Activation of the <em>Wnt</em>/β-catenin signaling pathway drives colorectal cancer growth by deregulating expression of downstream target genes, including the c-myc proto-oncogene. The critical targets that mediate the functions of oncogenic c-Myc in colorectal cancer have yet to be fully elucidated. Previously, we showed that activation of PI3K/Akt/mTOR contributes to colorectal cancer growth and metastasis. Here, we show that Deptor, a suppressor of mTOR, is a direct target of <em>Wnt</em>/β-catenin/c-Myc signaling in colorectal cancer cells. Inhibition of <em>Wnt</em>/β-catenin or knockdown of c-Myc decreased, while activation of <em>Wnt</em>/β-catenin or overexpression of c-Myc increased the expression of Deptor. c-Myc bound the promoter of Deptor and transcriptionally regulated Deptor expression. Inhibition of <em>Wnt</em>/β-catenin/c-Myc signaling increased mTOR activation, and the combination of <em>Wnt</em> and Akt/mTOR inhibitors enhanced inhibition of colorectal cancer cell growth in vitro and in vivo Deptor expression was increased in colorectal cancer cells; knockdown of Deptor induced differentiation, decreased expression of B lymphoma Mo-MLV insertion region 1 (Bmi1), and decreased proliferation in colorectal cancer cell lines and primary human colorectal cancer cells. Importantly, our work identifies Deptor as a downstream target of the <em>Wnt</em>/β-catenin/c-Myc signaling pathway, acting as a tumor promoter in colorectal cancer cells. Moreover, we provide a molecular basis for the synergistic combination of <em>Wnt</em> and mTOR inhibitors in treating colorectal cancer with elevated c-Myc.Significance: The mTOR inhibitor DEPTOR acts as a tumor promoter and could be a potential therapeutic target in colorectal cancer. Cancer Res; 78(<em>12</em>); 3163-75. ©2018 AACR.

OBJECTIVE

The prognosis of hepatocellular carcinoma (HCC) treated by radiofrequency ablation (RFA) is mainly linked to tumor recurrence. So far, no tissue biomarker of recurrence has been validated in biopsy samples. We aimed at investigating the prognostic value of tissue biomarkers in HCC biopsy samples of patients treated with RFA.

METHODS

All consecutive naive patients from 3 university hospitals, with compensated cirrhosis, early-stage (BCLC 0/A) uninodular HCC treated with RFA, and available tumor biopsy, were included. Edmondson's grade, and the expression of cytokeratin 19, glutamine synthase, beta-catenin, epithelial cell adhesion molecule (EpCAM), and endothelial cell-specific molecule 1 (ESM-1) were assessed. Main clinical end points were overall and early recurrence. Statistical analyses were performed using Kaplan Meier, Log-rank test, and Cox models.

RESULTS

150 patients were included. Recurrence, death or liver transplantation occurred in 85, 51, and <em>12</em> patients, respectively. Median follow-up was 27months. ESM-1 expression by HCC stromal endothelial cells was observed in 58 patients (40%) and was associated with higher serum AFP levels, larger tumor, and more frequent expression of EpCAM and surrogate markers of activation of the <em>Wnt</em>-ß-catenin pathway. The 2 independent predictive factors of overall recurrence were serum AFP (HR 1.11 [1.002; 1.22], p=0.045) and ESM-1 expression (HR 1.56 [1.004; 2.43], p=0.048). ESM-1 expression was also an independent predictive factor of early recurrence (HR 1.81 [1.02; 3.21], p=0.042).

CONCLUSIONS

ESM-1 expression by stromal endothelial cells, in tumor biopsy samples, has an independent predictive value of early recurrence after RFA.

Recent genome-wide association studies (GWAS) have identified several gene variants associated with sporadic chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Many of these CLL/SLL susceptibility loci are located in non-coding or intergenic regions, posing a significant challenge to determine their potential functional relevance. Here, we review the literature of all CLL/SLL GWAS and validation studies, and apply eQTL analysis to identify putatively functional SNPs that affect gene expression that may be causal in the pathogenesis of CLL/SLL. We tested <em>12</em> independent risk loci for their potential to alter gene expression through cis-acting mechanisms, using publicly available gene expression profiles with matching genotype information. Sixteen SNPs were identified that are linked to differential expression of SP140, a putative tumor suppressor gene previously associated with CLL/SLL. Three additional SNPs were associated with differential expression of DACT3 and GNG8, which are involved in the <em>WNT</em>/β-catenin- and G protein-coupled receptor signaling pathways, respectively, that have been previously implicated in CLL/SLL pathogenesis. Using in silico functional prediction tools, we found that 14 of the 19 significant eQTL SNPs lie in multiple putative regulatory elements, several of which have prior implications in CLL/SLL or other hematological malignancies. Although experimental validation is needed, our study shows that the use of existing GWAS data in combination with eQTL analysis and in silico methods represents a useful starting point to screen for putatively causal SNPs that may be involved in the etiology of CLL/SLL.

Prenatal exposure to infectious or inflammatory insults increases the risk of neurodevelopmental disorders. Using a well-established mouse model of prenatal viral-like immune activation, we examined whether this pathological association involves genome-wide DNA methylation differences at single nucleotide resolution.

