The aim of the present study was to investigate the effect and possible mechanism of pioglitazone (PIO) on the calcification of rat vascular smooth muscle cells (VSMCs) in vitro. β‑glycerophosphate (β‑GP; 10 mmol/l) was used to induce calcification of VSMCs treated with a range of concentrations (5, 10, 15 and 20 µmol/l) of PIO for <em>12</em> days. Calcium deposits were revealed by Alizarin red staining. Extracellular calcium content was detected using a calcium assay kit. Western blotting was used to measure the expression of α‑smooth muscle actin (α‑SMA), runt‑related transcription factor 2 (Runx2), bone morphogenetic protein‑2 (BMP2), β‑catenin, glycogen synthase kinase‑3β (GSK‑3β), phosphorylated (p)‑GSK‑3β and cyclin‑D1. A total of 10 mmol/l β‑GP, 20 µmol/l PIO and 20 µmol/l peroxisome proliferator‑activated receptor γ (PPAR γ) antagonist GW9662, was added to the cell culture media. The changes of the above indexes were observed. The calcium content in the calcification group, treated with high phosphorus, increased significantly compared with the controls (P<0.05) and all different concentrations of PIO reduced extracellular calcium content (P<0.05). Alizarin red staining was positive in calcified VSMCs and PIO (20 µmol/l) intervention group was almost negative. The expressions of Runx2, β‑catenin, p‑GSK‑3β, BMP2 and cyclin‑D1 increased significantly in the calcification group, and treatment with 20 µmol/l PIO downregulated the expression of all the above proteins, while upregulating the expression of α‑SMA. The PPAR γ antagonist GW9662 could partly inhibit the effect of PIO on calcified VSMCs. The results of the present study indicated that PIO can alleviate the calcification of rat aortic VSMCs induced by β‑GP via inhibiting the activity of the <em>Wnt</em>/β‑catenin signaling pathway.

Lithium has been the first-line treatment for bipolar disorder (BD) for more than six decades. Although the molecular effects of lithium have been studied extensively and gene expression changes are generally believed to be involved, the specific mechanisms of action that mediate mood regulation are still not known. In this study, a multi-step approach was used to explore the transcriptional changes that may underlie lithium's therapeutic efficacy. First, we identified genes that are associated both with lithium exposure and with BD, and second, we performed differential expression analysis of these genes in brain tissue samples from BD patients (n = 42) and healthy controls (n = 42). To identify genes that are regulated by lithium exposure, we used high-sensitivity RNA-sequencing of corpus callosum (CC) tissue samples from lithium-treated (n = 8) and non-treated (n = 9) rats. We found that lithium exposure significantly affected 1108 genes (FDR < 0.05), 702 upregulated and 406 downregulated. These genes were mostly enriched for molecular functions related to signal transduction, including well-established lithium-related pathways such as mTOR and <em>Wnt</em> signaling. To identify genes with differential expression in BD, we performed expression quantitative trait loci (eQTL) analysis on BD-associated genetic variants from the most recent genome-wide association study (GWAS) using three different gene expression databases. We found 307 unique eQTL genes regulated by BD-associated variants, of which <em>12</em> were also significantly modulated by lithium treatment in rats. Two of these showed differential expression in the CC of BD cases: RPS23 was significantly downregulated (p = 0.0036, fc = 0.80), while GRIN2A showed suggestive evidence of downregulation in BD (p = 0.056, fc = 0.65). Crucially, GRIN2A was also significantly upregulated by lithium in the rat brains (p = 2.2e-5, fc = 1.6), which suggests that modulation of GRIN2A expression may be a part of the therapeutic effect of the drug. These results indicate that the recent upsurge in research on this central component of the glutamatergic system, as a target of novel therapeutic agents for affective disorders, is warranted and should be intensified.

OBJECTIVE

The gut-vascular barrier (GVB) has recently been depicted to dampen the bacterial invasion of the bloodstream. The intestinal mucosa is a tissue rich in small vessels including capillaries. In this study, the protective effect of berberine on GVB in small bowel mucosa was investigated.

METHODS

The rat cecal ligation and puncture (CLP) sepsis model was employed to evaluate the effect of berberine on serum endotoxin level and intestinal vascular permeability to Evans blue in vivo. The rat intestinal microvascular endothelial cells (RIMECs) treated by lipopolysaccharide (LPS) were used to assess the effect of berberine on endothelial permeability to FITC-labeled dextran, transendothelial electrical resistance (TEER), and tight junction (TJ) and adherens junction (AJ) expression in vitro.

RESULTS

After 24-hr CLP operation the serum endotoxin concentration and gut vascular permeability were significantly increased, while berberine markedly reduced endotoxin level and vascular leakage. In vitro, LPS not only dramatically increased endothelial permeability of RIMECs to FITC-dextran, but also decreased TEER and inhibited claudin-<em>12</em>, beta-catenin and VE-cadherin expression. These effects of LPS were antagonized by berberine. In addition, our in vivo and vitro studies also confirmed that the effect of berberine on GVB could be partially abolished by ICG001.

