Yin Yang 1 is associated with cancer stem cell transcription factors (SOX2, OCT4, BMI1) and clinical implication.

The transcription factor Yin Yang 1 (YY1) is frequently overexpressed in cancerous tissues compared to normal tissues and has regulatory roles in cell proliferation, cell viability, epithelial-mesenchymal transition, metastasis and drug/immune resistance. YY1 shares many properties with cancer stem cells (CSCs) that drive tumorigenesis, metastasis and drug resistance and are regulated by overexpression of certain transcription factors, including SOX2, OCT4 (POU5F1), BMI1 and NANOG. Based on these similarities, it was expected that YY1 expression would be associated with SOX2, OCT4, BMI1, and NANOG's expressions and activities. Data mining from the proteomic tissue-based datasets from the Human Protein Atlas were used for protein expression patterns of YY1 and the four CSC markers in 17 types of cancer, including both solid and hematological malignancies. A close association was revealed between the frequency of expressions of YY1 and SOX2 as well as SOX2 and OCT4 in all cancers analyzed. Two types of dynamics were identified based on the nature of their association, namely, inverse or direct, between YY1 and SOX2. These two dynamics define distinctive patterns of BMI1 and OCT4 expressions. The relationship between YY1 and SOX2 expressions as well as the expressions of BMI1 and OCT4 resulted in the classification of four groups of cancers with distinct molecular signatures: (1) Prostate, lung, cervical, endometrial, ovarian and glioma cancers (YY1(lo)SOX2(hi)BMI1(hi)OCT4(hi)) (2) Skin, testis and breast cancers (YY1(hi)SOX2(lo)BMI1(hi)OCT4(hi)) (3) Liver, stomach, renal, pancreatic and urothelial cancers (YY1(lo)SOX2(lo)BMI1(hi)OCT4(hi)) and (4) Colorectal cancer, lymphoma and melanoma (YY1(hi)SOX2(hi)BMI1(lo)OCT4(hi)). A regulatory loop is proposed consisting of the cross-talk between the NF-kB/PI3K/AKT pathways and the downstream inter-regulation of target gene products YY1, OCT4, SOX2 and BMI1

Bmi1 loss produces an increase in astroglial cells and a decrease in neural stem cell population and proliferation

The polycomb transcriptional repressor Bmi1 promotes cell cycle progression, controls cell senescence, and is implicated in brain development. Loss of Bmi1 leads to a decreased brain size and causes progressive ataxia and epilepsy. Recently, Bmi1 was shown to control neural stem cell (NSC) renewal. However, the effect of Bmi1 loss on neural cell fate in vivo and the question whether the action of Bmi1 was intrinsic to the NSCs remained to be investigated. Here, we show that Bmi1 is expressed in the germinal zone in vivo and in NSCs as well as in progenitors proliferating in vitro, but not in differentiated cells. Loss of Bmi1 led to a decrease in proliferation in zones known to contain progenitors: the newborn cortex and the newborn and adult subventricular zone. This decrease was accentuated in vitro, where we observed a drastic reduction in NSC proliferation and renewal because of NSC-intrinsic effects of Bmi1 as shown by the means of RNA interference. Bmi1(-/-) mice also presented more astrocytes at birth, and a generalized gliosis at postnatal day 30. At both stages, colocalization of bromodeoxyuridine and GFAP demonstrated that Bmi1 loss did not prevent astrocyte precursor proliferation. Supporting these observations, Bmi1(-/-) neurospheres generate preferentially astrocytes probably attributable to a different responsiveness to environmental factors. Bmi1 is therefore necessary for NSC renewal in a cell-intrinsic mode, whereas the altered cell pattern of the Bmi1(-/-) brain shows that in vivo astrocyte precursors can proliferate in the absence of Bmi1

Dissecting BMI1 Protein-Protein Interactions Through Chemical Biology.

BMI1 has emerged as a key oncogenic factor in many cancers, associated with unregulated cellular proliferation, tumor metastasis and cancer-initiating cell self-renewal. BMI1 is best characterized as a component of the canonical vertebrate polycomb repression complex 1 (PRC1) which negatively regulate transcription of hundreds of genes through ubiquitination of histone H2A. Previous work suggested that BMI1 has multiple protein binding partners within the PRC1 complex and we were motivated by the prospects to target these protein-protein interactions (PPIs) with small molecule inhibitors. This dissertation describes a multi-pronged campaign to: 1) characterize BMI1 PPIs at the molecular level and 2) develop novel chemical tools to explore BMI1 function in both normal and cancer biology. Using X-ray crystallography and solution NMR approaches we solved the 3D structure of BMI1 in complex with its PRC1 binding partner protein PHC2. Supporting biochemical and biophysical characterization of the BMI1 PPI domain demonstrated a novel mode of self-association of this domain. Mutagenic disruption of both BMI1-PHC2 and BMI1-BMI1 interactions blocks cellular proliferation demonstrating that multiple PPIs are critical for BMI1 function. To identify small molecule inhibitors of BMI1 we designed two biochemical assays to quantify the BMI1-PHC2 interaction and these assays were used as a platform for high-throughput screening. Through this screen we identified three classes of small molecule inhibitors that bind directly to BMI1 to disrupt the BMI1-PHC2 interaction, representing three different strategies for BMI1 inhibitor development. As a complementary approach to inhibit BMI1 we developed a specific inhibitor of Ring1B/BMI1- mediated H2A ubiquitination with potent inhibitory activity both in vitro and in cells. Mechanistic characterization demonstrates that Ring1B/BMI1 inhibitors induce significant protein conformational change and the inhibitor-bound conformation is incompatible with nucleosome binding by Ring1B. These molecules represent the first direct-binding inhibitors of Ring1B/BMI1 and have a novel mechanism of action to block direct protein-nucleosome interaction. Overall, this work contributes to the understanding of BMI1 function through characterization of its multiple PPIs and demonstrates that these interactions can be inhibited by small molecules representing novel strategies to target this protein for development of new chemical tools or potential therapeutics for cancer.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113363/1/flvgray_1.pd