Prenatal immune activation was induced by maternal treatment with the viral mimetic polyriboinosinic-polyribocytidylic acid in middle or late gestation. Following behavioral and cognitive characterization of the adult offspring (n = <em>12</em> per group), unbiased capture array bisulfite sequencing was combined with subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and quantitative real-time polymerase chain reaction analyses to quantify DNA methylation changes and transcriptional abnormalities in the medial prefrontal cortex of immune-challenged and control offspring. Gene ontology term enrichment analysis was used to explore shared functional pathways of genes with differential DNA methylation.

Adult offspring of immune-challenged mothers displayed hyper- and hypomethylated CpGs at numerous loci and at distinct genomic regions, including genes relevant for gamma-aminobutyric acidergic differentiation and signaling (e.g., Dlx1, Lhx5, Lhx8), Wnt signaling (Wnt3, Wnt8a, Wnt7b), and neural development (e.g., Efnb3, Mid1, Nlgn1, Nrxn2). Altered DNA methylation was associated with transcriptional changes of the corresponding genes. The epigenetic and transcriptional effects were dependent on the offspring's age and were markedly influenced by the precise timing of prenatal immune activation.

Prenatal viral-like immune activation is capable of inducing stable DNA methylation changes in the medial prefrontal cortex. These long-term epigenetic modifications are a plausible mechanism underlying the disruption of prefrontal gene transcription and behavioral functions in subjects with prenatal infectious histories.

Curcumin, the major phytochemical in turmeric, exerts anti‑proliferative, anticancer and anti‑inflammatory activities in various types of cancer cells. Curcumin has been demonstrated to induce apoptosis through multiple signaling pathways; however, its association with survival pathways, including the <em>Wnt</em> signaling pathway, is not fully understood. The <em>Wnt</em> signaling pathway is involved in diverse functions, including cell development, growth and proliferation. This pathway is important for cancer cell survival and metastasis. β‑catenin and GSK3β play a key role in the <em>Wnt</em> signaling pathway and therefore, various members of the <em>Wnt</em> signaling pathway have been hypothesized to represent potential targets for anticancer therapy. In the present study, the effect of curcumin on the suppression of migration and proliferation of Hep3B hepatocarcinoma cells was investigated via suppression of <em>Wnt</em> signaling in vitro and in vivo. <em>12</em>‑O‑tetradecanoylphorbol‑13‑acetate (TPA)‑induced cell migration was observed to be suppressed by curcumin treatment. In addition, curcumin suppressed TPA‑induced activation of <em>Wnt</em> signaling. These results indicate that curcumin induces anti‑migratory activity, which functions via the <em>Wnt</em> signaling pathway.

BACKGROUND

Wnt-4 is a mitogen expressed during postnatal repair and scar formation; however, its expression profile during scarless repair is unknown. Transforming growth factor (TGF)-beta1 has high expression during healing with scar formation. Whether TGF-beta1 directly influences Wnt-4 expression in fetal or postnatal fibroblasts has not been examined.

METHODS

Primary fetal and postnatal mouse fibroblasts were stimulated with TGF-beta1 and Wnt-4 expression quantitated by real-time polymerase chain reaction. Fetal E17 and postnatal mouse excisional wounds were also analyzed for Wnt-4 expression by real-time polymerase chain reaction.

RESULTS

In E17 fibroblasts after TGF-beta1 stimulation, Wnt-4 expression increased 4-fold at 1 hour (p < 0.05) and peaked with an 11-fold increase at 2 hours (p < 0.05). By 24 hours, expression decreased to 2-fold baseline levels (p < 0.05). In postnatal fibroblasts, Wnt-4 expression also increased after TGF-beta stimulation, but peak expression was larger and relatively delayed, with a 17-fold increase at 12 hours (p < 0.005). Expression levels at 24 hours were still 4-fold greater than baseline (p < 0.05). In E17 fetal skin, Wnt-4 expression was 3.5-fold greater compared with 3-week-old mice (p < 0.005). Small increases in Wnt-4 expression (less than 2-fold) occurred during both fetal scarless and postnatal scarring mouse wound repair.

CONCLUSIONS

The authors' data suggest that TGF-beta directly increases Wnt-4 expression in fetal and postnatal fibroblasts and that Wnt-4 is increased in both fetal and postnatal repair.

BACKGROUND

Sclerostin levels have been reported to be low in ankylosing spondylitis (AS), but there is no data regarding the possible role of this Wnt inhibitor during anti-tumor necrosis factor (TNF) therapy. The present study longitudinally evaluated sclerostin levels, inflammatory markers and bone mineral density (BMD) in AS patients under anti-TNF therapy.

METHODS

Thirty active AS patients were assessed at baseline, 6 and 12 months after anti-TNF therapy regarding clinical parameters, inflammatory markers, BMD and baseline radiographic damage (mSASSS). Thirty age- and sex-matched healthy individuals comprised the control group. Patients' sclerostin levels, sclerostin binding low-density lipoprotein receptor-related protein 6 (LRP6) and BMD were evaluated at the same time points and compared to controls.

