pathological stage (3.827 +/- 2.673 vs 1.590 +/- 0.735, P = 0.0001). The patients were divided into high-expression and low-expression groups based on the median expression level of Bmi1 mRNA, and a shorter overall survival time in the former group was observed. Immunohistochemistry for Bmi1 oncoprotein showed diffusely positive, focally positive and negative expression in 44, 16 and 10 of 70 ESCC cases, respectively, compared with three, two and five of 10 adjacent non-cancerous cases (P = 0.027). The positive rate of the oncoprotein in samples of histological grade III was higher than that of grade II (P = 0.031), but its expression had no relation to the lymph node metastasis and pathological staging. In 70 ESCC samples, Bmi1 showed high intense expression in the cytoplasm and less or even no expression in the nucleus. Bmi1 was over-expressed in ESCC. Increased Bmi1 mRNA expression was significantly associated with ESCC progression, and the oncoprotein was largely distributed in the cytoplasm of tumor cells.

Generation of CD8+ Cytotoxic T Cell Clones Recognizing BMI1-Derived Peptides

Leukemic stem cells (LSCs) resistant to conventional chemotherapeutic drugs can cause relapse of disease even after intensive treatment. Therefore, novel therapeutic strategies targeting LSCs are needed. The polycomb group gene BMI1 was initially identified as a cooperating oncogene in Eμ/myc transgenic mice. The protein product of BMI1 has been shown to repress the products of the CDKN2 tumor suppressor gene, p16 (INK4A) and p14 (ARF), and is required for self-renewal of both normal hematopoietic stem cells as well as LSCs. BMI1 expression level has been correlated with prognosis in myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML). Furthermore, higher BMI1 expression in the tumorigenic cancer stem cell population has been demonstrated in brain tumor, breast cancer, and head and neck cancer. Thus, BMI1 represents a potential attractive target for cancer stem cell-specific immunotherapy. To further evaluate BMI1 potential, we generated CD8+BMI1-specific cytotoxic T cell clones. Two BMI1-encoded peptides (CLPSPSTPV, BMI1271-279, and TLQDIVYKL, BMI174-82) with predicted high affinity binding to the class I MHC molecule HLA-A*0201 were identified using publicly available prediction algorithms, and high-affinity binding to HLA-A*0201 was confirmed using an MHC stabilization assay on T2 cells. CD8+ T cells were negatively enriched from peripheral blood mononuclear cells (PBMC) from three normal human HLA-A 0201+ donors and stimulated at 10-day intervals with BMI1 peptide-pulsed autologous dendritic cells or PBMC for two or three cycles. Reactivity of the resulting T cell lines for the corresponding BMI1 peptides was subsequently assessed by ELISPOT assay, and was detected in lines from two of the three donors for CLPSPSTPV and in a line from one of the three donors for TLQDIVYKL. A CTL clone specific for CLPSPSTPV was isolated from one of the donors and evaluated in further studies. A peptide titration assay demonstrated that the clone had high affinity (peptide concentration required for half maximal lysis at E/T=10:1, 0.1 nM). In standard cytotoxicity assays, this CTL clone efficiently killed CLPSPSTPV peptide-pulsed T2 cells and 6 of 9 HLA-A*0201-positive or -transduced BMI1+ myeloid leukemia cell lines, and did not kill nonmalignant HLA-A2+ cells, including fibroblasts, PHA-blasts, and EBV-transformed B cells. Quantitative RT-PCR analysis of BMI1 expression was performed on a commercial multiple tissue cDNA panel, 39 primary leukemia/lymphoma samples (4 ALL, 22 AML, 4 CLL, 4 DLBCL, 4 FL, 1 MCL), and 31 solid tumor lines (21 Renal cell ca., 1 Ovarian ca., 3 Breast ca., 4 Melanoma, 1 Colon ca., 1 Pancreas ca.). All samples had detectable levels of BMI1 expression. Among normal tissues, testis had the highest level of BMI1 expression. Of the primary leukemia/lymphoma samples, four of 22 AML cells and one of four CLL cells expressed high levels of BMI1, and all solid tumor lines had low levels of BMI1 expression. When tested for cytotoxicity against five HLA-A*0201+ primary leukemia cells (3 AML, 1 ALL, 1 CML-BC), the BMI1-specific CTL clone killed 3 of 5 efficiently. The clone was also tested for recognition of 14 HLA-A*0201+ solid tumor lines (6 Renal cell ca., 2 Breast ca. 2 Melanoma, 1 Ovarian ca., 1 Colon ca., 2 Medulloblastoma), but only killed 2 of the 14 lines. These results suggest that CTL therapy targeting BMI1 may have utility as a novel strategy for eliminating LSCs. Future studies will determine whether this clone can specifically recognize leukemic stem cells and inhibit the engraftment of human AML in NOD/SCID mice.

