👤 Gail A Hecht

For the advancements of photoresponsive materials with tunable properties, the usage of multidimensional signals is desired. Using the polarization of the light in addition to the wavelength represents a further parameter to control the materials properties. Here, the first-time dynamic and reversible manipulation of the guest-host properties of a nanoporous material by linearly polarized light (LPL) is reported. The material is based on a metal-organic framework (MOF) with photoresponsive azobenzene side groups covalently connected to the MOF structure. The azobenzene moieties are reversibly reoriented by LPL, making the MOF structure and, thus, the pores anisotropic. As a result, the mobility of the guest molecules in the pores of the initially isotropic material becomes anisotropic, which can be dynamically controlled by the light polarization. The experiments by impedance spectroscopy are supported by molecular dynamics (MD) simulations. The study shows that the light polarization can be a further parameter to modify the material properties, allowing a more complex and more refined level of control for smart materials. Show less

Local invasion is the initial step towards metastasis, the main cause of cancer mortality. In human colorectal cancer (CRC), malignant cells predominantly invade as cohesive collectives and may undergo partial epithelial-mesenchymal transition (pEMT) at the invasive front. How this particular mode of stromal infiltration is generated is unknown. Here we investigated the impact of oncogenic transformation and the microenvironment on tumor cell invasion using genetically engineered organoids as CRC models. We found that inactivation of the Apc tumor suppressor combined with expression of oncogenic Kras Show less

Transforming growth factor beta (TGFβ) superfamily signaling is a prime inducer of epithelial-mesenchymal transitions (EMT) that foster cancer cell invasion and metastasis, a major cause of cancer-related deaths. Yet, TGFβ signaling is frequently inactivated in human tumor entities including colorectal cancer (CRC) and pancreatic adenocarcinoma (PAAD) with a high proportion of mutations incapacitating SMAD4, which codes for a transcription factor (TF) central to canonical TGFβ and bone morphogenetic protein (BMP) signaling. Beyond its role in initiating EMT, SMAD4 was reported to crucially contribute to subsequent gene regulatory events during EMT execution. It is therefore widely assumed that SMAD4-mutant (SMAD4 Show less

The transcription factor SNAIL1 is a master regulator of epithelial-to-mesenchymal transition (EMT), a process entailing massive gene expression changes. To better understand SNAIL1-induced transcriptional reprogramming we performed time-resolved transcriptome analysis upon conditional SNAIL1 expression in colorectal cancer cells. Gene set variation analyses indicated that SNAIL1 strongly affected features related to cell cycle and Wnt/β-Catenin signalling. This correlated with upregulation of LEF1, a nuclear binding partner of β-Catenin. Likewise, transcriptomes of cell lines and colorectal cancers, including poor-prognosis mesenchymal tumours, exhibit positively correlated SNAI1 and LEF1 expression, and elevated LEF1 levels parallel increased patient mortality. To delineate the functional contribution of LEF1 to SNAIL1-induced EMT, we used the CRISPR/Cas9 system to knock-out LEF1 in colorectal cancer cells, and to engineer cells that express LEF1 mutants unable to interact with β-Catenin. Both complete LEF1-deficiency and prevention of the β-Catenin-LEF1 interaction impaired the ability of SNAIL1 to elicit expression of an alternative set of Wnt/β-catenin targets, and to promote cancer cell invasion. Conversely, overexpression of wildtype, but not of mutant LEF1, stimulated alternative Wnt/β-Catenin target gene expression, and caused cell-cycle arrest. Moreover, like SNAIL1, LEF1 retarded tumour growth in xenotransplantations. Thus, LEF1 phenocopies SNAIL1 with respect to several critical aspects of EMT. Indeed, comparative transcriptomics suggested that 35% of SNAIL1-induced transcriptional changes are attributable to LEF1. However, LEF1 did not autonomously induce EMT. Rather, LEF1 appears to be a strictly β-Catenin-dependent downstream effector of SNAIL1. Apparently, SNAIL1 employs β-Catenin-LEF1 complexes to redirect Wnt/β-Catenin pathway activity towards pro-invasive and anti-proliferative gene expression. Show less

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system to inject effector proteins into host intestinal epithelial cells causing diarrhoea. EPEC infection redistributes basolateral proteins β1-integrin and Na Show less