RESULTS

At baseline, AS patients had lower sclerostin levels (60.5 ± 32.7 vs. 96.7 ± 52.9 pmol/L, P = 0.002) and comparable sclerostin binding to LRP6 (P = 0.387) than controls. Improvement of Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), Bath Ankylosing Spondylitis Metrology Index (BASMI), Ankylosing Spondylitis quality of life (ASQoL) was observed at baseline vs. 6 vs. 12 months (P < 0.01). Concomitantly, a gradual increase in spine BMD (P < 0.001) and a positive correlation between baseline mSASSS and spine BMD was found (r = 0.468, P < 0.01). Inflammatory parameters reduction was observed comparing baseline vs. 6 vs. 12 months (P <0.01). Sclerostin levels progressively increased [baseline (60.5 ± 32.7) vs. 6 months (67.1 ± 31.9) vs. 12 months (72.7 ± 32.3) pmol/L, P <0.001]. At 12 months, the sclerostin levels remained significantly lower in patients compared to controls (72.7 ± 32.3 vs. 96.70 ± 52.85 pmol/L, P = 0.038). Moreover, sclerostin serum levels at 12 months were lower in the 10 patients with high C reactive protein (CRP) (≥ 5 mg/l) compared to the other 20 patients with normal CRP (P = 0.004). Of note, these 10 patients with persistent inflammation also had lower sclerostin serum levels at baseline compared to the other patients (P = 0.023). Univariate logistic regression analysis demonstrated that AS patients with lower sclerostin serum levels had an increased risk to have high CRP at 12 months (odds ratio = 7.43, 95% CI 1.23 to 45.01, P = 0.020) than those with higher sclerostin values.

CONCLUSIONS

Persistent low sclerostin levels may underlie continuous inflammation in AS patients under anti-TNF therapy.

The related transcriptional co-factors YAP (Yes-associated protein) and TAZ (transcriptional co-activator with PDZ-binding motif) have been proposed to either promote or inhibit osteoblast differentiation. Here we investigated the skeletal consequences of deleting YAP and TAZ at different stages of the osteoblast lineage using Prx1-Cre, Osx1-Cre, and Dmp1-Cre transgenic mice. Prx1-Cre-mediated deletion resulted in embryonic lethality. Mice lacking both copies of TAZ and one copy of YAP in cells targeted by Prx1-Cre were viable and displayed elevated bone mass associated increased bone formation. Deletion of YAP and TAZ using Osx1-Cre mice led to perinatal lethality. Suppression of Osx1-Cre activity until 21 days of age permitted postnatal deletion of YAP and TAZ, which resulted in increased osteoblast number at <em>12</em> weeks of age but no change in bone mass. Mechanistic studies revealed that YAP and TAZ suppress canonical <em>Wnt</em> signaling and Runx2 activity in osteoblast progenitors. Consistent with this, deletion of YAP and TAZ from osteoprogenitor cells increased osteoblast differentiation in vitro. Deletion of YAP and TAZ from mature osteoblasts and osteocytes using Dmp1-Cre mice led to reduced osteoblast number and bone formation, as well as increased osteoclast number, but no changes in known regulators of bone turnover such as RANKL, OPG, and Sost. Together these results suggest that YAP and TAZ in osteoblast progenitors oppose differentiation towards the osteoblast lineage but in mature osteoblasts and osteocytes, they promote bone formation and inhibit bone resorption.

Postmenopausal osteoporosis induced by estrogen deficiency causes inadequate new bone formation and affects millions of women worldwide. Melatonin can improve bone mineral density at the femoral neck in postmenopausal women with osteopenia. This study aimed to investigate the mechanism of melatonin in estrogen deficiency-induced osteoporosis by focusing on osteoblast differentiation. <em>12</em>-week-old female C57BL/6J mice were ovariectomized (OVX) and intraperitoneally injected with 10 or 50 mg/kg of melatonin for 8 weeks. Micro-computerized tomography scanning demonstrated that melatonin alleviated OVX-induced bone loss in a dose-dependent manner. Serum levels of ALP and osteocalcin (OCN) were further increased, whereas tartrate-resistant acid phosphatase level was decreased by melatonin in OVX-treated mice. Melatonin promoted osteoblast differentiation in primary bone marrow mesenchymal stem cells from OVX mice. It also inhibited activation of NLRP3 inflammasome in femoral bone protein and in induced osteoblasts stimulated by OVX. Knockdown of NLRP3 attenuated OVX-induced repression of osteogenic differentiation. The NLRP3 inflammasome activator monosodium urate partly abrogated the effect of melatonin on the expression of osteoblastogenic markers, including Runx2 and OCN. Additionally, the results showed that melatonin suppressed NLRP3 inflammasome activation by regulating <em>Wnt</em>/β-catenin signaling, which was confirmed by the <em>Wnt</em>/β-catenin inhibitor recombinant DKK1. These results indicated that melatonin ameliorates estrogen deficiency-induced osteoporosis and impaired osteogenic differentiation potential by suppressing activation of the NLRP3 inflammasome via mediating the <em>Wnt</em>/β-catenin pathway.

