RON is a tyrosine kinase receptor that triggers scattering of normal cells and invasive growth of cancer cells on ligand binding. We identified a short RON mRNA, which is expressed in human lung, ovary, tissues of the gastrointestinal tract, and also in several human cancers, including ovarian carcinomas and cell lines from pancreatic carcinomas and leukemias. This transcript encodes a truncated protein (short-form RON; sf-RON), lacking most of the RON receptor extracellular domain but retaining the whole transmembrane and intracellular domains. Sf-RON shows strong intrinsic tyrosine kinase activity and is constitutively phosphorylated. Epithelial cells transduced with sf-RON display an aggressive phenotype; they shift to a nonepithelial morphology, are unable to form aggregates, grow faster in monolayer cultures, show anchorage-independent growth, and become motile. We show that in these cells, E-cadherin expression is lost through a dominant transcriptional repression pathway likely mediated by the transcriptional factor SLUG. Altogether, these data show that expression of a naturally occurring, constitutively active truncated RON kinase results in loss of epithelial phenotype and aggressive behavior and, thus, it might contribute to tumor progression.

The MET oncogene was causally involved in the pathogenesis of a rare tumor, i.e., the papillary renal cell carcinoma, in which activating mutations, either germline or somatic, were identified. MET activating mutations are rarely found in other human tumors, whereas at higher frequencies, MET is amplified and/or overexpressed in sporadic tumors of specific histotypes, including osteosarcoma. In this work, we provide experimental evidence that overexpression of the MET oncogene causes and sustains the full-blown transformation of osteoblasts. Overexpression of MET, obtained by lentiviral vector-mediated gene transfer, resulted in the conversion of primary human osteoblasts into osteosarcoma cells, displaying the transformed phenotype in vitro and the distinguishing features of human osteosarcomas in vivo. These included atypical nuclei, aberrant mitoses, production of alkaline phosphatase, secretion of osteoid extracellular matrix, and striking neovascularization. Although with a lower tumorigenicity, this phenotype was superimposable to that observed after transfer of the MET gene activated by mutation. Both transformation and tumorigenesis were fully abrogated when MET expression was quenched by short-hairpin RNA or when signaling was impaired by a dominant-negative MET receptor. These data show that MET overexpression is oncogenic and that it is essential for the maintenance of the cancer phenotype. (Cancer Res 2006; 66(9): 4750-7)

The TrkA receptor tyrosine kinase is crucial for differentiation and survival of nerve-growth-factor-dependent neurons. Paradoxically, TrkA also induces cell death in pediatric tumor cells of neural origin, via an unknown mechanism. Here, we show that CCM2, a gene product associated with cerebral cavernous malformations, interacts with the juxtamembrane region of TrkA via its phosphotyrosine binding (PTB) domain and mediates TrkA-induced death in diverse cell types. Both the PTB and Karet domains of CCM2 are required for TrkA-dependent cell death, such that the PTB domain determines the specificity of the interaction, and the Karet domain links to death pathways. Downregulation of CCM2 in medulloblastoma or neuroblastoma cells attenuates TrkA-dependent death. Combined high expression levels of CCM2 and TrkA are correlated with long-term survival in a large cohort of human neuroblastoma patients. Thus, CCM2 is a key mediator of TrkA-dependent cell death in pediatric neuroblastic tumors.

