Wijnen, André J. van

Ewing sarcoma is an aggressive pediatric small round cell tumor that predominantly occurs in bone. Approximately 85% of Ewing sarcomas harbor the EWS/FLI fusion protein, which arises from a chromosomal translocation, t(11:22)(q24:q12). EWS/FLI interacts with numerous lineage‐essential transcription factors to maintain mesenchymal progenitors in an undifferentiated state. We previously showed that EWS/FLI binds the osteogenic transcription factor RUNX2 and prevents osteoblast differentiation. In this study, we investigated the role of another Runt‐domain protein, RUNX3, in Ewing sarcoma. RUNX3 participates in mesenchymal‐derived bone formation and is a context dependent tumor suppressor and oncogene. RUNX3 was detected in all Ewing sarcoma cells examined, whereas RUNX2 was detected in only 73% of specimens. Like RUNX2, RUNX3 binds to EWS/FLI via its Runt domain. EWS/FLI prevented RUNX3 from activating the transcription of a RUNX‐responsive reporter, p6OSE2. Stable suppression of RUNX3 expression in the Ewing sarcoma cell line A673 delayed colony growth in anchorage independent soft agar assays and reversed expression of EWS/FLI‐responsive genes. These results demonstrate an important role for RUNX3 in Ewing sarcoma. J. Cell. Physiol. 229: 2049–2056, 2014. © 2014 Wiley Periodicals, Inc.

ABSTRACTThe chromatin remodeling complex SWI/SNF and the transcription factor C/EBPβ play critical roles in osteoblastic cells as they jointly control transcription of a number of bone‐related target genes. The largest C/EBPβ isoform, LAP*, possesses a short additional N‐terminal domain that has been proposed to mediate the interaction of this factor with SWI/SNF in myeloid cells. Here we examine the requirement of a functional N‐terminus in C/EBPβ‐LAP* for binding SWI/SNF and for recruiting this complex to the Ric‐8B gene to mediate transcriptional repression. We find that both C/EBPβ‐LAP* and SWI/SNF simultaneously bind to the Ric‐8B promoter in differentiating osteoblasts that repress Ric‐8B expression. This decreased expression of Ric‐8B is not accompanied by significant changes in histone acetylation at the Ric‐8B gene promoter sequence. A single aminoacid change at the C/EBPβ‐LAP* N‐terminus (R3L) that inhibits C/EBPβ‐LAP*‐SWI/SNF interaction, also prevents SWI/SNF recruitment to the Ric‐8B promoter as well as C/EBPβ‐LAP*‐dependent repression of the Ric‐8B gene. Inducible expression of the C/EBPβ‐LAP*R3L protein in stably transfected osteoblastic cells demonstrates that this mutant protein binds to C/EBPβ‐LAP*‐target promoters and competes with the endogenous C/EBPβ factor. Together our results indicate that a functional N‐terminus in C/EBPβ‐LAP* is required for interacting with SWI/SNF and for Ric‐8B gene repression in osteoblasts. J. Cell. Physiol. 229: 1521–1528, 2014. © 2014 Wiley Periodicals, Inc.

AbstractCore Binding Factor β (CBFβ) is complexed with the RUNX family of transcription factors in the nucleus to support activation or repression of genes related to bone (RUNX2), hematopoiesis (RUNX1) and gastrointestinal (RUNX3) development. Furthermore, RUNX proteins contribute to the onset and progression of different types of cancer. Although CBFβ localizes to cytoskeletal architecture, its biological role in the cytoplasmic compartment remains to be established. Additionally, the function and localization of CBFβ during the cell cycle are important questions relevant to its biological role. Here we show that CBFβ dynamically distributes in different stages of cell division and importantly is present during telophase at the midbody, a temporal structure important for successful cytokinesis. A functional role for CBFβ localization at the midbody is supported by striking defects in cytokinesis that include polyploidy and abscission failure following siRNA‐mediated downregulation of endogenous CBFβ or overexpression of the inv(16) fusion protein CBFβ‐SMMHC. Our results suggest that CBFβ retention in the midbody during cytokinesis reflects a novel function that contributes to epigenetic control. J. Cell. Physiol. 229: 1466–1474, 2014. © 2014 Wiley Periodicals, Inc.

Abstract The regulatory information for phenotype, proliferation, and growth of normal and tumor cells must be maintained through genome replication in the S phase and cell division during mitosis. Epigenetic mechanisms that include DNA methylation, posttranslational modifications of histones, selective utilization of histone variants, and inheritable RNA molecules play pivotal roles in maintaining cellular identity through mitotic divisions. Recent studies demonstrate that mitotic occupancy of genes, which are determinants of cell fate, growth, and proliferation, by lineage-restricted transcription factors is a key epigenetic mechanism for retention and transmission of cellular expression memory. Evidence is emerging for the presence of distinct transcriptional regulatory microenvironments in mitotic chromosomes in which the genes bookmarked for reactivation postmitotically reside. Importantly, some oncoproteins are present in mitotic microenvironments where they occupy target genes during mitosis and may contribute to perpetuating the transformed phenotype. We discuss emerging regulatory implications of epigenetically bookmarking genes during mitosis for physiologic control as well as for the onset and progression of cancer. Cancer Res; 74(2); 420–5. ©2014 AACR.

