The Central Region of the Drosophila Co-repressor Groucho as a Regulatory Hub

Groucho (Gro) is a Drosophila co-repressor that regulates the expression of a large number of genes, many of which are involved in developmental control. Previous studies have shown that its central region is essential for function even though its three domains are poorly conserved and intrinsically disordered. Using these disordered domains as affinity reagents, we have now identified multiple embryonic Gro-interacting proteins. The interactors include protein complexes involved in chromosome organization, mRNA processing, and signaling. Further investigation of the interacting proteins using a reporter assay showed that many of them modulate Gro-mediated repression either positively or negatively. The positive regulators include components of the spliceosomal subcomplex U1 small nuclear ribonucleoprotein (U1 snRNP). A co-immunoprecipitation experiment confirms this finding and suggests that a sizable fraction of nuclear U1 snRNP is associated with Gro. The use of RNA-seq to analyze the gene expression profile of cells subjected to knockdown of Gro or snRNP-U1-C (a component of U1 snRNP) showed a significant overlap between genes regulated by these two factors. Furthermore, comparison of our RNA-seq data with Gro and RNA polymerase II ChIP data led to a number of insights, including the finding that Gro-repressed genes are enriched for promoter-proximal RNA polymerase II. We conclude that the Gro central domains mediate multiple interactions required for repression, thus functioning as a regulatory hub. Furthermore, interactions with the spliceosome may contribute to repression by Gro.     

Overexpression of α2,3sialyl T-antigen in breast cancer determined by miniaturized glycosyltransferase assays and confirmed using tissue microarray immunohistochemical analysis

Glycan structure alterations during cancer regulate disease progression and represent clinical biomarkers. The study determined the degree to which changes in glycosyltransferase activities during cancer can be related to aberrant cell-surface tumor associated carbohydrate structures (TACA). To this end, changes in sialyltransferase (sialylT), fucosyltransferase (fucT) and galactosyltransferase (galT) activity were measured in normal and tumor tissue using a miniaturized enzyme activity assay and synthetic glycoconjugates bearing terminal LacNAc Type-I (Galβ1-3GlcNAc), LacNAc Type-II (Galβ1-4GlcNAc), and mucin core-1/Type-III (Galβ1-3GalNAc) structures. These data were related to TACA using tissue microarrays containing 115 breast and 26 colon cancer specimen. The results show that primary human breast and colon tumors, but not adjacent normal tissue, express elevated β1,3GalT and α2,3SialylT activity that can form α2,3SialylatedType-IIIglycans (Siaα2-3Galβ1-3GalNAc). Prostate tumors did not exhibit such elevated enzymatic activities. α1,3/4FucT activity was higher in breast, but not in colon tissue. The enzymology based prediction of enhanced α2,3sialylated Type-III structures in breast tumors was verified using histochemical analysis of tissue sections and tissue microarrays. Here, the binding of two markers that recognize Galβ1-3GalNAc (peanut lectin and mAb A78-G/A7) was elevated in breast tumor, but not in normal control, only upon sialidase treatment. These antigens were also upregulated in colon tumors though to a lesser extent. α2,3sialylatedType-III expression correlated inversely with patient HER2 expression and breast metastatic potential. Overall, enzymology measurements of glycoT activity predict truncated O-glycan structures in tumors. High expression of the α2,3sialylated T-antigen O-glycans occur in breast tumors. A transformation from linear core-1 glycan to other epitopes may accompany metastasis.     

A FAK scaffold inhibitor disrupts FAK and VEGFR-3 signaling and blocks melanoma growth by targeting both tumor and endothelial cells

Melanoma has the highest mortality rate of all skin cancers and a major cause of treatment failure is drug resistance. Tumors heterogeneity requires novel therapeutic strategies and new drugs targeting multiple pathways. One of the new approaches is targeting the scaffolding function of tumor related proteins such as focal adhesion kinase (FAK). FAK is overexpressed in most solid tumors and is involved in multiple protein-protein interactions critical for tumor cell survival, tumor neovascularization, progression and metastasis. In this study, we investigated the anticancer activity of the FAK scaffold inhibitor C4, targeted to the FAK-VEGFR-3 complex, against melanomas. We compared C4 inhibitory effects in BRAF mutant vs BRAF wild type melanomas. C4 effectively caused melanoma tumor regression in vivo, when administered alone and sensitized tumors to chemotherapy. The most dramatic effect of C4 was related to reduction of vasculature of both BRAF wild type and V600E mutant xenograft tumors. The in vivo effects of C4 were assessed in xenograft models using non-invasive multimodality imaging in conjunction with histologic and molecular biology methods. C4 inhibited cell viability, adhesion and motility of melanoma and endothelial cells, specifically blocked phosphorylation of VEGFR-3 and FAK and disrupted their complexes. Specificity of in vivo effects for C4 were confirmed by a decrease in tumor FAK and VEGFR-3 phosphorylation, reduction of vasculogenesis and reduced blood flow. Our collective observations provide evidence that a small molecule inhibitor targeted to the FAK protein-protein interaction site successfully inhibits melanoma growth through dual targeting of tumor and endothelial cells and is effective against both BRAF wild type and mutant melanomas.     