No abstract

BackgroundThe poor prognosis for glioblastoma patients is caused by the diffuse infiltrative growth pattern of the tumor. Therefore, the molecular and cellular processes underlying cell migration continue to be a major focus of glioblastoma research. Emerging evidence supports the concept that the tumor microenvironment has a profound influence on the functional properties of tumor cells. Accordingly, substantial effort must be devoted to move from traditional two-dimensional migration assays to three-dimensional systems that more faithfully recapitulate the complex in vivo tumor microenvironment.MethodsIn order to mimic the tumor microenvironment of adult gliomas, we used adult organotypic brain slices as an invasion matrix for implanted, fluorescently labeled tumor spheroids. Cell invasion was imaged by confocal or epi-fluorescence microscopy and quantified by determining the average cumulative sprout length per spheroid. The tumor microenvironment was manipulated by treatment of the slice with small molecule inhibitors or using different genetically engineered mouse models as donors.ResultsBoth epi-fluorescence and confocal microscopy were applied to precisely quantify cell invasion in this ex vivo approach. Usage of a red-emitting membrane dye in addition to tissue clearing drastically improved epi-fluorescence imaging. Preparation of brain slices from of a genetically engineered mouse with a loss of a specific cell surface protein resulted in significantly impaired tumor cell invasion. Furthermore, jasplakinolide treatment of either tumor cells or brain slice significantly reduced tumor cell invasion.ConclusionWe present an optimized invasion assay that closely reflects in vivo invasion by the implantation of glioma cells into organotypic adult brain slice cultures with a preserved cytoarchitecture. The diversity of applications including manipulation of the tumor cells as well as the microenvironment, permits the investigation of rate limiting factors of cell migration in a reliable context. This model will be a valuable tool for the discovery of the molecular mechanisms underlying glioma cell invasion and, ultimately, the development of novel therapeutic strategies.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is caused by mutations in the Krebs cycle enzyme fumarate hydratase (FH). It has been proposed that "pseudohypoxic" stabilization of hypoxiainducible factor-α (HIF-α) by fumarate accumulation contributes to tumorigenesis in HLRCC. We hypothesized that an additional direct consequence of FH deficiency is the establishment of a biosynthetic milieu. To investigate this hypothesis, we isolated primary mouse embryonic fibroblast (MEF) lines from Fh1-deficient mice. As predicted, these MEFs upregulated Hif-1α and HIF target genes directly as a result of FH deficiency. In addition, detailed metabolic assessment of these MEFs confirmed their dependence on glycolysis, and an elevated rate of lactate efflux, associated with the upregulation of glycolytic enzymes known to be associated with tumorigenesis. Correspondingly, Fh1-deficient benign murine renal cysts and an advanced human HLRCC-related renal cell carcinoma manifested a prominent and progressive increase in the expression of HIF-α target genes and in genes known to be relevant to tumorigenesis and metastasis. In accord with our hypothesis, in a variety of different FH-deficient tissues, including a novel murine model of Fh1-deficient smooth muscle, we show a striking and progressive upregulation of a tumorigenic metabolic profile, as manifested by increased PKM2 and LDHA protein. Based on the models assessed herein, we infer that that FH deficiency compels cells to adopt an early, reversible, and progressive protumorigenic metabolic milieu that is reminiscent of that driving the Warburg effect. Targets identified in these novel and diverse FH-deficient models represent excellent potential candidates for further mechanistic investigation and therapeutic metabolic manipulation in tumors. Cancer Res; 70(22); 9153-65. ©2010 AACR.

Automated analyses of neuronal morphology are important for quantifying connectivity and circuitry in vivo, as well as in high content imaging of primary neuron cultures. The currently available tools for quantification of neuronal morphology either are highly expensive commercial packages or cannot provide automated image quantifications at single cell resolution. Here, we describe a new software package called WIS-NeuroMath, which fills this gap and provides solutions for automated measurement of neuronal processes in both in vivo and in vitro preparations. Diverse image types can be analyzed without any preprocessing, enabling automated and accurate detection of neurites followed by their quantification in a number of application modules. A cell morphology module detects cell bodies and attached neurites, providing information on neurite length, number of branches, cell body area, and other parameters for each cell. A neurite length module provides a solution for images lacking cell bodies, such as tissue sections. Finally, a ganglion explant module quantifies outgrowth by identifying neurites at different distances from the ganglion. Quantification of a diverse series of preparations with WIS-NeuroMath provided data that were well matched with parallel analyses of the same preparations in established software packages such as MetaXpress or NeuronJ. The capabilities of WIS-NeuroMath are demonstrated in a range of applications, including in dissociated and explant cultures and histological analyses on thin and whole-mount sections. WIS-NeuroMath is freely available to academic users, providing a versatile and cost-effective range of solutions for quantifying neurite growth, branching, regeneration, or degeneration under different experimental paradigms.

Glioblastoma frequently exhibits therapy-associated subtype transitions to mesenchymal phenotypes with adverse prognosis. Here, we perform multi-omic profiling of 60 glioblastoma primary tumours and use orthogonal analysis of chromatin and RNA-derived gene regulatory networks to identify 38 subtype master regulators, whose cell population-specific activities we further map in published single-cell RNA sequencing data. These analyses identify the oligodendrocyte precursor marker and chromatin modifier SOX10 as a master regulator in RTK I-subtype tumours. In vitro functional studies demonstrate that SOX10 loss causes a subtype switch analogous to the proneural–mesenchymal transition observed in patients at the transcriptomic, epigenetic and phenotypic levels. SOX10 repression in an in vivo syngeneic graft glioblastoma mouse model results in increased tumour invasion, immune cell infiltration and significantly reduced survival, reminiscent of progressive human glioblastoma. These results identify SOX10 as a bona fide master regulator of the RTK I subtype, with both tumour cell-intrinsic and microenvironmental effects.