BACKGROUND

Cancer of unknown primary (CUP) possesses distinct biology and peculiar natural history, in which the roles of the winged and hedgehog signalling pathways are unclear.

METHODS

We constructed tissue microarrays and studied the immunohistochemical (IHC) expression of β-catenin, smoothened (SMO) and the transcription factors TCF, LEF, GLI1 in 87 CUP cases for prognostic significance.

RESULTS

A low rate of IHC expression of proteins was seen, the cut-off used being any expression in ≥ 1% of tumour cells. At univariate analysis, only nuclear IHC SMO expression displayed a statistically significant association with favourable outcome [median Overall survival (OS) of 19 months in SMO-positive vs. <em>12</em> months in SMO-negative cases, P = 0·01]. An activated <em>Wnt</em> pathway, defined as IHC expression of any of nuclear β-catenin, TCF and LEF, was significantly associated with favourable progression free survival (median 9 vs. 5 months, P = 0·037) and OS (median 19 vs. 13 months, P = 0·04). This prognostic impact on OS was mainly driven by nuclear expression of TCF and/or LEF (P = 0·03). No prognostic significance of the hedgehog pathway activation status, defined as IHC expression of SMO or nuclear GLI1, could be established. A favourable prognostic impact of the concurrent activation of both pathways was observed. A trend for association of activated <em>Wnt</em> with response to chemotherapy (responders 67% among activated <em>Wnt</em> cases vs. 35% among nonactivated <em>Wnt</em> cases, P = 0·07) was observed in CUP adenocarcinomas.

CONCLUSIONS

Activation of the Wnt pathway was a positive prognostic factor in a small CUP series, possibly via enhanced chemosensitivity. Independent validation is warranted.

BACKGROUND

Age, pre-existing renal osteodistrophy, impaired renal function, and chronic use of immunosuppressive drugs are the main factors involved in the onset and development of bone metabolism disturbances and skeletal alterations occurring after renal transplantation. However, at the state of the art, no reports have analyzed the additional post-menopausal physiological mechanisms associated with the onset and development of bone complications in renal transplant recipients.

METHODS

We measured by means of molecular strategies (enzyme-linked immunoassay, chemiluminescence) the serum levels of Sclerostin and Dickkopf-1 (DKK1), two major antagonists of the <em>Wnt</em>/β-catenin pathway, and several bone resorption/formation biomarkers (N-terminal procollagen type 1, bone-specific alkaline phosphatase, and serum C-terminal telopeptides of type I collagen) in 19 post-menopausal kidney transplant patients and <em>12</em> post-menopausal chronic kidney disease patients (CKD group) matched for age and renal function.

RESULTS

Our results showed that the levels of both Wnt antagonists were similar in the two study groups (P=.15 and .96, respectively). Additionally, no correlation was found between Sclerostin and DKK1 serum levels in all patients included in the study (R2=0.03, P=.2). After statistical analysis, we found no differences in the bone resorption/formation biomarkers between renal transplant and CKD patients. Multivariate analysis showed that Sclerostin levels were significantly positively correlated with serum phosphorus levels (P=.008) and inversely correlated with renal function (P=.026). Surprisingly, no significant correlation was found between all the analyzed demographic and clinical parameters and DKK1.

CONCLUSIONS

Our study demonstrated for the first time that renal transplantation per se and immunosuppressive treatments do not represent additional factors contributing to bone metabolic/biochemical alterations in post-menopausal women. However, our results emphasized that a better preservation of the graft function could significantly slow down the progression of bone metabolic deregulations and prevent clinical bone complications.

OBJECTIVE

To determine the effects of interleukin (IL)-6 and IL-1β stimulation on expression of growth differentiation factor (GDF)-5 and Wnt signaling pathway genes in equine chondrocytes.

METHODS

Macroscopically normal articular cartilage samples from 6 horses and osteochondral fragments (OCFs) from 3 horses.

METHODS

Chondrocyte pellets were prepared and cultured without stimulation or following stimulation with IL-6 or IL-1β for 1, 2, 12, and 48 hours; expression of GDF-5 was determined with a quantitative real-time PCR assay. Expression of genes in various signaling pathways was determined with microarrays for pellets stimulated for 1 and 2 hours. Immunohistochemical analysis was used to detect GDF-5, glycogen synthase kinase 3β (GSK-3β), and β-catenin proteins in macroscopically normal cartilage samples and OCFs.

RESULTS

Chondrocytes stimulated with IL-6 had significantly higher GDF-5 expression within 2 hours versus unstimulated chondrocytes. Microarray analysis of Wnt signaling pathway genes indicated expression of GSK-3β and coiled-coil domain containing 88C increased after 1 hour and expression of β-catenin decreased after 2 hours of IL-6 stimulation. Results of immunohistochemical detection of proteins were similar to microarray analysis results. Chondrocytes in macroscopically normal articular cartilage and OCFs had immunostaining for GDF-5.