Expression of Biomarkers That Predict Progression in Chronic Myeloid Leukemia

Chronic Myelogenous Leukemia (CML) is associated with a chromosomal translocation, t(9;22)(q34;q11.2), that produces the Philadelphia chromosome (Ph).The molecular consequence of this translocation is the generation of the BCR/ABL oncogene, which encodes a chimeric protein of 210 kDa (p210Bcr/Abl) with elevated tyrosine kinase activity. BCR/ABL exerts its oncogenic effect in CML cells essentially by stimulating cell proliferation, inhibiting apoptosis and altering cell adhesion to bone marrow stroma. Despite of this consistent molecular abnormality, a marked heterogeneity in prognosis and response to treatment has been reported.Different molecular markers have been studied, such as: BMI1, ELA2, PR3, E2F1 and apoptotic genes (BCL-2, BCL-XL, BAX, BAD, BAK) in order to predict progression and overall survival in myeloid leukemia.The polycomb group gene BMI1 plays an essential role in regulating the proliferative activity in leukemic stem cell. The expression of this gene is related to a higher degree of malignancy. On the other hand, BCL-2 family genes involved in the mitochondrial-apoptotic pathway are related with clinical response and treatment failure. Enhanced expression of the apoptotic inhibitor BCL-2 or its homolog BCL-XL lead to tumor cells having a decreased susceptibility to cell death. Other BCL-2 family members such as BAX are able to induced apoptosis, so that the ratio of expression of proapoptotic and anti-apoptotic members might determine the apoptotic potencial of cancer cells.In this study we evaluated the expression of BMI1 and BAX/BCL-XL ratio (apoptotic index) to determine whether these genes could behave as biomarkers to predict disease aggressiveness and progression from chronic phase to more advanced phases.Total RNA was extracted from leucocytes of peripheral blood. using Trizol method. cDNA was synthesized with random hexamer primers and reverse transcriptase. The expression was assessed by quantitative real time (QRT-PCR) using the LightCycler 2.0 instrument (Roche), based on the Syber-Green method.All QRT-PCR reactions were performed in 20ul volume. The β-actin expression was used as the endogenous cDNA quality control. Groups of patients were compared using the Mann-Whitney test.The study was performed in 31 patients: 16 in chronic phase (CP), 15 in advanced phases (accelerated and blast crisis) and 10 healthy donors (control group).BMI1 expression levels were significantly lower in CP (mean ± SEM: 0.54±0.15) than in more advanced stages of CML (mean ± SEM: 4.54±1.4) (P<0.0005). In peripherical blood of healthy donors, the expression of this gene was similar to CML-CP patients (0.4±0.13). The relationship of BAX/BCL-XL values were higher in CP (mean ± SEM: 13.81± 1.85) and lower in advanced phase (mean ± SEM: 0.88±0.17) than in the control group (mean ± SEM: 4.82 ± 0.49) (P<0.0044 and P< 0.0002, respectively).The CP patients showed a low BMI1 expression level and a high apoptotic index, this inverse correlation is associated with a benign stage of the disease and good treatment response. On the contrary, cases in more advance stage displayed overexpression of BMI1 gene and low BAX/BCL-XL ratio suggesting an aggressive stage and poor response. The identification of a genetic hostile profile in CP phase could predict an impending disease progression. Our results show that the simultaneous use of two biomarkers: BMI1 and the ratio BAX/BCL-XL represent sensitive indicators of clinical outcome in CML-CP. Therefore, the prospective screening of these biomarkers would help to refine CML disease staging and would be useful prognostic indicators for optimizing therapeutic strategies.

