FSCN1 and epithelial mesenchymal transformation transcription factor expression in human pancreatic intraepithelial neoplasia and ductal adenocarcinoma

Background: The actin regulatory protein fascin (FSCN1) and epithelial mesenchymal transition (EMT) transcription factor (TF) SLUG/SNAI2 have been shown to be expressed in PDAC and its precursor lesions (pancreatic intraepithelial neoplasia (PanIN), graded 1-3) in in vitro and murine in vivo studies. Our aim was to investigate the expression of FSCN1 and EMT-TFs and their association with survival in human PanIN and PDAC. Methods: Expression was investigated in silico using TCGA PanCancer Atlas data (177 PDAC samples with mRNA data) and immunohistochemical staining of a tissue microarray (TMA) (59 PDAC patients). Results: High FSCN1 expression was associated with poorer overall survival (p = 0.02) in the TCGA data. EMT-TF expression was not associated with survival, however FSCN1 expression correlated with that of the EMT-TFs SLUG/SNAI2 (rho = 0.49, p<0.001) and TWIST1 (rho = 0.52, p<0.001). TMA IHC showed low expression of SNAI2 and TWIST1 in normal ductal epithelium, while FSCN1 was not expressed. SNAI2 increased slightly in PanIN1-2, then decreased in higher grade lesions. TWIST1 increased in PanIN2-3 and was retained in PDAC. FSCN1 was increasingly expressed from PanIN2 onwards. SNAI2 and TWIST1 expression positively correlated in all grades of PanIN and PDAC (rho = 0.52, p<0.001). FSCN1 correlated positively with SNAI2 in PanIN1 (rho = 0.56, p<0.01). Conclusions: Increased expression of EMT-TFs in low-grade PanIN followed by FSCN1 in PanIN3 and PDAC suggests EMT-TFs may trigger FSCN1 expression and are potential early diagnostic markers. FSCN1 expression correlated with overall survival in PDAC and may have value as a prognostic marker

Regulation of Glucose Homeostasis by KSR1 and MARK2

Protein scaffolds control the intensity and duration of signaling and dictate the specificity of signaling through MAP kinase pathways. KSR1 is a molecular scaffold of the Raf/MEK/ERK MAP kinase cascade that regulates the intensity and duration of ERK activation. Relative to wild-type mice, ksr1-/- mice are modestly glucose intolerant, but show a normal response to exogenous insulin. However, ksr1-/- mice also demonstrate a three-fold increase in serum insulin levels in response to a glucose challenge, suggesting a role for KSR1 in insulin secretion. The kinase MARK2 is closely related to C-TAK1, a known regulator of KSR1. Mice lacking MARK2 have an increased rate of glucose disposal in response to exogenous insulin, increased glucose tolerance, and are resistant to diet-induced obesity. mark2-/-ksr1-/- (DKO) mice were compared to wild type, mark2-/-, and ksr1-/- mice for their ability to regulate glucose homeostasis. Here we show that disruption of KSR1 in mark2-/- mice reverses the increased sensitivity to exogenous insulin resulting from MARK2 deletion. DKO mice respond to exogenous insulin similarly to wild type and ksr1-/- mice. These data suggest a model whereby MARK2 negatively regulates insulin sensitivity in peripheral tissue through inhibition of KSR1. Consistent with this model, we found that MARK2 binds and phosphorylates KSR1 on Ser392. Phosphorylation of Ser392 is a critical regulator of KSR1 stability, subcellular location, and ERK activation. These data reveal an unexpected role for the molecular scaffold KSR1 in insulin-regulated glucose metabolism

New Functions of Ctf18-RFC in Preserving Genome Stability outside Its Role in Sister Chromatid Cohesion

Expansion of DNA trinucleotide repeats causes at least 15 hereditary neurological diseases, and these repeats also undergo contraction and fragility. Current models to explain this genetic instability invoke erroneous DNA repair or aberrant replication. Here we show that CAG/CTG tracts are stabilized in Saccharomyces cerevisiae by the alternative clamp loader/unloader Ctf18-Dcc1-Ctf8-RFC complex (Ctf18-RFC). Mutants in Ctf18-RFC increased all three forms of triplet repeat instability—expansions, contractions, and fragility—with effect over a wide range of allele lengths from 20–155 repeats. Ctf18-RFC predominated among the three alternative clamp loaders, with mutants in Elg1-RFC or Rad24-RFC having less effect on trinucleotide repeats. Surprisingly, chl1, scc1-73, or scc2-4 mutants defective in sister chromatid cohesion (SCC) did not increase instability, suggesting that Ctf18-RFC protects triplet repeats independently of SCC. Instead, three results suggest novel roles for Ctf18-RFC in facilitating genomic stability. First, genetic instability in mutants of Ctf18-RFC was exacerbated by simultaneous deletion of the fork stabilizer Mrc1, but suppressed by deletion of the repair protein Rad52. Second, single-cell analysis showed that mutants in Ctf18-RFC had a slowed S phase and a striking G2/M accumulation, often with an abnormal multi-budded morphology. Third, ctf18 cells exhibit increased Rad52 foci in S phase, often persisting into G2, indicative of high levels of DNA damage. The presence of a repeat tract greatly magnified the ctf18 phenotypes. Together these results indicate that Ctf18-RFC has additional important functions in preserving genome stability, besides its role in SCC, which we propose include lesion bypass by replication forks and post-replication repair