ABSTRACTThe bone marrow provides a protective environment for acute myeloid leukemia (AML) cells that often allows leukemic stem cells to survive standard chemotherapeutic regimens. Targeting these leukemic stem cells within the bone marrow is critical for preventing relapse. We recently demonstrated that SDF‐1, a chemokine abundant in the bone marrow, induces apoptosis in AML cell lines and in patient samples expressing high levels of its receptor, CXCR4. Here we show that a subset of osteoblast lineage cells within the bone marrow can protect AML cells from undergoing apoptosis in response to the SDF‐1 naturally present in that location. In co‐culture systems, osteoblasts at various stages of differentiation protected AML cell lines and patient isolates from SDF‐1‐induced apoptosis. The differentiation of the osteoblast cell lines, MC3T3 and W‐20‐17, mediated this protection via a cell contact‐independent mechanism. In contrast, bone marrow‐derived mesenchymal cells, the precursors of osteoblasts, induced apoptosis in AML cells via a CXCR4‐dependent mechanism and failed to protect AML cells from exogenously added SDF‐1. These results indicate that osteoblasts in the process of differentiation potently inhibit the SDF‐1‐driven apoptotic pathway of CXCR4‐expressing AML cells residing in the bone marrow. Drugs targeting this protective mechanism could potentially provide a new approach to treating AML by enhancing the SDF‐1‐induced apoptosis of AML cells residing within the bone marrow microenvironment. J. Cell. Biochem. 115: 1128–1137, 2014. © 2013 Wiley Periodicals, Inc.

ABSTRACTHeparan sulfate proteoglycans (HSPGs) are key components of the extracellular matrix that mediate cell proliferation, invasion, and cellular signaling. The biological functions of HSPGs are linked to their co‐stimulatory effects on extracellular ligands (e.g., WNTs) and the resulting activation of transcription factors that control mammalian development but also associated with tumorigenesis. We examined the expression profile of HSPG core protein syndecans (SDC1–4) and glypicans (GPC1–6) along with the enzymes that initiate or modify their glycosaminoglycan chains in human breast cancer (HBC) epithelial cells. Gene expression in relation to cell proliferation was examined in the HBC cell lines MCF‐7 and MDA‐MB‐231 following treatment with the HS agonist heparin. Heparin increased gene expression of chain initiation and modification enzymes including EXT1 and NDST1, as well as core proteins SDC2 and GPC6. With HS/Wnt interactions established, we next investigated WNT pathway components and observed that increased proliferation of the more invasive MDA‐MB‐231 cells is associated with activation of the Wnt signaling pathway. Specifically, there was substantial upregulation (>5‐fold) of AXIN1, WNT4A, and MYC in MDA‐MB‐231 but not in MCF‐7 cells. The changes in gene expression observed for HSPG core proteins and related enzymes along with the associated Wnt signaling components suggest coordinated interactions. The influence of HSPGs on cellular proliferation and invasive potential of breast cancer epithelial cells are cell and niche specific. Further studies on the interactions between HSPGs and WNT ligands may yield clinically relevant molecular targets, as well as new biomarkers for characterization of breast cancer progression. J. Cell. Biochem. 115: 967–976, 2014. © 2013 Wiley Periodicals, Inc.

Abstract Signaling through fibroblast growth factor receptor one (FGFR1) is a known inducer of proliferation in both embryonic and human adult mesenchymal stem cells (hMSCs) and positively regulates maintenance of stem cell viability. Leveraging the mitogenic potential of FGF2/FGFR1 signaling in stem cells for therapeutic applications necessitates a mechanistic understanding of how this receptor stimulates cell cycle progression. Using small interfering RNA (siRNA) depletion, antibody-inhibition, and small molecule inhibition, we establish that FGFR1 activity is rate limiting for self-renewal of hMSCs. We show that FGFR1 promotes stem cell proliferation through multiple mechanisms that unite to antagonize cyclin-dependent kinase (CDK) inhibitors. FGFR1 not only stimulates c-Myc to suppress transcription of the CDK inhibitors p21Waf1 and p27Kip1, thus promoting cell cycle progression but also increases the activity of protein kinase B (AKT) and the level of S-phase kinase-associated protein 2 (Skp2), resulting in the nuclear exclusion and reduction of p21Waf1. The in vivo importance of FGFR1 signaling for the control of proliferation in mesenchymal progenitor populations is underscored by defects in ventral mesoderm formation during development upon inhibition of its signaling. Collectively, these studies demonstrate that FGFR1 signaling mediates the continuation of MSC growth and establishes a receptor target for enhancing the expansion of mesenchymal progenitors while maintaining their multilineage potential. Stem Cells 2013;31:2724–2736

AbstractRunx2 is the master transcription factor for bone formation. Haploinsufficiency of RUNX2 is the genetic cause of cleidocranial dysplasia (CCD) that is characterized by hypoplastic clavicles and open fontanels. In this study, we found that Pin1, peptidyl prolyl cis–trans isomerase, is a critical regulator of Runx2 in vivo and in vitro. Pin1 mutant mice developed CCD‐like phenotypes with hypoplastic clavicles and open fontanels as found in the Runx2+/− mice. In addition Runx2 protein level was significantly reduced in Pin1 mutant mice. Moreover Pin1 directly interacts with the Runx2 protein in a phosphorylation‐dependent manner and subsequently stabilizes Runx2 protein. In the absence of Pin1, Runx2 is rapidly degraded by the ubiquitin‐dependent protein degradation pathway. However, Pin1 overexpression strongly attenuated uniquitin‐dependent Runx2 degradation. Collectively conformational change of Runx2 by Pin1 is essential for its protein stability and possibly enhances the level of active Runx2 in vivo. J. Cell. Physiol. 228: 2377–2385, 2013. © 2013 Wiley Periodicals, Inc.