Mutations in MECP2, encoding methyl CpG binding protein 2 (MeCP2), cause most cases of Rett syndrome (RTT), an X-linked neurodevelopmental disorder. Both RTT and autism are "pervasive developmental disorders" and share a loss of social, cognitive and language skills and a gain in repetitive stereotyped behavior, following apparently normal perinatal development. Although MECP2 coding mutations are a rare cause of autism, MeCP2 expression defects were previously found in autism brain. To further study the role of MeCP2 in autism spectrum disorders (ASDs), we determined the frequency of MeCP2 expression defects in brain samples from autism and other ASDs. We also tested the hypotheses that MECP2 promoter mutations or aberrant promoter methylation correlate with reduced expression in cases of idiopathic autism. MeCP2 immunofluorescence in autism and other neurodevelopmental disorders was quantified by laser scanning cytometry and compared with control postmortem cerebral cortex samples on a large tissue microarray. A significant reduction in MeCP2 expression compared to age-matched controls was found in 11/14 autism (79%), 9/9 RTT (100%), 4/4 Angelman syndrome (100%), 3/4 Prader-Willi syndrome (75%), 3/5 Down syndrome (60%), and 2/2 attention deficit hyperactivity disorder (100%) frontal cortex samples. One autism female was heterozygous for a rare MECP2 promoter variant that correlated with reduced MeCP2 expression. A more frequent occurrence was significantly increased MECP2 promoter methylation in autism male frontal cortex compared to controls. Furthermore, percent promoter methylation of MECP2 significantly correlated with reduced MeCP2 protein expression. These results suggest that both genetic and epigenetic defects lead to reduced MeCP2 expression and may be important in the complex etiology of autism.

Ewing sarcoma family tumors (ESFT) are aggressive bone and soft tissue tumors that express EWS-ETS fusion genes as driver mutations. Although the histogenesis of ESFT is controversial, mesenchymal (MSC) and/or neural crest (NCSC) stem cells have been implicated as cells of origin. For the current study we evaluated the consequences of EWS-FLI1 expression in human embryonic stem cell-derived NCSC (hNCSC). Ectopic expression of EWS-FLI1 in undifferentiated hNCSC and their neuro-mesenchymal stem cell (hNC-MSC) progeny was readily tolerated and led to altered expression of both well established as well as novel EWS-FLI1 target genes. Importantly, whole genome expression profiling studies revealed that the molecular signature of established ESFT is more similar to hNCSC than any other normal tissue, including MSC, indicating that maintenance or reactivation of the NCSC program is a feature of ESFT pathogenesis. Consistent with this hypothesis, EWS-FLI1 induced hNCSC genes as well as the polycomb proteins BMI-1 and EZH2 in hNC-MSC. In addition, up-regulation of BMI-1 was associated with avoidance of cellular senescence and reversible silencing of p16. Together these studies confirm that, unlike terminally differentiated cells but consistent with bone marrow-derived MSC, NCSC tolerate expression of EWS-FLI1 and ectopic expression of the oncogene initiates transition to an ESFT-like state. In addition, to our knowledge this is the first demonstration that EWS-FLI1-mediated induction of BMI-1 and epigenetic silencing of p16 might be critical early initiating events in ESFT tumorigenesis.

