Abstract: There is increasing evidence that a variety of cancers arise from transformation of normal stem cells to cancer stem cells (CSCs). CSCs are thought to sustain cancer progression, invasion, metastasis, and recurrence after therapy. Reports suggest that CSCs are highly resistant to conventional therapy. Emerging evidences show that the chemoresistance of CSCs are in part due to the activation of B cell-specific Moloney murine leukemia virus integration site 1 (BMI1), a stem cell factor, and a polycomb group family member. BMI1 is reported to regulate the proliferation activity of normal, stem, and progenitor cells. BMI1 plays a role in cell cycle, cell immortalization, and senescence. Numerous studies demonstrate that BMI1, which is upregulated in a variety of cancers, has a positive correlation with clinical grade/stage and poor prognosis. Although evidences are in support of the role of BMI1 as a factor in chemoresistance displayed by CSCs, its mechanism of action is not fully understood. In this review, we provide summary of evidences (with mechanism of action established) suggesting the significance of BMI1 in chemoresistance and recurrence of CSCs. STEM CELLS 2012;30:372–378

Abstract: IL-1β is a pleiotropic pro-inflammatory cytokine and its up-regulation is closely associated with various cancers including gastrointestinal tumors. However, it remains unclear how IL-1β may contribute to the initiation and development of these inflammation-associated cancers. Here we investigated the role of IL-1β in colon cancer stem cell (CSC) development. Using self-renewal assay, soft-agar assay, invasion assay, real-time PCR analysis, immunoblot assay and shRNA knockdown, we determined the effects of IL-1β on cancer stem cell development and epithelial-mesenchymal transition (EMT) in human primary colon cancer cells and colon cancer cell line HCT-116. We found that IL-1β can increase sphere-forming capability of colon cancer cells in serum-free medium. IL-1β-induced spheres displayed an up-regulation of stemness factor genes (Bmi1 and Nestin) and increased drug resistance, hallmarks of CSCs. Importantly, expression of EMT activator Zeb1 was increased in IL-1β-induced spheres, indicating that there might be a close association between EMT and IL-1β-induced CSC self-renewal. Indeed, IL-1β treatment led to EMT of colon cancer cells with loss of E-cadherin, up-regulation of Zeb1, and gain of the mesenchymal phenotype. Furthermore, shRNA-mediated knockdown of Zeb1 in HCT-116 cells reversed IL-1β-induced EMT and stem cell formation. Our findings indicate that IL-1β may promote colon tumor growth and invasion through activation of CSC self-renewal and EMT, and Zeb1 plays a critical role in these two processes. Thus, IL-1β and Zeb1 might be new therapeutic targets against colon cancer stem cells.

Abstract: SOX9 [sex-determining region Y (SRY)-box 9 protein], a high mobility group box transcription factor, plays critical roles during embryogenesis and its activity is required for development, differentiation, and lineage commitment in various tissues including the intestinal epithelium. Here, we present functional and clinical data of a broadly important role for SOX9 in tumorigenesis. SOX9 was overexpressed in a wide range of human cancers, where its expression correlated with malignant character and progression. Gain of SOX9 copy number is detected in some primary colorectal cancers. SOX9 exhibited several pro-oncogenic properties, including the ability to promote proliferation, inhibit senescence, and collaborate with other oncogenes in neoplastic transformation. In primary mouse embryo fibroblasts and colorectal cancer cells, SOX9 expression facilitated tumor growth and progression whereas its inactivation reduced tumorigenicity. Mechanistically, we have found that Sox9 directly binds and activates the promoter of the polycomb Bmi1, whose upregulation represses the tumor suppressor Ink4a/Arf locus. In agreement with this, human colorectal cancers showed a positive correlation between expression levels of SOX9 and BMI1 and a negative correlation between SOX9 and ARF in clinical samples. Taken together, our findings provide direct mechanistic evidence of the involvement of SOX9 in neoplastic pathobiology, particularly, in colorectal cancer.

