Osteoarthritis (OA), or degenerative arthritis, is characterized by mechanical stress-induced changes in cartilage and bone. OA is a leading cause of chronic disability in North America and Europe. A recent study written by Blom and colleagues (Arthritis and Rheumatism 2009; 60:501-<em>12</em>) showed that elevated <em>wnt</em> signaling was observed in experimental OA as well as in patient samples. The authors found that the known <em>wnt</em> target WISP-1 (CCN4) was also overexpressed; CCN4 was sufficient to recapitulate an OA phenotype in vitro and in vivo, suggesting that CCN4 may be a novel target for drug intervention in OA. This commentary summarizes these exciting findings.

Hyperlipidemia has been shown to stimulate vascular smooth muscle cell (VSMC) proliferation. <em>Wnt</em> signaling pathway plays a critical role in embryonic development and cell proliferation. In this study, Sprague-Dawley rats fed with high-fat or normal diet for <em>12</em> weeks were sacrificed, and the thoracic aorta was harvested to determine wnt3a, β-catenin, T-cell factor 4 (TCF4), and cyclin D1 expressions. VSMC proliferation within thoracic aorta and lipid accumulation within VSMCs were detected. Rat aortic VSMCs were cultured in serum from rats with hyperlipidemia or DKK-1; <em>Wnt</em>3a, β-catenin, TCF4, and cyclin D1 expressions, and cell cycle distribution were determined. The findings demonstrated that increased number of VSMCs, lipid droplets, and vacuoles within thoracic aorta in the high-fat-fed group. Compared with controls, VSMCs from high-fat-fed rats showed higher mRNA expressions of wnt3a, β-catenin, TCF4, and cyclin D1, as well as in VSMCs cultured with hyperlipidemic serum. After 24 h, VSMCs stimulated with hyperlipidemic serum showed significantly increased cell number and S-phase entry compared with cells exposed to normolipidemic serum. These effects were blocked by DKK-1. These results suggest that <em>Wnt</em>/β-catenin signaling plays an important role in hyperlipidemia-induced VSMC proliferation.

OBJECTIVE

Hypoxic-ischemic encephalopathy (HIE) is a common brain injury disease in neonates, which can lead to neonatal disability and death. Geniposide (GEN) is a main ingredient of Gardenia jasminoides whose anti-tumor, anti-inflammatory and anti-apoptotic effects have been reported in various diseases. However, the effect of GEN on HIE remains uninvestigated. This study aimed to clarify the protective effect of GEN on PC-<em>12</em> cells against oxygen and glucose deprivation (OGD)-induced injury.

METHODS

PC-<em>12</em> cells were subjected to OGD treatment, cell viability, cell cycle-associated factors, apoptosis and apoptosis-associated factors were then determined. The different concentrations of GEN were used to stimulate PC-<em>12</em> cells, and the effects of GEN on cell proliferation and apoptosis in OGD-treatment cells were assessed. Subsequently, relative expression level of H19 was analyzed in PC-<em>12</em> cells after treatment with GEN. After this, si-H19 was transfected into PC-<em>12</em> cells to explore the regulatory effect of H19 on PC-<em>12</em> cells after treatment with GEN and OGD. Besides, PI3K/AKT and Wnt/β-catenin pathways were examined by western blot assay.

RESULTS

OGD significantly inhibited cell viability, decreased CyclinD1, CDK4 and CDK6 expression, induced apoptosis and up-regulated Cleaved-Caspase-9/-7/-3 expression in PC-<em>12</em> cells. GEN treatment obviously alleviated OGD-induced cell injury. Additionally, H19 expression was up-regulated by GEN, and H19 knockdown reversed the protective effect of GEN on PC-<em>12</em> cells against OGD-induced injury. Finally, GEN activated PI3K/AKT and Wnt/β-catenin pathways by regulating H19 in OGD-insulted PC-<em>12</em> cells.

CONCLUSIONS

The findings suggested that GEN protected PC-<em>12</em> cells against OGD-induced injury by up-regulation of H19.

BACKGROUND

As Wnt/β-catenin/glycogen synthase kinase 3β (GSK3β) signaling has been implicated in myocardial injury and diabetic cardiomyopathy (DCM) is a major part of diabetic cardiovascular complications, we therefore investigated the alterations of Wnt/β-catenin/GSK3β signaling during the development of DCM.

METHODS

The rat model of diabetes mellitus (DM) was established using a single intraperitoneal injection of streptozotocin (STZ, 60 mg/kg). The alterations of Wnt/β-catenin/GSK3β signaling were determined 4, 8, and 12 weeks following DM using Western blotting, immunohistochemistry, and quantitative real-time reverse transcriptase polymerase chain reaction. Cardiac pathology changes were evaluated using hematoxylin and eosin, Masson trichromatic, and terminal dUTP nick-end labeling staining.