BMI-1 expression is enhanced through transcriptional and posttranscriptional regulation during the progression of chronic myeloid leukemia

BMI-1 plays a critical role in regulating the activity of hematopoietic stem and progenitor cells. Patients with chronic myeloid leukemia (CML) are at a risk of developing blastic crisis (BC) even after the emergence of imatinib mesylate. In this study, to determine the relevance of BMI-1 to BC, we investigated the expression of BMI-1 in CD34(+) cells at each of the chronic phase (CP), the accelerated phase (AP), and BC by flow cytometry. Interestingly, the level of BMI-1 expression was significantly higher in CP than in controls and was further increased during the course of the disease progression (control--5.66%; CP--36.93%; AP and BC--76.41%). Curiously, mRNA levels for BMI-1 were almost consistent during the disease progression from CP to BC (control--2.21; CP--9.77; AP and BC--9.70 (BMI-1/glyceraldehyde-3-phosphate dehydrogenase ratio)). Since we further found that overexpression of BCR-ABL in human embryonic kidney-293 cells enhanced BMI-1 expression and that BMI-1 expression was increased in K562 cells, derived from patients with BC, in the presence of proteasomal inhibitors, BMI-1 was presumed to be positively regulated by BCR-ABL and further by posttranscriptional modification in the course of the disease progression. We suggest the usefulness of BMI-1 expression in CD34(+) cells as a molecular marker for monitoring patients with CML.

Loss of BMI-1 expression is associated with clinical progress of malignant melanoma

BMI-1 is a member of the Polycomb group of genes (PcGs) and is involved in embryonic gene regulation and maintenance of adult stem cells. It has been suggested that BMI-1 protein is important in cell cycle regulation, since both p16/INK4a and p14/ARF are downstream BMI-1 targets. BMI-1 has been implicated in the development and progression of several malignancies, but its role in melanocytic tumors of the skin is unknown. In the present study, using immunohistochemistry on 178 benign and malignant melanocytic lesions and two different antibodies, BMI-1 expression was reduced in melanomas compared with benign nevi. In established melanomas, loss of BMI-1 expression was associated with features of aggressive tumors, such as increased tumor cell proliferation, presence of necrosis and increased expression of both N-cadherin and β3-integrin, indicating a more invasive and mesenchymal phenotype. Low BMI-1 expression was associated with low p14 and CDK4 but not with p16 expression. Low levels of BMI-1 expression were also significantly associated with decreased patient survival.

Correlation of Bmi-1 expression and telomerase activity in human ovarian cancer

This study investigates the correlation between the oncoprotein Bmi-1 and telomerase activity in ovarian cancer. A real-time polymerase chain reaction (PCR) method is used to detect the messenger RNA (mRNA) expression of Bmi-1 protein in 47 ovarian epithelial cancer cases, and immunohistochemistry is used to detect Bmi-1 protein expression in the tissues. A modified telomeric repeat amplification protocol (TRAP) is used to detect telomerase activity. Western blotting is used to detect the expression of telomerase hTERT in the tissues studied. Compared to normal ovarian epithelial tissue, Bmi-1 protein in the 47 ovarian epithelial cancer cases showed higher expression and was related to pathological grade and clinical stage. Significantly higher Bmi-1 levels were found among different clinocopathological types of the cancer (P<0.05). Grade G3 cases expressed Bmi-1 at a higher rate (93.10%) than did grade G2 cases (61.11%). Expression in phase II and phase III cases was lower (66.67%) than in phase IV (92.31%). In ovarian epithelial cancer tissues, 87.23% (41/47) cases demonstrated positive telomerase activity, whereas no activity was observed in normal tissues. The majority (90.24%) of specimens with positive telomerase activity showed high Bmi-1 expression levels. Spearman correlation analysis indicated that expression of Bmi-1 protein correlated positively with elevated telomerase activity. Bmi-1 protein is highly expressed in ovarian epithelial cancer tissues, and expression correlates with histological grade and clinical phase. Elevated Bmi-1 expression correlates closely with increased telomerase activity and plays a significant role in the pathogenesis of ovarian cancer.