CONCLUSIONS

Berberine exerted a protective effect on GVB function in sepsis, which was strictly related to the modulation of the Wnt/beta-catenin signaling pathway.

Smaller cross-sectional studies and bariatric surgery trials suggest that weight loss may change the expression of genes in adipose tissue that have been implicated in the development of metabolic diseases, but well-powered intervention trials are lacking. In post hoc analyses of data from a <em>12</em>-week dietary intervention trial initially designed to compare metabolic effects of intermittent vs. continuous calorie restriction, we analyzed the effects of overall weight loss on the subcutaneous adipose tissue (SAT) transcriptome. Changes in the transcriptome were measured by microarray using SAT samples of 138 overweight or obese individuals (age range: 35⁻65 years, BMI range: 25⁻40, non-smokers, non-diabetics). Participants were grouped post hoc according to the degree of their weight loss by quartiles (average weight loss in quartiles 1 to 4: 0%, -3.2%, -5.9%, and -10.7%). Candidate genes showing differential expression with weight loss according to microarray analyses were validated by reverse transcription quantitative polymerase chain reaction (RT-qPCR), and fold changes (FCs) were calculated to quantify differences in gene expression. A comparison of individuals in the highest vs. the lowest weight loss quartile revealed 681 genes to be differentially expressed (corrected <i>p</i> < 0.05), with 40 showing FCs of at least 0.4. Out of these, expression changes in secreted frizzled-related protein 2 (SFRP2, FC = 0.65, <i>p</i> = 0.006), stearoyl-CoA desaturase (SCD, FC = -1.00, <i>p</i> < 0.001), and hypoxia inducible lipid droplet-associated (HILPDA, FC = -0.45, <i>p</i> = 0.001) with weight loss were confirmed by RT-qPCR. Dietary weight loss induces significant changes in the expression of genes implicated in lipid metabolism (SCD and HILPDA) and <em>WNT</em>-signaling (SFRP2) in SAT.

Cardiotoxicity is a serious adverse effect of doxorubicin (DOX) treatment in cancer patients. Currently, there is a lack of sensitive biomarkers to predict the risk of DOX-induced cardiotoxicity. Using SOMAmer-based proteomic technology, 1<em>12</em>9 proteins were profiled to identify potential early biomarkers of cardiotoxicity in plasma from male B6C3F₁ mice given a weekly intravenous dose of 3 mg/kg DOX or saline (SAL) for 2, 3, 4, 6, or 8 weeks (6, 9, <em>12</em>, 18, or 24 mg/kg cumulative DOX doses, respectively). Also, a group of mice received the cardio-protectant, dexrazoxane (DXZ; 60 mg/kg; intraperitoneal) 30 min before a weekly DOX or SAL dose. Proteomic analysis in plasma collected a week after the last dose showed a significant ≥1.2-fold change in level of 18 proteins in DOX-treated mice compared to SAL-treated counterparts during 8-week exposure. Of these, neurogenic locus notch homolog protein 1 (NOTCH1), von Willebrand factor (vWF), mitochondrial glutamate carrier 2, <em>Wnt</em> inhibitory factor 1, legumain, and mannan-binding lectin serine protease 1 were increased in plasma at 6 mg/kg cumulative DOX dose, prior to the release of myocardial injury marker, cardiac troponin I at <em>12</em> mg/kg and higher cumulative doses. These six proteins also remained significantly elevated following myocardial injury or pathology at 24 mg/kg. Pretreatment of mice with DXZ significantly attenuated DOX-induced elevated levels of only NOTCH1 and vWF with mitigation of cardiotoxicity. This suggests NOTCH1 and vWF as candidate early biomarkers of DOX cardiotoxicity, which may help in addressing a clinically important question of identifying cancer patients at risk for cardiotoxicity.

Lymphoepithelioma-like hepatocellular carcinoma (LEL-HCC) is a distinct variant of HCC that is characterized by dense tumor-infiltrating lymphocytes (TILs). Patients with LEL-HCC also show better clinical outcomes compared to conventional HCC (c-HCC), which is commonly presented with low TIL. Emerging evidence has begun to highlight tumor-intrinsic genetic abnormalities in the tumor-host immune interfaces. However, genome-wide characterization of LEL-HCC remains largely unexplored. Here, we defined the genomic landscape of <em>12</em> LEL-HCC using whole-exome sequencing, and further underpinned those genetic alterations related to an immune active microenvironment by comparing findings to 15 c-HCC that were sequenced in parallel. Overall, the mutational load between LEL-HCC and c-HCC was similar. Interestingly, SNV incidences of specific genes (CTNNB1, AXIN1, NOTCH1, and NOTCH2) were significantly higher in c-HCC than LEL-HCC, suggesting a plausible link between activated <em>Wnt</em>/β-catenin and Notch signaling pathways and immune avoidance. Marked focal amplification of chromosome 11q13.3 was prevalent in LEL-HCC. Using The Cancer Genome Atlas dataset, we further established oncogenes expressed from chromosome 11q13.3 (CCND1, FGF19, and FGF4) to be strongly associated with the immune checkpoint signature (CD274, PDCD1, BTLA, CTLA4, HAVCR2, IDO1, and LAG3). Our results have illustrated for the first time the somatic landscape of LEL-HCC, and highlighted molecular alterations that could be exploited in combinatory therapy with checkpoint inhibitors in targeting HCC. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