Down syndrome (DS) is caused by a triplication of chromosome 21 (HSA21). Increased oxidative stress, decreased neurogenesis and synaptic dysfunction from HSA21 gene overexpression are thought to cause mental retardation, dementia and seizure in this disorder. Recent epigenetic studies have raised the possibility that DNA methylation has significant effects on DS neurodevelopment. Here, we performed methylome profiling in normal and DS fetal cortices and observed a significant hypermethylation in ∼4% of probes in the DS samples compared with age-matched normals. The probes with differential methylation were distributed across all chromosomes, with no enrichment on HSA21. Functional annotation and pathway analyses showed that genes in the ubiquitination pathway were significantly altered, including: BRCA1, TSPYL5 and PEX10 HSA21 located DNMT3L was overexpressed in DS neuroprogenitors, and this overexpression increased the promoter methylation of TSPYL5 potentially through DNMT3B, and decreased its mRNA expression. DNMT3L overexpression also increased mRNA levels for TP53 and APP, effectors of TSPYL5 Furthermore, DNMT3L overexpression increased APP and PSD95 expression in differentiating neurons, whereas DNMT3LshRNA could partially rescue the APP and PSD95 up-regulation in DS cells. These results provide some of the first mechanistic insights into causes for epigenetic changes in DS, leading to modification of genes relevant for the DS neural endophenotype. Show less

Hereditary Multiple Exostoses (HME) is an autosomal-dominant disorder characterized by benign cartilage tumors (exostoses) forming near the growth plates, leading to severe health problems. EXT1 and EXT2 are the two genes known to harbor heterozygous loss-of-function mutations that account for the vast majority of the primary genetic component of HME. However, patients present with wide clinical heterogeneity, suggesting that modifier genes play a role in determining severity. Our previous work has pointed to an imbalance of β-catenin signaling being involved in the pathogenesis of osteochondroma formation. TCF7L2 is one of the key 'gate-keeper' TCF family members for Wnt/β-catenin signaling pathway, and TCF7L2 and EXT2 are among the earliest associated loci reported in genome wide appraisals of type 2 diabetes (T2D). Thus we investigated if the key T allele of single nucleotide polymorphism (SNP) rs7903146 within the TCF7L2 locus, which is strongly over-represented among T2D cases, was also associated with HME. We leveraged genotype data available from ongoing GWAS efforts from genomics and orthopedic centers in the US, Canada and Italy. Collectively 213 cases and 1890 controls were analyzed and, surprisingly, the T allele was in fact significantly under-represented in the HME patient group [P = 0.009; odds ratio = 0.737 (95% C.I. 0.587-0.926)]; in addition, the direction of effect was consistent within each individual cohort. Immunohistochemical analyses revealed that TCF7L2 is differentially expressed and distributed in normal human growth plate zones, and exhibits substantial variability in human exostoses in terms of staining intensity and distribution. In summary, the data indicate that there is a putative genetic connection between TCF7L2 and EXT in the context of HME. Given this observation, we suggest that these loci could possibly modulate shared pathways, in particular with respect to β-catenin, and their respective variants interplay to influence HME pathogenesis as well as T2D. Show less

An exostosis or osteochondroma is an aberrant bony growth occurring next to the growth plate either as an isolated growth abnormality or as part of the Hereditary Multiple Exostosis (HME) syndrome. Mutations in either exostosin 1 (EXT1) or exostosin 2 (EXT2) gene cause the HME syndrome and also some isolated osteochondromas. The EXT1 and EXT2 genes are glycosyltransferases that function as hetero-oligomers in the Golgi to add repeating glycosaminoglycans (GAGs) to heparan sulfate (HS) chains. Previously, we demonstrated that HS is markedly diminished in the exostosis cartilage cap and that the HS proteoglycan, perlecan, has an abnormal distribution in these caps. The present studies were undertaken to evaluate which chondrocyte-specific functions are associated with diminished HS synthesis in human chondrocytes harboring either EXT1 or EXT2 mutations. Systematic evaluation of exostosis cartilage caps and chondrocytes, both in vitro and in vivo, suggests that chondrocyte-specific cell functions account for diminished HS levels. In addition, we provide evidence that perichondrial cells give rise to chondrocytes that clonally expand and develop into an exostosis. Undifferentiated EXT chondrocytes synthesized amounts of HS similar to control chondrocytes; however, EXT chondrocytes displayed very poor survival in vitro under conditions that promote normal chondrocyte differentiation with high efficiency. Collectively, these observations suggest that loss of one copy of either the EXT1 or EXT2 gene product compromises the perichondrial chondrocytes' ability to differentiate normally and to survive in a differentiated state in vitro. In vivo, these compromised responses may lead to abnormal chondrocyte growth, perhaps from a perichondrial stem cell reserve. Show less