RESULTS

Histological analyses revealed that DM induced significant myocardial injury and progressive cardiomyocyte apoptosis. The protein and mRNA levels of WntWnt inhibitor Dickkopf-1 was increased after STZ injection and then decreased as DCM developed.

CONCLUSIONS

Wnt/β-catenin/GSK3β signaling pathway is activated in the development of DCM. Further investigation into the role of Wnt signaling during DCM will functionally find novel therapeutic target for DCM.

Methyl-amoorain (methyl-25-hydroxy-3-oxoo-lean-<em>12</em>-en-28-oate, AMR-Me), a novel synthetic oleanane triterpenoid, exerts a striking chemopreventive effect against 7,<em>12</em>-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumorigenesis through antiproliferative and proapoptotic actions. Nevertheless, the underlying mechanisms of action remain to be established. As estrogen receptor (ER) and canonical <em>Wnt</em>/b-catenin signaling are involved in the development and progression of breast cancer, the current study was designed to investigate the effects of AMR-Me treatment on the expressions of ER-a, ER-b, b-catenin and cyclin D1 in rat mammary tumors induced by DMBA. Mammary tumor samples were harvested from an 18-week chemopreventive study in which AMR-Me (0.8–1.6 mg/kg) was shown to inhibit mammary carcinogenesis in a dose–response manner. The expressions of ER-a, ER-b, b-catenin, and cyclin D1 were determined by immunohistochemistry and reverse transcription-polymerase chain reaction. AMR-Me downregulated the expression of intratumor ER-a and ER-b and lowered the ratio of ER-a to ER-b. AMR-Me also reduced the expression, cytoplasmic accumulation, and nuclear translocation of b-catenin, the essential transcriptional cofactor for <em>Wnt</em> signaling. Furthermore, AMR-Me modulated the expression of cell growth regulatory gene cyclin D1, which is a downstream target for both ER and <em>Wnt</em> signaling. AMR-Me at 1.6 mg/kg for 18 weeks did not exhibit any hepatotoxicity or renotoxicity. The results of the present study coupled with our previous findings indicate that simultaneous disruption of ER and <em>Wnt</em>/b-catenin signaling possibly contributes to antiproliferative and apoptosis-inducing effects implicated in AMR-Me-mediated chemoprevention of DMBA-induced breast tumorigenesis in rats. Our results also suggest a possible crosstalk between two key regulatory pathways, namely ER and <em>Wnt</em>/b-catenin signaling, involved in mammary carcinogenesis and the value of simultaneously targeting these pathways to achieve breast cancer chemoprevention.

The Hippo pathway is involved in intestinal epithelial homeostasis with <em>Wnt</em>, BMP, Notch, and EGF signaling. We investigated the relationship between Hippo and other signaling pathways and the role of MOB kinase activator 1A/1B (MOB1A/B) in intestinal homeostasis. Mice with intestinal epithelial cell (IEC)-specific depletion of MOB1A/B showed hyperproliferation in IECs, defects in secretory lineage differentiation and loss of intestinal stem cells and eventually died at 10-<em>12</em> days after tamoxifen treatment. In MOB1A/B-depleted IECs, expression of <em>Wnt</em> target genes were downregulated but Bmp2 and Tgfbr2 were transcriptionally activated with enhanced YAP activity. In in vivo and in vitro experiments with several signaling inhibitors, it has been shown that the BMP inhibitor LDN193189 or TGF-β inhibitor SB431542 had effects on partial restoration of the intestinal degenerative phenotype. Treatment with these inhibitors restored differentiation of secretory lineage cells in MOB1A/B-deficient mice, but not ISC pools in the crypt region. These studies reveal that IEC-specific depletion of MOB1A/B induced overexpression of Bmp2 and Tgfbr2 and inhibited <em>Wnt</em> activity, finally leading to loss of ISCs and functional epithelia in the mouse intestine. These results suggest that MOB1A/B has an essential function for intestinal epithelial homeostasis by regulating YAP, <em>Wnt</em> activity, and BMP/TGF-β signaling.