The transition metal cadmium (Cd) is an environmental pollutant which damages the kidneys. Chronic Cd exposure may induce renal fibrosis and/or cancer, but the signaling pathways involved are not understood. The <em>Wnt</em> pathway is a key signaling cascade responsible for renal development, fibrosis and cancer. Hence the effect of chronic in vivo Cd exposure (100 mg/l drinking water for <em>12</em> weeks) on transcriptional activation of the <em>Wnt</em> pathway and markers of epithelial-to-mesenchymal transition (EMT) was investigated in mouse kidneys. Cd exposure increased kidney Cd content from 0.023+/-0.001 microg/g to 61+/-7 microg/g wet weight (means+/-S.D. of 6-7 animals). This was accompanied by increased expression of <em>Wnt</em> ligands (<em>Wnt</em>3a/6/7a/7b/9a/9b/10a/11), as determined by RT-PCR. The <em>Wnt</em> receptors Frizzled (Fz1/2/4,5,7-10) were also upregulated, as were the co-receptors low-density lipoprotein receptor-related proteins 5/6. Immunoblots with <em>Wnt</em>10a and Fz7 antibodies also revealed increased protein expression induced by Cd exposure. In contrast, <em>Wnt</em> antagonists were largely unaffected. Upregulation of <em>Wnt</em> signaling components induced by Cd was corroborated by increased expression of <em>Wnt</em> target genes, i.e. cell proliferation and survival genes c-Myc, cyclin D1 and the multidrug transporter P-glycoprotein Abcb1b, which promote malignancy. Lastly the EMT markers Twist, fibronectin and collagen I, but not alpha-smooth muscle actin, were also upregulated, suggesting that Cd-induced changes of renal epithelial tissue characteristics towards fibrosis and cancer may be mediated by <em>Wnt</em> signaling.

OBJECTIVE

To investigate the expression and role of long non-coding RNA (lncRNA) small nucleolar RNA host gene <em>12</em> (SNHG<em>12</em>) in papillary thyroid carcinoma (PTC).

METHODS

The relative expression levels of lncRNA SNHG<em>12</em> (hereinafter referred to as SNHG<em>12</em>) in 42 pairs of PTC tissues and para-carcinoma tissues were detected via quantitative reverse transcription polymerase chain reaction (qRT-PCR). SNHG<em>12</em> specific interference sequences were designed and synthesized. The relative expression level and transfection efficiency of SNHG<em>12</em> in PTC cells were detected via qRT-PCR. After the interference in SNHG<em>12</em> expression, the change in cell proliferation capacity was detected via methyl thiazolyl tetrazolium (MTT) assay, the change in cell cycle distribution was detected via flow cytometry, the changes in cell migration and invasion capacities were detected via Transwell assay and wound healing assay, and the changes in expressions of molecular markers of Wnt/β-catenin pathway were detected via Western blotting. The pulmonary metastasis model of nude mice was established, and the changes in migration and invasion capacities of tumor cells were studied via the in-vivo experiment after the interference in SNHG<em>12</em> expression.

RESULTS

The results of qRT-PCR showed that the SNHG<em>12</em> expression was up-regulated in 30 pairs of PTC tissues and cells. The results of MTT assay showed that the cell proliferation capacity was inhibited after the interference in SNHG<em>12</em>. The results of flow cytometry showed that the cell cycle progression was blocked in G1-G0 phase after the knockdown of SNHG<em>12</em> expression. The results of Transwell assay and Western blotting showed that the interference in SNHG<em>12</em> could inhibit the invasion and metastasis capacities of tumor cells through influencing the Wnt/β-catenin signaling pathway. Metastatic tumor model of nude mice showed that SNHG<em>12</em> could affect the invasion and metastasis of tumor cells in vivo.

CONCLUSIONS

The SNHG<em>12</em> expression is relatively high in PTC tissues and cells. In-vivo/in-vitro experiments prove that SNHG<em>12</em> can promote the proliferation and metastasis of PTC cells through influencing the Wnt/β-catenin signaling pathway.

OBJECTIVE

To investigate the injury of murine mesenchymal stem cells (mMSC) exposed to 4 Gy X-radiation and the role of canonical and non-canonical wingless-type (Wnt) signaling in the radiation injury.

METHODS

C3H10T1/2 cells were submitted to 4 Gy X-radiation. At different time points after radiation, Hoechst33258 staining and Annexin V-fluorescein isothiocyanate (FITC) flow cytometry analysis were performed to assess cellular apoptosis. Senescence-associated β-galactosidase (SA-β-gal) staining was performed to analyze cellular senescence. Cell cycle was measured by flow cytometry. P53, p21, <em>Wnt</em>3a, <em>Wnt</em>5a, sonic hedgehog (Shh) mRNA was detected by Real time polymerase chain reaction (PCR) and <em>Wnt</em>5a protein was determined by Western blot.

RESULTS

A time-dependent cellular apoptosis was observed with a peak level 12 hours after radiation. Cellular senescence was detected 72 h after radiation. A remarkable up-regulation of <em>Wnt</em>5a mRNA expression (∼ 269-fold) and protein expression was seen 72 h after radiation.