EXT1 and EXT2 are genes that have been shown to cause hereditary multiple exostosis (HME), a syndrome marked by the formation of bony growths juxtaposed to the growth plate. These genes are members of a growing family of proteins with glycosyltransferase activity required for the synthesis of heparan sulfate chains. This protein activity is predicted to play a role in the expression of proteoglycans on the cell surface and in the extracellular matrix. We and others have previously suggested that a two-hit mutational model applies to the development of an exostosis where a germline mutation coupled with a somatic mutation results in the loss of EXT1 or EXT2 function and subsequent tumor formation. We report the direct sequencing and loss of heterozygosity (LOH) analysis of 12 exostoses from 10 HME families, 4 solitary exostoses, and their corresponding constitutional DNA. Of the 16 exostoses screened, we find only one solitary case in which two somatic mutations, a deletion and an LOH, are present. This provides limited support for the two-hit hypothesis involving the EXT1 and EXT2 genes for the development of an exostosis. Alternative models are developed based on the functional significance of EXT proteins in heparan sulfate biosynthesis. Show less

Hereditary multiple exostoses (HME), a condition associated with development and growth of bony exostoses at the ends of the long bones, is caused by germline mutations in the EXT genes. EXT1 and EXT2 function as glycosyltransferases that participate in the biosynthesis of heparan sulfate (HS) to modify proteoglycans. HS proteoglycans, synthesized by chondrocytes and secreted to the extracellular matrix of the growth plate, play critical roles in growth plate signaling and remodeling. As part of studies to delineate the mechanism(s) by which an exostosis develops, we have systematically evaluated four growth plates from two HME and two solitary exostoses. Mutational events were correlated with the presence/absence and distribution of HS and the normally abundant proteoglycan, perlecan (PLN). DNA from the HME exostoses demonstrated heterozygous germline EXT1 or EXT2 mutations, and DNA from one solitary exostosis demonstrated a somatic EXT1 mutation. No loss of heterozygosity was observed in any of these samples. The chondrocyte zones of four exostosis growth plates showed absence of HS, as well as diminished and abnormal distribution of PLN. These results indicate that, although multiple mutational events do not occur in the EXT1 or EXT2 genes, a complete loss of HS was found in the exostosis growth plates. This functional knockout of the exostosis chondrocytes' ability to synthesize HS chains further supports the observations of cytoskeletal abnormalities and chondrocyte disorganization associated with abnormal cell signaling. Show less

The EXT family of putative tumor suppressor genes affect endochondral bone growth, and mutations in EXT1 and EXT2 genes cause the autosomal dominant disorder Hereditary Multiple Exostoses (HME). Loss of heterozygosity (LOH) of these genes plays a role in the development of exostoses and chondrosarcomas. In this study, we characterized EXT genes in 11 exostosis chondrocyte strains using LOH and mutational analyses. We also determined subcellular localization and quantitation of EXT1 and EXT2 proteins by immunocytochemistry using antibodies raised against unique peptide epitopes. In an isolated non-HME exostosis, we detected three genetic hits: deletion of one EXT1 gene, a net 21-bp deletion within the other EXT1 gene and a deletion in intron 1 causing loss of gene product. Diminished levels of EXT1 and EXT2 protein were found in 9 (82%) and 5 (45%) exostosis chondrocyte strains, respectively, and 4 (36%) were deficient in levels of both proteins. Although we found mutations in exostosis chondrocytes, mutational analysis alone did not predict all the observed decreases in EXT gene products in exostosis chondrocytes, suggesting additional genetic mutations. Moreover, exostosis chondrocytes exhibit an unusual cellular phenotype characterized by abnormal actin bundles in the cytoplasm. These results suggest that multiple mutational steps are involved in exostosis development and that EXT genes play a role in cell signaling related to chondrocyte cytoskeleton regulation. Show less

The EXT genes are a group of putative tumor suppressor genes that previously have been shown to participate in the development of hereditary multiple exostoses (HME), HME-associated and isolated chondrosarcomas. Two HME disease genes, EXT1 and EXT2, have been identified and are expressed ubiquitously. However, the only known effect of mutations in the EXT genes is on chondrocyte function as evidenced by aberrant proliferation of chondrocytes leading to formation of bony, cartilage-capped projections (exostoses). In this study, we have characterized exostosis chondrocytes from three patients with HME (one with EXT1 and two with EXT2 germline mutations) and from one individual with a non-HME, isolated exostosis. At the light microscopic level, exostosis chondrocytes have a stellate appearance with elongated inclusions in the cytoplasm. Confocal and immunofluorescence of in vitro and in vivo chondrocytes showed that these massive accumulations are composed of actin bundled by 1.5-microm repeat cross-bridges of alpha-actinin. Western blot analysis shows that exostosis chondrocytes from two out of three patients aberrantly produce high levels of muscle-specific alpha-actin, whereas beta-actin levels are similar to normal chondrocytes. These findings suggest that mutations in the EXT genes cause abnormal processing of cytoskeleton proteins in chondrocytes. Show less

