iPSC-derived progenitor stromal cells provide new insights into aberrant musculoskeletal development and resistance to cancer in down syndrome.

Down syndrome (DS) is a congenital disorder caused by trisomy 21 (T21). It is associated with cognitive impairment, muscle hypotonia, heart defects, and other clinical anomalies. At the same time, individuals with Down syndrome have lower prevalence of solid tumor formation. To gain new insights into aberrant DS development during early stages of mesoderm formation and its possible connection to lower solid tumor prevalence, we developed the first model of two types of DS iPSC-derived stromal cells. Utilizing bioinformatic and functional analyses, we identified over 100 genes with coordinated expression among mesodermal and endothelial cell types. The most significantly down-regulated processes in DS mesodermal progenitors were associated with decreased stromal progenitor performance related to connective tissue organization as well as muscle development and functionality. The differentially expressed genes included cytoskeleton-related genes (actin and myosin), ECM genes (Collagens, Galectin-1, Fibronectin, Heparan Sulfate, LOX, FAK1), cell cycle genes (USP16, S1P complexes), and DNA damage repair genes. For DS endothelial cells, our analysis revealed most down-regulated genes associated with cellular response to external stimuli, cell migration, and immune response (inflammation-based). Together with functional assays, these results suggest an impairment in mesodermal development capacity during early stages, which likely translates into connective tissue impairment in DS patients. We further determined that, despite differences in functional processes and characteristics, a significant number of differentially regulated genes involved in tumorigenesis were expressed in a highly coordinated manner across endothelial and mesodermal cells. These findings strongly suggest that microRNAs (miR-24-4, miR-21), cytoskeleton remodeling, response to stimuli, and inflammation can impact resistance to tumorigenesis in DS patients. Furthermore, we also show that endothelial cell functionality is impaired, and when combined with angiogenic inhibition, it can provide another mechanism for decreased solid tumor development. We propose that the same processes, which specify the basis of connective tissue impairment observed in DS patients, potentially impart a resistance to cancer by hindering tumor progression and metastasis. We further establish that cancer-related genes on Chromosome 21 are up-regulated, while genome-wide cancer-related genes are down-regulated. These results suggest that trisomy 21 induces a modified regulation and compensation of many biochemical pathways across the genome. Such downstream interactions may contribute toward promoting tumor resistant mechanisms.

Regulation of GLI1 by cis DNA elements and epigenetic marks.

GLI1 is one of three transcription factors (GLI1, GLI2 and GLI3) that mediate the Hedgehog signal transduction pathway and play important roles in normal development. GLI1 and GLI2 form a positive-feedback loop and function as human oncogenes. The mouse and human GLI1 genes have untranslated 5' exons and large introns 5' of the translational start. Here we show that Sonic Hedgehog (SHH) stimulates occupancy in the introns by H3K27ac, H3K4me3 and the histone reader protein BRD4. H3K27ac and H3K4me3 occupancy is not significantly changed by removing BRD4 from the human intron and transcription start site (TSS) region. We identified six GLI binding sites (GBS) in the first intron of the human GLI1 gene that are in regions of high sequence conservation among mammals. GLI1 and GLI2 bind all of the GBS in vitro. Elimination of GBS1 and 4 attenuates transcriptional activation by GLI1. Elimination of GBS1, 2, and 4 attenuates transcriptional activation by GLI2. Eliminating all sites essentially eliminates reporter gene activation. Further, GLI1 binds the histone variant H2A.Z. These results suggest that GLI1 and GLI2 can regulate GLI1 expression through protein-protein interactions involving complexes of transcription factors, histone variants, and reader proteins in the regulatory intron of the GLI1 gene. GLI1 acting in trans on the GLI1 intron provides a mechanism for GLI1 positive feedback and auto-regulation. Understanding the combinatorial protein landscape in this locus will be important to interrupting the GLI positive feedback loop and providing new therapeutic approaches to cancers associated with GLI1 overexpression.

p53 modulates the activity of the GLI1 oncogene through interactions with the shared coactivator TAF9.