Bmi‐1 expression predicts prognosis for patients with gastric carcinoma

The Bmi-1 gene is a transcriptional repressor involved in oncogenesis in various human cancers. Here, we examine Bmi-1 expression in gastric carcinoma (GC) and investigates whether its expression correlates with patient prognosis. Immunohistochemistry was performed using an anti-Bmi-1 antibody on primary tumor samples of 146 cases of GC. The association between Bmi-1 expression and the clinicopathological status and prognosis of GC patients was statistically analyzed. Furthermore, reverse transcription-PCR (RT-PCR) and Western blotting were performed to determine the expression levels of Bmi-1 in an additional 8 GC and the adjacent non-cancerous samples. Using immunohistochemistry, we found that 99 of 146 paraffin-embedded GC samples expressed Bmi-1 extensively. Statistical analysis showed that Bmi-1 overexpression was highly correlated with tumor size, clinical stage, lymph node metastasis and T classification (P < 0.05), Patients with Bmi-1 expression had shorter overall survival time than those without Bmi-1 expression (P < 0.01). Multivariate analysis indicated that Bmi-1 expression is an independent prognostic factor of GC. RT-PCR and Western blotting showed that Bmi-1 was up-regulated at both the transcriptional and translational levels in the GC tissues compared with the adjacent non-cancerous tissues. Bmi-1 may serve as a valuable marker for diagnosis and prognosis of GC.

Association between the Polycomb Group (PcG) BMI-1 Gene Expression and Outcome in Chronic Myeloid Leukemia (CML) Patients Receiving Allogeneic Stem Cell Transplantation (allo-SCT).

Despite a consistent molecular abnormality, the BCR-ABL oncogene, and the success of tyrosine kinase inhibitors, CML still exhibits a marked heterogeneity in prognosis. The PcG gene BMI-1 plays an essential role in regulating the proliferative activity of both normal and leukemic stem cells. BMI-1 correlation with outcome was described in different malignancies. We have previously shown that levels of BMI-1 RNA were significantly higher in advanced phase than in chronic phase (CP) CML. In addition, the level of BMI-1 at diagnosis correlated with time to transformation to blast crisis and, in patients treated with hydroxyurea and IFN-a, low BMI-1 expression was associated with an improved overall survival (OS), suggesting that BMI-1 may be a biomarker for the intrinsic heterogeneity of CML (Mohty et al., Blood 2007). Here, we investigated whether BMI-1 and the other previously established prognostic genes (CD7, PR-3 and ELA-2) are implicated in the prognosis of CML in the context of allo-SCT. For this purpose, we studied 84 CP-CML patients who received allo-SCT from HLA-identical related donors. CD7, PR-3, ELA-2 and BMI-1 expression was assessed by Q-RT/PCR in the recipient's PBMCs collected before allo-SCT. The median expression level for each gene was used to segregate the patients into 2 groups (“low”: gene expression <median, and “high”: gene expression >median). The median follow-up post-allo-SCT was 9.9 (range, 1.7–23.9) years. The median EBMT-Gratwohl score was 3. None of the 4 tested genes showed any significant association with neutrophil or platelet engraftment, or with graft rejection. CD7, PR-3 and ELA-2 expression was not associated with OS. However, in sharp contrast to our previous findings in the non-allo-SCT setting, patients displaying a “high” BMI-1 expression level prior to allo-SCT had significantly better OS than those with “low” expression (P=0.005). When BMI-1 was included in a multivariate survival model and adjusted for the other prognostic variables (EBMT-Gratwohl score, allo-SCT era, and other relevant parameters), a high expression was found to be an independent marker associated with better survival (RR=2.72, 95%CI; 1.1–6.9; P=0.034). Given the impact of BMI-1 expression level on OS, without a significant association with relapse, and since neither BMI-1, nor the other genes showed any significant association with leukemia-free survival, we assessed their impact on transplant-related mortality (TRM). There was a striking and significant association between acute GVHD and BMI-1 expression, not only in overall incidence (“low” BMI-1: grade 0–1 (n=21), grade 2 (n=10), grade 3–4 (n=9); “high” BMI-1: grade 0–1 (n=32), grade 2 (n=9), grade 3–4 (n=1); P=0.005), but also in cumulative incidence at day 100 (48% vs. 24%, P=0.016). In multivariate analysis, a “low” BMI-1 expression level was associated with an increased risk of grade 2–4 acute GVHD (RR=2.85, 95%CI; 1.3–6.4; P=0.011). These results suggest that BMI-1 measured prior to allo-SCT can serve as a biomarker for predicting outcome in CP-CML patients receiving allo-SCT. The presence of a potential donor immune response against BMI-1, which is a genuine tumor-associated antigen, may abrogate any neoplastic proliferative advantage within the leukemia stem cell pool conferred by higher expression of BMI-1. Such measurement allows for tailored therapeutic intervention, including informed recommendation for allo-SCT in patients failing tyrosine-kinase inhibitors.

