BACKGROUND

Idiopathic pulmonary fibrosis (IPF) is a chronic fibrosing interstitial lung disease of unknown etiology that is currently untreatable. In this study we aim to characterize the potential of extracellular/circulating microRNAs (miRNAs) in serum as biomarkers for IPF.

METHODS

Total serum RNAs were isolated from serum from healthy control subjects (n=<em>12</em>), rapid progressive (n=<em>12</em>) and slowly progressive IPF patients (n=<em>12</em>). Serum RNA was analyzed by using TaqMan microRNA assays containing probes for 366 human miRNAs, and selected findings were validated with quantitative RT-PCR. Target prediction and pathway analysis on the significant differential miRNAs were performed using DIANA-mirPath.

RESULTS

We found 47 significantly differentially expressed serum miRNAs (p<0.05) in rapid progressive or slowly progressive IPF patients compared to healthy controls, including 21 up-regulated miRNAs and 26 down-regulated miRNAs. Bioinformatic analysis by DIANA-mirPath demonstrated that 53 KEGG biological processes were significantly enriched (p < 0.05, FDR corrected) among differentially expressed serum miRNAs, including TGF-beta signaling pathway (p < 0.0001), MAPK signaling pathway (p < 0.0001), PI3K-Akt signaling pathway (p < 0.0001), Wnt signaling pathway (p < 0.0001), HIF-1 signaling pathway (p < 0.0001), Regulation of actin cytoskeleton (p < 0.0001), Jak-STAT signaling pathway (p < 0.0001), Notch signaling pathway (p < 0.0001), and Cytokine-cytokine receptor interaction (p = 0.0062). We further validated six miRNAs (miR-21, miR-199a-5p, miR-200c, miR-31, let-7a, and let-7d) for further validation using an independent cohort of 20 rapid progressive IPF, 24 slowly progressive IPF patients and 20 healthy controls. In agreement with the preliminary data from miRNA assay, miR-21, miR-199a-5p, and miR-200c were significantly increased in serums of IPF patients while miR-31, let-7a, and let-7d were significantly under expressed in serums of IPF patients compared to healthy controls.

CONCLUSIONS

These studies demonstrated that extracellular/circulating miRNAs in serum could be potentially served as novel regulators influencing disease progression of IPF.

T cells resistant to the immunosuppressive drug cyclosporin A (CsA) may be important mediators of chronic graft rejection. We previously reported that T cells activated in the presence of endothelial cells (EC) develop resistance to CsA, and initiate IL-2 secretion within 8-<em>12</em> h of triggering. CsA normally blocks the phosphatase, calcineurin, thus preventing nuclear translocation of the transcription factor, NFAT. We find that in the presence but not the absence of EC, NFAT1 can be detected in the nuclei of CsA-treated T cells within 8 h of triggering, reaching a maximal level of 60% of control by 24 h. Glycogen synthase kinase-3beta (GSK-3beta), which rephosphorylates NFAT and promotes nuclear export, is inhibited by EC costimulation. GSK-3beta is a component of the <em>wnt</em> signaling pathway, and EC express <em>wnt</em>-5a and T cells express frizzled-5, a <em>wnt</em>-5a receptor. <em>Wnt</em>-5a promotes T cell NFAT nuclear accumulation in the presence of CsA, an effect mimicked by Li(+), a potent inhibitor of GSK-3beta. The protein kinase C agonist PMA dramatically synergizes with both EC and <em>wnt</em>-5a in stimulating T cell IL-2 synthesis, and inhibition of either protein kinase C by Ro-31-8425 or G-proteins by pertussis toxin effectively blocks the actions of <em>wnt</em>-5a on T cells. Finally, a secreted, dominant-negative form of frizzled-5 blocks EC-mediated CsA resistance. Thus, EC promote CsA-resistant nuclear localization of NFAT and subsequent IL-2 synthesis through a noncanonical <em>wnt</em>-dependent pathway.

β-Catenin transduces the <em>Wnt</em> signal from the membrane to nucleus, and certain gene mutations trigger its nuclear accumulation leading to cell transformation and cancer. β-Catenin shuttles between the nucleus and cytoplasm independent of classical Ran/transport receptor pathways, and this movement was previously hypothesized to involve the central Armadillo (Arm) domain. Fluorescence recovery after photobleaching (FRAP) assays were used to delineate functional transport regions of the Arm domain in living cells. The strongest nuclear import/export activity was mapped to Arm repeats R10-<em>12</em> using both in vivo FRAP and in vitro export assays. By comparison, Arm repeats R3-8 of β-catenin were highly active for nuclear import but displayed a comparatively weak export activity. We show for the first time using purified components that specific Arm sequences of β-catenin interact directly in vitro with the FG repeats of the nuclear pore complex (NPC) components Nup62, Nup98, and Nup153, indicating an independent ability of β-catenin to traverse the NPC. Moreover, a proteomics screen identified RanBP2/Nup358 as a binding partner of Arm R10-<em>12</em>, and β-catenin was confirmed to interact with endogenous and ectopic forms of Nup358. We further demonstrate that knock-down of endogenous Nup358 and Nup62 impeded the rate of nuclear import/export of β-catenin to a greater extent than that of importin-β. The Arm R10-<em>12</em> sequence facilitated transport even when β-catenin was bound to the Arm-binding partner LEF-1, and its activity was stimulated by phosphorylation at Tyr-654. These findings provide functional evidence that the Arm domain contributes to regulated β-catenin transport through direct interaction with the NPC.

