05-175 Sigma-AldrichAnti-Myb Antibody, clone 1-1

Decades of studies provided a detailed view of the mechanism of estrogen receptor-α (ERα) regulated gene transcription and the physio-pathological relevance of the genetic programs controlled by this receptor in a variety of tissues. However, still limited is our knowledge on the regulation of ERα synthesis. Preliminary observations showed that the expression of ERα is cell cycle regulated. Here, we have demonstrated that a well described polymorphic sequence in the first intron of ERα (PvuII and XbaI) has a key role in regulating the ERα content in cycling cells. We have shown that the RNA Pol II (Pol II) elongation is blocked at the polymorphic site and that the proto-oncogene c-MYB modulates the release of the pausing polymerase. It is well known that the two SNPs are associated to an increased risk, progression, survival and mortality of endocrine-related cancers, here we have demonstrated that the c-MYB-dependent release of Pol II at a specific phase of the cell cycle is facilitated by the px haplotype, thus leading to a higher ERα mitogenic signal. In breast cancer, this mechanism is disrupted when the hormone refractory phenotype is established; therefore, we propose this oscillator as a novel target for the development of therapies aimed at sensitizing breast cancer resistant to hormonal treatments. Because PvuII and XbaI were associated to a broad range physio-pathological conditions beside neoplastic transformation, we expect that the ERα oscillator contributes to the regulation of the estrogen signal in several tissues.

Satellite cells represent a heterogeneous population of stem and progenitor cells responsible for muscle growth, repair and regeneration. We investigated whether c-Myb could play a role in satellite cell biology because our previous results using satellite cell-derived mouse myoblast cell line C2C12 showed that c-Myb was expressed in growing cells and downregulated during differentiation. We detected c-Myb expression in activated satellite cells of regenerating muscle. c-Myb was also discovered in activated satellite cells associated with isolated viable myofiber and in descendants of activated satellite cells, proliferating myoblasts. However, no c-Myb expression was detected in multinucleated myotubes originated from fusing myoblasts. The constitutive expression of c-Myb lacking the 3' untranslated region (3' UTR) strongly inhibited the ability of myoblasts to fuse. The inhibition was dependent on intact c-Myb transactivation domain as myoblasts expressing mutated c-Myb in transactivation domain were able to fuse. The absence of 3' UTR of c-Myb was also important because the expression of c-Myb coding region with its 3' UTR did not inhibit myoblast fusion. The same results were repeated in C2C12 cells as well. Moreover, it was documented that 3' UTR of c-Myb was responsible for downregulation of c-Myb protein levels in differentiating C2C12 cells. DNA microarray analysis of C2C12 cells revealed that the expression of several muscle-specific genes was downregulated during differentiation of c-Myb-expressing cells, namely: ACTN2, MYH8, TNNC2, MYOG, CKM and LRRN1. A detailed qRT-PCR analysis of MYOG, TNNC2 and LRRN1 is presented. Our findings thus indicate that c-Myb is involved in regulating the differentiation program of myogenic progenitor cells as its expression blocks myoblast fusion.

Epithelial-to-mesenchymal transition (EMT) promotes cell migration and is important in metastasis. Cellular proliferation is often downregulated during EMT, and the reverse transition (MET) in metastases appears to be required for restoration of proliferation in secondary tumors. We studied the interplay between EMT and proliferation control by MYB in breast cancer cells.MYB, ZEB1, and CDH1 expression levels were manipulated by lentiviral small-hairpin RNA (shRNA)-mediated knockdown/overexpression, and verified with Western blotting, immunocytochemistry, and qRT-PCR. Proliferation was assessed with bromodeoxyuridine pulse labeling and flow cytometry, and sulforhodamine B assays. EMT was induced with epidermal growth factor for 9 days or by exposure to hypoxia (1% oxygen) for up to 5 days, and assessed with qRT-PCR, cell morphology, and colony morphology. Protein expression in human breast cancers was assessed with immunohistochemistry. ZEB1-MYB promoter binding and repression were determined with Chromatin Immunoprecipitation Assay and a luciferase reporter assay, respectively. Student paired t tests, Mann-Whitney, and repeated measures two-way ANOVA tests determined statistical significance (P less than 0.05).Parental PMC42-ET cells displayed higher expression of ZEB1 and lower expression of MYB than did the PMC42-LA epithelial variant. Knockdown of ZEB1 in PMC42-ET and MDA-MB-231 cells caused increased expression of MYB and a transition to a more epithelial phenotype, which in PMC42-ET cells was coupled with increased proliferation. Indeed, we observed an inverse relation between MYB and ZEB1 expression in two in vitro EMT cell models, in matched human breast tumors and lymph node metastases, and in human breast cancer cell lines. Knockdown of MYB in PMC42-LA cells (MYBsh-LA) led to morphologic changes and protein expression consistent with an EMT. ZEB1 expression was raised in MYBsh-LA cells and significantly repressed in MYB-overexpressing MDA-MB-231 cells, which also showed reduced random migration and a shift from mesenchymal to epithelial colony morphology in two dimensional monolayer cultures. Finally, we detected binding of ZEB1 to MYB promoter in PMC42-ET cells, and ZEB1 overexpression repressed MYB promoter activity.This work identifies ZEB1 as a transcriptional repressor of MYB and suggests a reciprocal MYB-ZEB1 repressive relation, providing a mechanism through which proliferation and the epithelial phenotype may be coordinately modulated in breast cancer cells.