CONCLUSIONS

Results indicated IL-6 stimulation decreased chondrocyte expression of the canonical Wnt signaling pathway transactivator β-catenin, induced expression of inhibitors of the Wnt pathway, and increased expression of GDF-5. This suggested IL-6 may inhibit the Wnt signaling pathway with subsequent upregulation of GDF-5 expression. Anabolic extracellular matrix metabolism in OCFs may be attributable to GDF-5 expression. This information could be useful for development of cartilage repair methods.

OBJECTIVE

To investigate the role of beta-catenin as a key element of Wnt/beta-catenin signaling pathway in the development of embryonic liver and liver tumorigenesis.

METHODS

Immunohistochemical method was used to examined beta-catenin proteins in 12-day (E12), 16-day (E16) embryonic rat liver, neonate rat liver, and in adult rat liver as well as in rat hepatoma. Beta-catenin mRNA was amplified in the above-mentioned samples by means of semiquantitative RT-PCR.

RESULTS

Beta-catenin proteins were found in cytoplasm of E12 and E16 embryonic liver as well as in hepatoma. The expression level of beta-catenin protein in E12 embryonic liver was higher than that in E16. There were much more positive cells in E12 embryonic liver than in E16. However, no positive cell was observed expressing beta-catenin proteins in neonate rat liver and adult rat liver. The quantity of beta-catenin mRNA was the same in all samples.

CONCLUSIONS

Wnt/beta-catenin signaling pathway was open during the development of embryonic liver and hepatic tumorigenesis. The accumulation of beta-catenin proteins in cytoplasm of embryonic liver and rat hepatic carcinoma cells might be caused not by the elevation of transcription of beta-catenin gene but by the avoidance of degeneration of beta-catenin protein.

Infant crying is a series of innate vocal patterns intended to elicit the attention of adult caregivers for fulfillment of specific needs such as pain, hunger, or hypostimulation. It is one of the earliest forms of observable communication. In neonatal rodents, this behavior has recently been investigated as a potential early behavioral marker of neural deficits in neurodevelopmental disorders. However, few studies have examined the effects of seizures on vocalization behavior during the neonatal period. The purpose of this study is to investigate the effect of a single kainate-induced early life seizure on vocalization behavior in mice. This study also investigates the subsequent effect of seizures on two pathways critical for early neural development and epileptogenesis: the phosphoinositide 3-kinase|serine/threonine kinase|mammalian target of rapamycin (PI3K-Akt-mTOR) and canonical (Wingless-Int Wnt) intracellular signaling pathways.

On postnatal day 10, male and female 129SvEvTac mice received a single intraperitoneal injection of kainic acid (2.5 mg/kg) or vehicle injection. The kainate administration resulted in 1-2 h of status epilepticus. On postnatal days 11 and 12, the quantity and duration of isolation-induced ultrasonic vocalizations were recorded. Western blotting analyses were performed using male and female pups on postnatal day 12.

There was significant, male-specific suppression in the quantity and total duration of 50-kHz calls on postnatal day 12 following seizures. The hippocampi of male mice on this postnatal day also revealed male-specific changes in the PI3K-Akt-mTOR intracellular signaling pathway, as well as changes in phosphorylated fragile × mental retardation protein.

These findings demonstrate that early life seizures can disrupt communication behavior in neonatal mice.

Avian pathogenic Escherichia coli (APEC) causes one of the most common bacterial diseases of poultry worldwide. Effective control methods are therefore desirable and will be facilitated by a better understanding of the host response to the pathogen. Currently, microRNAs (miRNAs) involved in host resistance to APEC are unknown. Here, we applied RNA sequencing to explore the changed miRNAs and deregulated genes in the spleen of three groups of broilers: nonchallenged (NC), APEC-challenged with mild pathology (CM), and APEC-challenged with severe pathology (CS). Twenty-seven differentially expressed miRNAs (fold change >1.5; P value <0.01) were identified, including 13 miRNAs between the NC and CM, 17 between the NC and CS, and 14 between the CM and CS groups. Through functional analysis of these miRNA targets, <em>12</em> immune-related biological processes were found to be significantly enriched. Based on combined analyses of differentially expressed miRNAs and mRNAs within each of the three groups, 43 miRNA-mRNA pairs displayed significantly negative correlations (r < -0.8). Notably, gga-miR-429 was greatly increased in the CS group compared to levels in both the CM and NC groups. In vitro, gga-miR-429 directly repressed luciferase reporter gene activity via binding to 3' untranslated regions of TMEFF2, NTRK2, and SHISA2. Overexpression of gga-miR-429 in the HD11 macrophage cell line significantly inhibited TMEFF2 and SHISA2 expression, which are involved in the lipopolysaccharide-induced platelet-derived growth factor (PDGF) and <em>Wnt</em> signaling pathways. In summary, we provide the first report characterizing the miRNA changes during APEC infection, which may help to shed light on the roles of these recently identified genetic elements in the mechanisms of host resistance and susceptibility to APEC.