OBJECTIVE

Preoperative chemoradiotherapy (CRT) represents the standard treatment for locally advanced rectal cancer. Tumor response and progression vary considerably. MicroRNAs represent master regulators of gene expression, and may therefore contribute to this diversity.

METHODS

Genome-wide microRNA (miRNA) profiling was performed for <em>12</em> colorectal cancer (CRC) cell lines and an individual in vitro signature of chemoradiosensitivity was established. Functional relevance of selected miRNAs was established by transfecting miRNA-mimics into SW480 and SW837 cells. The prognostic value of selected miRNAs was assessed in <em>12</em>8 pretherapeutic patient biopsies.

RESULTS

Thirty-six miRNAs were identified to significantly correlate with sensitivity to CRT (Q < 0.05) including miR-320a and other miRNAs involved in the MAPK-, TGF- and Wnt-pathway. Transfection of selected miRNAs (let-7g, miR-132, miR-224, miR-320a) each induced a shift of sensitivity. High expression of let-7 g was associated with a good prognosis in rectal cancer patients (P = 0.03).

CONCLUSIONS

This is the first report of a miRNA expression signature for in vitro chemoradiosensitivity of CRC cell lines. Many of the identified miRNAs have not been linked to the response to CRT and may represent potential molecular targets to sensitize resistant cancers. If further validated, let7g expression may serve as predictive biomarker.

Chronic obstructive pulmonary disease (COPD) is characterised by a progressive loss of lung tissue. Inducing repair processes within the adult diseased lung is of major interest and <em>Wnt</em>/β-catenin signalling represents a promising target for lung repair. However, the translation of novel therapeutic targets from model systems into clinical use remains a major challenge.We generated murine and patient-derived three-dimensional (3D) ex vivo lung tissue cultures (LTCs), which closely mimic the 3D lung microenvironment in vivo. Using two well-known glycogen synthase kinase-3β inhibitors, lithium chloride (LiCl) and CHIR 99021 (CT), we determined <em>Wnt</em>/β-catenin-driven lung repair processes in high spatiotemporal resolution using quantitative PCR, Western blotting, ELISA, (immuno)histological assessment, and four-dimensional confocal live tissue imaging.Viable 3D-LTCs exhibited preserved lung structure and function for up to 5 days. We demonstrate successful <em>Wnt</em>/β-catenin signal activation in murine and patient-derived 3D-LTCs from COPD patients. <em>Wnt</em>/β-catenin signalling led to increased alveolar epithelial cell marker expression, decreased matrix metalloproteinase-<em>12</em> expression, as well as altered macrophage activity and elastin remodelling. Importantly, induction of surfactant protein C significantly correlated with disease stage (per cent predicted forced expiratory volume in 1 s) in patient-derived 3D-LTCs.Patient-derived 3D-LTCs represent a valuable tool to analyse potential targets and drugs for lung repair. Enhanced <em>Wnt</em>/β-catenin signalling attenuated pathological features of patient-derived COPD 3D-LTCs.

The delineation of regulatory networks involved in early endocrine pancreas specification will play a crucial role in directing the differentiation of embryonic stem cells toward the mature phenotype of beta cells for cell therapy of type 1 diabetes. The transcription factor Ngn3 is required for the specification of the endocrine lineage, but its direct targets and the scope of biological processes it regulates remain elusive. We show that stepwise differentiation of embryonic stem cells using successive in vivo patterning signals can lead to simultaneous induction of Ptf1a and Pdx1 expression. In this cellular context, Ngn3 induction results in upregulation of its known direct target genes within <em>12</em> hours. Microarray gene expression profiling at distinct time points following Ngn3 induction suggested novel and diverse roles of Ngn3 in pancreas endocrine cell specification. Induction of Ngn3 expression results in regulation of the <em>Wnt</em>, integrin, Notch, and transforming growth factor beta signaling pathways and changes in biological processes affecting cell motility, adhesion, the cytoskeleton, the extracellular matrix, and gene expression. Furthermore, the combination of in vivo patterning signals and inducible Ngn3 expression enhances ESC differentiation toward the pancreas endocrine lineage. This is shown by strong upregulation of endocrine lineage terminal differentiation markers and strong expression of the hormones glucagon, somatostatin, and insulin. Importantly, all insulin(+) cells are also C-peptide(+), and glucose-dependent insulin release was 10-fold higher than basal levels. These data suggest that bona fide pancreas endocrine cells have been generated and that timely induction of Ngn3 expression can play a decisive role in directing ESC differentiation toward the endocrine lineage.