Abstract: Polycomb repressive complexes (PRCs) play key roles in developmental epigenetic regulation. Yet the mechanisms that target PRCs to specific loci in mammalian cells remain incompletely understood. In this study we show that Bmi1, a core component of Polycomb Repressive Complex 1 (PRC1), binds directly to the Runx1/CBFβ transcription factor complex. Genome-wide studies in megakaryocytic cells demonstrate significant chromatin occupancy overlap between the PRC1 core component Ring1b and Runx1/CBFβ and functional regulation of a considerable fraction of commonly bound genes. Bmi1/Ring1b and Runx1/CBFβ deficiencies generate partial phenocopies of one another in vivo. We also show that Ring1b occupies key Runx1 binding sites in primary murine thymocytes and that this occurs via PRC2-independent mechanisms. Genetic depletion of Runx1 results in reduced Ring1b binding at these sites in vivo. These findings provide evidence for site-specific PRC1 chromatin recruitment by core binding transcription factors in mammalian cells.

Abstract: Deregulated miRNAs participate in colorectal carcinogenesis. In this study, miR-218 was found to be downregulated in human colorectal cancer (CRC) by miRNA profile assay. miR-218 was silenced or downregulated in all five colon cancer cells (Caco2, HT29, SW620, HCT116 and LoVo) relative to normal colon tissues. miR-218 expression was significantly lower in 46 CRC tumor tissues compared with their adjacent normal tissues (P < 0.001). Potential target genes of miR-218 were predicted and BMI1 polycomb ring finger oncogene (BMI-1), a polycomb ring finger oncogene, was identified as one of the potential targets. Upregulation of BMI-1 was detected in CRC tumors compared with adjacent normal tissues (P < 0.001) and in all five colon cancer cell lines. Transfection of miR-218 in colon cancer cell lines (HCT116, HT29) significantly reduced luciferase activity of the wild-type construct of BMI-1 3′ untranslated region (3′UTR) (P < 0.001), whereas this effect was not seen in the construct with mutant BMI-1 3′UTR, indicating a direct and specific interaction of miR-218 with BMI-1. Ectopic expression of miR-218 in HCT116 and HT29 cells suppressed BMI-1 mRNA and protein expression. In addition, miR-218 suppressed protein expression of BMI-1 downstream targets of cyclin-dependent kinase 4, a cell cycle regulator, while upregulating protein expression of p53. We further revealed that miR-218 induced apoptosis (P < 0.01), inhibited cell proliferation (P < 0.05) and promoted cell cycle arrest in the G2 phase (P < 0.01). In conclusion, miR-218 plays a pivotal role in CRC development through inhibiting cell proliferation and cycle progression and promoting apoptosis by downregulating BMI-1.

Abstract: Prostate cancer (PCa) is a major lethal malignancy in men, but the molecular events and their interplay underlying prostate carcinogenesis remain poorly understood. Epigenetic events and the upregulation of polycomb group silencing proteins including Bmi1 have been described to occur during PCa progression. Here, we found that conditional overexpression of Bmi1 in mice induced prostatic intraepithelial neoplasia, and elicited invasive adenocarcinoma when combined with PTEN haploinsufficiency. In addition, Bmi1 and the PI3K/Akt pathway were coactivated in a substantial fraction of human high-grade tumors. We found that Akt mediated Bmi1 phosphorylation, enhancing its oncogenic potential in an Ink4a/Arf-independent manner. This process also modulated the DNA damage response and affected genomic stability. Together, our findings demonstrate the etiological role of Bmi1 in PCa, unravel an oncogenic collaboration between Bmi1 and the PI3K/Akt pathway, and provide mechanistic insights into the modulation of Bmi1 function by phosphorylation during prostate carcinogenesis.