Colon cancer is one of the most common malignant tumors in the human body, ranking second as a gastrointestinal tumor. It has a high incidence in Europe, America and China and more than 1 million new cases of colon cancer are reported worldwide each year. The incidence of colon cancer in China has increased from <em>12</em>/0.1 million in the early 1970s to 56/0.1 million at present with an annual growth rate of 4.2%, which far exceeds the international level (2%). Rhotekin (RTKN) 2, a Rho-guanosine triphosphatase (GTPase) effector, has been reported to be anti-apoptotic. However, the molecular mechanism underlying the biological function of RTKN2 in colon cancer remains unknown. The present study investigated whether the mRNA expression level of RTKN2 was markedly higher in 30 human colon cancer specimens compared with adjacent non-cancerous tissues. The results showed that the protein expression level of RTKN2 was significantly higher in SW480 and HCT116 cells, compared with HIEC cells. Knockdown of RTKN2 in the SW480 and HCT116 colon cancer cells, by lentivirus-mediated RNA interference led to the notable inhibition of cell proliferation and cell cycle progression, by reducing the expression levels of the PCDA, Cyclin D1 and c-myc cell cycle-associated proteins. The inhibitory effect of RTKN2 silencing on the proliferation of colon cancer cells may be partially realized by inhibiting the <em>Wnt</em>/β-catenin signaling pathway. Furthermore, the silencing of RTKN2 in the cells induced apoptosis by reducing the expression level of Bax and increasing the expression level of Bcl2. These results show that RTKN2 is involved in the carcinogenesis and progression of human colon cancer, indicating that RTKN2 may be a molecular target in colon cancer therapy.

BACKGROUND

Growing evidence suggests that the Wnt/β-catenin signaling cascade is implicated in the control of stem cell activity, cell proliferation, lineage commitment, and cell survival during normal development and tissue regeneration of the gastrointestinal epithelium. The roles of this signaling cascade in stimulation of cell proliferation after massive small bowel resection are unknown. The purpose of this study was to evaluate the role of Wnt/β-catenin signaling during late stages of intestinal adaptation in a rat model of short bowel syndrome (SBS).

METHODS

Male rats were divided into two groups: sham rats underwent bowel transection and SBS rats underwent a 75 % bowel resection. Parameters of intestinal adaptation, enterocyte proliferation and apoptosis were determined 2 weeks after operation. Illumina's digital gene expression analysis was used to determine Wnt/β-catenin signaling gene expression profiling. Twelve Wnt/β-catenin-related genes and β-catenin protein expression were determined using real-time PCR, western blotting and immunohistochemistry.

RESULTS

From the total number of 20,000 probes, 20 genes related to Wnt/β-catenin signaling were investigated. From these genes, seven genes were found to be up-regulated and eight genes to be down-regulated in SBS vs. sham animals with a relative change in gene expression level of 20 % or more. From 12 genes determined by real-time PCR, nine genes were down-regulated in SBS rats compared to control animals including target gene c-Myc. SBS rats also showed a significant decrease in β-catenin protein compared to control animals.

CONCLUSIONS

Two weeks following massive bowel resection in rats, Wnt/β-catenin signaling pathway is inhibited. In addition, it appears that cell differentiation rather than proliferation is most important in the late stages of intestinal adaptation.

The murine mutant Splotch (Sp) is a well-established model for studying neural tube closure defects. In the current investigation, the progression through neural tube closure (NTC) as well as the expression patterns of <em>12</em> developmentally regulated genes were examined in the neural tissue of wildtype (+/+), Splotch heterozygous (Sp/+), and Splotch homozygous (Sp/Sp) embryos during neurulation. The overall growth of the embryos, as measured by the number of somite pairs, did not differ significantly between the three genotypes at any of the collection time-points. There was, however, a significant delay in the progression through NTC for both the Sp/+ and Sp/Sp embryos. A univariate analysis on the expression of the <em>12</em> candidate genes (bcl-2, FBP-2, Hmx-2, Msx-3, N-cam, N-cad, noggin, p53, Pax-3, Shh, Wee-1, <em>wnt</em>-1) revealed that although 11 were statistically altered, across time or by genotype, there were no significant interactions between gestation age and genotype for any of these genes during NTC. However, a multivariate statistical analysis on the simultaneous expression of these genes revealed interactions at both gestation day (GD) 8:<em>12</em> (day:hour) and 9:00 among Pax-3, N-cam, N-cad, bcl-2, p53, and Wee-1 that could potentially explain the aberrant NTC. The data from these studies suggest that a disruption in the genes that govern the cell cycle or extracellular matrices of the developing neural tube might play a critical role in the occurrence of the NTDs observed in Splotch embryos.