Mir-29 microRNA families are involved in regulation of various types of cancers. Although Mir-29 was shown to play an inhibitory role in tumorigenesis, the role of Mir-29 in breast cancer still remains obscure. In this study, we showed that Mir-29a is the dominant isoform in its family in mammary cells and expression of Mir-29a was down-regulated in different types of breast cancers. Furthermore, over-expression of Mir-29a resulted in significant slower growth of breast cancer cells and caused higher percentage of cells at G0/G1 phase. Consistent with this over-expression data, knockdown of Mir-29a in normal mammary cells lead to higher cell growth rate, and higher percentage of cells entering S phase. We further found that Mir-29a negatively regulated expression of B-Myb, which is a transcription factor associated with tumorigenesis. The protein levels of Cyclin A2 and D1 are consistent with the protein level of B-Myb. Taken together, our data suggests Mir-29a plays an important role in inhibiting growth of breast cancer cells and arresting cells at G0/G1 phase. Our data also suggests that Mir-29a may suppress tumor growth through down-regulating B-Myb.

In this study we report that expression of glioma pathogenesis-related protein 1 (GLIPR1) regulated numerous apoptotic, cell cycle, and spindle/centrosome assembly-related genes, including AURKA and TPX2, and induced apoptosis and/or mitotic catastrophe (MC) in prostate cancer (PCa) cells, including p53-mutated/deleted, androgen-insensitive metastatic PCa cells. Mechanistically, GLIPR1 interacts with heat shock cognate protein 70 (Hsc70); this interaction is associated with SP1 and c-Myb destabilization and suppression of SP1- and c-Myb-mediated AURKA and TPX2 transcription. Inhibition of AURKA and TPX2 using siRNA mimicked enforced GLIPR1 expression in the induction of apoptosis and MC. Recombinant GLIPR1-ΔTM protein inhibited AURKA and TPX2 expression, induced apoptosis and MC, and suppressed orthotopic xenograft tumor growth. Our results define a novel GLIPR1-regulated signaling pathway that controls apoptosis and/or mitotic catastrophe in PCa cells and establishes the potential of this pathway for targeted therapies.

Children with Down syndrome develop a unique congenital clonal megakaryocytic proliferation disorder (transient myeloproliferative disorder [TMD]). It is caused by an expansion of fetal megakaryocyte-erythroid progenitors (MEPs) triggered by trisomy of chromosome 21 and is further enhanced by the somatic acquisition of a mutation in GATA1. These mutations result in the expression of a short-isoform GATA1s lacking the N-terminal domain. To examine the hypothesis that the Hsa21 ETS transcription factor ERG cooperates with GATA1s in this process, we generated double-transgenic mice expressing hERG and Gata1s. We show that increased expression of ERG by itself is sufficient to induce expansion of MEPs in fetal livers. Gata1s expression synergizes with ERG in enhancing the expansion of fetal MEPs and megakaryocytic precursors, resulting in hepatic fibrosis, transient postnatal thrombocytosis, anemia, a gene expression profile that is similar to that of human TMD and progression to progenitor myeloid leukemia by 3 months of age. This ERG/Gata1s transgenic mouse model also uncovers an essential role for the N terminus of Gata1 in erythropoiesis and the antagonistic role of ERG in fetal erythroid differentiation and survival. The human relevance of this finding is underscored by the recent discovery of similar mutations in GATA1 in patients with Diamond-Blackfan anemia.