The murine <em>Wnt</em> family of proteins consists of at least <em>12</em> members that possess significant amino acid homology. Current evidence suggests that these proteins are secreted cell-signaling molecules which are likely to have multiple roles during both embryonic development and oncogenesis. Although the biochemical properties of <em>Wnt</em>-1 have been thoroughly examined, less is known about the characteristics of other <em>Wnt</em> family members. We have compared the properties of six murine <em>Wnt</em> proteins (<em>Wnt</em>-1, <em>Wnt</em>-3a, <em>Wnt</em>-5a, <em>Wnt</em>-5b, <em>Wnt</em>-6, and <em>Wnt</em>-7b) transiently expressed in COS cells. All members enter the endoplasmic reticulum (ER) and are glycosylated. However, all six <em>Wnt</em> proteins are primarily retained in the ER in association with BiP, a resident ER protein that binds to improperly folded proteins and prevents their secretion and/or promotes proper folding. Although all <em>Wnt</em> family members examined are similarly processed, one notable difference was identified. Whereas addition of suramin to COS cell cultures significantly increases the levels of all six <em>Wnts</em> in the medium, the addition of heparin only influences the levels of <em>Wnt</em>-1, <em>Wnt</em>-6, and <em>Wnt</em>-7b.

Adrenal tumors are common, with an estimated incidence of 7.3% in autopsy cases, while adrenocortical carcinomas (ACCs) are rare, with an estimated prevalence of 4-<em>12</em> per million population. Because the prognoses for adrenocortical adenomas (ACAs) and ACCs are vastly different, it is important to be able to accurately differentiate the two tumor types. Advancement in the understanding of the pathophysiology of ACCs is essential for the development of more sensitive means of diagnosis and treatment, resulting in better clinical outcome. Adrenocortical tumors (ACTs) occur as a component of several hereditary tumor syndromes, which include the Li-Fraumeni syndrome, Beckwith-Wiedemann syndrome, multiple endocrine neoplasia 1, Carney complex, and congenital adrenal hyperplasia. The genes involved in these syndromes have also been shown to play a role in the pathogenesis of sporadic ACTs. The adrenocorticotropic hormone-cAMP-protein kinase A and <em>Wnt</em> pathways are also implicated in adrenocortical tumorigenesis. The aim of this review is to summarize the current knowledge on the molecular mechanisms involved in adrenocortical tumorigenesis, including results of comparative genomic hybridization, loss of heterozygosity, and microarray gene-expression profiling studies.

<em>Wnt</em>/β-catenin signaling pathway plays an important role in the genesis and development of Alzheimer's disease. The study aims to investigate the effect of Curcumin on the expression of GSK-3β, β-catenin and CyclinD1 in vitro, which are tightly correlated with <em>Wnt</em>/β-catenin signaling pathway, and also to explore the mechanisms, which will provide a novel therapeutic intervention for treatment of Alzheimer's disease. Plasmid APPswe and BACE1-mychis were transiently co-transfected into SHSY5Y cells by Liposfectamin™2000. The cells were treated with Curcumin at 0, 1.25, 5.0, 20.0 μmol/L for 24 h, or with Curcumin at 5.0 μmol/L for 0, and <em>12</em>, 24 and 48 h for time course assay. Cell lysates were collected for RT-PCR, Western blot assay and immunofluorescent staining were carried out for detecting the effect of Curcumin on the expression of GSK-3β, β-catenin and CyclinD1. RT-PCR and Western blot results showed that the expression of GSK-3β mRNA and protein significantly decreased in the transfected cells treated with Curcumin, and that the changes were in a dose and time-dependent manner (P<0.05); however, the protein expression of GSK-3β-Ser9 was increased (P<0.05). Meanwhile, the expressions of β-catenin and transcriptional factors CyclinD1 mRNA and protein increased and the changes were also in a dose and time-dependent manner (P<0.05). Immunofluorescent staining results not only confirmed the above changes, but also showed that β-catenin had translocated into the nucleus gradually with the increased dosage of Curcumin. Therefore, GSK-3β is a potential target for treatment of AD. Curcumin could activate the <em>Wnt</em>/β-catenin signaling pathway through inhibiting the expression of GSK-3β and inducing the expression of β-catenin and CyclinD1, which will provide a new theory for treatment of neurodegenerative diseases by Curcumin.