CONCLUSIONS

The effect of 4 Gy X-radiation to mMSC was time-dependent in the form of cellular apoptosis in the early period and cellular senescence in the late period. Non-canonical Wnt signaling may be involved in mMSC senescence induced by 4 Gy X-radiation.

Recently, we analysed the 8p11-<em>12</em> genomic region for copy number and gene expression changes in a panel of human breast cancer cell lines and primary specimens. We found that SFRP1 (Secreted frizzled related protein 1) is frequently under expressed even in breast tumours with copy number increases in this genomic region. SFRP1 encodes a <em>WNT</em> signalling antagonist, and plays a role in the development of multiple solid tumour types. In this study, we analysed methylation-associated silencing of the SFRP1 gene in breast cancer cells with the 8p11-<em>12</em> amplicon, and investigated the tumour suppressor properties of SFRP1 in breast cancer cells. SFRP1 expression was markedly reduced in both the breast cancer cell lines and primary tumour specimens relative to normal primary human mammary epithelial cells even when SFRP1 is amplified. Suppression of SFRP1 expression in breast cancer cells with an SFRP1 gene amplification is associated with SFRP1 promoter methylation. Furthermore, restoration of SFRP1 expression suppressed the growth of breast cancer cells in monolayer, and inhibited anchorage independent growth. We also examined the relationship between the silencing of SFRP1 gene and <em>WNT</em> signalling in breast cancer. Ectopic SFRP1 expression in breast cancer cells suppressed both canonical and non-canonical <em>WNT</em> signalling pathways, and SFRP1 expression was negatively associated with the expression of a subset of <em>WNT</em> responsive genes including RET and MSX2. Thus, down-regulation of SFRP1 can be triggered by epigenetic and/or genetic events and may contribute to the tumourigenesis of human breast cancer through both canonical and non-canonical <em>WNT</em> signalling pathways.

Lymphoid-enhancer-binding factor 1 (LEF1), coupling with β-catenin, functions as a key nuclear mediator of <em>WNT</em>/β-catenin signaling, which regulates cell proliferation and survival. LEF1 has an important role in lymphopoiesis, and is normally expressed in T and pro-B cells but not mature B cells. However, gene expression profiling demonstrates overexpression of LEF1 in chronic lymphocytic leukemia, and knockdown of LEF1 decreases the survival of the leukemic cells. So far, the data on LEF1 expression in B-cell lymphomas are limited. This study represents the first attempt to assess LEF1 by immunohistochemistry in a large series (290 cases) of B-cell lymphomas. Strong nuclear staining of LEF1 was observed in virtually all neoplastic cells in 92 of 92 (100%) chronic lymphocytic leukemia/small lymphocytic lymphomas including two CD5- cases, with strongest staining in cells with Richter's transformation. LEF1 also highlighted the morphologically inconspicuous small lymphocytic lymphoma component in three composite lymphomas. All 53 mantle cell lymphomas, 31 low-grade follicular lymphomas and 31 marginal zone lymphomas, including 3 CD5+ cases, were negative. In <em>12</em> grade 3 follicular lymphomas, LEF1 was positive in a small subset (5-15%) of cells. Diffuse large B-cell lymphoma, however, demonstrated significant variability in LEF1 expression with overall positivity in 27 of 71 (38%) cases. Our results demonstrate that nuclear overexpression of LEF1 is highly associated with chronic lymphocytic leukemia/small lymphocytic lymphoma, and may serve as a convenient marker for differential diagnosis of small B-cell lymphomas. The expression of β-catenin, the coactivator of LEF1 in <em>WNT</em> signaling, was examined in 50 chronic lymphocytic leukemia/small lymphocytic lymphomas, of which 44 (88%) showed negative nuclear staining. The findings of universal nuclear overexpression of LEF1 but lack of nuclear β-catenin in the majority of chronic lymphocytic leukemia/small lymphocytic lymphoma suggest that the pro-survival function of LEF1 in this disease may be independent of <em>WNT</em>/β-catenin signaling.

Aberrant activation of <em>Wnts</em> is common in human cancers, including prostate. Hypermethylation associated transcriptional silencing of <em>Wnt</em> antagonist genes SFRPs (Secreted Frizzled-Related Proteins) is a frequent oncogenic event. The significance of this is not known in prostate cancer. The objectives of our study were to (i) profile <em>Wnt</em> signaling related gene expression and (ii) investigate methylation of <em>Wnt</em> antagonist genes in prostate cancer. Using TaqMan Low Density Arrays, we identified 15 <em>Wnt</em> signaling related genes with significantly altered expression in prostate cancer; the majority of which were upregulated in tumors. Notably, histologically benign tissue from men with prostate cancer appeared more similar to tumor (r = 0.76) than to benign prostatic hyperplasia (BPH; r = 0.57, p < 0.001). Overall, the expression profile was highly similar between tumors of high (≥ 7) and low (≤ 6) Gleason scores. Pharmacological demethylation of PC-3 cells with 5-Aza-CdR reactivated 39 genes (≥ 2-fold); 40% of which inhibit <em>Wnt</em> signaling. Methylation frequencies in prostate cancer were 10% (2/20) (SFRP1), 64.86% (48/74) (SFRP2), 0% (0/20) (SFRP4) and 60% (<em>12</em>/20) (SFRP5). SFRP2 methylation was detected at significantly lower frequencies in high-grade prostatic intraepithelial neoplasia (HGPIN; 30%, (6/20), p = 0.0096), tumor adjacent benign areas (8.82%, (7/69), p < 0.0001) and BPH (11.43% (4/35), p < 0.0001). The quantitative level of SFRP2 methylation (normalized index of methylation) was also significantly higher in tumors (116) than in the other samples (HGPIN = 7.45, HB = 0.47, and BPH = 0.<em>12</em>). We show that SFRP2 hypermethylation is a common event in prostate cancer. SFRP2 methylation in combination with other epigenetic markers may be a useful biomarker of prostate cancer.