The GLI1 oncogene and p53 tumor suppressor gene function in an inhibitory loop that controls stem cell and tumor cell numbers. Since GLI1 and p53 both interact with the coactivator TATA Binding Protein Associated Factor 9 (TAF9), we hypothesized that competition between these transcription factors for TAF9 in cancer cells may contribute to the inhibitory loop and directly affect GLI1 function and cellular phenotype. We showed that TAF9 interacts with the oncogenic GLI family members GLI1 and GLI2 but not GLI3 in cell-free pull-down assays and with GLI1 in rhabdomyosarcoma and osteosarcoma cell lines. Removal of the TAF9-binding acidic alpha helical transactivation domain of GLI1 produced a significant reduction in the ability of GLI1 to transform cells. We then introduced a point mutation into GLI1 (L1052I) that eliminates TAF9 binding and a point mutation into GLI3 (I1510L) that establishes binding. Wild-type and mutant GLI proteins that bind TAF9 showed enhanced transactivating and cell transforming activity compared with those that did not. Therefore, GLI-TAF9 binding appears important for oncogenic activity. We then determined whether wild-type p53 down-regulates GLI function by sequestering TAF9. We showed that p53 binds TAF9 with greater affinity than does GLI1 and that co-expression of p53 with GLI1 or GLI2 down-regulated GLI-induced transactivation, which could be abrogated using mutant forms of GLI1 or p53. This suggests that p53 sequesters TAF9 from GLI1, which may contribute to inhibition of GLI1 activity by p53 and potentially impact therapeutic success of agents targeting GLI-TAF9 interactions in cancer.

Noncanonical regulation of the Hedgehog mediator GLI1 by c-MYC in Burkitt lymphoma.

Although Hedgehog signaling plays a major role in GLI1 transcription, there is now evidence suggesting that other pathways/genes, such as c-MYC, may also regulate GLI1 expression. We initiated studies in Burkitt lymphoma cells, which constitutively express c-MYC due to a chromosomal translocation, to determine whether Hedgehog or c-MYC regulates GLI1 expression. We show that all Burkitt lymphoma cell lines tested express GLI1, PTCH1, and SMO and that five of six Burkitt lymphomas express GLI1. Exposure to Sonic or Indian Hedgehog or cyclopamine (SMO inhibitor) does not modulate GLI1 expression, cell proliferation, or apoptosis in most Burkitt lymphoma cell lines. Sequence analysis of PTCH1, SMO, and SuFu failed to show mutations that might explain the lack of Hedgehog responsiveness, and we did not detect primary cilia, which may contribute to it. We show that c-MYC interacts with the 5'-regulatory region of GLI1, using chromatin immunoprecipitation (ChIP) assay, and E-box-dependent transcriptional activation of GLI1 by c-MYC in NIH3T3 and HeLa cells. The c-MYC small-molecule inhibitor 10058-F4 downregulates GLI1 mRNA and protein and reduces the viability of Burkitt lymphoma cells. Inhibition of GLI1 by GANT61 increases apoptosis and reduces viability of some Burkitt lymphoma cells. Collectively, our data provide evidence that c-MYC directly regulates GLI1 and support an antiapoptotic role for GLI1 in Burkitt lymphoma. Burkitt lymphoma cells do not seem to be Hedgehog responsive. These findings suggest a mechanism for resistance to SMO inhibitors and have implications for using SMO inhibitors to treat human cancers.

Three dimensional visualization and fractal analysis of mosaic patches in rat chimeras: cell assortment in liver, adrenal cortex and cornea.

The production of organ parenchyma in a rapid and reproducible manner is critical to normal development. In chimeras produced by the combination of genetically distinguishable tissues, mosaic patterns of cells derived from the combined genotypes can be visualized. These patterns comprise patches of contiguously similar genotypes and are different in different organs but similar in a given organ from individual to individual. Thus, the processes that produce the patterns are regulated and conserved. We have previously established that mosaic patches in multiple tissues are fractal, consistent with an iterative, recursive growth model with simple stereotypical division rules. Fractal dimensions of various tissues are consistent with algorithmic models in which changing a single variable (e.g. daughter cell placement after division) switches the mosaic pattern from islands to stripes of cells. Here we show that the spiral pattern previously observed in mouse cornea can also be visualized in rat chimeras. While it is generally held that the pattern is induced by stem cell division dynamics, there is an unexplained discrepancy in the speed of cellular migration and the emergence of the pattern. We demonstrate in chimeric rat corneas both island and striped patterns exist depending on the age of the animal. The patches that comprise the pattern are fractal, and the fractal dimension changes with the age of the animal and indicates the constraint in patch complexity as the spiral pattern emerges. The spiral patterns are consistent with a loxodrome. Such data are likely to be relevant to growth and cell division in organ systems and will help in understanding how organ parenchyma are generated and maintained from multipotent stem cell populations located in specific topographical locations within the organ. Ultimately, understanding algorithmic growth is likely to be essential in achieving organ regeneration in vivo or in vitro from stem cell populations.