The <em>Wnt</em> genes encode a set of conserved glycoproteins regulating early development, cell proliferation and differentiation, and tissue regeneration in metazoans. In some mollusks, the knowledge of <em>Wnt</em> gene family has been limited because of the short of the genomic and transcriptomic resources. Ruditapes philippinarum is an economically important bivalve with a variety of shell coloration patterns and ability to regenerate its siphon. To gain a greater understanding of the evolutionary dynamics of <em>Wnt</em> gene family, we carried out a genome-wide identification and phylogenetic analysis of <em>Wnt</em> gene family in R. philippinarum and other four mollusks. A total of <em>12</em> <em>Wnt</em> genes were identified in the genome of R. philippinarum, and the dynamic patterns of gene conservation, loss and duplication of <em>Wnt</em> genes were analyzed in mollusks and model organisms. Furthermore, the transcriptome analyses demonstrated the expression profiles of the <em>Wnt</em> genes at different developmental stage, in adult tissues, during siphon regeneration, in four different shell color strains, and at uncolored and colored developmental stages in two different shell color strains. These findings suggest that the expansion of <em>Wnt</em> genes may play vital roles in the larval development, the formation of shell color pattern and siphon regeneration in R. philippinarum. This study provides a valuable insight into <em>Wnt</em> function and evolution in mollusks.

Keywords: Expression profiling; Mollusk; Phylogenetic analysis; Ruditapes philippinarum; Wnt gene family.

<AbstractText>Icariin (IC) promotes osteogenic differentiation, and it may be a potential small molecule drug for local application in bone regeneration. Icariin-loaded hydroxyapatite/alginate (IC/HAA) porous composite scaffolds were designed in this study for the potential application of the sustainable release of icariin and subsequent bone regeneration.</AbstractText><AbstractText>An icariin-loaded hydroxyapatite/alginate porous composite scaffold was prepared and characterized by SEM and HPLC for morphology and release behavior, respectively. The mechanical properties, degradation in PBS and cytotoxicity on BMSCs were also evaluated by MTT assay, compression strength and calculation of weight remaining ratio, respectively. Rabbit BMSCs were cocultured with IC/HAA scaffolds, and ALP activity and Alizarin Red staining were performed to evaluate osteogenic differentiation induction. The mRNA and protein expression level of an osteogenic gene was detected by RT-PCR and Western blotting, respectively. In vivo animal models of critical bone defects in the radius of rabbit were used. Four and <em>12</em> weeks after the implantation of IC/HAA scaffolds in the bone defect, radiographic images of the radius were obtained and scored by using the Lane and Sandhu X-ray scoring system. Tissue samples were also evaluated using H&E and Masson staining, and an osteogenic gene and <em>Wnt</em> signaling pathway genes were detected.</AbstractText><AbstractText>A hydroxyapatite/alginate (HAA) porous composite scaffold-loaded icariin was fabricated using a freeze-drying method. Our data indicated that the icariin was loaded in alginate scaffold without compromising the macro/microstructure or mechanical properties of the scaffold. Notably, the IC/HAA promoted the proliferation of rBMSCs without exerting cytotoxicity on rBMSCs. In vivo, rabbit radius bone defect experiments demonstrated that the IC/HAA scaffold exhibited better capacity for bone regeneration than HAA, and IC/HAA upregulated the relative expression levels of an osteogenic gene and the <em>Wnt</em> signaling pathway genes. Most notably, the IC/HAA scaffold also inhibited osteoclast activity in vivo.</AbstractText><AbstractText>Our data suggests a promising application for the use of HAA scaffolds to load icariin and promote bone regeneration in situ through mediation of the coupling processes of osteogenesis induction and osteoclast activity inhibition.</AbstractText>

Histone deacetylase 8 (HDAC8) is a class 1 histone deacetylase and a member of the cohesin complex. HDAC8 is expressed in smooth muscles, but its expression in skeletal muscle has not been described. We have shown for the first time that HDAC8 is expressed in human and zebrafish skeletal muscles. Using RD/<em>12</em> and RD/18 rhabdomyosarcoma cells with low and high differentiation potency, respectively, we highlighted a specific correlation with HDAC8 expression and an advanced stage of muscle differentiation. We inhibited HDAC8 activity through a specific PCI-34051 inhibitor in murine C2C<em>12</em> myoblasts and zebrafish embryos, and we observed skeletal muscles differentiation impairment. We also found a positive regulation of the canonical <em>Wnt</em> signaling by HDAC8 that might explain muscle differentiation defects. These findings suggest a novel mechanism through which HDAC8 expression, in a specific time window of skeletal muscle development, positively regulates canonical <em>Wnt</em> pathway that is necessary for muscle differentiation.