BACKGROUND

Recent work led to recognize sessile serrated adenomas (SSA) as precursor to many of the sporadic colorectal cancers with microsatellite instability (MSI). However, comprehensive analyses of DNA methylation in SSA and MSI cancer have not been conducted.

METHODS

With an array-based methylation sensitive amplified fragment length polymorphism (MS-AFLP) method we analyzed 8 tubular (TA) and 19 serrated (SSA) adenomas, and 14 carcinomas with (MSI) and <em>12</em> without (MSS) microsatellite instability. MS-AFLP array can survey relative differences in methylation between normal and tumor tissues of 9,654 DNA fragments containing all NotI sequences in the human genome.

RESULTS

Unsupervised clustering analysis of the genome-wide hypermethylation alterations revealed no major differences between or within these groups of benign and malignant tumors regardless of their location in intergenic, intragenic, promoter, or 3' end regions. Hypomethylation was less frequent in SSAs compared with MSI or MSS carcinomas. Analysis of variance of DNA methylation between these four subgroups identified 56 probes differentially altered. The hierarchical tree of this subset of probes revealed two distinct clusters: Group 1, mostly composed by TAs and MSS cancers with KRAS mutations; and Group 2 with BRAF mutations, which consisted of cancers with MSI and MLH1 methylation (Group 2A), and SSAs without MLH1 methylation (Group 2B). AXIN2, which cooperates with APC and β-catenin in Wnt signaling, had more methylation alterations in Group 2, and its expression levels negatively correlated with methylation determined by bisulfite sequencing. Within group 2B, low and high AXIN2 expression levels correlated significantly with differences in size (P = 0.01) location (P = 0.05) and crypt architecture (P = 0.01).

CONCLUSIONS

Somatic methylation alterations of AXIN2, associated with changes in its expression, stratify SSAs according to some clinico-pathological differences. We conclude that hypermethylation of MLH1, when occurs in an adenoma cell with BRAF oncogenic mutational activation, drives the pathway for MSI cancer by providing the cells with a mutator phenotype. AXIN2 inactivation may contribute to this tumorigenic pathway either by mutator phenotype driven frameshift mutations or by epigenetic deregulation contemporary with the unfolding of the mutator phenotype.

WntWnt signaling, is involved in inflammatory responses, however the mechanism is not well understood. We examined the role of WntWntWntWntWntWnt expression. Taken together, these results suggest that Wnt expression in DCs, and thereby inducing Th1 differentiation in colitis.

<em>Wnt</em> signaling is important for bone formation and osteoblastic differentiation. Recent findings indicate a stimulating role of <em>Wnt</em> signaling in bone mechanotransduction. However, negative effects of <em>Wnt</em> signaling on osteoblast differentiation and mineralization have been described as well. We conducted in vitro stretch experiments using human pre-osteoblasts to study short- and long-term effects of mechanical loading on <em>Wnt</em>/beta-catenin signaling. As the extracellular regulated kinase (ERK) pathway is known to be involved in mechanotransduction in osteoblasts, we also evaluated its role in <em>Wnt</em>/beta-catenin signaling. Stretch experiments up to 21 days (using stretch episodes of 15 min, alternated with 90 min rest) resulted in higher mineralization compared to static control cultures. We found that 15 min of stretch initially increased nuclear beta-catenin, but ultimately resulted in significant decrease at <em>12</em> and 40 h after stretch. Downregulation of <em>Wnt</em>-responsive element activity 16 h after stretch, using a luciferase construct, further supported these findings. The presence of the ERK inhibitor U0<em>12</em>6 did not alter the stretch-induced decrease of beta-catenin levels. Our data indicate a biphasic effect of mechanical loading on beta-catenin in mineralizing human differentiating osteoblasts, which is independent of the ERK pathway. The osteogenic potential of our loading regime was confirmed by an increase in osteogenic differentiation markers such as alkaline phosphatase activity and calcium deposition after 3 weeks of culture. We conjecture that the biphasic aspect of <em>Wnt</em>/beta-catenin signaling with a strong decrease up to 40 h after the stretch induction, is important for the anabolic effects of mechanical stretch on bone.