Abstract: Polycomb group (PcG) proteins have recently been shown related to cancer development. The PcG protein EZH2 is involved in progression of prostate and breast cancers, and has been identified as a molecular marker in breast cancer. Nevertheless, the molecular mechanism by which PcG proteins regulate cancer progression and malignant metastasis is still unclear. PcG proteins methylate H3K27 in undifferentiated epithelial cells, resulting in the repression of differentiation genes such as HOX. FOXC1 is a member of the Forkhead box transcription factor family, which plays an important role in differentiation, and is involved in eye development. We discovered in this study that the expression of FOXC1 gene was negatively correlated to that of PcG genes, i.e., Bmi1, EZH2, and SUZ12, in MCF-7 and MDA-MB-231 cells. To investigate the regulatory effects of PcG proteins on FOXC1 gene, the two cell lines were transfected with either expression plasmids or siRNA plasmids of Bmi1, EZH2, and SUZ12, and we found that PcGs, especially EZH2, could repress the transcription of FOXC1 gene. Chromatin immunoprecipitation (ChIP) assay showed that histone methylation and acetylation modifications played critical roles in this regulatory process. When FOXC1 was stably transfected into MDA-MB-231 cells, the migration and invasion of the cells were repressed. Moreover, the tumorigenicity and the spontaneous metastatic capability regulated by FOXC1 were determined by using an orthotropic xenograft tumor model of athymic mice with the FOXC1-MDA-MB-231HM and the GFP-MDA-MB-231HM cells, and the results showed that FOXC1 in MDA-MB-231HM cells inhibited migration and invasion in vitro and reduced the pulmonary metastasis in vivo. Data presented in this report contribute to the understanding of the mechanisms by which EZH2 participates in tumor development.

Abstract: Polycomb-group (PcG) proteins form the multiprotein polycomb repressive complexes (PRC) 1 and 2, and function as transcriptional repressors through histone modifications. They maintain the proliferative capacity of hematopoietic stem and progenitor cells by repressing the transcription of tumor suppressor genes, namely Ink4a and Arf , and thus have been characterized as oncogenes. However, the identification of inactivating mutations in the PcG gene, EZH2 , unveiled a tumor suppressor function in myeloid malignancies, including primary myelofibrosis (PMF). Here, we show that loss of another PcG gene, Bmi1 , causes pathological hematopoiesis similar to PMF. In a mouse model, loss of Bmi1 in Ink4a-Arf −/− hematopoietic cells induced abnormal megakaryocytopoiesis accompanied by marked extramedullary hematopoiesis, which eventually resulted in lethal myelofibrosis. Absence of Bmi1 caused derepression of a cohort of genes, including Hmga2 , which is an oncogene overexpressed in PMF. Chromatin immunoprecipitation assays revealed that Bmi1 directly represses the transcription of Hmga2 . Overexpression of Hmga2 in hematopoietic stem cells induced a myeloproliferative state with enhanced megakaryocytopoiesis in mice, implicating Hmga2 in the development of pathological hematopoiesis in the absence of Bmi1. Our findings provide the first genetic evidence of a tumor suppressor function of Bmi1 and uncover the role of PcG proteins in restricting growth by silencing oncogenes.

Abstract: Neuroblastoma (NB) is the most common pediatric solid malignant tumor derived from the sympathetic nervous system. High-risk NB is still one of the most difficult tumors to cure, with only 40% long-term survival despite intensive multimodal therapy. The clinical presentation and treatment response of advanced NB, which results in relapse and a refractory state after a good response to the initial chemotherapy, suggests that cancer stem cells (CSCs) likely exist in NB tumors. Putative CSCs using primary tumor sphere formation from NB patients were reported previously, and several molecules will be elucidated from the tumor sphere to develop CSC-targeting therapies. Recently, our group reported that a CSC marker for several malignancies, CD133, and the stemness-related polycomb BMI1 have functions to repress NB cell differentiation. Depletion of CD133 or BMI1 effectively induced neurite elongation and marker molecules for differentiation in NB cells. Of note, CD133-related NB cell differentiation and RET (rearranged during transfection) repression were considerably dependent on p38MAPK and phosphoinositide 3-kinase (PI3K)/AKT pathways. Intriguingly, both CD133 and BMI1 also have a role in xenograft tumor formation and tumor sphere formation. These observations suggest that CD133 and BMI1 may be candidates for the development of CSC-targeting therapies for refractory NB patients.