Bmi1 Is Expressed in Postnatal Myogenic Satellite Cells, Controls Their Maintenance and Plays an Essential Role in Repeated Muscle Regeneration

PMCID: PMC3212532This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Age-dependent decline in beta-cell proliferation restricts the capacity of beta-cell regeneration in mice.

ObjectiveThe aim of this study was to elucidate whether age plays a role in the expansion or regeneration of beta-cell mass.Research design and methodsWe analyzed the capacity of beta-cell expansion in 1.5- and 8-month-old mice in response to a high-fat diet, after short-term treatment with the glucagon-like peptide 1 (GLP-1) analog exendin-4, or after streptozotocin (STZ) administration.ResultsYoung mice responded to high-fat diet by increasing beta-cell mass and beta-cell proliferation and maintaining normoglycemia. Old mice, by contrast, did not display any increases in beta-cell mass or beta-cell proliferation in response to high-fat diet and became diabetic. To further assess the plasticity of beta-cell mass with respect to age, young and old mice were injected with a single dose of STZ, and beta-cell proliferation was analyzed to assess the regeneration of beta-cells. We observed a fourfold increase in beta-cell proliferation in young mice after STZ administration, whereas no changes in beta-cell proliferation were observed in older mice. The capacity to expand beta-cell mass in response to short-term treatment with the GLP-1 analog exendin-4 also declined with age. The ability of beta-cell mass to expand was correlated with higher levels of Bmi1, a polycomb group protein that is known to regulate the Ink4a locus, and decreased levels of p16(Ink4a)expression in the beta-cells. Young Bmi1(-/-) mice that prematurely upregulate p16(Ink4a)failed to expand beta-cell mass in response to exendin-4, indicating that p16(Ink4a)levels are a critical determinant of beta-cell mass expansion.Conclusionsbeta-Cell proliferation and the capacity of beta-cells to regenerate declines with age and is regulated by the Bmi1/p16(Ink4a)pathway

Inhibition of chemotherapy resistant breast cancer stem cells by a ROR1 specific antibody.

Breast cancers enduring treatment with chemotherapy may be enriched for cancer stem cells or tumor-initiating cells, which have an enhanced capacity for self-renewal, tumor initiation, and/or metastasis. Breast cancer cells that express the type I tyrosine kinaselike orphan receptor ROR1 also may have such features. Here we find that the expression of ROR1 increased in breast cancer cells following treatment with chemotherapy, which also enhanced expression of genes induced by the activation of Rho-GTPases, Hippo-YAP/TAZ, or B lymphoma Mo-MLV insertion region 1 homolog (BMI1). Expression of ROR1 also enhanced the capacity of breast cancer cells to invade Matrigel, form spheroids, engraft in Rag2-/-[Formula: see text] mice, or survive treatment with paclitaxel. Treatment of mice bearing breast cancer patient-derived xenografts (PDXs) with the humanized anti-ROR1 monoclonal antibody cirmtuzumab repressed expression of genes associated with breast cancer stemness, reduced activation of Rho-GTPases, Hippo-YAP/TAZ, or BMI1, and impaired the capacity of breast cancer PDXs to metastasize or reengraft Rag2-/-[Formula: see text] mice. Finally, treatment of PDX-bearing mice with cirmtuzumab and paclitaxel was more effective than treatment with either alone in eradicating breast cancer PDXs. These results indicate that targeting ROR1 may improve the response to chemotherapy of patients with breast cancer

BMI1 and Mel-18 oppositely regulate carcinogenesis and progression of gastric cancer