OBJECTIVE

To employ the classical Wnt/β-catenin signaling pathway interference to explore the effects on the functional changes of eutopic endometrium stromal cells and the differences between endometriosis in a murine model.

METHODS

Two out of three mouse groups received an injection of either Wnt/β-catenin signaling pathway activator or blocker. Later the endometrial tissue samples were obtained to develop endometrial stromal cell cultures for the detection of cell invasion ability via Boyden chamber invasion assay and Western blot (WB). Then the methods of WB and Immunohistochemical staining (IHC) were used to examine the factors of eutopic endometrium. And an endometriosis model was established to investigate the factors of signaling pathway via quantitative polymerase chain reaction (QPCR) and IHC.

RESULTS

According to WB test, the level of β-catenin, GSK-3β and APC in the activation group were significantly higher than in the inhibition group (P < 0.01). In Boyden chamber invasion assay, the number of cells on membranes in the trial group was significantly higher than the control group [(113 ± 12) vs (64 ± 13)]. The expressions of VEGF and MMP-9 in the endometrial stromal cells culture from Boyden chamber assay analyzed via WB were ranked from highest to lowest respectively as activation group (vs control group was 35.6% and 27.4% higher), control group and inhibition group (vs control group was 12.3% and 30.4% lower). Furthermore, the endometrial E-cadherin and VEGF examined via IHC respectively showed a positive expression in inhibitor group and strong positive expression in activation group. QPCR showed the level of <em>Wnt</em>3, <em>Wnt</em>7, GSK3β, Lef and E-cadherin in the activation group was higher than those in the inhibition group (P < 0.05).

CONCLUSIONS

The intervention of WNT signaling pathway in vivo cause the changes of eutopic endometrial invasion and adhesion function, and further affect the development of endometriosis. Wnt/β-catenin signaling pathway may promote the eutopic endometrial cell proliferation and improve the ability of eutopic endometrial implantation, invasion, metastasis and angiogenesis.

Long non-coding RNAs (lncRNAs) are under active investigation in the development of cancers including gastric cancer (GC). Oncogenic autophagy is required for cancer cell survival. The present study aimed to investigate the regulatory role of lncRNA small nucleolar host gene 11 (SNHG11) in GC. We show that SNHG11 is upregulated in GC, and that its upregulation correlated with dismal patient outcomes. Functionally, SNHG11 aggravated oncogenic autophagy to facilitate cell proliferation, stemness, migration, invasion, and epithelial-to-mesenchymal transition (EMT) in GC. Mechanistically, SNHG11 post-transcriptionally upregulated catenin beta 1 (CTNNB1) and autophagy related <em>12</em> (ATG<em>12</em>) through miR-483-3p/miR-<em>12</em>76, while the processing of pre-miR-483/pre-miR-<em>12</em>76 was hindered by SNHG11. SNHG11 induced GSK-3β ubiquitination through interacting with Cullin 4A (CUL4A) to further activate the <em>Wnt</em>/β-catenin pathway. Intriguingly, SNHG11 regulated autophagy in a manner dependent upon ATG<em>12</em> rather than the <em>Wnt</em>/β-catenin pathway, whereas SNHG11 contributed to the malignant behaviors of GC cells via both pathways. Finally, SNHG11 upregulation in GC cells was shown to be transcriptionally induced by TCF7L2. In conclusion, we reveal that SNHG11 is an onco-lncRNA in GC and might be a promising prognostic and therapeutic target for GC.

<strong class="sub-title"> Keywords: </strong> ATG<em>12</em>; SNHG11; <em>Wnt</em>/β-catenin; gastric cancer; miR-<em>12</em>67; miR-483-3p.

