Lloyd Pereira

Myb Controls Intestinal Stem Cell Genes and Self-Renewal

Rapid advances have been made in the understanding of how the highly proliferative gastrointestinal tract epithelium is regulated under homeostasis and disease. The identification of putative intestinal stem cell (ISC) genes and the ability to culture ISC capable of generating all four lineages plus the architecture of small intestinal (SI) crypts has been transformative. Here, we show that transcription factor Myb governs ISC gene expression, particularly Lgr5. Lgr5 is associated with cells that have the capacity to generate all cell lineages in SI organoid cultures and colorectal cancer cells, which overexpress Myb. Furthermore, Wnt signaling and Myb cooperate in maximal Lgr5 promoter activation while hypomorphic Myb (plt4/plt4) mice have decreased Lgr5 expression. After ionizing radiation (IR), ISC genes are elevated; but in plt4/plt4 mice, this response is substantially subdued. ISC genes bmi‐1 and olfm4 are expressed at subnormal levels in plt4/plt4 mice, and bmi‐1 is induced with IR to half the level in mutant mice. dcamkl‐1 and olfm4 failed to recover after IR in both wild‐type (wt) and mutant mice. Although not considered as an ISC gene, cyclinE1 is nevertheless used to assist cells in the emergence from a quiescent state (an expectation of ISC following IR) and is overexpressed after IR in wt mice but does not change from a very low base in plt4/plt4 mice. Self‐renewal assays using organoid cultures and inducible Myb knockout studies further highlighted the dependence of ISC on Myb consistent with role in other stem cell‐containing tissues. Stem Cells 2011;29:2042–2050.

Brachyury and Related Tbx Proteins Interact with the Mixl1 Homeodomain Protein and Negatively Regulate Mixl1 Transcriptional Activity

Mixl1 is a homeodomain transcription factor required for mesoderm and endoderm patterning during mammalian embryogenesis. Despite its crucial function in development, co-factors that modulate the activity of Mixl1 remain poorly defined. Here we report that Mixl1 interacts physically and functionally with the T-box protein Brachyury and related members of the T-box family of transcription factors. Transcriptional and protein analyses demonstrated overlapping expression of Mixl1 and Brachyury during embryonic stem cell differentiation. In vitro protein interaction studies showed that the Mixl1 with Brachyury associated via their DNA-binding domains and gel shift assays revealed that the Brachyury T-box domain bound to Mixl1-DNA complexes. Furthermore, luciferase reporter experiments indicated that association of Mixl1 with Brachyury and related T-box factors inhibited the transactivating potential of Mixl1 on the Gsc and Pdgfrα promoters. Our results indicate that the activity of Mixl1 can be modulated by protein-protein interactions and that T-box factors can function as negative regulators of Mixl1 activity.

MYB Is Essential for Mammary Tumorigenesis

MYB oncogene upregulation is associated with estrogen receptor (ER)-positive breast cancer, but disease requirements for MYB function in vivo have not been explored. In this study, we provide evidence of a critical requirement for MYB functions in models of human and murine breast cancer. In human breast cancer, we found that MYB expression was critical for tumor cell growth both in vitro and in vivo in xenograft settings. In transgenic knockout mice, tissue-specific deletion of the murine MYB gene caused a transient defect in mammary gland development that was reflected in delayed ductal branching and defective apical bud formation. In mouse mammary tumor virus (MMTV)-NEU mice where tumors are initiated by activation of HER2, MYB deletion was sufficient to abolish tumor formation. In the more aggressive MMTV-PyMT model system, MYB deletion delayed tumorigenesis significantly. Together, the findings in these transgenic knockout models implied that MYB was critical during an early window in mammary development when it was essential for tumor initiation, even though MYB loss did not exert a lasting impact upon normal mammary function. Two important MYB-target genes that promote cell survival, BCL2 and GRP78/BIP, were each elevated compared with nontransformed mammary epithelial cells, thereby promoting survival as confirmed in colony formation assays in vitro. Taken together, our findings establish a role for MYB at the hub of ER- and HER2-dependent pathways in mammary carcinogenesis.