Abstract: Acute myeloid leukemia (AML) is a highly malignant disease that is not curable in the majority of patients. Numerous non-random genetic abnormalities are known, among which several translocations such as PLZF/RARα or AML1/ETO are known to aberrantly recruit histone deacetylases. Deacetylase inhibitors (DACi) are promising drugs leading to growth inhibition, cell cycle arrest, premature senescence and apoptosis in malignant cells. It is believed that DACi may have clinical efficacy by eradicating the most primitive population of leukemic stem and progenitor cells, possibly by interfering with self-renewal. The aim of the study was to investigate the effects of DACi on leukemic stem and progenitor cells using murine transduction-transplantation models of hematopoietic cells harboring the leukemia-associated fusion proteins (LAFP) PLZF/RARα or a truncated AML1/ETO protein (AML1/ETO exon 9). We show that the self-renewal and short-term repopulation capacity of AML1/ETO- or PLZF/RARα-expressing Sca1+/lin- stem and progenitor cells are profoundly inhibited by clinically applicable concentrations of the DACi dacinostat and vorinostat. To further investigate the mechanisms underlying these effects, we examined the impact of DACi on the transcription factor c-MYC and the Polycomb group protein BMI1, which are induced by LAFP and involved in leukemic transformation. In AML1/ETO or PLZF/RARα-positive 32D cells, DACi-mediated antiproliferative effects were associated with downregulation of BMI1 and c-MYC protein levels. Similar effects were demonstrated in primary samples of cytogenetically defined high-risk AML patients. In conclusion, DACi may be effective as maintenance therapy by negatively interfering with signaling pathways that control survival and proliferation of leukemic stem and progenitor cells.

Abstract: Background The polycomb group (PcG) family BMI1, acting downstream of the hedgehog (Hh) pathway, plays an essential role in the self-renewal of haematopoietic, neural, and intestinal stem cells, and is dysregulated in many types of cancer. Our recent report has demonstrated that Hh signalling activation can predict very earlier relapse of oesophageal cancers. As data were not available on the clinical role of BMI1 expression in oesophageal cancers after chemoradiotherapy (CRT), we analysed whether it could be also used to predict disease progression and prognosis in oesophageal cancer patients undergoing trimodality therapy of preoperative CRT and oesophagectomy.

Abstract: Bmi1 is a member of the polycomb repressive complex 1 and plays different roles during embryonic development, depending on the developmental context. Bmi1 over expression is observed in many types of cancer, including tumors of astroglial and neural origin. Although genetic depletion of Bmi1 has been described to result in tumor inhibitory effects partly through INK4A/Arf mediated senescence and apoptosis and also through INK4A/Arf independent effects, it has not been proven that Bmi1 can be causally involved in the formation of these tumors. To see whether this is the case, we developed two conditional Bmi1 transgenic models that were crossed with GFAP-Cre mice to activate transgenic expression in neural and glial lineages. We show here that these mice generate intermediate and anterior lobe pituitary tumors that are positive for ACTH and beta-endorphin. Combined transgenic expression of Bmi1 together with conditional loss of Rb resulted in pituitary tumors but was insufficient to induce medulloblastoma therefore indicating that the oncogenic function of Bmi1 depends on regulation of p16INK4A/Rb rather than on regulation of p19ARF/p53. Human pituitary adenomas show Bmi1 overexpression in over 50% of the cases, which indicates that Bmi1 could be causally involved in formation of these tumors similarly as in our mouse model.