Increased polycomb-group oncogene Bmi-1 expression correlates with poor prognosis in hepatocellular carcinoma

Recent studies have identified polycomb-group gene Bmi-1 as oncogene in the generation of mouse pre-cell lymphomas, and overexpression of Bmi-1 has been found in several human tumor with the disease progress and poor prognosis of the cancer patients. In present study, we investigated Bmi-1 expression and its prognostic significance in hepatocellular carcinoma (HCC) by performing immunohistochemical analysis, using a total of 137 HCC clinical tissue samples. High Bmi-1 expression (Bmi-1 2+ or 3+) was shown in 29.9% cases. The positive immuno-staining of Bmi-1 was not only in well/moderately-differentiated tumor cells, but also in surrounding noncancerous or cirrhotic liver tissue. Bmi-1 expression level did not correlate with any clinicopathological parameters. However, survival analysis showed that the high-Bmi-1 group had a significantly shorter overall survival time than the low-Bmi-1 group (P=0.047). Multivariate analysis after 24 months revealed that Bmi-1 expression was a significant and independent prognostic parameter (P=0.002) for HCC patients. Our study indicated that Bmi-1 could be a candidate biomarker for long-term survival in HCC.

Circulating Bmi-1 mRNA as a possible prognostic factor for advanced breast cancer patients

IntroductionDeregulation of Polycomb member Bmi-1 is involved in cell proliferation and human oncogenesis. Modulation of Bmi-1 is found in several tumor tissues, including primary breast carcinomas; however, analysis of Bmi-1 in plasma of cancer patients has not been reported. This is the first study that evaluates Bmi-1 in plasma by using a large series of primary breast carcinomas to investigate the presence at diagnosis of detectable Bmi-1 mRNA in plasma and possible correlations between this event and a series of clinical-pathological parameters of the tumors.MethodsBmi-1 expression levels were quantified in plasma of 111 breast cancer patients and in 20 healthy controls by real-time quantitative polymerase chain reaction.ResultsCancer patients with the presence of Bmi-1 mRNA in plasma had higher levels of Bmi-1 expression than healthy controls with Bmi-1 mRNA in plasma. The higher expression levels of Bmi-1 correlated with well-established markers of poor clinical outcome in breast cancer such as positive p53 immunostaining and negative progesterone receptors. Moreover, we described for the first time a statistically significant correlation between Bmi-1 expression in plasma of breast cancer patients and disease-free and overall survival in advanced stages.ConclusionOur results suggest that levels of Bmi-1 expression may be a surrogate marker of poor prognosis and may become clinically useful as noninvasive diagnostic markers.

Essential role for activation of the Polycomb group (PcG) protein chromatin silencing pathway in metastatic prostate cancer: Berezovska OP, Glinskii AB, Yang Z, Li XM, Hoffman RM, Glinsky GV, Translational and Functional Genomics Laboratory, Ordway Cancer Center, Ordway Research Institute, Inc., Center for Medical Sciences, Albany, NY