<AbstractText>Endothelial-mesenchymal transition (EndMT) is a complex process whereby differentiated endothelial cells undergo phenotypic transition to mesenchymal cells. EndMT can be stimulated by several factors and the most common are the transforming growth factor-beta (TGF-β) and SNAIL transcription factor. Given the diversity of the vascular system, it is unclear whether endothelial cells lining different vessels are able to undergo EndMT through the same mechanisms. Here we evaluate the molecular and functional changes that occur in different types of endothelial cells following induction of EndMT by overexpression of SNAIL and TGF-β2.</AbstractText><AbstractText>We found that responses to induction by SNAIL are determined by cell origin and marker expression. Human coronary endothelial cells (HCAECs) showed the greatest EndMT responses evidenced by significant reciprocal changes in the expression of mesenchymal and endothelial markers, effects that were potentiated by a combination of SNAIL and TGF-β2. Key molecular events associated with EndMT driven by SNAIL/TGF-β2 involved extracellular-matrix remodeling and inflammation (IL-8, IL-<em>12</em>, IGF-1, and TREM-1 signaling). Notch signaling pathway members DLL4, NOTCH3 and NOTCH4 as well as members of the <em>Wnt</em> signaling pathway FZD2, FZD9, and WNT5B were altered in the combination treatment strategy, implicating Notch and <em>Wnt</em> signaling pathways in the induction process.</AbstractText><AbstractText>Our results provide a foundation for understanding the roles of specific signaling pathways in mediating EndMT in endothelial cells from different anatomical origins.</AbstractText>

Antiresorptive agents, such as bisphosphonates and denosumab, are frequently used for the management of osteoporosis. Indeed, both medications decrease the risk of osteoporotic fractures; however, these medications are associated with rare but potentially severe side effects, such as osteonecrosis of the jaw (ONJ). ONJ, defined as an area of exposed bone in the maxillofacial region that lasts for 8 weeks, often presents with significant pain and infection and can lead to serious complications. Interestingly, other treatments for osteoporosis have been developed, such as antibodies against the osteocyte-secreted protein, sclerostin. Sclerostin functions to inhibit the <em>Wnt</em> signaling cascade, leading to inhibition of bone formation. In clinical trials, a sclerostin antibody (romosozumab, Amgen Inc., UCB Brussels) increases bone formation and lowers the risk of osteoporotic fractures. However, in conjunction with increased osteoblastic activity, a reduction in bone resorption markers is observed. This antiresorptive effect raises the concern of possible ONJ development in patients treated with sclerostin antibodies. Here, utilizing ligature-induced experimental periodontitis (EP), we evaluated the effects of sclerostin inhibition on the development of ONJ-like lesions in ovariectomized rats. Beginning 8 weeks post-ovariectomy, rats were treated for 22 weeks with weekly injections of vehicle (Veh), 200 μg/kg zoledronic acid (ZA), a potent bisphosphonate at 100-fold the osteoporosis dose, or 5 mg/kg sclerostin antibody (Scl-Ab) at the osteoporotic dose. EP was initiated at week <em>12</em> and maintained for the remainder of the study. Scl-Ab treatment transiently increased serum P1NP, a bone formation marker, increased BV/TV, and decreased eroded surfaces in lumbar vertebrae. ZA-treated rats developed histologic features of ONJ, whereas Veh-treated controls did not. Scl-Ab animals lost less periodontal bone in sites with EP. However, these animals presented with no histologic signs of ONJ. In conclusion, sclerostin inhibition enhanced structural bone parameters, without inducing ONJ-like lesions, in ovariectomized rats with EP. © 2018 American Society for Bone and Mineral Research.

Recent progress in the field of molecular biology has allowed us to identify at least two different molecular mechanisms implicated in colorectal carcinogenesis (CRC): chromosomal instability (CIN) and genetic instability. Even though the two molecular mechanisms differ, their signalling pathways, implicated in malignant transformation of colonic epithelial cells, appear to be similar. The most frequent group of CRC, which represents 80% of sporadic CRC, is characterized by allelic losses on the short arm of chromosome 17 and 8 and on the long arm of chromosome 5, 18 and 22. These allelic losses are associated with mutations in TP53, APC, SMAD2 and SMAD4 genes. All of these alterations are grouped under the phenotype CIN. A genetic instability termed MSI (microsatellite instability), which results from a mismatch repair (MMR) deficiency, appears in <em>12</em>-15% of CRC cases. The presence of MMR deficiency leads to the accumulation of mutations in genes controlling cell cycle and apoptosis (TGFBRII, BAX or CASPASE5). More recently, the existence of a third phenotype was suggested. The main alteration associated with this group of tumors is the hypermethylation of the promoter region of numerous genes, leading to their inactivation. An activating mutation of BRAF is frequently associated with this phenotype. As described above, CRC shows genetic heterogeneity, however the consequences in terms of signalling pathway alterations are similar. For example, the activation of <em>Wnt</em> signalling pathways can result from the inactivation of the APC gene in the CIN phenotype or from an activating mutation in the β-catenin gene in MSI tumors. The inactivation of TGFβ pathways is also present in both tumor types and is driven by SMAD4, and more rarely by a SMAD2 inactivating mutation in CIN tumors, or by the existence of a frame-shift mutation occurring in a polyG coding track of the TGFβ (transforming growth factor) receptor type II in MSI tumors. The RAS-MAP kinase pathway is activated by KRAS mutations in CIN tumors or by BRAF mutations in MSI tumors. The p53 pathway is inactivated by TP53 inactivation in CIN tumors or by BAX inactivating mutations in MSI tumors.