Abstract: Bmi1 polycomb ring finger oncogene (Bmi1) and the enhancer of zeste homolog 2 (EZH2) are members of polycomb repressive complex (PRC) 1 and PRC2, respectively. PRC1 represses tumor suppressor genes such as p16INK4a and p14ARF in a PRC2-dependent manner. There have been few studies on Bmi1 or EZH2 expression in esophageal squamous cell carcinoma (ESCC). We investigated Bmi1 and EZH2 expression in 164 cases of ESCCs using immunohistochemistry, and evaluated the correlation with clinicopathologic features and their prognostic significance. Bmi1 and EZH2 were more highly expressed in tumor than in adjacent normal tissue (p High expression of both Bmi1 and EZH2, not each alone, is an independent poor prognostic factor in ESCCs, supporting the repression of tumor suppressor gene by Bmi1 in an EZH2-dependent manner. This result suggests that both Bmi1 and EZH2, not each alone, could be potent candidates of new target therapy in ESCCs.

Abstract: Preservation of hematopoietic hierarchy requires a constant and reciprocal interplay between chromatin-specific epigenetic regulators and lineage-modifying transcription factors. The polycomb member Bmi1 is a key factor in hematopoietic stem cell (HSC) maintenance, but its specific physiological role in subsequent hematopoietic lineage-specific commitments is unclear. Here, we generated conditional Bmi1 knockout (Bmi1-KO) mice. Selective ablation of Bmi1 in the hematopoietic system induced extensive upregulation of Ikaros and concomitant Ikaros-dependent lymphoid-lineage transcriptional priming, which is marked by their loss of H2A ubiquitination and increased H3K4 trimethylation in Bmi1-KO long-term HSCs (LT-HSCs). Removal of Ikaros in Bmi1-null LT-HSCs significantly diminished the hematopoietic defects seen in conditional Bmi1-KO mice. These alterations resulted in recovering the Bmi1-KO exhausted quiescent stem-cell pool, whereas the block in Bmi1-KO lymphoid-progenitor differentiation was rescued, allowing the development of mature lymphoid cells. Together, our results indicate that Ikaros is a critical Bmi1 target in vivo that prevents premature lineage specification of HSCs.