The Polycomb group (PcG) gene BMI1 is required for the proliferation and self-renewal of normal and leukemic stem cells. Overexpression of Bmi1 oncogene causes neoplastic transformation of lymphocytes and plays essential role in pathogenesis of myeloid leukemia. Another PcG protein, Ezh2, was implicated in metastatic prostate and breast cancers, suggesting that PcG pathway activation is relevant for epithelial malignancies. Whether an oncogenic role of the BMI1 and PcG pathway activation may be extended beyond the leukemia and may affect progression of solid tumors as well remains unknown. Here we demonstrate that activation of the BMI1 oncogene-associated PcG pathway plays an essential role in metastatic prostate cancer, thus mechanistically linking the pathogenesis of leukemia, self-renewal of stem cells, and prostate cancer metastasis. To characterize the functional status of the PcG pathway in metastatic prostate cancer, we utilized advanced cell- and whole animal-imaging technologies, gene and protein expression profiling, stable siRNA-gene targeting, and tissue microarray (TMA) analysis in relevant experimental and clinical settings. We demonstrate that in multiple experimental models of metastatic prostate cancer both BMI1 and Ezh2 genes are amplified and gene amplification is associated with increased expression of corresponding mRNAs and proteins. For the first time, we provide images of human prostate carcinoma metastasis precursor cells isolated from blood and shown to overexpress both BMI1 and Ezh2 oncoproteins. Consistent with the PcG pathway activation hypothesis, increased BMI1 and Ezh2 expression in metastatic cancer cells is associated with elevated levels of H2AubiK119 and H3metK27 histones. Quantitative immunofluorescence colocalization analysis and expression profiling experiments documented increased BMI1 and Ezh2 expression in clinical prostate carcinoma samples and demonstrated that high levels of BMI1 and Ezh2 expression are associated with markedly increased likelihood of therapy failure and disease relapse after radical prostatectomy. Gene-silencing analysis reveals that activation of the PcG pathway is mechanistically linked with highly malignant behavior of human prostate carcinoma cells and is essential for in vivo growth and metastasis of human prostate cancer. We conclude that the results of experimental and clinical analyses indicate the important biological role of the PcG pathway activation in metastatic prostate cancer. Our work suggests that the PcG pathway activation is a common oncogenic event in pathogenesis of metastatic solid tumors and provides justification for development of small molecule inhibitors of the PcG chromatin silencing pathway as a novel therapeutic modality for treatment of metastatic prostate cancer.

Bmi-1 Is Essential for the Tumorigenicity of Neuroblastoma Cells

Activation of oncogenes underlies the pathogenesis of most human cancers. In neuroblastoma, amplification of the oncogene MYCN occurs in approximately 22% of cases and is associated with advanced stages of the disease and poor prognosis. Identification of other oncogenes that are consistently mutated or overexpressed in neuroblastoma is crucial for a molecular understanding of the pathogenic process. Here, we report that the oncogene Bmi-1 is highly expressed in human neuroblastoma cell lines and primary tumors. Neuroblastoma development in MYCN transgenic mice, an animal model for the human disease, was associated with a marked increase in the levels of Bmi-1 expression. Bmi-1 cooperated with MYCN in transformation of benign S-type neuroblastoma cells and avian neural crest cells by inhibiting the apoptotic activity of MYCN. Importantly, down-regulation of Bmi-1 impaired the ability of neuroblastoma cells to grow in soft agar and induce tumors in immunodeficient mice. Moreover, Bmi-1-knockdown neuroblastoma xenografts were characterized by a significant increase in the amount of Schwannian stroma, a histological feature associated with clinically favorable neuroblastomas. These findings suggest a crucial role for Bmi-1 in neuroblastoma pathogenesis and provide insights into the molecular basis of neuroblastoma heterogeneity.

Implication of Polycomb Members Bmi-1, Mel-18, and Hpc-2 in the Regulation of p16INK4a, p14ARF, h-TERT, and c-Myc Expression in Primary Breast Carcinomas

Deregulation of mammalian Polycomb group (PcG) members may contribute to human carcinogenesis. p16INK4a and p14ARF tumor suppressors, human telomerase reverse transcriptase (h-TERT), and oncoprotein c-Myc have been implicated in the regulation of the cell cycle and proliferation mediated by PcG proteins, mainly Bmi-1, in mice and in cell culture experiments. Here, we examine whether these in vitro findings can be extrapolated to the in vivo situation. We measure the expression of PcG members Bmi-1, Mel-18, and Hpc-2 and their potential targets by reverse transcription-PCR, immunostaining, and Western blotting in a series of 134 breast carcinomas and correlate the data with several clinical-pathologic variables of the tumors. Expression of PcG genes was variably detected, but overexpression of Bmi-1 was the most frequent PcG alteration observed. In addition, statistical direct correlation in expression level of the three PcG members was detected. A correlation between c-Myc and Bmi-1 expression levels was observed; however, there was no correlation between expression of Bmi-1 and p16INK4a, p14ARF, or h-TERT. However, expression of the other PcG members Mel-18 and Hpc-2 correlated with the cell cycle regulators. Moreover, PcG mRNA-altered expression correlated significantly with certain clinical-pathologic variables associated with poor prognosis. Our data suggest that the oncogenic role of Bmi-1 in human primary breast carcinomas is not determined by its capacity to inhibit INK4a/ARF proteins or to induce telomerase activity.