Zinc oxide nanoparticles (ZnO NPs) are increasingly used in various products as coating and additive materials for household goods, personal-care products, and drug delivery systems. Because of their broad applications, the potential risks to nontarget organisms associated with their input into aquatic environments have generated much concern. We investigated the acute toxicity, morphological responses, and potential impact on physiology and metabolism in polyps exposed to spherical ZnO NPs of either 20 nm (ZnO NP20) or 100 nm (ZnO NP100). The median lethal concentrations (LC50) of ZnO NP20 were 55.3, 8.7, and 7.0 μg/mL after exposure for 48, 72, and 96 h, respectively; and those of ZnO NP100 were 262.0, 14.9, and 9.9 μg/mL, respectively. The morphological responses of the hydra polyps to a range of ZnO NP concentrations suggest that ZnO NPs may negatively affect neurotransmission in Hydra. ZnO NPs may also induce abnormal regeneration in the polyps by affecting the expression of several genes related to the <em>Wnt</em> signaling pathway. The presence of ZnO NP20 in the hydra tissue was confirmed with electron microscopy. A Gene Ontology analysis of the genes differentially expressed in hydra polyps after exposure to ZnO NP20 for <em>12</em> or 24 h revealed changes in various processes, including cellular and metabolic process, stress response, developmental process, and signaling. A KEGG pathway analysis of hydra polyps after exposure of ZnO NP20 or ZnO NP100 for <em>12</em> or 24 h demonstrated various changes, including in the DNA replication and repair, endocytosis, lysosomes, <em>Wnt</em> signaling, and natural killer-cell-mediated cytotoxicity pathways, suggesting the mechanisms that maintain cellular homeostasis in response to ZnO NPs. Progesterone-mediated oocyte maturation was also affected by the ZnO NPs nanoparticles, suggesting that they are potential endocrine disruptors. This study should increase our concern regarding the dispersal of ZnO NPs in aquatic environments.

Endoplasmic reticulum (ER) stress and the related apoptosis and inflammation damage play key roles in osteoarthritis development. The aim of the present work was to investigate the exact role and potential underlying mechanism of pyruvate kinase M2 (PKM2) in rat chondrocytes exposed to interleukin-Iβ (IL-1β). We observed that IL-1β stimulation resulted in an apparent enhancement in PKM2 expression. Additionally, loss of PKM2 evidently ascended cell viability in response to IL-1β exposure. Simultaneously, elimination of PKM2 manifestly repressed IL-1β-stimulated chondrocyte apoptosis, concomitant with attenuated in the proapoptotic protein markers Bax and cleaved caspase-3, and elevated the antiapoptotic protein Bcl-2. In the meanwhile, knockdown of PKM2 ameliorated ER stress in IL-1β-treated chondrocytes, as evidenced by reduced expression of the ER stress-associated proteins GRP78, CHOP, and cleaved caspase-<em>12</em>. Furthermore, PKM2 silencing protected chondrocytes against IL-1β-triggered inflammatory response, as reflected by the downregulated release of proinflammatory mediators, including tumor necrosis factor-α, IL-6, inducible nitric oxide synthase, cyclooxygenase-2, and prostaglandin E2, as well as decreased nitric oxide generation. More important, abrogating PKM2 expression caused a marked decline in Rspo2 expression, and subsequently blocked <em>Wnt</em>/β-catenin signaling. Mechanistically, the <em>Wnt</em>/β-catenin signaling activator Licl effectively impeded the beneficial effects of PKM2 ablation on IL-1β-stimulated apoptosis and inflammatory response. These findings collectively implicated that PKM2 inhibition protected against ER stress-mediated cell apoptosis and inflammatory injury in rat chondrocytes stimulated with IL-1β by inactivating Rspo2-mediated <em>Wnt</em>/β-catenin pathway, and may represented a novel therapeutic target for osteoarthritis.