Abstract: Recent articles in the Journal of Experimental Medicine and in Cell Cycle by the laboratory of A. Iwama established a role for polycomb gene BMI1 in leukemogenesis using a mouse model.1,2 Polycomb proteins regulate gene expression through the control of chromatin structure and repressive histone marks. They play major roles in development and stem cell biology. They distribute into two complexes called polycomb repressive complexes 1 and 2 (PRC1 and PRC2). PRC1 contains several proteins, including BMI1. PRC1 monoubiquitylates lysine 119 of histone H2A via the ubiquitin ligases RING1 and RNF2.3 PRC2 contains EED, EZH2 and SUZ12 proteins. PRC2 is a histone methyltransferase that trimethylates lysine 27 of histone H3, resulting in the H3K27me3 mark, which specifies transcriptional repression. Recent studies have shown that components of PRC2, especially EZH2, are targeted by inactivating mutations in human myeloid malignancies.4-7 PRC2 genes are mutated in acute stages, such as acute myeloid leukemia (AML), and chronic stages, such as myelodysplastic syndromes (MDS) or myeloproliferative neoplasms (MPN). The latter comprise polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). EZH2 is mutated in 13% of PMF cases and in 12% of cases with MDS/MPN features.5 Actually, through these direct inactivating mutations in its components or through alterations in PRC2-interacting proteins such as ASXL1,8 PRC2 appears as a key tumor suppressor complex in myeloid diseases. No such results have been reported yet for PRC1 genes in human samples. In contrast, because the loss of Bmi1 gene induces a defect in the self-renewal capacity of hematopoietic stem cells9 it was thought that PRC1 and PRC2 played opposite roles in leukemogenesis. Oguro and colleagues1 have just demonstrated that in an Ink4a/Arf-l- mouse the loss of the Bmi1 gene generated a disease similar to PMF. Mice repopulated with Bmi1-/-Ink4a-Arf−/− hematopoietic cells developed a lethal disease with clinical features observed in human PMF. This exciting result suggests that alterations of PRC1 complex components could, according to the genetic context (an abrogation of p16 and p19 tumor-suppressor control in the mouse model of Oguro and colleagues), play a role in the development of myeloid diseases similar to that of PRC2. PRC1 and PRC2 silencing functions are coordinated, and the two complexes may exert their leukemogenic effect through the deregulation of the same loci. Indeed, Oguro and colleagues identified oncogenic loci repressed by PRC1, such as Hmga2, and further demonstrated that Hmga2 promoter is also under the control of EZH2.1,2 A consequence of these results in human medicine is that alterations or deregulation of PRC1 components and controlled loci may substantially increase the proportion of hematopoietic diseases with defects in polycomb network genes. To determine whether BMI1 may be altered in human MPNs, we studied the genome of 35 PMF cases by using array-comparative genomic hybridization (aCGH) on high-density oligonucleotide microarrays (Hu-244A, Agilent Technologies), as previously described.10 We also searched for mutations in BMI1 in 77 MPN cases comprising 4 PV, 20 ET and 36 myelofibrosis (MF) (25 PMF, 8 post-ET MF, 3 post-PV MF), 3 MPN/MDS, 3 MPN-unclassifiable, 3 blast phase post-ET, 5 blast phase post-ET MF, 3 blast phase PMF using Sanger DNA sequencing of BMI1 coding exons 2 to 10. We did not find any mutations in BMI1. We found a heterozygous deletion of chromosome region 10p12.31 encompassing the BMI1 locus and seven other genes (Fig. 1) in case HD-1095, a 64-year-old man diagnosed with PMF. The HD-1095 sample had a JAK2V617F mutation (50–60% allele burden), was not mutated in ASXL1, DNMT3A, IDH1, IDH2 and TET2, and displayed other small chromosomal deletions that had led to the loss of SOCS2, FBXL18 and MYB, but not of CDKN2A/INK4A (not shown). To our knowledge, this is the first example of BMI1 locus alteration in human cancer. BMI1 deletion could lead to haploinsufficiency; unfortunately, RNA was not available for this sample. We studied BMI1 mRNA expression in 25 PMF profiled by Affymetrix microarrays. BMI1 expression in PMF was similar to that in normal blood (not shown), suggesting that the BMI1 locus is not inactivated by hypermethylation as a general rule. In this series studied by aCGH we did not find alterations of the HMGA2 locus, but we had previously reported one case of HMGA2 3′UTR breakage and mRNA overexpression in an MPN case.11 Finally, to determine whether BMI1 alteration could be found in other myeloid diseases, we searched for a deletion of the locus in 253 non-PMF samples (73 MPNs, 53 chronic myelomonocytic leukemias, 63 MDSs, and 64 AMLs) by using aCGH but found no other deleted case. Our results show that, although structural alterations of BMI1 and downstream effectors are rare, they do occur in human MPN samples. This supports both the findings of Oguro and colleagues in the mouse and the potential role of PRC1 in leukemogenesis. Whether other mechanisms of downregulation of BMI1 or alterations in other PRC1 components can be found in hematopoietic diseases remains to be demonstrated. Figure 1. aCGH profile of chromosome 10 in one PMF case showing loss of the BMI1 region. (A) Chromosome 10 ideogram and aCGH profile of HD-1095 case. (B) zoom on the 10p12.31 band showing the deletion that spans chr10:21,274,386–23,428,386, ...

Abstract: The Polycomb group protein Bmi1 is a constituent of the Polycomb repressive complex 1, and it is an important molecule for the regulation of the self-renewal of hematopoietic stem cells. In the field of clinical hematology, there are reports that the level of Bmi1 expression in blast cells is related to the prognosis of acute myeloid leukemia, chronic myeloid leukemia, and myelodysplastic syndrome. We investigated whether the level of Bmi1 expression in leukemic cells is related to the prognosis and the characteristics of childhood acute lymphoblastic leukemia. In all the leukemic blast cells, Bmi1 gene expression was lower value than that in normal B cells. There were no correlations between the level of Bmi1 gene expression in leukemic blast cells and other parameters, including prognosis. Here, we report that the level of Bmi1 expression in blast cells is not related to the prognosis of pediatric acute lymphoblastic leukemia.

