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.
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. Lastly, 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.
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.
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.
Ewing Sarcoma is an aggressive malignant neoplasm affecting children and young adults. Ewing Sarcoma is driven by transcription factor fusion oncoproteins, most commonly EWS/Fli1. While some patients can be cured with high-dose, multi-agent, chemotherapy, those that cannot currently have few options. Targeting of the driver oncofusion remains a logical therapeutic approach, but has proven difficult. Recent work has pointed to epigenetic mechanisms as key players, and potential new therapeutic targets, in Ewing Sarcoma. In this study we examined the activity of the pan-JHDM pharmacologic inhibitor JIB-04 in this disease. We show that JIB-04 potently inhibits the growth and viability of Ewing Sarcoma cells, and also impairs tumor xenograft growth. Effects on histone methylation at growth-inhibitory doses vary among cell lines, with most cell lines exhibiting increased total H3K27me3 levels, and some increased H3K4me3 and H3K9me3. JIB-04 treatment widely alters expression of oncogenic and tumor suppressive pathways, including downregulation of known oncogenic members of the Homeobox B and D clusters. JIB-04 also disrupts the EWS/Fli1 expression signature, including downregulation of pro-proliferative pathways normally under positive oncofusion control. Interestingly, these changes are accompanied by increased levels of the EWS/Fli1 oncofusion, suggesting that the drug could be uncoupling EWS/Fli1 from its oncogenic program. All Ewing Sarcoma cell lines examined also manifest increased DNA damage upon JIB-04 treatment. Together, the findings suggest that JIB-04 acts via multiple mechanisms to compromise Ewing Sarcoma cell growth and viability.
Ewing Sarcoma is an aggressive malignancy of bone and soft tissue affecting children and young adults. Ewing Sarcoma is driven by EWS/Ets fusion oncoproteins, which cause widespread alterations in gene expression in the cell. Dysregulation of receptor tyrosine kinase signaling, particularly involving IGF-1R, also plays an important role in Ewing Sarcoma pathogenesis. However, the basis of this dysregulation, including the relative contribution of EWS/Ets-dependent and independent mechanisms, is not well understood. In the present study, we identify variable expression of two modifiers of PI3K signaling activity, PIK3R3 and PTEN, in Ewing Sarcoma, and examine the consequences of this on PI3K pathway regulation and oncogenic phenotypes. Our findings indicate that PIK3R3 plays a growth-promotional role in Ewing Sarcoma, but suggest that this role is not strictly dependent on regulation of PI3K pathway activity. We further show that expression of PTEN, a well-established, potent tumor suppressor, is lost in a subset of Ewing Sarcomas, and that this loss strongly correlates with high baseline PI3K pathway activity in cell lines. In support of functional importance of PTEN loss in Ewing Sarcoma, we show that re-introduction of PTEN into two different PTEN-negative Ewing Sarcoma cell lines results in downregulation of PI3K pathway activity, and sensitization to the IGF-1R small molecule inhibitor OSI-906. Our findings also suggest that PTEN levels may contribute to sensitivity of Ewing Sarcoma cells to the microtubule inhibitor vincristine, a relevant chemotherapeutic agent in this cancer. Our studies thus identify PIK3R3 and PTEN as modifiers of oncogenic phenotypes in Ewing Sarcoma, with potential clinical implications.
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.
Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy of childhood. RMS exists as two major disease subtypes, with oncofusion-positive RMS (FP-RMS) typically carrying a worse prognosis than oncofusion-negative RMS (FN-RMS), in part due to higher propensity for metastasis. Epigenetic mechanisms have recently emerged as critical players in the pathogenesis of pediatric cancers, as well as potential new therapeutic vulnerabilities. Herein, we show that the epigenetic regulator KDM3A, a member of the Jumonji-domain histone demethylase (JHDM) family, is overexpressed, potently promotes colony formation and transendothelial invasion, and activates the expression of genes involved in cell growth, migration and metastasis, in both FN-RMS and FP-RMS. In mechanistic studies, we demonstrate that both RMS subtypes utilize a KDM3A/Ets1/MCAM disease-promoting axis recently discovered in Ewing Sarcoma, another aggressive pediatric cancer of distinct cellular and molecular origin. We further show that KDM3A depletion in FP-RMS cells inhibits both tumor growth and metastasis in vivo, and that RMS cells are highly sensitive to colony growth inhibition by the pan-JHDM inhibitor JIB-04. Together, our studies reveal an important role for the KDM3A/Ets1/MCAM axis in pediatric sarcomas of distinct cellular and molecular ontogeny, and identify new targetable vulnerabilities in RMS.
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