Hereditary multiple exostoses (EXT) is an autosomal dominant disorder characterized by the formation of cartilage-capped prominences that develop from the growth centers of the long bones. EXT is genetically heterogeneous, with three loci, currently identified on chromosomes 8q24.1, 11p13, and 19q. The EXT1 gene, located on chromosome 8q24.1, has been cloned and is encoded by a 3.4-kb cDNA. Five mutations in the EXT1 gene have been identified--four germ-line mutations, including two unrelated families with the same mutation, and one somatic mutation in a patient with chondrosarcoma. Four of the mutations identified resulted in frameshifts and premature termination codons, while the fifth mutation resulted in a substitution of leucine for arginine. Loss of heterozygosity (LOH) analysis of chondrosarcomas and chondroblastomas revealed multiple LOH events at loci on chromosomes 3q, 8q, 10q, and 19q. One sporadic chondrosarcoma demonstrated LOH for EXT1 and EXT3, while a second underwent LOH for EXT2 and chromosome 10. A third chondrosarcoma underwent LOH for EXT1 and chromosome 3q. These results agree with previous findings that mutations at EXT1 and multiple genetic events that include LOH at other loci may be required for the development of chondrosarcoma. Show less

Hereditary multiple exostoses (EXT) is an autosomal dominant condition characterized by short stature and the development of bony protuberances at the ends of all the long bones. Three genetic locl have been identified by genetic linkage analysis at chromosomes 8q24.1, 11p11-13 and 19p. The EXT1 gene on chromosome 8 was recently identified and characterized. Here, we report the isolation and characterization of the EXT2 gene. This gene shows striking sequence similarity to the EXT1 gene, and we have identified a four base deletion segregating with the phenotype. Both EXT1 and EXT2 show significant homology with one additional expressed sequence tag, defining a new multigene family of proteins with potential tumour suppressor activity. Show less

Hereditary multiple exostoses (EXT) is an autosomal dominant skeletal disorder characterized by the formation of multiple exostoses on the long bones. EXT is genetically heterogeneous, with at least three loci involved: one (EXT1) in the Langer-Giedion region on 8q23-q24, a second (EXT2) in the pericentromeric region of chromosome 11, and a third (EXT3) on chromosome 19p. In this study, linkage analysis in seven extended EXT families, all linked to the EXT2 locus, refined the localization of the EXT2 gene to a 3-cM region flanked by D11S1355 and D11S1361/D11S554. This implies that the EXT2 gene is located at the short arm of chromosome 11, in band 11p11-p12. The refined localization of EXT2 excludes a number of putative candidate genes located in the pericentromeric region of chromosome 11 and facilitates the process of isolating the EXT2 gene. Show less

Hereditary multiple exostosis (EXT) is an autosomal dominant disorder characterized by bony exostoses at the ends of the long bones. Linkage studies have recently suggested that there are three chromosomal locations for EXT genes, 8q24.1 (EXT1), the pericentric region of 11 (EXT2), and 19p (EXT3). As part of a larger study to determine the frequencies of the three EXT types in the United States, we have ascertained a large multigenerational family with EXT and one family member with a chondrosarcoma. This family demonstrated linkage of the disease to chromosome 11 markers. The constitutional and tumor DNAs from the affected family member were compared using short-tandem-repeat markers from chromosomes 8, 11, and 19. Loss of heterozygosity (LOH) in the tumor was observed for chromosome 8 and 11 markers, but chromosome 19 markers were intact. An apparent deletion of the marker D11S903 was observed in constitutional DNA from all affected individuals and in the tumor sample. These results indicate that the EXT2 gene maps to the region containing marker D11S903, which is flanked by markers D11S1355 and D11S1361. Additional constitutional and chondrosarcoma DNA pairs from six unrelated individuals, two of whom had EXT, were similarly analyzed. One tumor from an individual with EXT demonstrated LOH for chromosome 8 markers, and a person with a sporadic chondrosarcoma was found to have tumor-specific LOH and a homozygous deletion of chromosome 11 markers. These findings suggest that EXT genes may be tumor-suppressor genes and that the initiation of tumor development may follow a multistep model. Show less