Purpose: To examine the relationship between immune activity, PD-L1 expression, and tumor cell signaling, in metastatic melanomas prior to and during treatment with targeted MAPK inhibitors.Experimental Design: Thirty-eight tumors from 17 patients treated with BRAF inhibitor (n = <em>12</em>) or combination BRAF/MEK inhibitors (n = 5) with known PD-L1 expression were analyzed. RNA expression arrays were performed on all pretreatment (PRE, n = 17), early during treatment (EDT, n = 8), and progression (PROG, n = 13) biopsies. HLA-A/HLA-DPB1 expression was assessed by IHC.Results: Gene set enrichment analysis (GSEA) of PRE, EDT, and PROG melanomas revealed that transcriptome signatures indicative of immune cell activation were strongly positively correlated with PD-L1 staining. In contrast, MAPK signaling and canonical <em>Wnt</em>/-β-catenin activity was negatively associated with PD-L1 melanoma expression. The expression of PD-L1 and immune activation signatures did not simply reflect the degree or type of immune cell infiltration, and was not sufficient for tumor response to MAPK inhibition.Conclusions: PD-L1 expression correlates with immune cells and immune activity signatures in melanoma, but is not sufficient for tumor response to MAPK inhibition, as many PRE and PROG melanomas displayed both PD-L1 positivity and immune activation signatures. This confirms that immune escape is common in MAPK inhibitor-treated tumors. This has important implications for the selection of second-line immunotherapy because analysis of mechanisms of immune escape will likely be required to identify patients likely to respond to such therapies. Clin Cancer Res; 23(20); 6054-61. ©2017 AACR.

Hepatoblastoma (HB) is the most common liver cancer in children. We aimed to characterize HB related to <i>APC</i> (Adenomatous Polyposis Coli) germline mutation (APC-HB). This French multicentric retrospective study included <em>12</em> APC-HB patients under 5 at diagnosis. Clinical features of APC-HB were compared to the French SIOPEL2-3 cohort of HB patients. Molecular and histopathological analyses of APC-HB were compared to 15 consecutive sporadic HB treated at Bicêtre hospital from 2013 to 2015 (non-APC-HB). APC-HB patients have a peculiar spectrum of germline <i>APC</i> mutations, with no events in the main hotspot of classical <i>APC</i> mutations at codon 1309 (<i>P</i> < .05). Compared to sporadic HB, they have similar clinical features including good prognosis since all patients are alive in complete remission at last follow-up. APC-HB are mostly well-limited tumors with fetal predominance and few mesenchymal components. All APC-HB have an activated <em>Wnt</em>/β-catenin pathway without <i>CTNNB1</i> mutation, confirming that germline <i>APC</i> and somatic <i>CTNNB1</i> mutations are mutually exclusive (<i>P</i> < .001). Pathological reviewing identified massive intratumor tertiary lymphoid structures (TLS) containing both lymphocytes and antigen-presenting cells in all 11 APC-HB cases who received cisplatin-based neoadjuvant chemotherapy but not in five pre-chemotherapy samples (four paired biopsies and one patient resected without chemotherapy), indicating that these TLS are induced by chemotherapy (<i>P</i> < .001). Conclusion: APC-HB show a good prognosis, they are all infiltrated by cisplatin-induced TLS, a feature only retrieved in a minority of non-APC-HB. This suggests that <i>AP</i>C inactivation can synergize with cisplatin to induce an immunogenic cell death that initiates an anti-tumor immune response.

UNASSIGNED

We aimed to establish an efficient method for retinal ganglion cell (RGC) differentiation from human pluripotent stem cells (hPSCs) using defined factors.

UNASSIGNED

To define the contribution of specific signal pathways to RGC development and optimize the differentiation of hPSCs toward RGCs, we examined RGC differentiation in three stages: (1) eye field progenitors expressing the eye field transcription factors (EFTFs), (2) RGC progenitors expressing MATH5, and (3) RGCs expressing BRN3B and ISLET1. By monitoring the condition that elicited the highest yield of cells expressing stage-specific markers, we determined the optimal concentrations and combinations of signaling pathways required for efficient generation of RGCs from hPSCs.

UNASSIGNED

Precise modulation of signaling pathways, including <em>Wnt</em>, insulin growth factor-1, and fibroblast growth factor, in combination with mechanical isolation of neural rosette cell clusters significantly enriched RX and PAX6 double-positive eye field progenitors from hPSCs by day <em>12</em>. Furthermore, Notch signal inhibition facilitated differentiation into MATH5-positive progenitors at 90% efficiency by day 20, and these cells further differentiated to BRN3B and ISLET1 double-positive RGCs at 45% efficiency by day 40. RGCs differentiated via this method were functional as exemplified by their ability to generate action potentials, express microfilament components on neuronal processes, and exhibit axonal transportation of mitochondria.