<em>Wnt</em>-1 and <em>Wnt</em>-3a have been postulated to share functional redundancy in spinal cord morphogenesis due to their homologies in protein structure and overlapping expression patterns. In this study, antisense oligonucleotides and a murine whole embryo culture system were used to examine functional interactions of <em>Wnt</em>-1 and <em>Wnt</em>-3a in late gastrulation and neurulation. Early somite mouse embryos were injected with combinations of <em>Wnt</em>-1 and <em>Wnt</em>-3a antisense oligonucleotides and then grown in vitro for up to 48 hr. Simultaneous inhibition of <em>Wnt</em>-1 and <em>Wnt</em>-3a expression resulted in pattern loss in the presumptive spinal cord, which was apparent within 4 hr following antisense treatment. The neural tube was wavy, there was a reduction in the number of nuclear layers in the walls of the neural tube, and evidence of decreased cell adhesion between neuroepithelial cells by <em>12</em> hr postinjection. In addition, notochord and primitive streak abnormalities accompanied neural tube abnormalities. The existence of regulatory interactions between <em>Wnt</em>-1, <em>Wnt</em>-3a, and engrailed genes was also examined in this study. Antisense inhibition of <em>Wnt</em>-1 or <em>Wnt</em>-3a expression resulted in reduction of engrailed protein levels in the brain, somites, and spinal cord. However, simultaneous inhibition of both <em>Wnt</em> genes resulted in more complete loss of engrailed protein in these regions. Herein, we present data suggesting functional redundancy of <em>Wnt</em>-1 and <em>Wnt</em>-3a in neural tube patterning and in regulation of engrailed expression.

The glutathione S-transferase GSTP is overexpressed in many human cancers and chemotherapy-resistant cancer cells, where there is evidence that GSTP may have additional functions beyond its known catalytic role. On the basis of evidence that Gstp-deficient mice have a comparatively higher susceptibility to skin carcinogenesis, we investigated whether this phenotype reflected an alteration in carcinogen detoxification or not. For this study, Gstp(-/-) mice were interbred with Tg.AC mice that harbor initiating H-ras mutations in the skin. Gstp(-/-)/Tg.AC mice exposed to the proinflammatory phorbol ester <em>12</em>-O-tetradecanoylphorbol-13-acetate (TPA) exhibited higher tumor incidence and multiplicity with a significant thickening of skin after treatment, illustrating hyperproliferative growth. Unexpectedly, we observed no difference in cellular proliferation or apoptosis or in markers of oxidative stress, although higher levels of the inflammatory marker nitrotyrosine were found in Gstp(-/-)/Tg.AC mice. Instead, gene set enrichment analysis of microarray expression data obtained from skin revealed a more proapoptotic and proinflammatory environment shortly after TPA treatment. Within 4 weeks of TPA treatment, Gstp(-/-)/Tg.AC mice displayed altered lipid/sterol metabolism and <em>Wnt</em> signaling along with aberrant processes of cytoskeletal control and epidermal morphogenesis at both early and late times. In extending the evidence that GSTP has a vital role in normal homeostatic control and cancer prevention, they also strongly encourage the emerging concept that GSTP is a major determinant of the proinflammatory character of the tumor microenvironment. This study shows that the GSTP plays a major role in carcinogenesis distinct from its role in detoxification and provides evidence that the enzyme is a key determinant of the proinflammatory tumor environment.

Canonical <em>Wnt</em>/β-catenin (hereafter <em>Wnt</em>) signaling regulates the proliferation and differentiation of various cell types. However, the role of non-canonical signaling including protein kinase C (PKC) signaling has not been investigated in intervertebral disc (IVD) cells. The aim of this study was to elucidate whether the activation of PKC signaling act to modulate <em>Wnt</em> signaling in IVD cells. We performed several reporter assays, real-time reverse transcription polymerase chain reaction (RT-PCR), immunohistochemical and immunofluorescence analyses, and western blot analyses using rat nucleus pulposus (NP) cells. We also examined the cell proliferation and cell cycle distribution under phorbol <em>12</em>-myristate 13-acetate (PMA) stimulation, a known activator of PKC signaling. We found that NP cells exhibited decreased β-catenin mRNA and protein levels upon stimulation with PMA. PMA treatment promoted proliferation and cell cycle progression in a time- and dose-dependent manner. In addition, activation of the PKC signaling also regulated the expression of aggrecan. Finally, activation by PMA induced the expression of several PKC isoforms in NP cells. It is concluded that activation of PKC signaling might lead to an increase in matrix synthesis and cell proliferation, thereby inhibiting IVD degeneration. Crosstalk in these signaling pathways plays an important role in the regulation of IVD homeostasis.

