van Wijnen, Andre J

Abstract The t(8;21) chromosomal translocation produces AML1-ETO, an oncogenic fusion protein that compromises the function of AML1, a transcription factor critical for the myeloid cell differentiation. The differentiation arrest and self-renewal phenotypes of cells expressing AML1-ETO have been chiefly studied at the single gene level. In order to understand how the genome-wide occupancy of AML1-ETO and associated co-regulators contributes to the leukemic properties of Kasumi-1 cells, a model system for human t(8;21) leukemia, chromatin immunoprecipitation-sequencing (ChIP-seq) experiments were employed. Genome-wide DNA binding profiles for AML1 and AML1-ETO in Kasumi-1 cells showed that both proteins were enriched at introns and distal upstream regulatory regions, the latter implying that distal elements are likely important for target gene regulation. Importantly, A substantial fraction of AML1/AML1-ETO-p300 co-localization occurs near transcriptional start sites (TSSs) in promoter regions associated with transcriptionally active loci, as indicated by an observed enrichment of the H3K4me3 histone modification. A significant discovery of the current study is the co-localization of AML1/AML1-ETO with N-CoR on regions that are primarily distal to TSSs. Analyses of both gene expression data and gene ontology suggest that the AML1/AML1-ETO co-localization with N-CoR, more so than with p300, is associated with the leukemic phenotype of Kasumi-1 cells. De novo motif discovery reveals that PU.1 motifs are over-represented in regions co-occupied by AML1-ETO and N-CoR compared to other AML1-ETO bound regions. Our findings establish a novel and predominant AML leukemia signature characterized by occupancy of the AML1-ETO/N-CoR complex at genomic regions enriched in motifs for myeloid differentiation factors. We propose that this property is critical for maintenance of the leukemic phenotype. Citation Format: Daniel J. Trombly, Troy W. Whitfield, Srivatsan Padmanabhan, Jonathan A. Gordon, Jane B. Lian, Andre J. van Wijnen, Sayyed K. Zaidi, Janet L. Stein, Gary S. Stein. Co-occupancy of AML1-ETO and N-CoR defines a dominant phenotypic signature in leukemic cells. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B28.

ABSTRACTThe organization of interphase chromosomes in chromosome territories (CTs) was first proposed more than one hundred years ago. The introduction of increasingly sophisticated microscopic and molecular techniques, now provide complementary strategies for studying CTs in greater depth than ever before. Here we provide an overview of these strategies and how they are being used to elucidate CT interactions and the role of these dynamically regulated, nuclear‐structure building blocks in directly supporting nuclear function in a physiologically responsive manner. J. Cell. Biochem. 117: 9–19, 2016. © 2015 Wiley Periodicals, Inc.

Three‐dimensional organization of the chromatin has important roles in transcription, replication, DNA repair, and pathologic events such as translocations. There are two fundamental ways to study higher‐order chromatin organization: microscopic and molecular approaches. In this review, we briefly introduce the molecular approaches, focusing on chromosome conformation capture or “3C” technology and its derivatives, which can be used to probe chromatin folding at resolutions beyond that provided by microscopy techniques. We further discuss the different types of data generated by the 3C‐based methods and how they can be used to answer distinct biological questions. J. Cell. Physiol. 230: 31–35, 2016. © 2015 Wiley Periodicals, Inc.

Abstract Ewing sarcomas are aggressive, poorly differentiated pediatric tumors of bone and soft tissue. Ewing sarcoma affects one to three people per million every year and is the second most common pediatric bone malignancy after osteosarcoma. 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. RUNX3 is highly homologous to RUNX2 and participates in the mesenchymal-derived bone formation. RUNX3 has been described as both a tumor suppressor and an oncogene in different tumor types. In this study, we investigated the role of RUNX3 in Ewing Sarcoma. RUNX3 was detected in all Ewing sarcoma cells examined (7/7 cell lines, and 4/4 primary tumors), whereas RUNX2 was detected in only 73% of these specimens. Immunoprecipitation experiments revealed that RUNX3 binds to EWS-FLI via its Runt domain, which is >91% identical to the Runt domain in RUNX2. The interaction between RUNX3 and EWS-FLI in cell nuclei was confirmed by immunofluorescence. Moreover, EWS-FLI interactions prevented RUNX3 from activating the transcription of a RUNX-responsive reporter, p6OSE2. To determine the role of RUNX3 in Ewing sarcoma cell growth, we stably suppressed RUNX3 expression in the Ewing sarcoma cell line A673 with shRNAs. In anchorage independent growth assays in soft agar, RUNX3-deficient A673 cells formed smaller colonies than cells expressing scrambled shRNAs. Consistent with the smaller colonies, RUNX3 suppressed cells had increased expression of the cyclin dependent kinase inhibitor p21. Taken together, these results suggest and oncogenic role for RUNX3 in Ewing sarcoma. Citation Format: Krista L. Bledsoe, Meghan E. McGee-Lawrence, Emily T. Camilleri, Andre M. Oliveira, Andre J. van Wijnen, Jennifer J. Westendorf. RUNX3 plays an oncogenic role in Ewing sarcoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1413. doi:10.1158/1538-7445.AM2014-1413