Enforced Expression of Mixl1 During Mouse ES Cell Differentiation Suppresses Hematopoietic Mesoderm and Promotes Endoderm Formation

The Mixl1 gene encodes a homeodomain transcription factor that is required for normal mesoderm and endoderm development in the mouse. We have examined the consequences of enforced Mixl1 expression during mouse embryonic stem cell (ESC) differentiation. We show that three independently derived ESC lines constitutively expressing Mixl1 (Mixl1C ESCs) differentiate into embryoid bodies (EBs) containing a higher proportion of E‐cadherin (E‐Cad)+ cells. Our analysis also shows that this differentiation occurs at the expense of hematopoietic mesoderm differentiation, with Mixl1C ESCs expressing only low levels of Flk1 and failing to develop hemoglobinized cells. Immunohistochemistry and immunofluorescence studies revealed that Mixl1C EBs have extensive areas containing cells with an epithelial morphology that express E‐Cad, FoxA2, and Sox17, consistent with enhanced endoderm formation. Luciferase reporter transfection experiments indicate that Mixl1 can transactivate the Gsc, Sox17, and E‐Cad promoters, supporting the hypothesis that Mixl1 has a direct role in definitive endoderm formation. Taken together, these studies suggest that high levels of Mixl1 preferentially allocate cells to the endoderm during ESC differentiation. STEM CELLS 2009;27:363–474

The Mix family of homeobox genes--key regulators of mesendoderm formation during vertebrate development.

The Mix/Bix family of paired-like homeobox genes encode evolutionarily conserved, sequence specific, DNA-binding transcription factors that have been implicated in the co-ordination of gene expression, axis formation and cell fate determination during gastrulation in vertebrates. When mutated, these genes give rise to dramatic phenotypes in amphibians, zebrafish and mice, that can be traced back to defects in the formation and specification of mesoderm and endoderm. We review here the biochemical properties of the Mix/Bix proteins and summarise genetic, molecular and embryological studies of Mix/Bix function in mesendoderm development. We emphasise recent data generated using embryonic stem cell differentiation systems that have provided important new insights into Mix/Bix function and the biological roles of these proteins in regulating the earliest phases of vertebrate development.

WNT3A Promotes Hematopoietic or Mesenchymal Differentiation from hESCs Depending on the Time of Exposure

We investigated the role of canonical WNT signaling in mesoderm and hematopoietic development from human embryonic stem cells (hESCs) using a recombinant human protein-based differentiation medium (APEL). In contrast to prior studies using less defined culture conditions, we found that WNT3A alone was a poor inducer of mesoderm. However, WNT3A synergized with BMP4 to accelerate mesoderm formation, increase embryoid body size, and increase the number of hematopoietic blast colonies. Interestingly, inclusion of WNT3A or a GSK3 inhibitor in methylcellulose colony-forming assays at 4 days of differentiation abrogated blast colony formation but supported the generation of mesospheres that expressed genes associated with mesenchymal lineages. Mesospheres differentiated into cells with characteristics of bone, fat, and smooth muscle. These studies identify distinct effects for WNT3A, supporting the formation of hematopoietic or mesenchymal lineages from human embryonic stem cells, depending upon differentiation stage at the time of exposure.

Estrogen receptor-α recruits P-TEFb to overcome transcriptional pausing in intron 1 of the MYB gene

The MYB proto-oncogene is expressed in most estrogen receptor-positive (ERα+) breast tumors and cell lines. Expression of MYB is controlled, in breast cancer and other cell types, by a transcriptional pausing mechanism involving an attenuation site located ∼1.7 kb downstream from the transcription start site. In breast cancer cells, ligand-bound ERα binds close to, and drives transcription beyond this attenuation site, allowing synthesis of complete transcripts. However, little is known, in general, about the factors involved in relieving transcriptional attenuation, or specifically how ERα coordinates such factors to promote transcriptional elongation. Using cyclin dependent kinase 9 (CDK9) inhibitors, reporter gene assays and measurements of total and intronic MYB transcription, we show that functionally active CDK9 is required for estrogen-dependent transcriptional elongation. We further show by ChIP and co-immunoprecipitation studies that the P-TEFb complex (CDK9/CyclinT1) is recruited to the attenuation region by ligand-bound ERα, resulting in increased RNA polymerase II Ser-2 phosphorylation. These data provide new insights into MYB regulation, and given the critical roles of MYB in tumorigenesis, suggest targeting MYB elongation as potential therapeutic strategy.