Uterine leiomyomas are common benign smooth muscle tumors that impose a major burden on women's health. Recent sequencing studies have revealed recurrent and mutually exclusive mutations in leiomyomas, suggesting the involvement of molecularly distinct pathways. In this study, we explored transcriptional differences among leiomyomas harboring different genetic drivers, including high mobility group AT-hook 2 (HMGA2) rearrangements, mediator complex subunit <em>12</em> (MED<em>12</em>) mutations, biallelic inactivation of fumarate hydratase (FH), and collagen, type IV, alpha 5 and collagen, type IV, alpha 6 (COL4A5-COL4A6) deletions. We also explored the transcriptional consequences of 7q22, 22q, and 1p deletions, aiming to identify possible target genes. We investigated 94 leiomyomas and 60 corresponding myometrial tissues using exon arrays, whole genome sequencing, and SNP arrays. This integrative approach revealed subtype-specific expression changes in key driver pathways, including <em>Wnt</em>/β-catenin, Prolactin, and insulin-like growth factor (IGF)1 signaling. Leiomyomas with HMGA2 aberrations displayed highly significant up-regulation of the proto-oncogene pleomorphic adenoma gene 1 (PLAG1), suggesting that HMGA2 promotes tumorigenesis through PLAG1 activation. This was supported by the identification of genetic PLAG1 alterations resulting in expression signatures as seen in leiomyomas with HMGA2 aberrations. RAD51 paralog B (RAD51B), the preferential translocation partner of HMGA2, was up-regulated in MED<em>12</em> mutant lesions, suggesting a role for this gene in the genesis of leiomyomas. FH-deficient leiomyomas were uniquely characterized by activation of nuclear factor erythroid 2-related factor 2 (NRF2) target genes, supporting the hypothesis that accumulation of fumarate leads to activation of the oncogenic transcription factor NRF2. This study emphasizes the need for molecular stratification in leiomyoma research and possibly in clinical practice as well. Further research is needed to determine whether the candidate biomarkers presented herein can provide guidance for managing the millions of patients affected by these lesions.

OBJECTIVE

The standard treatment of patients with locally advanced rectal cancers comprises preoperative 5-fluorouracil-based chemoradiotherapy followed by standardized surgery. However, tumor response to multimodal treatment has varied greatly, ranging from complete resistance to complete pathologic regression. The prediction of the response is, therefore, an important clinical need.

METHODS

To establish in vitro models for studying the molecular basis of this heterogeneous tumor response, we exposed <em>12</em> colorectal cancer cell lines to 3 μM of 5-fluorouracil and 2 Gy of radiation. The differences in treatment sensitivity were then correlated with the pretherapeutic gene expression profiles of these cell lines.

RESULTS

We observed a heterogeneous response, with surviving fractions ranging from 0.28 to 0.81, closely recapitulating clinical reality. Using a linear model analysis, we identified 4,796 features whose expression levels correlated significantly with the sensitivity to chemoradiotherapy (Q <.05), including many genes involved in the mitogen-activated protein kinase signaling pathway or cell cycle genes. These data have suggested a potential relevance of the insulin and Wnt signaling pathways for treatment response, and we identified STAT3, RASSF1, DOK3, and ERBB2 as potential therapeutic targets. The microarray measurements were independently validated for a subset of these genes using real-time polymerase chain reactions.

CONCLUSIONS

We are the first to report a gene expression signature for the in vitro chemoradiosensitivity of colorectal cancer cells. We anticipate that this analysis will unveil molecular biomarkers predictive of the response of rectal cancers to chemoradiotherapy and enable the identification of genes that could serve as targets to sensitize a priori resistant primary tumors.

Secreted Frizzled-related protein-1 (sFRP-1), a soluble protein that binds to <em>Wnts</em> and modulates <em>Wnt</em> signaling, contains an N-terminal domain homologous to the putative <em>Wnt</em>-binding site of Frizzled (Fz domain) and a C-terminal heparin-binding domain with weak homology to netrin. Both domains are cysteine-rich, having 10 and 6 cysteines in the Fz and heparin-binding domains, respectively. In this study, the disulfide linkages of recombinant sFRP-1 were determined. Numbering sFRP-1 cysteines sequentially from the N terminus, the five disulfide linkages in the Fz domain are 1-5, 2-4, 3-8, 6-10, and 7-9, consistent with the disulfide pattern determined for homologous domains of several other proteins. The disulfide linkages of the heparin-binding domain are 11-14, <em>12</em>-15, and 13-16. This latter set of assignments provides experimental verification of one of the disulfide patterns proposed for netrin (NTR) modules and thereby supports the prediction that the C-terminal heparin-binding domain of sFRP-1 is an NTR-type domain. Interestingly, two subsets of sFRPs appear to have alternate disulfide linkage patterns compared with sFRP-1, one of which involves the loss of a disulfide due to deletion of a single cysteine from the NTR module, whereas the remaining cysteine may pair with a new cysteine introduced in the Fz domain of the protein. Analysis of glycosylation sites showed that sFRP-1 contains a relatively large carbohydrate moiety on Asn(172) (approximately 2.8 kDa), whereas Asn(262), the second potential N-linked glycosylation site, is not modified. No O-linked carbohydrate groups were detected. There was evidence of heterogeneous proteolytic processing at both the N and C termini of the recombinant protein. The predominant N terminus was Ser(31), although minor amounts of the protein with Asp(41) and Phe(50) as the N termini were observed. The major C-terminal processing event was removal of the terminal amino acid (Lys(313)) with only a trace amount of unprocessed protein detected.