The dermonecrotic toxins from Pasteurella multocida (PMT), Bordetella (DNT), Escherichia coli (CNF1-3), and Yersinia (CNFY) modulate their G-protein targets through deamidation and/or transglutamination of an active site Gln residue, which results in activation of the G protein and its cognate downstream signaling pathways. Whereas DNT and the CNFs act on small Rho GTPases, PMT acts on the α subunit of heterotrimeric G(q), G(i), and G(<em>12</em>/13) proteins. We previously demonstrated that PMT potently blocks adipogenesis and adipocyte differentiation in a calcineurin-independent manner through downregulation of Notch1 and stabilization of β-catenin and Pref1/Dlk1, key proteins in signaling pathways strongly linked to cell fate decisions, including fat and bone development. Here, we report that similar to PMT, DNT, and CNF1 completely block adipogenesis and adipocyte differentiation by preventing upregulation of adipocyte markers, PPARγ and C/EBPα, while stabilizing the expression of Pref1/Dlk1 and β-catenin. We show that the Rho/ROCK inhibitor Y-27632 prevented or reversed these toxin-mediated effects, strongly supporting a role for Rho/ROCK signaling in dermonecrotic toxin-mediated inhibition of adipogenesis and adipocyte differentiation. Toxin treatment was also accompanied by downregulation of Notch1 expression, although this inhibition was independent of Rho/ROCK signaling. We further show that PMT-mediated downregulation of Notch1 expression occurs primarily through G(<em>12</em>/13) signaling. Our results reveal new details of the pathways involved in dermonecrotic toxin action on adipocyte differentiation, and the role of Rho/ROCK signaling in mediating toxin effects on <em>Wnt</em>/β-catenin and Notch1 signaling, and in particular the role of G(q) and G(<em>12</em>/13) in mediating PMT effects on Rho/ROCK and Notch1 signaling.

In Caenorhabditis elegans, two lateral blast cells called P11/<em>12</em>L and P11/<em>12</em>R are symmetric left-right homologs at hatching, migrate subsequently in opposite anteroposterior directions during the first larval stage, and adopt two different fates, thus breaking the symmetry between them. Our results show that, unlike most other cell fate decisions in C. elegans, the orientation of P11/<em>12</em>L/R migration is highly biased, but not fixed. The handedness of their migration is linked to whole body handedness and is randomized in lin-<em>12</em>/Notch mutants and by ablation of the Y cell. Migration handedness is independent of P11 and P<em>12</em> fate determination, previously shown to require the LIN-44/<em>Wnt</em> and the LIN-3/EGF pathways (L. I. Jiang and P. W. Sternberg, 1998, Development <em>12</em>5, 2337-2347). We further show that several changes in P11/<em>12</em>L/R asymmetry have occurred during nematode evolution: loss of asymmetry or reversals in orientation of migration. Strikingly, for most species studied, handedness of migration is highly biased but not fixed. Thus, whereas the final cell fate pattern of P11/<em>12</em> is invariant, the developmental route leading to it is subject both to developmental indeterminacy and to evolutionary variations.

Beta-catenin is a key mediator in the canonical <em>Wnt</em> signaling pathway, which plays important roles in multiple developmental processes. Inappropriate activation of this pathway leads to developmental defects and development of certain cancers. Upon <em>Wnt</em> signaling, beta-catenin binds TCF/LEF transcription factors. The TCF/LEF-beta-catenin complex then recruits a variety of transcriptional coactivators to the promoter/enhancer region of <em>Wnt</em>-responsive genes and activates target gene transcription. In this article, we demonstrate that GRIP1-associated coactivator 63 (GAC63), a recently identified nuclear receptor (NR) coactivator, interacts with beta-catenin. The N-terminus of GAC63 is the binding site for beta-catenin, whereas a C-terminal fragment of beta-catenin including armadillo repeats 10-<em>12</em> binds to GAC63. Over-expression of GAC63 enhanced the transcriptional activity of beta-catenin, and also greatly enhanced TCF/LEF-regulated reporter gene activity in a beta-catenin-dependent manner. Endogenous GAC63 was recruited to TCF/LEF-responsive enhancer elements when beta-catenin levels were induced by LiCl. In addition, reduction of endogenous GAC63 level by small interfering RNA (siRNA) inhibited TCF/LEF-mediated gene transcription. Our findings reveal a new function of GAC63 in transcriptional activation of <em>Wnt</em>-responsive genes.

In Hodgkin lymphoma (HL) we recently identified deregulated expression of homeobox genes MSX1 and OTX2 which are physiologically involved in development of the embryonal neural plate border region. Here, we examined in HL homeobox gene SIX1 an additional regulator of this embryonal region mediating differentiation of placodal precursors. SIX1 was aberrantly activated in <em>12</em> % of HL patient samples in silico, indicating a pathological role in a subset of this B-cell malignancy. In addition, SIX1 expression was detected in HL cell lines which were used as models to reveal upstream factors and target genes of this basic developmental regulator. We detected increased copy numbers of the SIX1 locus at chromosome 14q23 correlating with enhanced expression while chromosomal translocations were absent. Moreover, comparative expression profiling data and pertinent gene modulation experiments indicated that the <em>WNT</em>-signalling pathway and transcription factor MEF2C regulate SIX1 expression. Genes encoding the transcription factors GATA2, GATA3, MSX1 and SPIB - all basic lymphoid regulators - were identified as targets of SIX1 in HL. In addition, cofactors EYA1 and TLE4, respectively, contrastingly mediated activation and suppression of SIX1 target gene expression. Thus, the protein domain interfaces may represent therapeutic targets in SIX1-positive HL subsets. Collectively, our data reveal a gene regulatory network with SIX1 centrally deregulating lymphoid differentiation and support concordance of lymphopoiesis/lymphomagenesis and developmental processes in the neural plate border region.