We report generation and characterization of pain‐related behavior in a minimally invasive facet joint degeneration (FJD) animal model in rats. FJD was produced by a non‐open percutaneous puncture‐induced injury on the right lumbar FJs at three consecutive levels. Pressure hyperalgesia in the lower back was assessed by measuring the vocalization response to pressure from a force transducer. After hyperalgesia was established, pathological changes in lumbar FJs and alterations of intervertebral foramen size were assessed by histological and imaging analyses. To investigate treatment options for lumber FJ osteoarthritis‐induced pain, animals with established hyperalgesia were administered with analgesic drugs, such as morphine, a selective COX‐2 inhibitor, a non‐steroidal anti‐inflammatory drug (NSAID) (ketorolac), or pregabalin. Effects were assessed by behavioral pain responses. One week after percutaneous puncture‐induced injury of the lumbar FJs, ipsilateral primary pressure hyperalgesia developed and was maintained for at least 12 weeks without foraminal stenosis. Animals showed decreased spontaneous activity, but no secondary hyperalgesia in the hind paws. Histopathological and microfocus X‐ray computed tomography analyses demonstrated that the percutaneous puncture injury resulted in osteoarthritis‐like structural changes in the FJs cartilage and subchondral bone. Pressure hyperalgesia was completely reversed by morphine. The administration of celecoxib produced moderate pain reduction with no statistical significance while the administration of ketorolac and pregabalin produced no analgesic effect on FJ osteoarthritis‐induced back pain. Our animal model of non‐open percutanous puncture‐induced injury of the lumbar FJs in rats shows similar characteristics of low back pain produced by human facet arthropathy. J. Cell. Physiol. 9999: 2837–2847, 2015. © 2015 Wiley Periodicals, Inc.

The Brahma (BRM) and Brahma‐related Gene 1 (BRG1) ATPases are highly conserved homologs that catalyze the chromatin remodeling functions of the multi‐subunit human SWI/SNF chromatin remodeling enzymes in a mutually exclusive manner. SWI/SNF enzyme subunits are mutated or missing in many cancer types, but are overexpressed without apparent mutation in other cancers. Here, we report that both BRG1 and BRM are overexpressed in most primary breast cancers independent of the tumor's receptor status. Knockdown of either ATPase in a triple negative breast cancer cell line reduced tumor formation in vivo and cell proliferation in vitro. Fewer cells in S phase and an extended cell cycle progression time were observed without any indication of apoptosis, senescence, or alterations in migration or attachment properties. Combined knockdown of BRM and BRG1 showed additive effects in the reduction of cell proliferation and time required for completion of cell cycle, suggesting that these enzymes promote cell cycle progression through independent mechanisms. Knockout of BRG1 or BRM using CRISPR/Cas9 technology resulted in the loss of viability, consistent with a requirement for both enzymes in triple negative breast cancer cells. J. Cell. Physiol. 9999: 2683–2694, 2015. © 2015 Wiley Periodicals, Inc.