Numerous genetic targets and some individual pathways associated with aging have been identified using the worm model. However, less is known about the genetic mechanisms of aging in genome wide, particularly at the level of multiple pathways as well as the regulatory networks during aging. Here, we employed the gene expression datasets of three time points during aging in Caenorhabditis elegans (C. elegans) and performed the approach of gene set enrichment analysis (GSEA) on each dataset between adjacent stages. As a result, multiple genetic pathways and targets were identified as significantly down- or up-regulated. Among them, 5 truly aging-dependent signaling pathways including MAPK signaling pathway, mTOR signaling pathway, <em>Wnt</em> signaling pathway, TGF-beta signaling pathway and ErbB signaling pathway as well as <em>12</em> significantly associated genes were identified with dynamic expression pattern during aging. On the other hand, the continued declines in the regulation of several metabolic pathways have been demonstrated to display age-related changes. Furthermore, the reconstructed regulatory networks based on three of aging related Chromatin immunoprecipitation experiments followed by sequencing (ChIP-seq) datasets and the expression matrices of 154 involved genes in above signaling pathways provide new insights into aging at the multiple pathways level. The combination of multiple genetic pathways and targets needs to be taken into consideration in future studies of aging, in which the dynamic regulation would be uncovered.

The ubiquitin ligase ring finger protein 5 (<i>RNF5</i>) has previously been associated with the development of breast cancer. Patients with breast cancer and high <i>RNF5</i> expression have been demonstrated to have a shorter survival time compared with patients with low <i>RNF5</i> expression. However, the role of RNF5 in human glioma has not been determined. The present study analyzed the role of RNF5 in gliomas using bioinformatics analysis. The results revealed that RNF5 was differentially expressed in non-cancerous brain tissues and different grades of glioma. Furthermore, a high <i>RNF5</i> expression in patients with glioma was associated with an improved prognosis compared with patients with low expression. Gene Set Enrichment Analysis revealed that <i>RNF5</i> was particularly associated with '<em>Wnt</em> signaling pathway', 'apoptosis', 'focal adhesion' and 'cytokine-cytokine receptor interaction' in patients with glioma. Additionally, 4 potential ubiquitination substrates for <i>RNF5</i> were predicted, including sorting nexin 10, proprotein convertase subtilisin/kexin type 1, leucine rich glioma inactivated 1 and solute carrier family 39 member <em>12</em>. These findings provided the basis for further investigation on the role of <i>RNF5</i> in tumors.

Dkk-3 is a member of the dickkopf protein family of secreted inhibitors of the <em>Wnt</em> pathway, which has been shown to enhance angiogenesis. The mechanism underlying this effect is currently unknown. Here, we used cultured HUVECs to study the involvement of the TGF-β and VEGF on the angiogenic effect of Dkk-3. Addition of hrDkk-3 peptide (1 or 10 ng/ml) to HUVECs for 6 or <em>12</em> h enhanced the intracellular and extracellular VEGF protein levels, as assessed by RTPCR, immunoblotting, immunocytochemistry and ELISA. The increase in the extracellular VEGF levels was associated to the VEGFR2 activation. Pharmacological blockade of VEGFR2 abrogated Dkk-3-induced endothelial cell tubes formation, indicating that VEGF is a molecular player of the angiogenic effects of Dkk-3. Moreover, Dkk-3 enhanced Smad1/5/8 phosphorylation and recruited Smad4 to the VEGF gene promoter, suggesting that Dkk-3 activated ALK1 receptor leading to a transcriptional activation of VEGF. This mechanism was instrumental to the increased VEGF expression and endothelial cell tubes formation mediated by Dkk-3, because both effects were abolished by siRNA-mediated ALK1 knockdown. In summary, we have found that Dkk-3 activates ALK1 to stimulate VEGF production and induce angiogenesis in HUVECs.

BACKGROUND

Activation of the <em>WNT</em>/β-catenin pathway has emerged as a potential therapeutic target in androgenetic alopecia (AGA). Methyl vanillate (MV) - a safe plant-derived ingredient - has been recently shown to activate the <em>WNT</em>/β-catenin signaling. Objectives Two distinct substudies were conducted. First, we designed a 6-month, uncontrolled, open-label clinical study to investigate whether topically applied MV may increase hair count and hair mass index (HMI) in female AGA. Second, we conducted a molecular study on the effect of MV on <em>WNT</em>10B mRNA expression in scalp biopsies of women with AGA.

METHODS

A total of 20 Caucasian women (age range: 25-57 years) with AGA (Sinclair grade 1-2) were included. The research product was an alcohol-free formulation supplied in the form of a spray containing 0.2% MV as the active ingredient.

RESULTS

In the clinical study, hair count and HMI were found to increase at 6 months by 6% (P < 0.01) and 12% (P < 0.001), respectively, compared with baseline. No participant discontinued treatment due to adverse effects, and the overall patient satisfaction was good. At the molecular level, the topical application of the research product resulted in a 32% increase in <em>WNT</em>10B mRNA expression levels in the temporal scalp area (P < 0.001).