Abstract: We have shown, that in particular tumor cells at the invasive front undergo an epithelial-mesenchymal transition (EMT) and aberrantly express EMT-associated transcriptional repressors, like ZEB1. The amount of such cells strongly correlates with metastasis formation and poor clinical outcome. Strikingly, metastases show again a differentiated phenotype, indicating a mesenchymal-epithelial re-transition (MET) and a support a regulatory role of the tumor environment for malignant tumor progression. We described that the EMT-activator ZEB1 is a crucial promoter of metastasis and demonstrated that ZEB1 inhibits expression of cell polarity factors and the microRNA-200 family, whose members are strong inducers of epithelial differentiation. These results indicate that ZEB1 triggers a microRNA-mediated feedback-loop, which stabilizes EMT and promotes dissemination of cancer cells. Moreover we detected that in addition ZEB1 is necessary for the tumor initiating capacity of pancreatic and colorectal cancer cells. ZEB1 inhibits expression of miR-200c, miR-203 and miR-183, which cooperate to suppress expression of stem cell factors, as demonstrated for the polycomb repressor Bmi1. We propose that ZEB1 links EMT-activation and stemness-maintenance by suppressing stemness-inhibiting microRNAs and thereby is a promoter of mobile, migrating cancer stem cells. Notably, these cells also acquired a drug-resistance phenotype. Thus, targeting the ZEB1 - miR-200 feedback loop might be a promising treatment option for fatal tumors, such as pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY05-03. doi:1538-7445.AM2012-SY05-03

Abstract: Interleukin-1 beta (IL-1β) is an important mediator of inflammatory response, and the elevated expression of IL-1β is correlated with tumor growth and metastasis. Epithelialmesenchymal transition (EMT) is a reversible transition between epithelial phenotype and mesenchymal phenotype. Usually, EMT can be identified by its unique morphology change and expression of EMT markers. In our study, we have found after treated HCT-116, a colon cancer cell line, and human primary colon cancer cells with IL-1β, cells began to display mesenchymal phenotype with highly down-regulated E-cadherin expression and up-regulated ZEB factors expression. For colon cancer cells, sphere formation assay in serum free medium (SFM) with the presence of growth factors is used to identify cancer stem cell population. We have shown that IL-1β can induce colon cancer stem cell proliferation and express stem cell markers (Bmi1, Nanog, and Nestin). In addition, besides the stem cell markers, we also found ZEB factors were highly up-regulated in spheroid cells as well. We silenced Zeb1 expression and investigated the effect of IL-1β on shZeb1 HCT-116 cells. The results indicated Zeb1 knockdown not only inhibited IL-1β-induced EMT but also reduced proliferation of spheroid cells and inhibited Bmi1 expression. Therefore, ZEB factors must play an important role in both EMT process and cancer stem cell development. From our data, we conclude that IL-1β promotes epithelial-mesenchymal transition and a stem cell phenotype in colon cancer via ZEB factors.