Rat embryonic d 14 (E14) mesencephalic cells, 2.5% of which are glioblasts, were incubated in medium containing 10% of fetal bovine serum for <em>12</em> h and subsequently expanded in a serum-free medium using basic fibroblast growth factor (bFGF) as the mitogen. On a single occasion, after more than 15 d in culture, several islets of proliferating, glial-like cells were observed in one dish. The cells, when isolated and passaged, proliferated rapidly in either a serum-free or serum-containing growth medium. Subsequent immunocytochemical analysis showed that they stained positive for GFAP and vimentin, and negative for A2B5, O4, GalC, and MAP2. Serum-free conditioned medium (CM) prepared from these cells caused a fivefold increase in survival and promoted neuritic expansion of E14 mesencephalic dopaminergic neurons in culture. These actions are similar to those exerted by CM derived from primary, mesencephalic type-1 astrocytes. The pattern of expression of the region-selective genes; <em>wnt</em>-1, en-1, sis showed that 70% of the cells were heteroploid, and of these, 50% were tetraploid. No apparent decline in proliferative capacity has been observed after 25 passages. The properties of this cell line, named ventral mesencephalic cell line one (VMCL1), are consistent with those of an immortalized, type-1 astrocyte. The mesencephalic origin of the cell line, and the pattern and potency of the neurotrophic activity exerted by the CM, strongly suggest that the neurotrophic factor(s) identified are novel, and will likely be strong candidates with clinical utility for the treatment of Parkinson's disease.

BACKGROUND

Activating mutations in exon 3 of the β-catenin gene are involved in the pathogenesis of adamantinomatous craniopharyngiomas. Recently, the interaction between β-catenin and PROP1 has been shown to be responsible for pituitary cell lineage determination. We hypothesized that dysregulated PROP1 expression could also be involved in the pathogenesis of craniopharyngiomas

OBJECTIVE

To determine whether dysregulated gene expression was responsible for tumor pathogenesis in adamantinomatous craniopharyngiomas, the β-catenin gene was screened for mutations, and the expression of the β-catenin gene and PROP1 was evaluated. β-catenin gene was amplified and sequenced from 14 samples of adamantinomatous craniopharyngiomas. PROP1 and β-catenin gene expression was assessed by real-time RT-PCR from <em>12</em> samples, and β-catenin immunohistochemistry was performed on 11 samples.

RESULTS

Mutations in the β-catenin gene were identified in 64% of the adamantinomatous craniopharyngiomas samples. Evidence of β-catenin gene overexpression was found in 71% of the tumors with β-catenin mutations and in 40% of the tumors without mutations, and β-catenin immunohistochemistry revealed a nuclear staining pattern for each of the analyzed samples. PROP1 expression was undetectable in all of the tumor samples.

CONCLUSIONS

We found evidence of β-catenin gene overexpression in the majority of adamantinomatous craniopharyngiomas, and we also detected a nuclear β-catenin staining pattern regardless of the presence of a β-catenin gene mutation. These results suggest that WNT signaling activation plays an important role in the pathogenesis of adamantinomatous craniopharyngiomas. Additionally, this study was the first to evaluate PROP1 expression in adamantinomatous craniopharyngiomas, and the absence of PROP1 expression indicates that this gene is not involved in the pathogenesis of this tumor, at least in this cohort.