The differentiation of mesenchymal stem cells (MSCs) into type II alveolar epithelial (AT II) cells is critical for reepithelization and recovery in acute respiratory distress syndrome (ARDS), and <em>Wnt</em> signaling was considered to be the underlying mechanisms. In our previous study, we found that canonical <em>Wnt</em> pathway promoted the differentiation of MSCs into AT II cells, however the role of the noncanonical <em>Wnt</em> pathway in this process is unclear. It was disclosed in this study that noncanonical <em>Wnt</em> signaling in mouse bone marrow-derived MSCs (mMSCs) was activated during the differentiation of mMSCs into AT II cells in a modified co-culture system with murine lung epithelial-<em>12</em> cells and small airway growth media. The levels of surfactant protein (SP) C, SPB and SPD, the specific markers of AT II cells, increased in mMSCs when <em>Wnt</em>5a was added to activate noncanonical <em>Wnt</em> signaling, while pretreatment with JNK or PKC inhibitors reversed the promotion of <em>Wnt</em>5a. The differentiation rate of mMSCs also depends on their abilities to accumulate and survive in inflammatory tissue. We found that the <em>Wnt</em>5a supplement promoted the vertical and horizontal migration of mMSCs, ameliorated the cell death and the reduction of Bcl-2/Bax induced by H2O2. The effect of <em>Wnt</em>5a on the migration of mMSCs and their survival after H2O2 exposure were partially inhibited with PKC or JNK blockers. In conclusion, <em>Wnt</em>5a through <em>Wnt</em>/JNK signaling alone or both <em>Wnt</em>/JNK and <em>Wnt</em>/PKC signaling promoted the differentiation of mMSCs into AT II cells and the migration of mMSCs; through <em>Wnt</em>/PKC signaling, <em>Wnt</em>5a increased the survival of mMSCs after H2O2 exposure in vitro.

We developed a slow structural relaxation model to describe cellular dynamics in the crypt of the mouse small intestine. Cells are arranged in a three dimensional spiral the size of which dynamically changes according to cell production demands of adjacent villi. Cell differentiation and proliferation is regulated through <em>Wnt</em> and Notch signals, the strength of which depends on the local cell composition. The highest level of <em>Wnt</em> activity is associated with maintaining equipotent stem cells (SC), Paneth cells and common goblet-Paneth cell progenitors (CGPCPs) intermingling at the crypt bottom. Low levels of <em>Wnt</em> signalling area are associated with stem cells giving rise to secretory cells (CGPCPs, enteroendocrine or Tuft cells) and proliferative absorptive progenitors. Deciding between these two fates, secretory and stem/absorptive cells, depends on Notch signalling. Our model predicts that Notch signalling inhibits secretory fate if more than 50% of cells they are in contact with belong to the secretory lineage. CGPCPs under high <em>Wnt</em> signalling will differentiate into Paneth cells while those migrating out from the crypt bottom differentiate into goblet cells. We have assumed that mature Paneth cells migrating upwards undergo anoikis. Structural relaxation explains the localisation of Paneth cells to the crypt bottom in the absence of active forces. The predicted crypt generation time from one SC is 4-5 days with 10-<em>12</em> days needed to reach a structural steady state. Our predictions are consistent with experimental observations made under altered <em>Wnt</em> and Notch signalling. Mutations affecting stem cells located at the crypt floor have a 50% chance of being propagated throughout the crypt while mutations in cells above are rarely propagated. The predicted recovery time of an injured crypt losing half of its cells is approximately 2 days.

Previous studies have demonstrated the beneficial effect of mechanical loading on in vitro tendon engineering. To understand the mechanism, human tenocytes and polyglycolic acid long fibers were used for in vitro tendon engineering in a bioreactor system for <em>12</em> weeks with and without dynamic loading. The engineered neo-tendons were subjected to proteomic analysis using mass spectrometry along with shotgun strategy. As expected, mechanical loading resulted in a more mature tendon tissue characterized by a firmer tissue texture and densely deposited matrices which formed longitudinally aligned collagen fibers in a highly compact fashion. In contrast, non-loaded neo-tendon revealed loosely and less deposited matrices in a relatively less organized pattern. Proteins isolated from two groups of tissues exhibited similar distribution of isoeletric point and molecular weight indicating the similarity and comparability of the tissue specimens. Further, proteomic analysis showed that total 758 proteins were identified from both groups with 194 and 177 proteins uniquely presented in loaded and non-loaded tendons, respectively. Comparison of loaded and non-loaded tendons revealed 195 significantly up-regulated proteins and 189 significantly down-regulated proteins. The differentially expressed proteins could generally be classified into the categories of extracellular matrix, intra-cellular signaling, cytoskeleton and inflammatory response. Among them, significantly up-regulated collagens I and VI, MMP-14, WNT5A, microfilament molecules and some inflammatory factors suggest that the possible mechanism for this particular biological phenomenon may involve increased production of tendon specific matrices, enhanced cross-link of collagens and other matrix molecules, proper matrix remodeling for tissue maturation and mechanotransduction (including non-canonical <em>Wnt</em> signal pathway) mediated other biological processes.