UNASSIGNED

This protocol offers highly defined culture conditions for RGC differentiation from hPSCs and in vitro disease model and cell source for transplantation for diseases related to RGCs.

<em>Wnt</em>/β-catenin signaling (<em>WNT</em>) has widespread roles during stem cell differentiation. Whether <em>WNT</em> suppresses or promotes insulin-producing cell (IPC) differentiation and function is still not known. In this study, we investigated the role of <em>WNT</em> signaling during human adipose-derived stem cell (hADSC) differentiation into IPCs. Western blot analysis revealed that several key components of <em>WNT</em> were dynamically regulated in a <em>12</em>-day IPC differentiation assay. Specifically, protein levels of <em>Wnt</em>1, β-catenin, and GSK3β steadily increased from day 0 to day 9 and rapidly decreased by day <em>12</em> of differentiation. Similarly, endonuclear β-catenin levels peaked at day 9 and then, fell to pre-differentiation levels. The expression of two <em>WNT</em> pathway targets, TCF-1 and cyclin D1, closely followed the same pattern of regulation, confirming that <em>WNT</em> signaling was transiently activated during IPC differentiation. Interestingly, the inhibition of <em>WNT</em> signaling did not block IPC differentiation; instead, it resulted in the upregulation of IPC-specific markers, including PDX-1, insulin, IRS-1, and IRS-2. Notably, another IPC marker, glucokinase, remained downregulated since it is a direct target of <em>WNT</em> signaling. Next, we examined the effect of maintaining active <em>WNT</em> signaling from day 9 to day <em>12</em> of IPC differentiation. Differentiating cells were treated with <em>Wnt</em>1 on day 9, when <em>WNT</em> signaling is typically turned off, and subjected to gene expression analysis on day <em>12</em>. Remarkably, <em>Wnt</em>1 treatment resulted in reduced expression of IPC-specific markers. Taken together, these data indicate that <em>WNT</em> may not be necessary for IPC differentiation but may be involved in IPC maturation.

Progression of chronic kidney disease (CKD) is characterized by deposition of extracellular matrix. This is an irreversible process that leads to tubulointerstitial fibrosis and finally loss of kidney function. <em>Wnt</em>/ β-catenin pathway was reported to be aberrantly activated in the progressive damage associated with chronic organ failure. Extensive renal ablation is an experimental model widely used to gain insight into the mechanisms responsible for the development of CKD, but it was not evaluated for <em>Wnt</em>/ β-catenin pathway. This study aimed to elucidate if the rat 5/6 renal mass reduction model (RMR) is a good model for the <em>Wnt</em>/ β-catenin activation and possible next modulation. RMR model was evaluated at <em>12</em> and 18 weeks after the surgery, when CKD is close to end-stage kidney disease demonstrated by molecular and histological studies. <em>Wnt</em> pathway components were analyzed at mRNA and protein level. Our results demonstrate that <em>Wnt</em> pathway is active by increase of β-catenin at mRNA level and nuclear translocation in tubular epithelium as well as some target genes. These results validate the RMR model for future modulation of <em>Wnt</em> pathway, starting at shorter time after the surgery.

XTcf-3 functions as a transcriptional regulator in the canonical <em>Wnt</em> signaling cascade and can repress or activate downstream target genes. Expression of XTcf-3 is differentially regulated in time and place during development (Molenaar et al. [1998] Mech Dev. 75:151-154), but little is known about the mechanisms that control transcriptional activation and repression. A 15-kb genomic fragment of Tcf-3 sequences from Xenopus tropicalis was cloned, including the 5' untranslated region; exons 1, 2, and 3; and intron sequences. We used 5' deletion constructs for transgenesis and episomal luciferase assays in Xenopus to examine temporal and spatial regulation of the promoter during early development. A -3054/+34-bp Tcf-3 upstream region was identified that drives a green fluorescent protein (GFP) reporter transgene in a pattern similar to endogenous expression of XtTcf-3 from gastrula to tail bud stages. At stage <em>12</em>, expression of the reporter is restricted to the middle and posterior neurectoderm. At stage 22, expression is strongest in the neural plate, the eye anlagen and branchial arches. At stage 35/36, expression is found in the head mesenchyme, the branchial arches, the heart, the mesencephalon, eyes, otic vesicles, notochord, somites and the lateral plate mesoderm. Part of the cis-acting elements driving this GFP reporter transgene expression map between -372 and -95 bp of the transcription start site. Furthermore, two TCF/LEF sites are necessary for full activity of the promoter during gastrula stages in episomal luciferase assays.

Backgroud: Accumulated evidence has demonstrated a significant role of the Wnt pathway in human prostate cancer. We hypothesize that genetic variants in the Wnt pathway effector, Transcription factor 7-like 2 (TCF7L2), may influence clinical outcomes in prostate cancer.