Developmental potential of rat extraembryonic stem cells.

We have previously found that certain stem cells that are derived from rat blastocysts and named extraembryonic endoderm precursor (XEN-P) cells show a unique molecular signature sharing some of the characteristics of embryonic stem cells (ES), trophoblast stem cells (TS), and extraembryonic endoderm stem cells (XEN). These XEN-P cells are positive for AP, SSEA1, Oct4, and Rex1 markers similar to ES cells and also express signature markers of TS-eomesodermin (Eomes) and XEN-Gata6. Here we show that these cells integrate into the visceral and parietal extraembryonic endoderm lineages as well as into the inner cell mass (ICM), the primitive endoderm, and the polar and mural trophectoderm (TE) of cultured embryos. In addition, we find that the XEN-P cells colonize yolk sac and contribute to trophoblast lineages of postimplantation embryos following transfer to surrogate mothers. We also find that the XEN-P cell culture propagates by shedding cell clusters into the media in addition to typical expansion of colonies. Interestingly, the cell cultures exist as mixed populations of two interconvertible phenotypes of flat and round cells with preferential expression of stem cell markers Oct4 and SSEA1 in round cells. We believe these cells represent a metastable stage during ICM cellular segregation. These results are important for developing hypotheses of cell fate plasticity in the ICM and provide a model for the study of development and differentiation along the extraembryonic lineages.

Bistable switches control memory and plasticity in cellular differentiation.

Development of stem and progenitor cells into specialized tissues in multicellular organisms involves a series of cell fate decisions. Cellular differentiation in higher organisms is generally considered irreversible, and the idea of developmental plasticity in postnatal tissues is controversial. Here, we show that inhibition of mitogen-activated protein kinase (MAPK) in a human bone marrow stromal cell-derived myogenic subclone suppresses their myogenic ability and converts them into satellite cell-like precursors that respond to osteogenic stimulation. Clonal analysis of the induced osteogenic response reveals ultrasensitivity and an "all-or-none" behavior, hallmarks of a bistable switch mechanism with stochastic noise. The response demonstrates cellular memory, which is contingent on the accumulation of an intracellular factor and can be erased by factor dilution through cell divisions or inhibition of protein synthesis. The effect of MAPK inhibition also exhibits memory and appears to be controlled by another bistable switch further upstream that determines cell fate. Once the memory associated with osteogenic differentiation is erased, the cells regain their myogenic ability. These results support a model of cell fate decision in which a network of bistable switches controls inducible production of lineage-specific differentiation factors. A competitive balance between these factors determines cell fate. Our work underscores the dynamic nature of cellular differentiation and explains mechanistically the dual properties of stability and plasticity associated with the process.

Defining a role for Sonic hedgehog pathway activation in desmoplastic medulloblastoma by identifying GLI1 target genes.

A subgroup of medulloblastomas shows constitutive activation of the Sonic hedgehog pathway with expression of GLI1. We identified the subset of GLI1 transforming target genes specifically expressed in medulloblastomas by comparing GLI1 targets in RK3E cells transformed by GLI1 with the gene expression profile of Sonic hedgehog signature medulloblastomas. We identified 1,823 genes whose expression was altered more than 2-fold in 2 independent RK3E + GLI1 cell lines. We identified 25 whose expression was altered similarly in medulloblastomas expressing GLI1. We identified potential GLI binding elements in the regulatory regions of 10 of these genes, confirmed that GLI1 binds the regulatory regions and activates transcription of select genes, and showed that GLI1 directly represses transcription of Krox-20. We identified upregulation of CXCR4, a chemokine receptor that plays roles in the proliferation and migration of granule cell neuron precursors during development, supporting the concept that reinitiation of developmental programs may contribute to medulloblastoma tumorigenesis. In addition, the targets suggest a pathway through which GLI1 may ultimately affect medulloblastoma cell proliferation, survival and genomic stability by converging on p53, SGK1, MGMT and NTRK2. We identify a p53 mutation in RK3E + GLI1 cells, suggesting that p53 mutations may sometimes shift the balance toward dysregulated tumor cell survival.