OBJECTIVE

Patients with familial hypercholesterolemia (FH) due mutations in the low-density lipoprotein receptor (LDLR) suffer premature aortic calcification, an effect that is age- and gene dosage-dependent and cholesterol level independent later in life. To better understand this process, we examined a murine model.

METHODS

We compared chow fed Ldlr(-/-) mice to controls at 6, <em>12</em> and 18 months and on a Western diet (WD) at 6 months. Additionally, we compared controls to Ldlr(-/-) mice and transgenic mice Tg(Pcsk9) overexpressing PCSK9, which promotes LDLR degradation. Aortas were perfused-fixed, embedded in paraffin, and sections were stained with alizarin red. Micro-computerized tomography (micro-CT) was used to quantify vascular calcification.

RESULTS

Ldlr(-/-) mice develop calcification in the ascending, transverse aorta and neck vessels with a distribution similar to that of human. Calcification was most prominent in 18-month-old Ldlr(-/-) mice fed a chow diet and in 6-month-old Ldlr(-/-) mice fed a WD. Interestingly, Tg(Pcsk9) mice fed a WD develop aortic calcifications as well. Histology confirmed that the calcification were predominantly sub-intimal. Marked expression of LRP5 and WNT was observed in the Ldlr(-/-) and Tg(Pcsk9) models, but not in age-matched controls.

CONCLUSIONS

The two mouse models develop aortic calcification in an age- and diet-dependent manner. Abnormal regulation of the LRP5/Wnt pathway may play a role in the calcification process. Further analysis of these aortic calcification models using this micro-CT imaging technique may provide a better understanding of the link between FH and arterial calcification.

Solid pseudopapillary neoplasms of the pancreas almost consistently show a beta-catenin mutation activating the <em>Wnt</em>-signaling pathway, resulting in overexpression of cyclin D1, but not in overt malignancy of this tumor. Besides cyclin D1, a set of markers (ie FLI-1, CD56 and progesterone receptor), whose genes map to chromosome 11q, are frequently expressed in solid pseudopapillary neoplasms. Chromosome 11q is a region that is also often affected in pancreatic neuroendocrine tumors. This immunohistochemical study was undertaken to gain insights into the downstream regulation of the <em>Wnt</em>-signaling pathway and the significance of overexpressed gene products belonging to chromosome 11q for the tumorigenesis in solid pseudopapillary neoplasms. Fourteen solid pseudopapillary neoplasms were analyzed for the expression of cyclin-dependent kinase inhibitors p21, p27, p16 and hyperphosphorylated retinoblastoma (pRb) proteins. In an extended series of 93 solid pseudopapillary neoplasms, beta-catenin, cyclin D1, FLI-1 and CD56 expression was examined and compared with that in 22 pancreatic neuroendocrine tumors. Solid pseudopapillary neoplasms (98%) showed aberrant expression of beta-catenin with a concomitant cyclin D1 expression in 69% of the cases, but no expression of pRb (0%) was found. p27 and p21 were expressed in 100% (14/14) and 86% (<em>12</em>/14) of the cases, but only 2/14 (14%) were positive for p16. FLI-1 was expressed in 63% of solid pseudopapillary neoplasms, but only in 1/22 pancreatic neuroendocrine tumors (5%), cyclin D1 expression was present in 14% of the latter. We conclude that in solid pseudopapillary neoplasms the activated <em>Wnt</em>-signaling pathway is disrupted, and that p21 and p27 are contributing to this fact by blocking of the hyperphosphorylation of the Rb protein, thus causing the very low proliferation rate characterizing the solid pseudopapillary neoplasms. The accumulation of high expression of proteins whose genes are located on chromosome 11q is characteristic of solid pseudopapillary neoplasms, but not of pancreatic neuroendocrine tumors.

<em>WNT</em> signaling pathway is implicated in carcinogenesis and embryogenesis. We have previously cloned and characterized <em>WNT</em>10A, and demonstrated up-regulation of <em>WNT</em>10A in gastric cancer. Here, we investigated expression of <em>WNT</em>10A mRNA in various types of human cancer. <em>WNT</em>10A mRNA was detected in 10 out of <em>12</em> esophageal cancer cell lines by cDNA-PCR, and was significantly up-regulated in esophageal cancer cell lines TE2, TE3, TE4, and a brain tumor cell line A-172. <em>WNT</em>10A mRNA was not up-regulated by retinoic acid in a teratocarcinoma cell line NT2. TFF1/pS2 mRNA, but not <em>WNT</em>10A mRNA, was up-regulated by beta-estradiol in a breast cancer cell line MCF-7. Expression of <em>WNT</em>10A mRNA in various types of primary cancers was next investigated by using Matched tumor/normal expression array filter. <em>WNT</em>10A mRNA was significantly up-regulated in 2 out of 8 cases of primary gastric cancer, and in 1 out of 7 cases of primary rectal cancer. Expression of <em>WNT</em>10A mRNA in esophageal cancer was not investigated, because such samples were not blotted on the expression array filter. Up-regulation of <em>WNT</em>10A mRNA might play key roles in some cases of esophageal, gastric, and colorectal cancer.