The ubiquitin fold modifier 1 (UFM1) cascade is a recently identified evolutionarily conserved ubiquitin-like modification system whose function and link to human disease have remained largely uncharacterized. By using exome sequencing in Finnish individuals with severe epileptic syndromes, we identified pathogenic compound heterozygous variants in UBA5, encoding an activating enzyme for UFM1, in two unrelated families. Two additional individuals with biallelic UBA5 variants were identified from the UK-based Deciphering Developmental Disorders study and one from the Northern Finland Intellectual Disability cohort. The affected individuals (n = 9) presented in early infancy with severe irritability, followed by dystonia and stagnation of development. Furthermore, the majority of individuals display postnatal microcephaly and epilepsy and develop spasticity. The affected individuals were compound heterozygous for a missense substitution, c.1111G>A (p.Ala371Thr; allele frequency of 0.28% in Europeans), and a nonsense variant or c.164G>A that encodes an amino acid substitution p.Arg55His, but also affects splicing by facilitating exon 2 skipping, thus also being in effect a loss-of-function allele. Using an in vitro thioester formation assay and cellular analyses, we show that the p.Ala371Thr variant is hypomorphic with attenuated ability to transfer the activated UFM1 to UFC1. Finally, we show that the CNS-specific knockout of Ufm1 in mice causes neonatal death accompanied by microcephaly and apoptosis in specific neurons, further suggesting that the UFM1 system is essential for CNS development and function. Taken together, our data imply that the combination of a hypomorphic p.Ala371Thr variant in trans with a loss-of-function allele in UBA5 underlies a severe infantile-onset encephalopathy.

BackgroundAn increasing number of studies support the presence of stem-like cells in human malignancies. These cells are primarily responsible for tumor initiation and thus considered as a potential target to eradicate tumors. CD133 has been identified as an important cell surface marker to enrich the stem-like population in various human tumors. To reveal the molecular machinery underlying the stem-like features in tumor cells, we analyzed a promoter of CD133 gene using human colon carcinoma Caco-2 and synovial sarcoma Fuji cells, which endogenously express CD133 gene.ResultsA reporter analysis revealed that P5 promoter, located far upstream in a human CD133 gene locus, exhibits the highest activity among the five putative promoters (P1 to P5). Deletion and mutation analysis identified two ETS binding sites in the P5 region as being essential for its promoter activity. Electrophoretic mobility shift assays demonstrated the specific binding between nuclear factors and the ETS binding sequence. Overexpression of dominant-negative forms of Ets2 and Elk1 resulted in the significant decrease of P5 activity. Furthermore, treatment of Fuji cells with a specific MEK/ERK inhibitor, U0126, also markedly decreased CD133 expression, but there was no significant effect in Caco-2 cells, suggesting cell type-specific regulation of CD133 expression. Instead, the side population, another hallmark of TSLCs, was dramatically diminished in Caco-2 cells by U0126. Finally, Ras-mediated oncogenic transformation in normal human astrocytes conferred the stem-like capability to form neurosphere-like colonies with the increase of CD133 mRNA expression.ConclusionsIn conclusion, the Ras/ERK pathway at least in part contributes to the maintenance and the acquisition of stem-like hallmarks, although the extent of its contribution is varied in a cell type-specific manner. These findings could help our comprehensive understanding of tumor stemness, and also improve the development of eradicative therapies against human malignancies.

Cancer stem cells (CSCs) are believed to be maintained within a microenvironmental niche. Here we used polymer microarrays for the rapid and efficient identification of glioma CSC (GSC) niche mimicries and identified a urethane-based synthetic polymer, upon which two groups of niche components, namely extracellular matrices (ECMs) and iron are revealed. In cultures, side population (SP) cells, defined as GSCs in the rat C6 glioma cell line, are more efficiently sustained in the presence of their differentiated progenies expressing higher levels of ECMs and transferrin, while in xenografts, ECMs are supplied by the vascular endothelial cells (VECs), including SP cell-derived ones with distinctively greater ability to retain xenobiotics than host VECs. Iron is stored in tumor infiltrating host macrophages (Mus), whose protumoral activity is potently enhanced by SP cell-secreted soluble factor(s). Finally, coexpression of ECM-, iron-, and Mu-related genes is found to be predictive of glioma patients' outcome. Our polymer-based approach reveals the intrinsic capacities of GSCs, to adapt the environment to organize a selfadvantageous microenvironment niche, for their maintenance and expansion, which redefines the current concept of anti-CSC niche therapy and has the potential to accelerate cancer therapy development. STEM CELLS 2016;34:1151-1162 SIGNIFICANCE STATEMENTA specialized microenvironment called niche that maintains cancer stem cells (CSCs) has been studied as a promising target of cancer therapy. However, as recapitulating the niche by conventional approaches is challenging due to its biological and physical complexity, some important aspects of CSC biology might remain unelucidated. We here provide the first demonstration of the utility of synthetic polymer as a niche mimic for GSCs. Our approach also reveals an important finding, that is, self-organization of the GSC niche, which appends a new aspect to the current concept of CSC regulation and could help development therapeutic strategies for cancer eradication.