Runx1 is a transcription factor essential for definitive hematopoiesis, and genetic abnormalities in Runx1 cause leukemia. Runx1 is functionally promiscuous and acts as either an oncogene or tumor suppressor gene in certain epithelial cancers. Recent evidence suggests that Runx1 is an important factor in breast cancer, however, its role remains ambiguous. Here, we addressed whether Runx1 has a specific pathological role during breast cancer progression and show that Runx1 has an oncogenic function. We observed elevated Runx1 expression in a subset of human breast cancers. Furthermore, throughout the course of disease progression in a classical mouse model of breast cancer (i.e., the MMTV‐PyMT transgenic model), Runx1 expression increases in the primary site (mammary gland) and is further upregulated in tumors and distal lung metastatic lesions. Ex vivo studies using tumor epithelial cells derived from these mice express significantly higher levels of Runx1 than normal mammary epithelial cells. The tumor cells exhibit increased rates of migration and invasion, indicative of an aggressive cancer phenotype. Inhibition of Runx1 expression using RNA interference significantly abrogates these cancer‐relevant phenotypic characteristics. Importantly, our data establish that Runx1 contributes to murine mammary tumor development and malignancy and potentially represents a key disease‐promoting and prognostic factor in human breast cancer progression and metastasis. J. Cell. Physiol. 230: 2522–2532, 2015. © 2015 Wiley Periodicals, Inc.

Circadian rhythm dysfunction is linked to many diseases, yet pathophysiological roles in articular cartilage homeostasis and degenerative joint disease including osteoarthritis (OA) remains to be investigated in vivo. Here, we tested whether environmental or genetic disruption of circadian homeostasis predisposes to OA‐like pathological changes. Male mice were examined for circadian locomotor activity upon changes in the light:dark (LD) cycle or genetic disruption of circadian rhythms. Wild‐type (WT) mice were maintained on a constant 12 h:12 h LD cycle (12:12 LD) or exposed to weekly 12 h phase shifts. Alternatively, male circadian mutant mice ( Clock Δ19 or Csnk1e tau mutants) were compared with age‐matched WT littermates that were maintained on a constant 12:12 LD cycle. Disruption of circadian rhythms promoted osteoarthritic changes by suppressing proteoglycan accumulation, upregulating matrix‐degrading enzymes and downregulating anabolic mediators in the mouse knee joint. Mechanistically, these effects involved activation of the PKCδ‐ERK‐RUNX2/NFκB and β‐catenin signaling pathways, stimulation of MMP‐13 and ADAMTS‐5, as well as suppression of the anabolic mediators SOX9 and TIMP‐3 in articular chondrocytes of phase‐shifted mice. Genetic disruption of circadian homeostasis does not predispose to OA‐like pathological changes in joints. Our results, for the first time, provide compelling in vivo evidence that environmental disruption of circadian rhythms is a risk factor for the development of OA‐like pathological changes in the mouse knee joint. J. Cell. Physiol. 230: 2174–2183, 2015. © 2015 Wiley Periodicals, Inc.

Abstract Progression of breast cancer to metastatic bone disease is linked to deregulated expression of the transcription factor Runx2. Therefore, our goal was to evaluate the potential for clinical use of Runx2-targeting miRNAs to reduce tumor growth and bone metastatic burden. Expression analysis of a panel of miRNAs regulating Runx2 revealed a reciprocal relationship between the abundance of Runx2 protein and two miRNAs, miR-135 and miR-203. These miRNAs are highly expressed in normal breast epithelial cells where Runx2 is not detected, and absent in metastatic breast cancer cells and tissue biopsies that express Runx2. Reconstituting metastatic MDA-MB-231-luc cells with miR-135 and miR-203 reduced the abundance of Runx2 and expression of the metastasis-promoting Runx2 target genes IL11, MMP-13, and PTHrP. In addition, tumor cell viability was decreased and migration suppressed in vitro. Orthotopic implantation of MDA-MB-231-luc cells delivered with miR-135 or miR-203, followed by an intratumoral administration of the synthetic miRNAs, reduced the tumor growth and spontaneous metastasis to bone. Furthermore, intratibial injection of these miRNA-delivered cells impaired tumor growth in the bone environment and inhibited bone resorption. Importantly, reconstitution of Runx2 in MDA-MB-231-luc cells delivered with miR-135 and miR-203 reversed the inhibitory effect of the miRNAs on tumor growth and metastasis. Thus, we have identified that aberrant expression of Runx2 in aggressive tumor cells is related to the loss of specific Runx2-targeting miRNAs and that a clinically relevant replacement strategy by delivery of synthetic miRNAs is a candidate for a therapeutic approach to prevent metastatic bone disease by this route. Cancer Res; 75(7); 1433–44. ©2015 AACR.