The long non-coding RNAs (lncRNAs) regulating encoding transcripts/genes involved in <em>Wnt</em> signalling pathway in keloids is largely unclear. We used a pathway-focused lncRNA microarray to detect the differentiated expression profiles of both lncRNAs and genes involved in <em>Wnt</em> pathway, thus a total of 116 <em>Wnt</em>-targeted genes and 69 <em>Wnt</em>-related lncRNAs aberrantly expressed in keloids were initially identified. A stepwise bioinformatics was further performed to find skin-related lncRNA/gene pairs in <em>Wnt</em> pathway in keloids. Firstly, an lncRNA/gene co-expression network with clustered functional modules was constructed; simultaneously, 114 <em>Wnt</em>-genes regarding to dermis were online enriched using Phenotype Enrichment. Secondly, 17 skin-related keloid-aberrant <em>Wnt</em>-genes were acquired by overlapping the 114 skin-related <em>Wnt</em>-genes with the 116 keloid-aberrant <em>Wnt</em>-genes. Thirdly, after co-expression coefficient of each lncRNA/gene profile being ranked respectively, 11 top co-expressed lncRNAs characterized with the highest co-expression coefficients to the 17 genes were identified. Fourthly, seven of the 11 top co-expressed lncRNAs exhibiting array-detected aberrant expression in keloids, together with their <em>12</em> most interactive <em>Wnt</em>-genes, were selected to undergo in-pair intracellularly quantitative PCR validation in keloids. As a result, four lncRNAs including CACNA1G-AS1, HOXA11-AS, LINC003<em>12</em> and RP11-91I11.1 with their six paired <em>Wnt</em>-genes undergoing both array-and-qPCR as well as lncRNA-and-gene double validation were finally identified as skin-related lncRNA/gene pairs that involved in <em>Wnt</em> signalling pathway in keloids. In conclusion, in-depth exploration on these easily-accessible lncRNAs in keloids might aid to find the novel target on how to maintain highly recurrent tumours benign via <em>Wnt</em>-involved network regulation.

The stemness hypothesis states that all stem cells use common mechanisms to regulate self-renewal and multi-lineage potential. However, gene expression meta-analyses at the single gene level have failed to identify a significant number of genes selectively expressed by a broad range of stem cell types. We hypothesized that stemness may be regulated by modules of homologs. While the expression of any single gene within a module may vary from one stem cell type to the next, it is possible that the expression of the module as a whole is required so that the expression of different, yet functionally-synonymous, homologs is needed in different stem cells. Thus, we developed a computational method to test for stem cell-specific gene expression patterns from a comprehensive collection of 49 murine datasets covering <em>12</em> different stem cell types. We identified 40 individual genes and 224 stemness modules with reproducible and specific up-regulation across multiple stem cell types. The stemness modules included families regulating chromatin remodeling, DNA repair, and <em>Wnt</em> signaling. Strikingly, the majority of modules represent evolutionarily related homologs. Moreover, a score based on the discovered modules could accurately distinguish stem cell-like populations from other cell types in both normal and cancer tissues. This scoring system revealed that both mouse and human metastatic populations exhibit higher stemness indices than non-metastatic populations, providing further evidence for a stem cell-driven component underlying the transformation to metastatic disease.

S100A4 promotes cancer metastasis, but its overall status and splicing manner during gastrocarcinogenesis remains less known. We therefore examined S100A4 frequencies, splicing pattern(s) and the underlying reason(s) for S100A4 expression in gastric cancers. Immunohistochemistry revealed frequent S100A4 expression in intestinal gastric cancers (37/45; 82%) and diffuse gastric cancers (<em>12</em>/20; 60%), but uncommon in noncancerous epithelia (0/<em>12</em>), chronic gastritis (2/24; 8%), and intestinal metaplasia (3/15; 20%). Of 65 primary tumors, 18 were found with focal S100A4 expression, while their LN metastases showed homogenous distribution. S100A4-oriented reverse transcription-polymerase chain reaction yielded a transcript containing exons 1, 3, and 4 (AS1) in 20% of noncancerous, 84% premalignant, and 92% tumor tissues and a transcript harboring exons 1 to 4 (AS2) in 65% of gastric cancers (GCs), 26% premalignant but none in noncancerous tissues. Further analyses found AS1 expression in stromal but not epithelial cells of premalignant tissues, absence of AS2 in endoscopic inflammatory mucosa, and the coexistence of AS1/AS2 in the cultured fibroblasts. Methylation DNA sequencing revealed hypermethylation of four critical CpG sites within S100A4 intron first among S100A4-negative gastric tissues and hypomethylation in S100A4-expressing GC tissues/cell lines. E-cadherin reduction and <em>Wnt</em> activation were common in gastric cancers, which were closely correlated but unnecessarily overlapped with S100A4 expression. Our findings suggest that S100A4 expression is closely related with GC formation, which, as a hypomethylation event, is accompanied with E-cadherin reduction and <em>Wnt</em> activation. The preferential S100A4 AS2 expression in GC cells would have potential values in GC surveillance and prognostic assessment.