METHODS

We comprehensively selected 12 tagged single-nucleotide polymorphisms (SNPs) to capture majority of common variants across TCF7L2, and genotyped in 458 localized prostate cancer patients treated with radical prostatectomy (RP). Kaplan-Meier analysis, Cox proportional hazard model, and survival tree analyses were performed to identify significant SNPs that correlated with biochemical recurrence (BCR) after surgery.

RESULTS

A higher-order SNP-SNP interaction profile consisting of TCF7L2 rs7094463, rs10749127, and rs11196224 was significantly associated with BCR (P trend = 0.001). After adjusting for possible confounders, the genetic profile remained significant (P trend = 0.007). None of the studied SNPs were individually associated with BCR.

CONCLUSIONS

Our results support a genetic interaction in the TCF7L2 SNPs as a predictor of disease recurrence after curative RP in localized prostate cancer patients.

BACKGROUND

Marsupials such as the tammar wallaby (M.Eugenii) have a short gestation (29.3 days) and at birth the altricial young resembles a fetus, and the major development occurs postnatally while the young remains in the mother's pouch. The essential functional factors for the maturation of the neonate are provided by the milk which changes in composition progressively throughout lactation (300 days). Morphologically the lungs of tammar pouch young are immature at birth and the majority of their development occurs during the first 100 days of lactation.

RESULTS

In this study mouse embryonic lungs (E-<em>12</em>) were cultured in media with tammar skim milk collected at key time points of lactation to identify factors involved in regulating postnatal lung maturation. Remarkably the embryonic lungs showed increased branching morphogenesis and this effect was restricted to milk collected at specific time points between approximately day 40 to 100 lactation. Further analysis to assess lung development showed a significant increase in the expression of marker genes Sp-C, Sp-B, <em>Wnt</em>-7b, BMP4 and Id2 in lung cultures incubated with milk collected at day 60. Similarly, day 60 milk specifically stimulated proliferation and elongation of lung mesenchymal cells that invaded matrigel. In addition, this milk stimulated proliferation of lung epithelium cells on matrigel, and the cells formed 3-dimensional acini with an extended lumen.

CONCLUSIONS

This study has clearly demonstrated that tammar wallaby milk collected at specific times in early lactation contains bioactives that may have a significant role in lung maturation of pouch young.

<AbstractText><em>12</em>-Lipoxygenase (<em>12</em>-LOX) plays a major role in the progression and metastasis of various types of cancer. In gastric cancer (GC), the expression level of <em>12</em>-LOX is significantly up-regulated; however, its function, and underlying mechanism of action remain unclear.</AbstractText><AbstractText>The mRNA and protein expression levels of <em>12</em>-LOX were assessed using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analyses, respectively, in GC cell lines. <em>12</em>-LOX expression was stably up-regulated using lentiviral vector in BGC823 and MGC803 cells, and cell-counting kit-8 (CCK8), colony formation, and invasion assays were performed to verify the function of <em>12</em>-LOX in proliferation and metastasis. In addition, the expression levels of epithelial-mesenchymal transition (EMT) differentiation markers and downstream targets of the <em>Wnt</em>/β-catenin signaling pathway were examined by Western blotting. A nude mouse model of tumor growth and metastasis was established to investigate the role of <em>12</em>-LOX in vivo.</AbstractText><AbstractText>Our findings demonstrate that <em>12</em>-LOX mRNA and protein were highly expressed in GC cell lines. <em>12</em>-LOX overexpression promoted GC cell proliferation, migration, and invasion both in vitro and in vivo. In addition, up-regulation of <em>12</em>-LOX promoted the EMT in GC cells, as reflected by a decrease in E-cadherin expression and an increase in N-cadherin and Snail expression. <em>12</em>-LOX overexpression in GC cells also increased the expression of multiple downstream targets of the <em>Wnt</em>/β-catenin signaling pathway.</AbstractText><AbstractText>These findings revealed that <em>12</em>-LOX functions as an oncogene in promoting GC cell proliferation and metastasis in vitro and in vivo. In addition, <em>12</em>-LOX might regulate the EMT via the <em>Wnt</em>/β-catenin signaling pathway, indicating a potential role for <em>12</em>-LOX as a target in GC treatment.</AbstractText>

OBJECTIVE

To investigate the associations of the Wnt modulators Wnt inhibitory factor 1 (WIF-1) and Dickkopf 3 (DKK-3) in the aqueous humor with neovascular age-related macular degeneration (nAMD) and to determine their clinical implications.

METHODS

Seventy-four nAMD patients initially treated with an intravitreal injection of ranibizumab (IVR) and 74 age- and sex-matched controls were studied. Aqueous humor WIF-1 and DKK-3 levels were measured by Western blotting and an ELISA before and 1 month after two consecutive IVRs (pre- and post-IVR). Visual acuity assessments and spectral domain optical coherence tomography were performed pre- and post-IVR.