BACKGROUND

PRDM (PRDI-BF1 and RIZ domain containing) proteins are zinc finger proteins involved in multiple cellular regulations by acting as epigenetic modifiers. We studied a recently identified PRDM member PRDM5 for its epigenetic abnormality and tumor suppressive functions in multiple tumorigeneses.

RESULTS

Semi-quantitative RT-PCR showed that PRDM5 was broadly expressed in human normal tissues, but frequently silenced or downregulated in multiple carcinoma cell lines due to promoter CpG methylation, including 80% (4/5) nasopharyngeal, 44% (8/18) esophageal, 76% (13/17) gastric, 50% (2/4) cervical, and 25% (3/<em>12</em>) hepatocellular carcinoma cell lines, but not in any immortalized normal epithelial cell lines. PRDM5 expression could be restored by 5-aza-2'-deoxycytidine demethylation treatment in silenced cell lines. PRDM5 methylation was frequently detected by methylation-specific PCR (MSP) in multiple primary tumors, including 93% (43/46) nasopharyngeal, 58% (25/43) esophageal, 88% (37/42) gastric and 63% (29/46) hepatocellular tumors. PRDM5 was further found a stress-responsive gene, but its response was impaired when the promoter was methylated. Ectopic PRDM5 expression significantly inhibited tumor cell clonogenicity, accompanied by the inhibition of TCF/β-catenin-dependent transcription and downregulation of CDK4, TWIST1 and MDM2 oncogenes, while knocking down of PRDM5 expression lead to increased cell proliferation. ChIP assay showed that PRDM5 bound to its target gene promoters and suppressed their transcription. An inverse correlation between the expression of PRDM5 and activated β-catenin was also observed in cell lines.

CONCLUSIONS

PRDM5 functions as a tumor suppressor at least partially through antagonizing aberrant WNT/β-catenin signaling and oncogene expression. Frequent epigenetic silencing of PRDM5 is involved in multiple tumorigeneses, which could serve as a tumor biomarker.

Nasopharyngeal carcinoma (NPC) is a common tumor in Southern China with marked ethnic and geographic distributions and concomitant EBV infection. However, the molecular basis of NPC remains largely unknown and the role of EBV genomic variations in the pathogenesis of NPC is unclear. Whole-genome sequencing of a collection of <em>12</em> EBV-positive paired NPC tumor/peripheral blood samples from Hunan province was performed, and the FBXO11 gene was subjected to further functional analyses. We identified 69 missense mutations in signaling pathways typically altered in cancer, including NF-κB and <em>Wnt</em>/Hedgehog/Notch. Additionally, <em>12</em>2 variations were identified in non-coding regions. Among these, a subset of genes was confirmed as dysregulated in NPC by mining the NPC cDNA microarray database. The randomly selected gene, FBXO11, could promote the malignant progression of NPC in vitro. Full-length EBV genomes from 8 of the <em>12</em> patients with NPC were also successfully assembled, and latent EBV infection is a primary cause of NPC. The various subtypes of EBV detected exhibited clear correlations with its geographical distribution. This study has explored novel biological markers and tumorigenic pathways with substantial potential to enhance therapeutic strategies for NPC.

Brg1 is a member of the SWI/SNF chromatin-remodeling complex, and in some organisms Brg1 has been shown to interact with beta-catenin and positively control the TCF/LEF transcription factor that is located downstream of the <em>Wnt</em> signal transduction pathway. During development, TCF/LEF activity is critical during neurogenesis and head induction. In zebrafish, Brg1-deficient embryos exhibit retinal cell differentiation and eye defects; however, the role of Brg1 in neurogenesis and neural crest cell induction remains elusive. We used zebrafish deficient in Brg1 (yng) or Brg1 specific-morpholino oligonucleotide-mediated knockdown to analyze the embryonic requirements of Brg1. Our results indicate that reduction in Brg1 expression leads to the expansion of the forebrain-specific transcription factor, six3, and marked reduction in expression of the mid/hind-brain boundary and hind-brain genes, engrailed2 and krox20, respectively. At <em>12</em> hpf, the expression of neural crest specifiers are severely affected in Brg1-morpholino-injected embryos. These results suggest that Brg1 is involved in neural crest induction, which is critical for the development of neurons, glia, pigment cells, and craniofacial structures. Brg1 is a maternal factor, and brg1-deficient embryos bearing the yng mutation derived from heterozygote intercrosses exhibit lesser effects on neural crest-specific gene expression, but show defects in neurogenesis and neural crest cell differentiation. This is exhibited by the aberrant brain patterning, a reduction in the sensory neurons, and craniofacial defects. These results further elucidate the critical role for Brg1 in neurogenesis, neural crest induction, and differentiation.