CONCLUSIONS

Our pilot data suggest that topical MV can increase hair count and HMI by inducing <em>WNT</em>10B expression in the scalp, potentially serving as a novel treatment strategy for female AGA.

MLL rearrangements were analysed in the blood of a patient receiving chemotherapy for diffuse large B-cell lymphoma using inverse polymerase chain reaction targeting exon <em>12</em>, parallel sequencing and a custom algorithm design. Of thirteen MLL rearrangements detected, five were capable of generating MLL fusion genes, including MLL-MLLT3, the most common fusion in acute myeloid leukaemia (AML). Other fusions, all previously clinically unobserved, included MLL-NKD1, a fusion to the negative regulator of <em>Wnt</em>/β-catenin signaling, a pathway linked to leukaemic cell proliferation. The majority of the fusions exhibited clonal persistence from before treatment until 6 months post-chemotherapy, suggesting the fusions may confer a survival advantage to the mutant clone. MLL breakpoints were partly clustered at a specific location, indicating commonality in the process of their formation. Further, the same MLL breakpoint location exhibited a 50-100-fold increase in C to T transitions, consistent with attack by activation-induced cytidine deaminase (AICDA). As is also observed in AML and acute lymphoblastic leukaemia, in this single patient setting, MLL is capable of interacting with multiple fusion partners. This finding defines a discrete site of MLL susceptibility to fragmentation, linked to possible deregulation of AICDA function.

Diabetic neuropathy pain (DNP), a spontaneous pain with hyperalgesia and allodynia, greatly compromises patients' quality of life. Our previous study suggested that dexmedetomidine (DEX) can relieve hyperalgesia in rats by inhibiting inflammation and apoptosis at the level of the spinal cord. In the present study, we aimed to evaluate the role of <em>Wnt</em> 10a/<i>β</i>-catenin signaling in DEX-induced alleviation of DNP in rats. Forty-eight rats were randomly allocated to four groups (n=<em>12</em>/group): control, DNP, DEX, and yohimbine groups. The DNP model was established by streptozotocin (STZ) injection. The effects of DEX with or without the <i>α</i>₂ adrenergic antagonist yohimbine were assessed by behavior tests (mechanical withdrawal threshold and thermal withdrawal latency). Spinal cord tissue was evaluated by immunofluorescence staining of astrocytes as well as for <em>Wnt</em> 10a and <i>β</i>-catenin expression, western blot analysis of <em>Wnt</em> 10a and <i>β</i>-catenin expression, and enzyme-linked immunosorbent assay measurement of proinflammatory cytokines (tumor necrosis factor-<i>α</i> and interleukin-1<i>β</i>). Rats with STZ-induced DNP had a decreased pain threshold, activated astrocytes, increased expression of <em>Wnt</em> 10a and <i>β</i>-catenin, and increased levels of proinflammatory cytokines compared to the control group, and these effects were ameliorated by treatment with DEX. Yohimbine administration partly abolished the protective effects of DEX in the DNP model rats. In conclusion, DEX alleviated DNP in rats by inhibiting inflammation and astrocyte activation, which may be attributed to downregulation of the <em>Wnt</em> 10a/<i>β</i>-catenin signaling pathway.

Estrogen receptor alpha (ESR1) is 1 of the 2 intracellular receptors for estrogen and is expressed by hepatocytes in the liver. The role of ESR1 in the regulation of toxicant-induced liver injury and compensatory regeneration is not completely clear. We investigated the role of ESR1 in liver regeneration after carbon tetrachloride (CCl4)-induced liver injury using wild type (WT) and ESR1 knockout (ESR1-KO) rats. Adult female WT and ESR1-KO rats were treated with 1 mL/kg CCl4 and euthanized over a time course of 0 to 48 hours. Liver injury measured by serum alanine amino transaminase, and histopathological analysis showed significantly higher liver injury in ESR1-KO as compared to WT rats. Hematoxylin and eosin staining revealed 2-fold higher necrosis and significant inflammatory cell infiltration in ESR1-KO rats. Chloracetate esterase staining revealed higher neutrophil infiltration in ESR1-KO rat livers. Interestingly, proliferating cell nuclear antigen immunohistochemistry showed that in spite of 2-fold higher liver injury, the ESR1-KO rats had equal liver regeneration as compared to WT rats. Western blot analysis of cyclin D1 and phosphorylated Rb, proteins involved in the initiation of the cell cycle, was significantly higher at all time points in ESR1-KO rats. Further analysis revealed faster activation of canonical <em>Wnt</em>/β-catenin and NF-κB signaling in ESR1-KO rats characterized by higher activated β-catenin and phosphorylated p65 at <em>12</em> hours after CCl4 treatment. Taken together, these data indicate that ESR1-mediated signaling inhibits liver regeneration by downregulation of <em>Wnt</em> signaling resulting in lower cyclin D1 activation after chemical-induced liver injury.