TAZ induces growth factor-independent proliferation through activation of EGFR ligand amphiregulin

The Hippo signaling pathway regulates cellular proliferation and survival, thus exerting profound effects on normal cell fate and tumorigenesis. We previously showed that the pivotal effector of this pathway, YAP, is amplified in tumors and promotes epithelial-to-mesenchymal transition (EMT) and malignant transformation. Here, we report that overexpression of TAZ, a paralog of YAP, in human mammary epithelial cells promotes EMT and, in particular, some invasive structures in 3D cultures. TAZ also leads to cell migration and anchorage-independent growth in soft agar. Furthermore, we identified amphiregulin (AREG), an epidermal growth factor receptor (EGFR) ligand, as a target of TAZ. We show that AREG functions in a non-cell-autonomous manner to mediate EGF-independent growth and malignant behavior of mammary epithelial cells. In addition, ablation of TEAD binding completely abolishes the TAZ-induced phenotype. Last, analysis of breast cancer patient samples reveals a positive correlation between TAZ and AREG in vivo. In summary, TAZ-dependent secretion of AREG indicates that activation of the EGFR signaling is an important non-cell-autonomous effector of the Hippo pathway, and TAZ as well as its targets may play significant roles in breast tumorigenesis and metastasis.     

Atomic force microscopy reveals important differences in axonal resistance to injury

Axonal degeneration after traumatic brain injury and nerve compression is considered a common underlying cause of temporary as well as permanent disability. Because a proper functioning of neural network requires phase coherence of all components, even subtle changes in circuitry may lead to network failure. However, it is still not possible to determine which axons will recover or degenerate after injury. Several groups have studied the pressure threshold for axonal injury within a nerve, but difficulty accessing the injured region; insufficient imaging methods and the extremely small dimensions involved have prevented the evaluation of the response of individual axons to injury. We combined microfluidics with atomic force microscopy and in vivo imaging to estimate the threshold force required to 1), uncouple axonal transport without impairing axonal survival, and 2), compromise axonal survival in both individual and bundled axons. We found that rat hippocampal axons completely recover axonal transport with no detectable axonal loss when compressed with pressures up to 65 ± 30 Pa for 10 min, while dorsal root ganglia axons can resist to pressures up to 540 ± 220 Pa. We investigated the reasons for the differential susceptibility of hippocampal and DRG axons to mechanical injury and estimated the elasticity of live axons. We found that dorsal root ganglia axons have a 20% lower elastic modulus than hippocampal axons. Our results emphasize the importance of the integrity of the axonal cytoskeleton in deciding the axonal fate after damage and open up new avenues to improve injury diagnosis and to identify ways to protect axons.     

Intrathyroidal parathyroid carcinoma: a case report with clinical and histological findings

Aims

To analyse the histo-morphology of cases of medullomyoblastoma and identifying its divergent differentiation.

Methods

A retrospective review of all cases reported as medulloblastoma between the period of Jan 2000 to Dec 2006 was carried out on Hematoxylin and eosin (H & E) stained slides. The cases were screened on light microscopy for primitive neuroectodermal component of a medulloblastoma accompanied by areas of "myoid" differentiation, identified on the basis of presence of strap cells (indicating a clear skeletal muscle differentiation) and/or large anaplastic cells with vescicular nuclei and moderate to abundant amount of eosinophilic cytoplasm. All these cases were subjected to a panel of immunohistochemical stains, including Desmin, GFAP, NFP, HMB45, SMA, S100, CK and EMA. Ultrastructral analysis was done on tissue obtained from paraffin blocks in 2 cases.

Results

Male predominance (M:F = 5:1) was noted with an incidence of five percent of all cases of medulloblastoma (6 out of 120 cases) over a period of 6 years. Primitive neuroectodermal areas were accompanied with areas of "myoid" differentiation, 5 cases showing strap cells. Two cases with epithelial and cartilaginous differentiation were seen. Three cases showed focal melanocytic differentiation, identified only on HMB45 immunostaining. Four cases showed glial differentiation. Neuronal differentiation again was very focally seen in two cases, of which one was identified only by NFP immunostain. Seventh case is included in the study, however it is not considered to calculate incidence as it occurred beyond the period of 6 years of records search.

Conclusion

Medullomyoblastoma is a rare childhood tumor of cerebellum. Majority of cases reveal divergent differentiation, which are identified with the help of panel of immunostains indicating multi-potential nature of primitive neuroectodermal cells.     

Differential vitamin D 24-hydroxylase/CYP24A1 gene promoter methylation in endothelium from benign and malignant human prostate

Epigenetic alterations occur in tumor-associated vessels in the tumor microenvironment. Methylation of the CYP24A1 gene promoter differs in endothelial cells isolated from tumors and non-tumor microenvironments in mice. The epigenetic makeup of endothelial cells of human tumor-associated vasculature is unknown due to difficulty of isolating endothelial cells populations from a heterogeneous tissue microenvironment. To ascertain CYP24A1 promoter methylation in tumor-associated endothelium, we utilized laser microdissection guided by CD31 immunohistochemistry to procure endothelial cells from human prostate tumor specimens. Prostate tissues were obtained following robotic radical prostatectomy from men with clinically localized prostate cancer. Adjacent histologically benign prostate tissues were used to compare endothelium from benign versus tumor microenvironments. Sodium bisulfite sequencing of CYP24A1 promoter region showed that the average CYP24A1 promoter methylation in the endothelium was 20% from the tumor microenvironment compared with 8.2% in the benign microenvironment (p < 0.05). A 2-fold to 17-fold increase in CYP24A1 promoter methylation was observed in the prostate tumor endothelium compared with the matched benign prostate endothelium in four patient samples, while CYP24A1 promoter methylation remained unchanged in two patient samples. In addition, there is no correlation of the level of CYP24A1 promoter methylation in prostate tumor-associated endothelium with that of epithelium/stroma. This study demonstrates that the CYP24A1 promoter is methylated in tumor-associated endothelium, indicating that epigenetic alterations in CYP24A1 may play a role in determining the phenotype of tumor-associated vasculature in the prostate tumor microenvironment.Key words: CYP24A1, DNA methylation, human prostate cancer, tumor endothelium, laser microdissection