The oncogenic transcription factor TAL1/SCL is aberrantly expressed in 60% of cases of human T cell acute lymphoblastic leukemia (T-ALL) and initiates T-ALL in mouse models. By performing global microRNA (miRNA) expression profiling after depletion of TAL1, together with genome-wide analysis of TAL1 occupancy by chromatin immunoprecipitation coupled to massively parallel DNA sequencing, we identified the miRNA genes directly controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3, and RUNX1. The most dynamically regulated miRNA was miR-223, which is bound at its promoter and up-regulated by the TAL1 complex. miR-223 expression mirrors TAL1 levels during thymic development, with high expression in early thymocytes and marked down-regulation after the double-negative-2 stage of maturation. We demonstrate that aberrant miR-223 up-regulation by TAL1 is important for optimal growth of TAL1-positive T-ALL cells and that sustained expression of miR-223 partially rescues T-ALL cells after TAL1 knockdown. Overexpression of miR-223 also leads to marked down-regulation of FBXW7 protein expression, whereas knockdown of TAL1 leads to up-regulation of FBXW7 protein levels, with a marked reduction of its substrates MYC, MYB, NOTCH1, and CYCLIN E. We conclude that TAL1-mediated up-regulation of miR-223 promotes the malignant phenotype in T-ALL through repression of the FBXW7 tumor suppressor.

Tumor progression to metastasis is a complex, sequential process that requires proliferation, resistance to apoptosis, motility and invasion to colonize at distant sites. The acquisition of these features implies a phenotypic plasticity by tumor cells that must adapt to different conditions by modulating several signaling pathways (1) during the journey to the final site of metastasis. Several transcription factors and microRNA play a role in tumor progression, but less is known about the control of their expression during this process. Here, we demonstrate by ectopic expression and gene silencing that the proto-oncogene c-Myb activates the expression of the 5 members of miR200 family (miR200b, miR200a, miR429, miR200c and miR141) that are involved in the control of epithelial-mesenchymal transition (EMT) and metastasis in many types of cancers. Transcriptional activation of miR200 by c-Myb occurs through binding to myb binding sites located in the promoter regions of miR200 genes on human chromosomes 1 and 12. Furthermore, when c-Myb and the transcriptional repressor ZEB1 are co-expressed, as at the onset EMT, the repression by ZEB1 prevails over the activation by c-Myb, and the expression of miR200 is inhibited. We also demonstrate that during EMT induced by TGF-β, the promoters of miR200 genes are methylated, and their transcription is repressed regardless of the presence of repressors such as ZEB1 and activators such as c-Myb. Finally, we find a correlation between the expression of c-Myb and that of four out of 5 miR200 in a data set of 207 breast cancer patients.

The temporal control of RAG (Rag) expression in developing lymphocytes prevents DNA breaks during periods of proliferation that could threaten genomic integrity. In developing B cells, the IL-7R and precursor B cell Ag receptor (pre-BCR) synergize to induce proliferation and the repression of Rag at the protein and mRNA levels for a brief period following successful Ig H chain gene rearrangement. Whereas the mechanism of RAG2 protein downregulation is well defined, little is known about the pathways and transcription factors that mediate transcriptional repression of Rag. Using Abelson murine leukemia virus-transformed B cells to model this stage of development, we identified early B cell factor 1 (Ebf1) as a strong repressor of Rag transcription. Short hairpin RNA-mediated knockdown of either Ebf1 or its downstream target c-Myb was sufficient to induce Rag transcription in these highly proliferative cells. Ebf1 and c-Myb antagonize Rag transcription by negatively regulating the binding of Foxo1 to the Rag locus. Ebf1 accomplishes this through both direct negative regulation of Foxo1 expression and direct positive regulation of Gfi1b expression. Ebf1 expression is driven by the IL-7R downstream effector Stat5, providing a link between the negative regulation of Rag transcription by IL-7 and a novel repressive pathway involving Ebf1 and c-Myb.

Glucocorticoid (GC) hormones are used in the treatment of hematopoietic malignancies. When the GC binds to the glucocorticoid receptor (GR) protein, c-Myb and GR are recruited at the Glucocorticoid Response Unit in the DNA. Here we demonstrate that c-Myb interacts with the GR and that decreasing c-Myb amounts reduces the levels of GR transcripts and protein in 697 pre-B-acute lymphoblastic leukemia (ALL) cells. Furthermore, the auto-upregulation of GR promoter 1C and promoter 1D is blunted at reduced c-Myb levels. Taken together, these data show that c-Myb is a direct, key regulator of the GR. Unexpectedly, the reduction in c-Myb levels increased the sensitivity of the cells to steroid-mediated apoptosis. This was because the reduction in c-Myb itself decreases cell viability, and the residual GR remained above the threshold needed to trigger apoptosis. These studies show the mutual importance of c-Myb and the GR in controlling survival of pre-B ALL cells.