Fatty acid metabolism governs multiple intracellular signaling pathways in many cell types, but its role in hematopoietic stem cells (HSCs) is largely unknown. Herein, we establish a critical role for <em>12</em>/15-lipoxygenase (<em>12</em>/15-LOX)-mediated unsaturated fatty acid metabolism in HSC function. HSCs from <em>12</em>/15-LOX-deficient mice are severely compromised in their capacity to reconstitute the hematopoietic compartment in competitive and serial reconstitution assays. Furthermore, we demonstrate that <em>12</em>/15-LOX is required for the maintenance of long-term HSC quiescence and number. The defect in HSCs is cell-autonomous and associated with a selective reduction in <em>12</em>/15-LOX-mediated generation of bioactive lipid mediators and reactive oxygen species and with a decrease in canonical <em>Wnt</em> signaling as measured by nuclear beta-catenin staining. These results have implications for development, aging, and transformation of the hematopoietic compartment.

BACKGROUND

Oral leukoplakia is a precancerous change developed in the oral mucosa, and the mechanism that oral leukoplakia becomes malignant through atypical epithelium is not known. Here we compared the beta-catenin expression detected by immunohistochemical staining in the normal oral epithelium and in the oral leukoplakia with or without dysplasia.

RESULTS

The normal oral epithelium showed beta-catenin expression only in the cell membrane, but not in the nuclei. In the oral leukoplakia without dysplasia, 7 out of 17 samples (41%) showed beta-catenin expression in the cell membrane, and 5 samples (29%) showed expression in the nuclei. In the oral leukoplakia with dysplasia, nuclear expression of beta-catenin was shown in 11 out of <em>12</em> samples (92%). Incidence of nuclear beta-catenin expression was significantly different between dysplasia and normal oral epithelium (P < 0.01), and also between oral leukoplakia with dysplasia and those without dysplasia (P < 0.01). Wnt3 expression was detected in the epithelial cell membrane or cytoplasm in oral leukoplakia where nuclear expression of beta-catenin was evident, but not in epithelial cells without nuclear expression of beta-catenin.

CONCLUSIONS

The components of canonical Wnt pathway, such as Wnt3, beta-catenin, and cyclin D1, were detected, implying that this pathway is potentially involved in the progression of dysplasia in oral leukoplakia.

To address the roles of <em>Wnts</em> in the development of the anterior eye, we used a chicken model to perform comprehensive expression analysis of all <em>Wnt</em> genes during anterior eye development. In analyzing the available genomic sequences, we found that the chicken genome encodes 18 <em>Wnt</em> proteins that are homologous to corresponding human and mouse proteins. The mRNA sequences for <em>12</em> chicken <em>Wnt</em> genes are available in GenBank, and mRNAs for six other <em>Wnt</em> genes (<em>Wnt</em>2, <em>Wnt</em>5b, <em>Wnt</em>7b, <em>Wnt</em>8b, <em>Wnt</em>9b, and <em>Wnt</em>16) were identified and cloned based on the homology to the genes from other species. In addition, we found that chicken <em>Wnt</em>3a and <em>Wnt</em>7b genes encode two alternative mRNA isoforms containing different first exons. Following in situ hybridization, we found that out of 18 <em>Wnt</em> genes, 11 genes were expressed in the anterior eye, exhibiting distinct temporal-spatial patterns. Several <em>Wnts</em> were expressed in the lens, including <em>Wnt</em>2 and <em>Wnt</em>2b in the anterior epithelium and <em>Wnt</em>5a, <em>Wnt</em>5b, <em>Wnt</em>7a, and <em>Wnt</em>7b in the differentiating lens fiber cells. In the cornea, we detected <em>Wnt</em>3a, <em>Wnt</em>6, and <em>Wnt</em>9b in the ocular surface ectoderm, including the corneal epithelium, and <em>Wnt</em>9a in the corneal endothelium from the onset of its differentiation. In the optic cup, <em>Wnt</em>2, <em>Wnt</em>2b, and <em>Wnt</em>9a were localized in the rim of the optic cup (presumptive iris), while <em>Wnt</em>5a and <em>Wnt</em>16 were detected in the ciliary epithelium/iris zone of the differentiated optic cup, and <em>Wnt</em>6 was expressed in the iridial mesenchyme. These data suggest that <em>Wnt</em> signaling might play important roles in anterior eye development.

