The histone demethylase KDM3A, and its downstream target MCAM, promote Ewing Sarcoma cell migration and metastasis.

Ewing Sarcoma is the second most common solid pediatric malignant neoplasm of bone and soft tissue. Driven by EWS/Ets, or rarely variant, oncogenic fusions, Ewing Sarcoma is a biologically and clinically aggressive disease with a high propensity for metastasis. However, the mechanisms underpinning Ewing Sarcoma metastasis are currently not well understood. In the present study, we identify and characterize a novel metastasis-promotional pathway in Ewing Sarcoma, involving the histone demethylase KDM3A, previously identified by our laboratory as a new cancer-promoting gene in this disease. Using global gene expression profiling, we show that KDM3A positively regulates genes and pathways implicated in cell migration and metastasis, and demonstrate, using functional assays, that KDM3A promotes migration in vitro and experimental, post-intravasation, metastasis in vivo. We further identify the melanoma cell adhesion molecule (MCAM) as a novel KDM3A target gene in Ewing Sarcoma, and an important effector of KDM3A pro-metastatic action. Specifically, we demonstrate that MCAM depletion, like KDM3A depletion, inhibits cell migration in vitro and experimental metastasis in vivo, and that MCAM partially rescues impaired migration due to KDM3A knock-down. Mechanistically, we show that KDM3A regulates MCAM expression both through a direct mechanism, involving modulation of H3K9 methylation at the MCAM promoter, and an indirect mechanism, via the Ets1 transcription factor. Finally, we identify an association between high MCAM levels in patient tumors and poor survival, in two different Ewing Sarcoma clinical cohorts. Taken together, our studies uncover a new metastasis-promoting pathway in Ewing Sarcoma, with therapeutically targetable components.

The histone demethylase KDM3A is a microRNA-22-regulated tumor promoter in Ewing Sarcoma.

Ewing Sarcoma is a biologically aggressive bone and soft tissue malignancy affecting children and young adults. Ewing Sarcoma pathogenesis is driven by EWS/Ets fusion oncoproteins, of which EWS/Fli1 is the most common. We have previously shown that microRNAs (miRs) regulated by EWS/Fli1 contribute to the pro-oncogenic program in Ewing Sarcoma. Here we show that miR-22, an EWS/Fli1-repressed miR, is inhibitory to Ewing Sarcoma clonogenic and anchorage-independent cell growth, even at modest overexpression levels. Our studies further identify the H3K9me1/2 histone demethylase KDM3A (JMJD1A/JHDM2A) as a new miR-22-regulated gene. We show that KDM3A is overexpressed in Ewing Sarcoma, and that its depletion inhibits clonogenic and anchorage-independent growth in multiple patient-derived cell lines, and tumorigenesis in a xenograft model. KDM3A depletion further results in augmentation of the levels of the repressive H3K9me2 histone mark, and downregulation of pro-oncogenic factors in Ewing Sarcoma. Together, our studies identify the histone demethylase KDM3A as a new, miR-regulated, tumor promoter in Ewing Sarcoma.

A novel oncogenic mechanism in Ewing sarcoma involving IGF pathway targeting by EWS/Fli1-regulated microRNAs.

MicroRNAs (miRs) are a novel class of cellular bioactive molecules with critical functions in the regulation of gene expression in normal biology and disease. MiRs are frequently misexpressed in cancer, with potent biological consequences. However, relatively little is known about miRs in pediatric cancers, including sarcomas. Moreover, the mechanisms behind aberrant miR expression in cancer are poorly understood. Ewing sarcoma is an aggressive pediatric malignancy driven by EWS/Ets fusion oncoproteins, which are gain-of-function transcriptional regulators. We employed stable silencing of EWS/Fli1, the most common of the oncogenic fusions, and global miR profiling to identify EWS/Fli1-regulated miRs with oncogenesis-modifying roles in Ewing sarcoma. In this report, we characterize a group of miRs (100, 125b, 22, 221/222, 27a and 29a) strongly repressed by EWS/Fli1. Strikingly, all of these miRs have predicted targets in the insulin-like growth factor (IGF) signaling pathway, a pivotal driver of Ewing sarcoma oncogenesis. We demonstrate that miRs in this group negatively regulate the expression of multiple pro-oncogenic components of the IGF pathway, namely IGF-1, IGF-1 receptor, mammalian/mechanistic target of rapamycin and ribosomal protein S6 kinase A1. Consistent with tumor-suppressive functions, these miRs manifest growth inhibitory properties in Ewing sarcoma cells. Our studies thus uncover a novel oncogenic mechanism in Ewing sarcoma, involving post-transcriptional derepression of IGF signaling by the EWS/Fli1 fusion oncoprotein via miRs. This novel pathway may be amenable to innovative therapeutic targeting in Ewing sarcoma and other malignancies with activated IGF signaling.

Temporal dissociation between lithium-induced changes in frontal lobe myo-inositol and clinical response in manic-depressive illness.