<p>Abstract</p> <p>Background</p> <p>The <it>BMI1 </it>oncogene is overexpressed in several human malignancies including gastric cancer. In addition to BMI1, mammalian cells also express Mel-18, which is closely related to BMI1. We have reported that Mel-18 functions as a potential tumor suppressor by repressing the expression of BMI1 and consequent downregulation of activated AKT in breast cancer cells. However, the mechanisms of BMI1 overexpression and the role of Mel-18 in other cancers are still not clear. The purpose of this study is to investigate the role of BMI1 and Mel-18 in gastric cancer.</p> <p>Results</p> <p>BMI1 was found to be overexpressed in gastric cancer cell lines and gastric tumors. Overexpression of BMI1 correlated with advanced clinical stage and lymph node metastasis; while the expression of Mel-18 negatively correlated with BMI1. BMI1 but not Mel-18 was found to be an independent prognostic factor. Downregulation of BMI1 by Mel-18 overexpression or knockdown of BMI1 expression in gastric cancer cell lines led to upregulation of p16 (p16INK4a or CDKN2A) in p16 positive cell lines and reduction of phospho-AKT in both p16-positive and p16-negative cell lines. Downregulation of BMI1 was also accompanied by decreased transformed phenotype and migration in both p16- positive and p16-negative gastric cancer cell lines.</p> <p>Conclusions</p> <p>In the context of gastric cancer, <it>BMI1 </it>acts as an oncogene and Mel-18 functions as a tumor suppressor via downregulation of BMI1. Mel-18 and BMI1 may regulate tumorigenesis, cell migration and cancer metastasis via both p16- and AKT-dependent growth regulatory pathways.</p

GUCY2C maintains intestinal LGR5+ stem cells by opposing ER stress

Long-lived multipotent stem cells (ISCs) at the base of intestinal crypts adjust their phenotypes to accommodate normal maintenance and post-injury regeneration of the epithelium. Their long life, lineage plasticity, and proliferative potential underlie the necessity for tight homeostatic regulation of the ISC compartment. In that context, the guanylate cyclase C (GUCY2C) receptor and its paracrine ligands regulate intestinal epithelial homeostasis, including proliferation, lineage commitment, and DNA damage repair. However, a role for this axis in maintaining ISCs remains unknown. Transgenic mice enabling analysis of ISCs (Lgr5-GFP) in the context of GUCY2C elimination (Gucy2c-/-) were combined with immunodetection techniques and pharmacological treatments to define the role of the GUCY2C signaling axis in supporting ISCs. ISCs were reduced in Gucy2c-/- mice, associated with loss of active Lgr5+ cells but a reciprocal increase in reserve Bmi1+ cells. GUCY2C was expressed in crypt base Lgr5+ cells in which it mediates canonical cyclic (c) GMPdependent signaling. Endoplasmic reticulum (ER) stress, typically absent from ISCs, was elevated throughout the crypt base in Gucy2c-/- mice. The chemical chaperone tauroursodeoxycholic acid resolved this ER stress and restored the balance of ISCs, an effect mimicked by the GUCY2C effector 8Br-cGMP. Reduced ISCs in Gucy2c-/-mice was associated with greater epithelial injury and impaired regeneration following sub-lethal doses of irradiation. These observations suggest that GUCY2C provides homeostatic signals that modulate ER stress and cell vulnerability as part of the machinery contributing to the integrity of ISCs. © Kraft et al

BMI1 and KAP1 interaction and function: BMI1 capped by KAP1?

The Polycomb-repressive complex 1 (PRC1) protein BMI1 is of major importance in the epigenetic regulation of gene expression. The repression of important tumour suppressor genes (such a P16INK4a and P14ARF) by means of chromatin remodeling has marked BMI1 as a proto-oncoprotein. We previously found evidence that posttranslational modification by phosphorylation may be implicated in the stability and functioning of BMI1. Furthermore, we found that KAP1, through direct interaction with BMI1, may be implicated in regulation of BMI1 functioning. I here begin to elucidate how phosphorylation affects BMI1 and how KAP1 regulates BMI1. Several U2OS or TIG3ER cell lines were created that overexpressed BMI1 wild type and mutants that either contain phospho-mimic or phospho-null mutations. shRNA’s were used to effectively knockdown KAP1 expression. The effect of BMI1 mutant overexpression and/or KAP1 knock down on proliferation was measured under cell stress conditions induced by arsenite, selenite or etoposide. The effect of KAP1 knock down and mutant KAP1 lacking the RingFinger domain (KAP1-DeltaRF) on sub-cellular localization was assessed in U2OS cells. Finally functional interaction between KAP1 and PRC1 was measured by analysis of transcriptional induction of the PRC1-target gene ATF3 upon mitogenic stimulation. BMI1 overexpression partially rescues arsenite induced senescence; this rescue activity is affected by its phosphorylation status. KAP1 knockdown increases the effect of BMI1 overexpression on proliferation under arsenite induced cell stress but ablates the differences observed between different BMI1 phospho-mutants. KAP1 induced increases of ATF3 induction point towards a functional interaction between KAP1 and PRC1. My experiments provide experimental indication that BMI1 affects proliferation under arsenite induced cell stress condition. This effect was enhanced by KAP1 knockdown suggesting that KAP1 inhibits the pro-proliferative effects of BMI1. Increased ATF3 induction in the presence of KAP1-DeltaRF mutant protein suggests that the KAP1 negatively controls expression of ATF3 in a RF-dependent manner. Further research is required to elucidate the exact molecular mechanisms underlying the function interaction of BMI1 and KAP1.