RESULTS

Western blotting showed increased WIF-1 and DKK-3 in 12 nAMD patients compared with 12 controls. The ELISA analysis demonstrated elevated WIF-1 (pre) and DKK-3 (pre) in 62 patients compared with 62 controls (54.7 vs. 23.0 and 114.3 vs. 93.0 ng/mL, respectively). In multivariate analyses, high WIF-1 (pre) levels were associated with increased disruption in the photoreceptor junction's inner and outer segments (IS/OS) (pre and post) and high WIF-1 (post) levels. Interestingly, WIF-1 (pre) levels were significantly higher in type 3 neovascularization (NV) patients than in type 1 or 2 NV (90.5 ± 36.7 vs. 48.3 ± 22.5 and 41.3 ± 28.8 ng/mL, respectively). However, choroidal thickness was not correlated with WIF-1 levels.

CONCLUSIONS

We report, for the first time, the possibility of phenotypic, anatomic, and ocular proteomic correlations, demonstrating correlated WIF-1 and DKK-3 upregulation in nAMD patients' aqueous humor. Secreted WIF-1, reflecting the degree of retinal structure damage, may be a new biomarker for the retina's healthy and disease states.

Colon cancer-associated transcript 2 (CCAT2) is a newly recognized lncRNA transcribed from the 8q24 genomic region. It functions as an oncogene in various types of cancers including breast cancer, in which it affects Wnt/β-catenin pathway. Previous studies have shown a putative interaction between this lncRNA and MYC proto-oncogene.

In the current study, we evaluated the expression of CCAT2 in breast cancer tissues with regards to the expression of its target MYC. In addition, we assessed the relationship between CCAT2 and MYC expression levels in tumor tissues and the clinical prognostic characteristics of breast cancer patients.

MYC expression levels were significantly up-regulated in tumor tissues compared with adjacent non-cancerous tissues (ANCTs), while such analysis showed no statistically significant difference between these two tissue types in CCAT2 expression. Starkly increased CCAT2 gene expression levels were found in 12/48 (25%) of cancer tissue samples compared with their corresponding ANCTs. Furthermore, significant inverse correlations were found between CCAT2 expression and stage, as well as lymph node involvement. Besides, a significant inverse correlation was found between the relative MYC expression in tumor tissues compared with their corresponding ANCTs and disease stage.

These results highlight the significance of MYC and CCAT2 expressions in the early stages of breast cancer development and suggest a potentially significant role for CCAT2 in a subset of breast cancer patients, which could be applied as a potential therapeutic target in these patients.

Heterocyclic amines are potent mutagens and carcinogens formed in cooked protein rich foods. In this study, we screened liver tumors induced by 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) in CDF1 mice for beta-catenin and APC mutations and other genetic alterations shown to occur in human hepatocellular carcinomas (HCC), including mutations in the p53 and H-ras genes, c-myc amplification and E-cadherin promoter methylation. SSCP followed by direct DNA sequencing revealed mutations in exon 2 of the beta-catenin gene in 2 of 16 liver tumors (<em>12</em>.5%). Promoter methylation of the E-cadherin gene was detected in one liver tumor induced by MeIQ. There were no mutations in the mutation cluster region of the APC gene, in exons 5-8 of the p53 gene, or in codons <em>12</em>, 13 and 61 of the H-ras gene, nor c-myc amplification in any of liver tumors induced by MeIQ. These data indicate that except for the occasional disruption of the <em>Wnt</em> pathway through beta-catenin mutations, the genetic pathways involved in the development of HCC differ significantly between human liver cancer and tumors induced in mice by MeIQ, but do not rule out the possibility that heterocyclic amines constitute a carcinogenic risk factor in humans.

Gene mutations impairing the functions of the <em>WNT</em> signaling transduction pathway have been found in approximately 15% of human sporadic medulloblastomas. To understand the functional role of the <em>WNT</em> pathway in medulloblastoma, we have investigated the intracellular distribution of beta-catenin in a series of 17 human medulloblastomas to correlate such expression with neuronal differentiation and in cultured cell models following functional silencing of the APC gene by small-interference RNA (siRNA). Transient siRNA transfection resulted in a 50% reduction of the APC gene product levels in both DAOY and D283MED cell lines. In the former, less-differentiated cell line, beta-catenin levels remained unchanged or were slightly reduced, but beta-catenin translocated in the nucleus following APC gene siRNA silencing. In contrast, in the more differentiated D283MED cells, beta-catenin levels increased about twofold while mainly maintaining the cytoplasmic and cell membrane localization. Cytoplasmic/nuclear localization of beta-catenin was present in <em>12</em> of 17 cases of medulloblastoma with a prevalent distribution in the classic, 6/7 cases, and large cell/anaplastic variant, 4/4 cases. The nodular/desmoplastic lesions showed strongly positivity in the cell membrane mainly of intranodular cells with advanced neuronal differentiation. These observations support an important functional role of <em>WNT</em>/beta-catenin pathway in neuronal differentiation in medulloblastoma.