In the present study we investigated the phosphorylation status of the <em>12</em> most important signaling cascades in glioblastomas. More than 60 tumor and control biopsies from tumor center and periphery (based on neuronavigation) were subjected to selective protein expression analysis using reverse-phase protein arrays (RPPA) incubated with antibodies against posttranslationally modified cancer pathway proteins. The ratio between phosphorylated (or modified) and non-phosphorylated protein was assessed. All samples were histopathologically validated and proteomic profiles correlated with clinical and survival data. By RPPA, we identified three distinct activation patterns within glioblastoma defined by the ratios of pCREB1/CREB1, NOTCH-ICD/NOTCH1, and pGSK3β/GSK3β, respectively. These subclasses demonstrated distinct overall survival patterns in a cohort of patients from a single-institution and in an analysis of publicly available data. In particular, a high pGSK3β/GSK3β-ratio was associated with a poor survival. <em>Wnt</em>-activation/GSK3β-inhibition in U373 and U251 cell lines halted glioma cell proliferation and migration. Gene expression analysis was used as an internal quality control of baseline proteomic data. The protein expression and phosphorylation had a higher resolution, resulting in a better class-subdivision than mRNA based stratification data. Patients with different proteomic profiles from multiple biopsies showed a worse overall survival. The CREB1-, NOTCH1-, GSK3β-phosphorylation status correlated with glioma grades. RPPA represent a fast and reliable tool to supplement morphological diagnosis with pathway-specific information in individual tumors. These data can be exploited for molecular stratification and possible combinatorial treatment planning. Further, our results may optimize current glioma grading algorithms.

The testis development-related protein-1 (TDRP1) gene was first isolated in spermatogenic cells of testis, and was related to the spermatogenesis and reproduction traits in mammals. This study was performed to further analyze the function of the pig TDRP1 gene by microarray screening of the TDRP1-overexpressed swine testis (ST) cell. After transfection of the recombinant pcDNA3.1-TDRP1 vector into the ST cell, the expression of the TDRP1 gene continued to increase at <em>12</em>, 24, and 36 h post-transfection, and then decreased at 48 h, as indicated by both the fluorescence signal and quantitative PCR. Microarray screening revealed 529 upregulated and 1086 downregulated genes in TDRP1-overexpressed ST cells at 24 h post-transfection (TD-24) versus untransfected control (TD-blank), and 764 upregulated and 858 downregulated genes at 36 h post-transfection (TD-36) versus TD-blank, as well as 720 upregulated and 375 downregulated genes in TD-36 versus TD-24. As far as three contrasts were totally considered, 29 common genes were identified, including 17 upregulated genes and <em>12</em> downregulated ones. Further, Kyoto encyclopedia of genes and genomes prediction analysis indicated that some of these 29 differently expressed genes were involved in a number of biological pathways. Among the 17 upregulated genes, the PMAIP1 gene was significantly enriched in the p53 signaling pathway, and the others were the DDIT3 gene in the mitogen-activated protein kinases (MAPK) signaling pathway, the PPP1R3C gene in the insulin signaling pathway, as well as the GADD45A gene in the p53 signaling pathway, MAPK signaling pathway, and the cell cycle. Among the <em>12</em> downregulated genes, the SFRP4 gene was significantly enriched in the <em>Wnt</em> signaling pathway. This study indicated that the TDRP1 gene regulated PMAIP1, GADD45A, DDIT3, and PPP1R3C expression, and these genes involving p53, MAPK, insulin, and <em>Wnt</em> signaling pathways are related to spermatogenesis or reproduction in pig.

Breast cancer is the leading cause of cancer-related death in the world, and it is of great value to reveal the molecular mechanisms of breast cancer progression and develop new therapeutic targets.

Transwell assay is used to analyze the migration and invasion of breast cancer cells. Real-time PCR and western blotting assay are applied to detect the expression levels of epithelial-mesenchymal transition markers and the key members of Wnt/β-catenin and PI3K/AKT signaling pathways.