We studied in vitro effects of glycogen synthase kinase 3beta (GSK3beta)-inhibitor lithium on the growth of hepatocellular carcinoma (HCC) cells. Lithium induced strong growth inhibition >> 70%) in 75% (n = 9 of <em>12</em>) of cell lines, apparently independent from the status of major genes that are mutated in HCC including p53, p16(INK4a), beta-catenin and Axin1. Comparative studies with a growth-sensitive Huh7 and growth-resistant Hep40 cell lines showed that lithium induces growth arrest in Huh7 cells but not in Hep40 cells. Lithium induced the accumulation of N-terminally phosphorylated inactive form of GSK3beta with concomitant increase in beta-catenin and beta-catenin/TCF transcriptional activity in both cell lines. This suggests that lithium-mediated HCC growth inhibition is independent of its well-known stimulatory effect on <em>Wnt</em>-beta-catenin signaling. The main differences between Huh7 and Hep40 responses to lithium treatment were observed at the levels PKB/Akt and cyclin E proteins. Lithium induced depletion of both proteins in growth-sensitive Huh7, but not in growth-resistant Hep40 cells. PKB/Akt and Cyclin E are 2 major proteins that are known to be constitutively active in HCC. The targeting of both proteins with lithium may be the main reason why most HCC cells are responsive to lithium-mediated growth inhibition, independent of their p53, retinoblastoma and <em>Wnt</em>-beta-catenin pathways. The exploration of molecular mechanisms involved in lithium-mediated growth inhibition in relation with PKB/Akt and cyclin E downregulation may provide new insights for therapy of liver tumors.

Aging diminishes bone formation engendered by mechanical loads, but the mechanism for this impairment remains unclear. Because <em>Wnt</em> signaling is required for optimal loading-induced bone formation, we hypothesized that aging impairs the load-induced activation of <em>Wnt</em> signaling. We analyzed dynamic histomorphometry of 5-month-old, <em>12</em>-month-old, and 22-month-old C57Bl/6JN mice subjected to multiple days of tibial compression and corroborated an age-related decline in the periosteal loading response on day 5. Similarly, 1 day of loading increased periosteal and endocortical bone formation in young-adult (5-month-old) mice, but old (22-month-old) mice were unresponsive. These findings corroborated mRNA expression of genes related to bone formation and the <em>Wnt</em> pathway in tibias after loading. Multiple bouts (3 to 5 days) of loading upregulated bone formation-related genes, e.g., Osx and Col1a1, but older mice were significantly less responsive. Expression of <em>Wnt</em> negative regulators, Sost and Dkk1, was suppressed with a single day of loading in all mice, but suppression was sustained only in young-adult mice. Moreover, multiple days of loading repeatedly suppressed Sost and Dkk1 in young-adult, but not in old tibias. The age-dependent response to loading was further assessed by osteocyte staining for Sclerostin and LacZ in tibia of TOPGAL mice. After 1 day of loading, fewer osteocytes were Sclerostin-positive and, corroboratively, more osteocytes were LacZ-positive (<em>Wnt</em> active) in both 5-month-old and <em>12</em>-month-old mice. However, although these changes were sustained after multiple days of loading in 5-month-old mice, they were not sustained in <em>12</em>-month-old mice. Last, <em>Wnt</em>1 and <em>Wnt</em>7b were the most load-responsive of the 19 <em>Wnt</em> ligands. However, 4 hours after a single bout of loading, although their expression was upregulated threefold to 10-fold in young-adult mice, it was not altered in old mice. In conclusion, the reduced bone formation response of aged mice to loading may be due to failure to sustain <em>Wnt</em> activity with repeated loading. © 2016 American Society for Bone and Mineral Research.

The development of parathyroid carcinoma has been associated with inactivating mutations of the Hyperparathyroidism type 2 (HRPT2) gene encoding parafibromin, a member of the human RNA Polymerase II-Associated Factor Complex (hPAF) and functionally linked to the Wingless type (<em>Wnt</em>) pathway. In this study, we characterized the expression of <em>Wnt</em> pathway molecules in parathyroid benign and malignant tumors. Tumors were investigated by immunohistochemistry supplemented with Western blot analyses using monoclonal antibodies. The study comprised 13 tumors from <em>12</em> cases of unequivocal parathyroid carcinoma, 18 cases of parathyroid adenoma, as well as non-tumorous parathyroid tissue. Adenomatous polyposis coli (APC) was uniformly expressed in non-tumorous parathyroid tissue and adenomas, but absent in carcinomas from 9 of <em>12</em> patients (75%). Expression of glycogen synthase kinase 3-beta (GSK3-beta) was lost in 4/<em>12</em> carcinomas and in 1/18 adenomas. The loss of APC and GSK3-beta did not lead to augmentation of the <em>Wnt</em> target protein cyclin D1 or the <em>Wnt</em> oncoprotein beta-catenin. Active beta-catenin showed cytoplasmic and nuclear expression in all non-tumorous tissues and tumors. Loss of APC immunoreactivity was significantly associated with parathyroid carcinoma as compared to adenomas (p<0.001), giving a high specificity (100%) and sensitivity (75%) for the detection of parathyroid malignancy. The results suggest the involvement of <em>Wnt</em>-pathway members APC and GSK3-beta in parathyroid carcinoma development. In addition, APC immunohistochemistry could become a useful tool for improved recognition of parathyroid carcinoma together with immunohistochemistry for parafibromin and proliferation index. Furthermore, the involvement of APC related pathways in the disease development opens possibilities to explore therapeutic routes complementary to surgery.