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BackgroundSome human cancers demonstrate cellular hierarchies in which tumor-initiating cancer stem cells generate progeny cells with reduced tumorigenic potential. This cancer stem cell population is proposed to be a source of therapy-resistant and recurrent disease. Ewing sarcoma family tumors (ESFT) are highly aggressive cancers in which drug-resistant, relapsed disease remains a significant clinical problem. Recently, the cell surface protein CD133 was identified as a putative marker of tumor-initiating cells in ESFT. We evaluated ESFT tumors and cell lines to determine if high levels of CD133 are associated with drug resistance.MethodsExpression of the CD133-encoding PROM1 gene was determined by RT-PCR in ESFT tumors and cell lines. CD133 protein expression was assessed by western blot, FACS and/or immunostaining. Cell lines were FACS-sorted into CD133+ and CD133- fractions and proliferation, colony formation in soft agar, and in vivo tumorigenicity compared. Chemosensitivity was measured using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assays.ResultsPROM1 expression was either absent or extremely low in most tumors. However, PROM1 was highly over-expressed in 4 of 48 cases. Two of the 4 patients with PROM1 over-expressing tumors rapidly succumbed to primary drug-resistant disease and two are long-term, event-free survivors. The expression of PROM1 in ESFT cell lines was similarly heterogeneous. The frequency of CD133+ cells ranged from 2-99% and, with one exception, no differences in the chemoresistance or tumorigenicity of CD133+ and CD133- cell fractions were detected. Importantly, however, the STA-ET-8.2 cell line was found to retain a cellular hierarchy in which relatively chemo-resistant, tumorigenic CD133+ cells gave rise to relatively chemo-sensitive, less tumorigenic, CD133- progeny.ConclusionsUp to 10% of ESFT express high levels of PROM1. In some tumors and cell lines the CD133+ fraction is relatively more drug-resistant, while in others there is no apparent difference between CD133+ and CD133- cells. These studies reveal heterogeneity in PROM1/CD133 expression in ESFT tumors and cell lines and confirm that high levels of PROM1 expression are, in at least some cases, associated with chemo-resistant disease. Further studies are required to elucidate the contribution of PROM1/CD133 expressing cells to therapeutic resistance in a large, prospective cohort of primary ESFT.

Ewing sarcoma family of tumors (ESFT) are aggressive bone and soft tissue tumors of unknown cellular origin. Most ESFT express EWS-FLI1, a chimeric protein which functions as a growth-promoting oncogene in ESFT but is toxic to most normal cells. A major difficulty in understanding EWS-FLI1 function has been the lack of an adequate model in which to study EWS-FLI1-induced transformation. Although the cell of origin of ESFT remains elusive, both mesenchymal (MSC) and neural crest (NCSC) have been implicated. We recently developed the tools to generate NCSC from human embryonic stem cells (hNCSC). In the current study we have used this model to test the hypothesis that NCSC are the cells of origin of ESFT and to evaluate the consequences of EWS-FLI1 expression on human NCSC biology. ESFT demonstrate variable degrees of neuro-mesenchymal differentiation potential. Similarly, we have found that hNCSC can be induced to differentiate into neural, glial and mesenchymal progeny. Significantly, Affymetrix whole genome expression profiling of 32 primary ESFT, 11 normal adult tissues, bone marrow-derived MSC and hNCSC revealed that ESFT are more similar to hNCSC than any other normal tissue, including MSC, thus further implicating NCSC in the origin of ESFT. To evaluate the consequences of EWS-FLI1 on these multipotent stem cells, hNCSC were stably transduced with an EWS-FLI1 lentivirus. Unlike most normal cells, hNCSC tolerated expression of the oncoprotein. Moreover, EWS-FLI1-transduced hNCSC continued to proliferate and maintain EWS-FLI1 expression in culture for several weeks after transduction. Affymetrix HuEx 1.0 expression profiling of hNCSC cells five days post-transduction with EWS-FLI1 demonstrated the expected induction and repression of well-established EWS-FLI1 targets and identified numerous other novel EWS-FLI1-regulated genes that are likely to be cell-type and situation specific. In contrast to control vector-transduced cells, EWS-FLI1 transduced hNCSC reproducibly maintained expression of the NCSC markers p75 and HNK-1, even after transfer to differentiation-inducing conditions. In addition, expression of the polycomb genes BMI1 and EZH2 was consistently and significantly upregulated in EWS-FLI1-transduced cells. In keeping with persistent expression of these stem cell-associated genes, EWS-FLI1-expressing cells retained the ability to form neurospheres upon transfer to non-adherent conditions and also the ability to differentiate into neural and mesenchymal lineages several weeks following transduction. In contrast, after six weeks in differentiation media, control cells had upregulated p16 and had undergone senescence. Together these data implicate NCSC in the origin of ESFT and suggest that EWS-FLI1 enables malignant transformation by inducing maintenance of their multipotent, stem cell state through deregulation of polycomb genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4239.