Radiotherapy for malignant pelvic disease is commonly accompanied by treatment-induced proctitis, and rarely by colorectal cancer. Translocation of the beta-catenin protein, which is a key downstream effector of the <em>Wnt</em> signal transduction pathway, is frequently found in colorectal cancer. Nuclear beta-catenin enhances transcriptional activity of the cyclin D1 gene in cancer cells. Here, we evaluate the involvement of the <em>Wnt</em> pathway in radiation-induced colon carcinogenesis with rats (n = 36). Beta-catenin, APC, and cyclin D1 expression profiles were analyzed by immunohistochemistry in radiation-induced chronic colon injury including cancers and ulcerative lesions in rats (n = <em>12</em> in treated group, n = <em>12</em> in control group). In total, 3 cases of invasive adenocarcinomas were developed in the irradiated portion 50 weeks after a single dose of 36 Gy irradiation. Nuclear translocation of beta-catenin was observed in all radiation-induced colon cancers, whereas this protein was also found in the cytoplasm and/or nucleus of 9 cases of non-neoplastic irradiated colonocytes. Nuclear translocation of beta-catenin correlated with loss of APC and gain of cyclin D1 expression, suggesting activation of the <em>Wnt</em> pathway during radiation-induced colorectal carcinogenesis. A single dose of 10 Gy was also given for acute injury (n = <em>12</em>: 3 each in days 0, 3, 5, and 7, respectively). Beta-catenin expression was distributed in the cytoplasm of degenerating glands at day 3 and 5, and was observed in the cell membrane of those glands with histological normalization at day 7 after irradiation. Because translocation of beta-catenin was found in irradiated-colonic mucosa as well as colon cancer, disruption of beta-catenin expression might be one of the early events in radiation-induced colonic carcinogenesis.

BACKGROUND

We have indicated previously that long-term feeding of beef tallow increases colorectal cancer in rats. In this study, we investigated the effects of conjugated linoleic acid (CLA) on colon carcinogenesis in rats under long-term feeding of beef tallow diets, pretreated with azoxymethane (AOM).

METHODS

Six-week-old male Sprague-Dawley rats were fed with 10% beef tallow diet only, 10% beef tallow with 1% CLA in triglyceride form (CLA-TG), or 10% beef tallow with 1% CLA in free fatty acid form (CLA-FFA). Colon carcinogenesis was induced by two intraperitoneal injections of AOM. Aberrant crypt foci (ACFs) were examined at <em>12</em> weeks. Cancer, cell proliferation, apoptosis, <em>Wnt</em> signaling, and the arachidonic acid cascade were examined at 44 weeks.

RESULTS

At <em>12</em> weeks, CLA-TG and CLA-FFA attenuated the increase in ACFs induced by 10% beef tallow and AOM pretreatment. At 44 weeks, both forms of CLA attenuated multiple colon cancers, and CLA-FFA reduced the incidence of colon cancer to 50% of that seen with CLA-TG. CLA-TG and CLA-FFA decreased the number of 5-bromo-2'-deoxyuridine-positive cells in AOM-pretreated rats fed with 10% beef tallow. CLA-FFA increased the number of apoptotic cells and the activity of caspase-3 in the colon mucosa, and CLA-TG enhanced the activity of caspase-3. Both forms of CLA suppressed <em>Wnt</em> signaling and the arachidonic acid cascade in rats treated with beef tallow and AOM.

CONCLUSIONS

These results suggested that CLA-TG and CLA-FFA suppressed colon carcinogenesis in rats with long-term feeding of a 10% beef tallow diet, through several mechanisms. The results of the present study with rats might be applicable to humans.

OBJECTIVE

Solid pseudopapillary tumor (SPT) of the pancreas is very rare. This study was performed to analyze the expression of Wnt signal target genes (matrix metalloproteinase-7 [MMP-7], cyclin-D1, and c-myc) and Ki-67 in resected SPTs to determine their clinicopathologic characteristics according to their expression.

METHODS

From January 1995 to December 2005, 23 patients underwent pancreatic resections for SPT of the pancreas. Among 23 formalin-fixed, paraffin-embedded tissues, 12 were evaluated as a pilot study. Immunohistochemistry was performed using various detection and antigen retrieval methods to detect MMP-7, cyclin-D1, c-myc, and Ki-67. The expression of Wnt target genes was correlated with clinicopathologic features of the patients.

RESULTS

Solid pseudopapillary tumors of the pancreas always showed cytoplasmic/nuclear accumulation of [beta]-catenin, frequent expression of cyclin-D1, and low proliferation index. MMP-7, cyclin-D1, c-myc, and Ki-67 were not correlated with microscopic features suggesting malignant potential (P>> 0.05). Tumor size was closely related to microscopic features of malignant potential and apparently has an inverse relationship with the expression of cyclin-D1 and Ki-67 (P < 0.05).

CONCLUSIONS

Low proliferative index and associated MMP-7 expression may cause an unpredictable clinical course in this tumor. Subtle changes in the intracellular environment, not pathologic (morphologic) changes, may elucidate the unpredictable clinical course of this tumor.