Pancreatic cancer is among the top five deadliest cancers in developed countries. Better knowledge of the molecular mechanisms contributing to its tumorigenesis is imperative to improve patient prognosis. Identification of novel tumor suppressor genes (TSGs) in pancreatic cancer will reveal new mechanisms of pathway deregulation and will ultimately help improve our understanding of this aggressive disease. According to Knudson's two-hit model, TSGs are classically disrupted by two concerted genetic events. In this study, we combined DNA methylation profiling with copy number and mRNA expression profiling to identify novel TSGs in a set of 20 pancreatic cancer cell lines. These data sets were integrated for each of ∼<em>12</em> 000 genes in each cell line enabling the elucidation of those genes that undergo DNA hypermethylation, copy-number loss and mRNA downregulation simultaneously in multiple cell lines. Using this integrative genomics strategy, we identified SOX15 (sex determining region Y-box 15) as a candidate TSG in pancreatic cancer. Expression of SOX15 in pancreatic cancer cell lines with undetectable expression resulted in reduced viability of cancer cells both in vitro and in vivo demonstrating its tumor suppressive capability. We also found reduced expression, homozygous deletion and aberrant DNA methylation of SOX15 in clinical pancreatic tumor data sets. Furthermore, we deduced a novel role for SOX15 in suppressing the <em>Wnt</em>/β-catenin signaling pathway, which we hypothesize is a pathway through which SOX15 may exert its tumor suppressive effects in pancreatic cancer.

Recently, de novo heterozygous loss-of-function mutations in beta-catenin (CTNNB1) were described for the first time in four individuals with intellectual disability (ID), microcephaly, limited speech and (progressive) spasticity, and functional consequences of CTNNB1 deficiency were characterized in a mouse model. Beta-catenin is a key downstream component of the canonical <em>Wnt</em> signaling pathway. Somatic gain-of-function mutations have already been found in various tumor types, whereas germline loss-of-function mutations in animal models have been shown to influence neuronal development and maturation. We report on 16 additional individuals from 15 families in whom we newly identified de novo loss-of-function CTNNB1 mutations (six nonsense, five frameshift, one missense, two splice mutation, and one whole gene deletion). All patients have ID, motor delay and speech impairment (both mostly severe) and abnormal muscle tone (truncal hypotonia and distal hypertonia/spasticity). The craniofacial phenotype comprised microcephaly (typically -2 to -4 SD) in <em>12</em> of 16 and some overlapping facial features in all individuals (broad nasal tip, small alae nasi, long and/or flat philtrum, thin upper lip vermillion). With this detailed phenotypic characterization of 16 additional individuals, we expand and further establish the clinical and mutational spectrum of inactivating CTNNB1 mutations and thereby clinically delineate this new CTNNB1 haploinsufficiency syndrome.

Radiation therapy induces DNA damage and inflammation leading to fibrosis. Fibrosis can occur 4 to <em>12</em> months after radiation therapy. This process worsens with time and years. Radiation-induced fibrosis is characterized by fibroblasts proliferation, myofibroblast differentiation, and synthesis of collagen, proteoglycans and extracellular matrix. Myofibroblasts are non-muscle cells that can contract and relax. Myofibroblasts evolve towards irreversible retraction during fibrosis process. In this review, we discussed the interplays between transforming growth factor-β1 (TGF-β1), canonical <em>WNT</em>/β-catenin pathway and peroxisome proliferator-activated receptor gamma (PPAR γ) in regulating the molecular mechanisms underlying the radiation-induced fibrosis, and the potential role of PPAR γ agonists. Overexpression of TGF-β and canonical <em>WNT</em>/β-catenin pathway stimulate fibroblasts accumulation and myofibroblast differentiation whereas PPAR γ expression decreases due to the opposite interplay of canonical <em>WNT</em>/β-catenin pathway. Both TGF-β1 and canonical <em>WNT</em>/β-catenin pathway stimulate each other through the Smad pathway and non-Smad pathways such as phosphatidylinositol 3-kinase/serine/threonine kinase (PI3K/Akt) signaling. <em>WNT</em>/β-catenin pathway and PPAR γ interact in an opposite manner. PPAR γ agonists decrease β-catenin levels through activation of inhibitors of the <em>WNT</em> pathway such as Smad7, glycogen synthase kinase-3 (GSK-3 β) and dickkopf-related protein 1 (DKK1). PPAR γ agonists also stimulate phosphatase and tensin homolog (PTEN) expression, which decreases both TGF-β1 and PI3K/Akt pathways. PPAR γ agonists by activating Smad7 decrease Smads pathway and then TGF-β signaling leading to decrease radiation-induced fibrosis. TGF-β1 and canonical <em>WNT</em>/β-catenin pathway promote radiation-induced fibrosis whereas PPAR γ agonists can prevent radiation-induced fibrosis.