Rap1b facilitates NK cell functions via IQGAP1-mediated signalosomes

Rap1 GTPases control immune synapse formation and signaling in lymphocytes. However, the precise molecular mechanism by which Rap1 regulates natural killer (NK) cell activation is not known. Using Rap1a or Rap1b knockout mice, we identify Rap1b as the major isoform in NK cells. Its absence significantly impaired LFA1 polarization, spreading, and microtubule organizing center (MTOC) formation in NK cells. Neither Rap1 isoform was essential for NK cytotoxicity. However, absence of Rap1b impaired NKG2D, Ly49D, and NCR1-mediated cytokine and chemokine production. Upon activation, Rap1b colocalized with the scaffolding protein IQGAP1. This interaction facilitated sequential phosphorylation of B-Raf, C-Raf, and ERK1/2 and helped IQGAP1 to form a large signalosome in the perinuclear region. These results reveal a previously unrecognized role for Rap1b in NK cell signaling and effector functions

Zfp521 controls bone mass by HDAC3-dependent attenuation of Runx2 activity

Zfp521 regulates osteoblast development during lineage commitment and osteoblast maturation by suppressing Runx2 transcriptional activity

Aurora-A overexpression enhances cell-aggregation of Ha-ras transformants through the MEK/ERK signaling pathway

<p>Abstract</p> <p>Background</p> <p>Overexpression of Aurora-A and mutant Ras (Ras^V12) together has been detected in human bladder cancer tissue. However, it is not clear whether this phenomenon is a general event or not. Although crosstalk between Aurora-A and Ras signaling pathways has been reported, the role of these two genes acting together in tumorigenesis remains unclear.</p> <p>Methods</p> <p>Real-time PCR and sequence analysis were utilized to identify Ha- and Ki-<it>ras </it>mutation (Gly -> Val). Immunohistochemistry staining was used to measure the level of Aurora-A expression in bladder and colon cancer specimens. To reveal the effect of overexpression of the above two genes on cellular responses, mouse NIH3T3 fibroblast derived cell lines over-expressing either Ras^V12and wild-type Aurora-A (designated WT) or Ras^V12and kinase-inactivated Aurora-A (KD) were established. MTT and focus formation assays were conducted to measure proliferation rate and focus formation capability of the cells. Small interfering RNA, pharmacological inhibitors and dominant negative genes were used to dissect the signaling pathways involved.</p> <p>Results</p> <p>Overexpression of wild-type Aurora-A and mutation of Ras^V12were detected in human bladder and colon cancer tissues. Wild-type Aurora-A induces focus formation and aggregation of the Ras^V12transformants. Aurora-A activates Ral A and the phosphorylation of AKT as well as enhances the phosphorylation of MEK, ERK of WT cells. Finally, the Ras/MEK/ERK signaling pathway is responsible for Aurora-A induced aggregation of the Ras^V12transformants.</p> <p>Conclusion</p> <p>Wild-type-Aurora-A enhances focus formation and aggregation of the Ras^V12transformants and the latter occurs through modulating the Ras/MEK/ERK signaling pathway.</p

Histone Deacetylase 3 Depletion in Osteo/Chondroprogenitor Cells Decreases Bone Density and Increases Marrow Fat

Histone deacetylase (Hdac)3 is a nuclear enzyme that contributes to epigenetic programming and is required for embryonic development. To determine the role of Hdac3 in bone formation, we crossed mice harboring loxP sites around exon 7 of Hdac3 with mice expressing Cre recombinase under the control of the osterix promoter. The resulting Hdac3 conditional knockout (CKO) mice were runted and had severe deficits in intramembranous and endochondral bone formation. Calvarial bones were significantly thinner and trabecular bone volume in the distal femur was decreased 75% in the Hdac3 CKO mice due to a substantial reduction in trabecular number. Hdac3-CKO mice had fewer osteoblasts and more bone marrow adipocytes as a proportion of tissue area than their wildtype or heterozygous littermates. Bone formation rates were depressed in both the cortical and trabecular regions of Hdac3 CKO femurs. Microarray analyses revealed that numerous developmental signaling pathways were affected by Hdac3-deficiency. Thus, Hdac3 depletion in osterix-expressing progenitor cells interferes with bone formation and promotes bone marrow adipocyte differentiation. These results demonstrate that Hdac3 inhibition is detrimental to skeletal health