The effects of Gremlin1 on human umbilical cord blood hematopoietic progenitors.

Bone morphogenetic proteins (BMPs) support malignant hematopoiesis in CML. Conversely, the multi-functional BMP antagonist Gremlin1 supports self-renewing cancer stem cells of other malignancies. Inhibition of BMP signaling in CML, or of Gremlin1 in solid tumors, may therefore have therapeutic potential. However, since BMPs regulate hematopoietic stem cell (HSC) decisions in the stem cell niche, it is necessary to determine how Gremlin1 influences normal HSC. We examined the effects of Gremlin1 on long-term culture-initiating cells (LTC-IC) and transplantable hematopoietic stem cells (SCID-repopulating cells: SRC) in human umbilical cord blood. Gremlin1 inhibited BMP signaling, downregulated BMP-6 and cyclin E2 expression and upregulated hairy and enhancer of split-1 (HES-1; a Notch transcriptional target) and Hedgehog interacting protein-1 (HHIP-1; an inhibitor of Hedgehog signaling). The functional effects of Gremlin1 on SRC, i.e. skewing of their myelopoietic:lymphopoietic potential towards B lymphopoiesis without affecting long-term engraftment potential, were entirely consistent with changes in gene expression induced by Gremlin1. Since both BMPs and Gremlin1 are secreted by osteoblasts in vivo, our studies provide potential insights into the molecular regulation of hematopoiesis in the stem cell niche. These results also suggest that Gremlin1 (and possibly its mimetics that may be developed for therapeutic use) may not adversely affect normal human hematopoietic stem cell survival, though they may reduce their myelopoietic potential.

Characterization of an immunodeficient mouse model of mucopolysaccharidosis type I suitable for preclinical testing of human stem cell and gene therapy.

Mucopolysaccharidosis type I (MPS-I or Hurler syndrome) is an inherited deficiency of the lysosomal glycosaminoglycan (GAG)-degrading enzyme alpha-l-iduronidase (IDUA) in which GAG accumulation causes progressive multi-system dysfunction and death. Early allogeneic hematopoietic stem cell transplantation (HSCT) ameliorates clinical features and extends life but is not available to all patients, and inadequately corrects its most devastating features including mental retardation and skeletal deformities. To test novel therapies, we characterized an immunodeficient MPS-I mouse model less likely to develop immune reactions to transplanted human or gene-corrected cells or secreted IDUA. In the liver, spleen, heart, lung, kidney and brain of NOD/SCID/MPS-I mice IDUA was undetectable, and reduced to half in heterozygotes. MPS-I mice developed marked GAG accumulation (3-38-fold) in these organs. Neuropathological examination showed GM(3) ganglioside accumulation in the striatum, cerebral peduncles, cerebellum and ventral brainstem of MPS-I mice. Urinary GAG excretion (6.5-fold higher in MPS-I mice) provided a non-invasive and reliable method suitable for serially following the biochemical efficacy of therapeutic interventions. We identified and validated using rigorous biostatistical methods, a highly reproducible method for evaluating sensorimotor function and motor skills development. This Rotarod test revealed marked abnormalities in sensorimotor integration involving the cerebellum, striatum, proprioceptive pathways, motor cortex, and in acquisition of motor coordination. NOD/SCID/MPS-I mice exhibit many of the clinical, skeletal, pathological and behavioral abnormalities of human MPS-I, and provide an extremely suitable animal model for assessing the systemic and neurological effects of human stem cell transplantation and gene therapeutic approaches, using the above techniques to measure efficacy.

Oxidored-nitro domain containing protein 1 (NOR1) expression suppresses slug/vimentin but not snail in nasopharyngeal carcinoma: Inhibition of EMT in vitro and in vivo in mice.

Oxidored-nitro domain containing protein 1 (NOR1) is a putative tumor suppressor gene. In this study, NOR1 expression was detected in NPC tissues and non-cancerous nasopharyngeal epithelium. The data showed that NOR1 protein was decreased in NPC tissues. Lost expression NOR1 protein was associated with poor overall and event-free survival of NPC patients. Overexpression of NOR1 in NPC cells resulted in a significant morphological change and decreased expression of epithelial-to-mesenchymal transition (EMT) mediators (e.g., slug and vimentin), but induced cytokeratin 13 expression. A nude mouse metastasis assay revealed that overexpression of NOR1 decreased NPC tumor cells metastasis capacity in vivo. Knockdown of NOR1 expression in HeLa cells was sufficient to abrogate epithelial traits and to enhance cell migration and invasion. Concomitant inhibition of slug or vimentin alleviated induction of EMT, migration or invasion by NOR1 siRNA in HeLa cells in vitro. In conclusion, the data from the current study suggest, for the first time, that NOR1 plays an important role in NPC in ex vivo, in vitro, and in vivo.

Sequential activation of Snail1 and N-Myc modulates sonic hedgehog-induced transformation of neural cells.

Activation of the Sonic hedgehog (Shh) pathway and increased expression of Gli1 play an important role in proliferation and transformation of granule cell progenitors (GCP) in the developing cerebellum. Medulloblastomas arising from cerebellar GCPs are frequently driven by Shh pathway-activating mutations; however, molecular mechanisms of Shh pathway dysregulation and transformation of neural progenitors remain poorly defined. We report that the transcription factor and oncogene Snail1 (Sna1) is directly induced by Shh pathway activity in GCPs, murine medulloblastomas, and human medulloblastoma cells. Enforced expression of Sna1 was sufficient to induce GCPs and medulloblastoma cell proliferation in the absence of Shh/Gli1 exposure. In addition, enforced expression of Sna1 increased transformation of medulloblastoma cells in vitro and in vivo. Analysis of potential Sna1 targets in neural cells revealed a novel Sna1 target, N-Myc, a transcription factor known to play a role in Shh-mediated GCP proliferation and medulloblastoma formation. We found that Sna1 directly induced transcription of N-Myc in human medulloblastoma cells and that depletion of N-Myc ablated the Sna1-induced proliferation and transformation. Taken together, these results provide further insight into the mechanism of Shh-induced transformation of neural progenitor cells and suggest that induction of Sna1 may serve to amplify the oncogenic potential of Shh pathway activation through N-Myc induction.

CSPG4, a potential therapeutic target, facilitates malignant progression of melanoma.

Chondroitin sulfate proteoglycan 4 (CSPG4), a transmembrane proteoglycan originally identified as a highly immunogenic tumor antigen on the surface of melanoma cells, is associated with melanoma tumor formation and poor prognosis in certain melanomas and several other tumor types. The complex mechanisms by which CSPG4 affects melanoma progression have started to be defined, in particular the association with other cell surface proteins and receptor tyrosine kinases (RTKs) and its central role in modulating the function of these proteins. CSPG4 is essential to the growth of melanoma tumors through its modulation of integrin function and enhanced growth factor receptor-regulated pathways including sustained activation of ERK 1,2. This activation of integrin, RTK, and ERK1,2 function by CSPG4 modulates numerous aspects of tumor progression. CSPG4 expression has further been correlated to resistance of melanoma to conventional chemotherapeutics. This review outlines recent advances in our understanding of CSPG4-associated cell signaling, describing the central role it plays in melanoma tumor cell growth, motility, and survival, and explores how modifying CSPG4 function and protein-protein interactions may provide us with novel combinatorial therapies for the treatment of advanced melanoma.