The Myb-p300-CREB axis modulates intestine homeostasis, radiosensitivity and tumorigenesis

The gastrointestinal (GI) epithelium is constantly renewing, depending upon the intestinal stem cells (ISC) regulated by a spectrum of transcription factors (TFs), including Myb. We noted previously in mice with a p300 mutation (plt6) within the Myb-interaction-domain phenocopied Myb hypomorphic mutant mice with regard to thrombopoiesis, and here, changes in GI homeostasis. p300 is a transcriptional coactivator for many TFs, most prominently cyclic-AMP response element-binding protein (CREB), and also Myb. Studies have highlighted the importance of CREB in proliferation and radiosensitivity, but not in the GI. This prompted us to directly investigate the p300–Myb–CREB axis in the GI. Here, the role of CREB has been defined by generating GI-specific inducible creb knockout (KO) mice. KO mice show efficient and specific deletion of CREB, with no evident compensation by CREM and ATF1. Despite complete KO, only modest effects on proliferation, radiosensitivity and differentiation in the GI under homeostatic or stress conditions were evident, even though CREB target gene pcna (proliferating cell nuclear antigen) was downregulated. creb and p300 mutant lines show increased goblet cells, whereas a reduction in enteroendocrine cells was apparent only in the p300 line, further resembling the Myb hypomorphs. When propagated in vitro, crebKO ISC were defective in organoid formation, suggesting that the GI stroma compensates for CREB loss in vivo, unlike in MybKO studies. Thus, it appears that p300 regulates GI differentiation primarily through Myb, rather than CREB. Finally, active pCREB is elevated in colorectal cancer (CRC) cells and adenomas, and is required for the expression of drug transporter, MRP2, associated with resistance to Oxaliplatin as well as several chromatin cohesion protein that are relevant to CRC therapy. These data raise the prospect that CREB may have a role in GI malignancy as it does in other cancer types, but unlike Myb, is not critical for GI homeostasis.Cell Death and Disease (2013) 4, e605; doi:10.1038/cddis.2013.119; published online 25 April 2013

Implications of epithelial-mesenchymal plasticity for heterogeneity in colorectal cancer

Colorectal cancer (CRC) is a genetically heterogeneous disease that develops and progresses through several distinct pathways characterized by genomic instability. In recent years, it has emerged that inherent plasticity in some populations of CRC cells can contribute to heterogeneity in differentiation state, metastatic potential, therapeutic response, and disease relapse. Such plasticity is thought to arise through interactions between aberrant signaling events, including persistent activation of the APC/β-catenin and KRAS/BRAF/ERK pathways, and the tumor microenvironment. Here, we highlight key concepts and evidence relating to the role of epithelial–mesenchymal plasticity as a driver of CRC progression and stratification of the disease into distinct molecular and clinicopathological subsets.

Intestinal-specific activatable Myb initiates colon tumorigenesis in mice

Transcription factor Myb is overexpressed in most colorectal cancers (CRC). Patients with CRC expressing the highest Myb are more likely to relapse. We previously showed that mono-allelic loss of Myb in an Adenomatous polyposis coli (APC)-driven CRC mouse model (ApcMin/+) significantly improves survival. Here we directly investigated the association of Myb with poor prognosis and how Myb co-operates with tumor suppressor genes (TSGs) (Apc) and cell cycle regulator, p27. Here we generated the first intestinal-specific, inducible transgenic model; a MybER transgene encoding a tamoxifen-inducible fusion protein between Myb and the estrogen receptor-α ligand-binding domain driven by the intestinal-specific promoter, Gpa33. This was to mimic human CRC with constitutive Myb activity in a highly tractable mouse model. We confirmed that the transgene was faithfully expressed and inducible in intestinal stem cells (ISCs) before embarking on carcinogenesis studies. Activation of the MybER did not change colon homeostasis unless one p27 allele was lost. We then established that MybER activation during CRC initiation using a pro-carcinogen treatment, azoxymethane (AOM), augmented most measured aspects of ISC gene expression and function and accelerated tumorigenesis in mice. CRC-associated symptoms of patients including intestinal bleeding and anaemia were faithfully mimicked in AOM-treated MybER transgenic mice and implicated hypoxia and vessel leakage identifying an additional pathogenic role for Myb. Collectively, the results suggest that Myb expands the ISC pool within which CRC is initiated while co-operating with TSG loss. Myb further exacerbates CRC pathology partly explaining why high MYB is a predictor of worse patient outcome.