Manganese-12 acetate (Mn12Ac) significantly inhibited the migration and invasion of MCF7 and MDA-MB-231 breast cancer cells. Western blotting assay further showed that Mn12Ac significantly upregulated E-cadherin, and downregulated N-cadherin and vimentin. We further found that Mn12Ac reduced the mRNA expressions of epithelial-mesenchymal transition-associated transcription factors snail, slug, twist1, and ZEB1 using real-time PCR assay. Importantly, we further found that Mn12Ac significantly reduced the <em>Wnt</em>1 and β-catenin protein expressions, and suppressed the phosphorylation of PI3K and AKT in MCF7 and MDA-MB-231 breast cancer cells. Very interestingly, we also showed that Mn12Ac decreased the mRNA and protein expressions of programmed cell death ligand 1.

Taken together, our results suggested that Mn12Ac inhibited the migration, invasion, and epithelial-mesenchymal transition by regulating Wnt/β-catenin and PI3K/AKT signaling pathways in breast cancer.

β-Catenin is a bifunctional protein related to cell adhesion and gene transcription when activated by <em>Wnt</em> pathway. Altered expression of β-catenin was related to loss of differentiation, more aggressive phenotype, increase of tumor invasion, and poor prognosis in a number of different cancers. Actinic cheilitis is caused by excessive exposure to ultraviolet radiation and has a high potential to suffer malignant transformation into squamous cell carcinoma (SCC) of the lip, the most frequent oral malignancy. Studies of oral cancer have shown the correlation of β-catenin expression and oral SCC prognosis, and loss of membrane expression may be considered as a potential marker for early tumor recurrence. Thirty-five cases of actinic cheilitis and <em>12</em> cases of SCC of the lip were select and submitted to immunohistochemical staining using β-catenin antibody. β-Catenin was positive on the membrane for all cases. Eighty-five percent of actinic cheilitis cases showed cytoplasmatic staining, and 22% nuclear staining. Eighty-three percent of SCC was positive for β-catenin, and none of them had nuclear staining. Cytoplasmatic and nuclear staining of β-catenin on studied cases point to pathway alterations. Results demonstrated that β-catenin expression is altered on epithelial dysplasia, and it is related to degree of alterations.

A wait and see approach for desmoid tumors (DT) has become part of the routine treatment strategy. However, predictive factors to select the risk of progressive disease are still lacking. A translational project was run in order to identify genomic signatures in patients enrolled within an Italian prospective observational study. Among <em>12</em> DT patients (10 CTNNB1-mutated and 2 wild type) enrolled from our institution only two patients (17%) showed a progressive disease. Tumor biopsies were collected for whole exome sequencing. Overall, DT exhibited low somatic sequence mutation rate and no additional recurrent mutation was found. In the two wild type (WT) cases, two novel alterations were detected: a complex deletion of APC and a pathogenic mutation of LAMTOR2. Focusing on WT DT subtype, deep sequencing of CTNNB1, APC and LAMTOR2 was conducted on a retrospective series of 11 WT DT using a targeted approach. No other mutation of LAMTOR2 was detected, while APC was mutated in two cases. Low-frequency (mean reads of 16%) CTNNB1 mutations were discovered in five samples (45%) and two novel intra-genic deletions in CTNNB1 were detected in two cases. Both deletions and low frequency mutations of CTNNB1 were highly expressed. In conclusion, a minority of DT is WT for either CTNNB1, APC or any other gene involved in the <em>WNT</em> pathway. In this subgroup novel and hard to be detected molecular alterations in APC and CTNNB1 were discovered, contributing to explain a portion of the allegedly WT DT cases.

As a common antagonist of <em>Wnt</em>/β-catenin signaling, <em>Wnt</em> inhibitory factor 1 (WIF1) plays an important role in the tumor progression. The aim of our meta-analysis was to summarize the diagnostic value of WIF1 methylation in colorectal cancer (CRC). Eligible studies were retrieved by a systemic search among PubMed, Embase, CNKI, and Wanfang literature databases. The diagnostic value of WIF1 methylation for CRC was assessed by the summary receiver operating characteristics (SROC) test. Our meta-analysis of <em>12</em> studies between 1420 CRC samples and 946 control samples showed that WIF1 hypermethylation was significantly associated with CRC (P < 0.001, OR = 30.10, 95% CI = 19.48-46.50). WIF1 hypermethylation, as a diagnostic biomarker for CRC, has a pooled sensitivity of 0.40 (95% CI: 0.37-0.42), a pooled specificity of 0.95 (95% CI: 0.93-0.96), a pooled positive-likelihood ratio (PLR) of 8.65 (95% CI, 4.47-16.73), and a pooled negative-likelihood ratio (NLR) of 0.41 (95% CI, 0.30-0.55), a diagnostic odds ratio (DOR) of 26.86 (95% CI: 15.73-45.89), and an area under the curve (AUC) of 0.9115. In conclusion, our study established that WIF1 hypermethylation might be a promising diagnostic biomarker for CRC.