Abstract: Neuroblastomas (NBs) are neuro-ectodermal tumors of embryonic neural crest-derived cells. The neural crest in normal development gives rise to nerve cells of the sympathetic nervous system. Amplification of the proto-oncogene MYCN is the most prototypic genetic aberration in NBs and is found in 20–25% of all NBs. MYCN-amplified tumors follow a very aggressive course and are strongly associated with additional structural abnormalities. Recent advances in NB research addressed whether epigenetic alterations, such as hypermethylation of promoter sequences with consequent silencing of tumor-suppressor genes, can have significant roles in the tumorigenesis of NB; however, the exact role of epigenetic alterations, except for DNA hypermethylation, remains to be elucidated in NB research. Recently, we identified the direct binding of MYCN to Bmi1 promoter and upregulation of Bmi1 transcription by MYCN (Ochiai et al., 2010, Oncogene 29:2681–2690). Bmi1 has an important role in NB cell proliferation and differentiation. Intriguingly, the above-mentioned Bmi1-related regulation of the NB cell phenotype seems not to be mediated only by p14ARF/p16INK4a in NB cells. Expression profiling analysis using a tumor-specific cDNA microarray addressed the Bmi1-dependent repression of KIF1Bβ and TSLC1 and found that it has important roles in predicting the prognosis of NB (Ando et al., 2008, Int J Cancer 123:2087–2094; Munirajan et al., 2008, J Biol Chem 283:24426–24434). These findings suggest that MYCN induces Bmi1 expression, resulting in the repression of tumor suppressors through Polycomb group gene-mediated epigenetic chromosome modification.

Abstract: The ability of cancer cells to divide indefinitely whilst supporting tumor growth, metastasis and invasiveness resembles the behavior of stem cells. Here, we overview the role of Polycomb (PcG)-dependent epigenetic silencing mechanisms in stem cell biology and cancer, focusing on two major PcG components, Ezh2 and Bmi1. In a recent patent, stem cell PcG targets were shown to be more prone to cancer-specific promoter DNA methylation than non-targets, indi- cating that reversible PcG-mediated gene repression becomes replaced by permanent silencing. This epigenetic switching keeps the cell in a sustained state of self-renewal, predisposing it to tumorigenic transformation. These findings might provide the means of identifying the stem-cell epigenetic signatures associated with the origin of specific types of cancer. Based on the reversibility of epigenetic histone modifications, PcG proteins have become established targets in clinical practice for the treatment of a variety of cancers, notably treatments with histone deacetylase and methyltransferase in- hibitors. A number of reports and patents highlight the potential of alternative approaches to targeting PcG for cancer therapy, including micro-RNA expression and the use of Hedgehog signaling pathway antagonists. The major shortcom- ing of current approaches is their lack of specificity. The identification of tumorigenic epigenetic alterations together with the development of inhibitors to target them promises to open the way toward personalized cancer treatment.

Abstract: Studies have shown that wild-type hTERT protein can functionally replace the HPV16E6 protein, which cooperates with the viral E7 protein in the immortalization of primary keratinocytes. Previously, we made the surprising finding that catalytically inactive hTERT (hTERT), elongation-defective hTERT (hTERT-HA), and telomere recruitment-defective (hTERT N+T) also cooperate with E7 in cell immortalization, indicating that hTERT has immortalizing activities independent of its telomere maintenance functions. Since reports show an hTERT role in gene activation, we performed microarray studies to discover that E6, hTERT and hTERT mutated proteins altered the expression of highly overlapping sets of cellular genes. Pursuing in-depth studies of these targets shared by E6 and hTERT, we focused on AIB1, a nuclear coactivator known to be elevated in some cancers, and BMI1, the core subunit of the Polycomb Group Repressor Complex (PRC) 1 which is known to play a role in iii immortalization and determining cell fate. We proved that AIB1 levels were increased in a number of cervical cancer cell lines. Additionally, both AIB1 and BMI1 are elevated in HPV-immortalized cell lines. We showed further that BMI1 can substitute for E6 or hTERT in cell immortalization. Finally, in vivo tissue studies revealed expression of AIB1 and BMI1 increase with the severity of cervical dysplasia, suggesting a potential role in cervical cancer. Whereas BMI1 appears to be a marker of progression differentiating between pre-neoplastic and neoplastic lesions, AIB1 appears to delineate invasion from earlier stages, significantly increasing in invasive carcinoma. Together, these data demonstrate that AIB1 may be a novel cellular target of the HPV E6 oncoprotein and that hTERT has extratelomeric activities in cell immortalization and that its induction of BMI1 is a potential mechanism for mediating this activity.