OBJECTIVE: The most widely accepted hypothesis regarding the mechanism underlying lithium's therapeutic efficacy in manic-depressive illness (bipolar affective disorder) is the inositol depletion hypothesis, which posits that lithium produces a lowering of myo-inositol in critical areas of the brain and the effect is therapeutic. Lithium's effects on in vivo brain myo-inositol levels were investigated longitudinally in 12 adult depressed patients with manic-depressive illness. METHOD: Medication washout (minimum 2 weeks) and lithium administration were conducted in a blinded manner. Regional brain myo-inositol levels were measured by means of quantitative proton magnetic resonance spectroscopy at three time points: at baseline and after acute (5-7 days) and chronic (3-4 weeks) lithium administration. RESULTS: Significant decreases (approximately 30%) in myoinositol levels were observed in the right frontal lobe after short-term administration, and these decreases persisted with chronic treatment. The severity of depression measured by the Hamilton Depression Rating Scale also decreased significantly over the study. CONCLUSIONS: This study demonstrates that lithium administration does reduce myo-inositol levels in the right frontal lobe of patients with manic-depressive illness. However, the acute myo-inositol reduction occurs at a time when the patient's clinical state is clearly unchanged. Thus, the short-term reduction of myo-inositol per se is not associated with therapeutic response and does not support the inositol depletion hypothesis as originally posited. The hypothesis that a short-term lowering of myo inositol results in a cascade of secondary signaling and gene expression changes in the CNS that are ultimately associated with lithium's therapeutic efficacy is under investigation.

Complexity, pattern, and evolutionary trade-offs in animal aggregation.

One of the most striking patterns in biology is the formation of animal aggregations. Classically, aggregation has been viewed as an evolutionarily advantageous state, in which members derive the benefits of protection, mate choice, and centralized information, balanced by the costs of limiting resources. Consisting of individual members, aggregations nevertheless function as an integrated whole, displaying a complex set of behaviors not possible at the level of the individual organism. Complexity theory indicates that large populations of units can self-organize into aggregations that generate pattern, store information, and engage in collective decision-making. This begs the question, are all emergent properties of animal aggregations functional or are some simply pattern? Solutions to this dilemma will necessitate a closer marriage of theoretical and modeling studies linked to empirical work addressing the choices, and trajectories, of individuals constrained by membership in the group.

Molecular characterization and embryonic expression of the even-skipped ortholog of Tribolium castaneum.

In short germ insects, the procephalon and presumptive anterior segments comprise most of the embryonic rudiment which lengthens as posterior segments are added during development (Sander, K. (1976) Adv. Insect Physiol. 12, 125-238). The expression pattern of a grasshopper ortholog of the primary pair-rule gene even-skipped (eve) suggests that it is not relevant to segmentation in this short germ insect (Patel, N.H., Ball, E.E. and Goodman, C.S. (1992) Nature 357, 339-342). However in Drosophila, a long germ insect that forms all segments simultaneously, eve plays a vital role in segment formation (Nüsslein-Volhard, C., Wieschaus, E. and Klüding, H. (1984) Roux's Arch. Dev. Biol. 193, 267-282). We have characterized the eve ortholog of the beetle Tribolium castaneum. The homeodomain sequence is highly conserved between beetle, fly, and grasshopper eve orthologs. Tc eve is expressed in stripes during segmentation, but in a pattern differing in some details from that of the fly gene. This pattern is coincident with that detected with a cross-reacting antibody (Patel, N.H., Condron, B.G. and Zinn, K. (1994) Nature 367, 429-434). Thus, an ancestral even-skipped gene appears to have evolved a role in segmentation in a common ancestor of flies and beetles. Unlike vertebrate orthologs but similar to eve, Tc eve is not linked to the homeotic complex.

Genetic control of early embryogenesis in the red flour beetle, Tribolium castaneum.

The power of genetic analysis possible with the fruit fly, Drosophila melanogaster, has yielded a detailed understanding of pattern formation controlled by homeotic and segmentation genes in early embryogenesis. We are studying the genetic regulation of embryogenesis in the red flour beetle, Tribolium castaneum. The dynamic process of germ rudiment formation and sequential segmentation exhibited by Tribolium provides a context different than Drosophila within which to assess the function of homeotic and segmentation gene homologs. Our analyses of the genes in the HOM-C suggest many similarities in structure and function with the well-characterized Drosophila genes. Abdominal resembles its Drosophila homolog abdominal-A in functioning to establish segmental identities in the abdomen, such that in each case mutations result in homeotic transformations to PS6. Although the anterior functional boundary of abdominal-A homologs is precisely conserved, the domain within which Abdominal is important extends more posterior than that of abdominal-A. The final expression pattern of the segmentation gene engrailed in Tribolium is identical to Drosophila, suggesting that these homologs are involved in a conserved developmental process. However, as expected the development of that pattern is different; engrailed stripes anticipate the formation of each new segment as they appear sequentially in the elongating germ band. Although the grasshopper even-skipped and fushi tarazu homologs are not apparently important in segmentation, the expression patterns of the Tribolium homologs strongly suggest that they have gained a role in segmentation in the lineage leading to beetles and flies. Nevertheless, differences between Tribolium and Drosophila in the dynamics of even-skipped expression and the fushi tarazu mutant phenotype indicate divergence in the regulation and roles of these genes.