MEK inhibitors have limited efficacy in treating RAS-RAF-MEK pathway-dependent cancers due to feedback pathway compensation and dose-limiting toxicities. Combining MEK inhibitors with other targeted agents may enhance efficacy. Here, codependencies of MEK, TAK1, and KRAS in colon cancer were investigated. Combined inhibition of MEK and TAK1 potentiates apoptosis in KRAS-dependent cells. Pharmacologic studies and cell-cycle analyses on a large panel of colon cancer cell lines demonstrate that MEK/TAK1 inhibition induces cell death, as assessed by sub-G1 accumulation, in a distinct subset of cell lines. Furthermore, TAK1 inhibition causes G2-M cell-cycle blockade and polyploidy in many of the cell lines. MEK plus TAK1 inhibition causes reduced G2-M/polyploid cell numbers and additive cytotoxic effects in KRAS/TAK1-dependent cell lines as well as a subset of BRAF-mutant cells. Mechanistically, sensitivity to MEK/TAK1 inhibition can be conferred by KRAS and BMP receptor activation, which promote expression of NF-κB-dependent proinflammatory cytokines, driving tumor cell survival and proliferation. MEK/TAK1 inhibition causes reduced mTOR, Wnt, and NF-κB signaling in TAK1/MEK-dependent cell lines concomitant with apoptosis. A Wnt/NF-κB transcriptional signature was derived that stratifies primary tumors into three major subtypes: Wnt-high/NF-κB-low, Wnt-low/NF-κB-high and Wnt-high/NF-κB-high, designated W, N, and WN, respectively. These subtypes have distinct characteristics, including enrichment for BRAF mutations with serrated carcinoma histology in the N subtype. Both N and WN subtypes bear molecular hallmarks of MEK and TAK1 dependency seen in cell lines. Therefore, N and WN subtype signatures could be utilized to identify tumors that are most sensitive to anti-MEK/TAK1 therapeutics.

This study describes a potential therapeutic strategy for a subset of colon cancers that are dependent on oncogenic KRAS signaling pathways, which are currently difficult to block with selective agents. Mol Cancer Res; 14(12); 1204-16. ©2016 AACR.

Alzheimer's disease (AD) is a disorder characterised by a progressive deterioration in memory and other cognitive functions. Neurofibrillary tangles (NFT) are a major pathological hallmark of AD, these are aggregations of paired helical filaments (PHF) comprised of the hyperphosphorylated microtubule associated protein tau. Several kinases, such as glycogen synthase kinase 3 beta (GSK3beta) and c-Jun N-terminal kinase (JNK), phosphorylate tau at sites that are phosphorylated in PHF. Dishevelled 1 (DVL1) is thought to act as a positive regulator of the <em>wnt</em> signalling pathway, and inhibits GSK3beta activity preventing beta-catenin degradation and thus allowing <em>wnt</em> target gene expression. JNK activation is also regulated by DVL1, however it is unclear if this is via the <em>wnt</em> signalling pathway. These observations suggest a central role for DVL1 in tau phosphorylation and AD and led us to investigate DVL1 as a candidate gene for this disorder. We determined the genomic structure of the DVL1 gene by sequencing and data mining and searched for sequence variations in the coding sequences and flanking introns. The DVL1 gene spans a region of approximately 13.8 kb (not including the 5' untranslated region) and is encoded by 15 exons. Analysis of over 4.3 kb of sequence, including 98% of exonic sequences and introns 2, 3, 6, 7, 9, 10, 11 and <em>12</em>, revealed there to be six rare (< or =6%) sequence variations. None of these had any association with late onset AD. This would suggest that polymorphic variations in the coding sequences of DVL1 are not important in AD. However further analysis of regulatory regions may lead to the identification of other sequence variations which may be implicated in AD.

Adrenocortical carcinoma is a rare cancer, but one that carries a poor prognosis due to its aggressive nature and unresponsiveness to conventional chemotherapeutic strategies. Over the past <em>12</em> years, there has been renewed interest in developing new therapies for this cancer, including identifying key signaling nodes responsible for cell proliferation.

Clinical trials of tyrosine kinase inhibitors as monotherapy have generally been disappointing, although the identification of exceptional responders may lead to the identification of targeted therapies that may produce responses in subsets of patients. Agents targeted to the Wnt signaling pathway, a known player in adrenal carcinogenesis, have been developed, although they have not yet been used specifically for adrenal cancer. There is current excitement about inhibitors of acetyl-coA cholesterol acetyl transferase 1, an enzyme required for intracellular cholesterol handling, although trials are still underway. Tools to target other proteins such as Steroidogenic Factor 1 and mechanistic target of rapamycin have been developed and are moving towards clinical application.

Progress is being made in the fight against adrenocortical carcinoma with the identification of new therapeutic targets and new means by which to attack them. Continued improvement in the prognosis for patients with adrenal cancer is expected as this research continues.

Most current treatments for osteoporosis inhibit bone resorption and reduce total fracture numbers by about one-quarter. The identification of the osteocytic protein sclerostin as a potent regulator of bone turnover and bone density has led to the development of a new therapeutic class-agents that inhibit sclerostin activity, resulting in increased bone formation and reduced bone resorption. Romosozumab and blosozumab are monoclonal antibodies that bind to sclerostin, reducing its inhibition of <em>Wnt</em> signaling. They have comparable activities in phase I and II studies, doubling formation markers, halving resorption indices, and increasing spine bone density by >10% over <em>12</em> months. Only romosozumab has progressed to phase III, where the first study showed a 73% reduction in vertebral fracture risk and a 36% reduction in clinical fractures at 1 year. It was well-tolerated. A further phase III study will conclude in 2017. Romosozumab is a very promising medication in the management of established osteoporosis, but much remains to be done to determine its optimal duration and sequence of administration.

The molecular pathogenesis of cemento ossifying fibroma (COF) is unclear. The purpose of this study was to investigate mutations in 50 oncogenes and tumor suppressor genes, including APC and CTNNB1, in which mutations in COF have been previously reported. In addition, we assessed the transcriptional levels of the Wnt/β-catenin pathway genes in COF.

We used a quantitative polymerase chain reaction array to evaluate the transcriptional levels of 44 Wnt/β-catenin pathway genes in 6 COF samples, in comparison with 6 samples of healthy jaws. By using next-generation sequencing (NGS) in 7 COF samples, we investigated approximately 2800 mutations in 50 genes.

The expression assay revealed 12 differentially expressed Wnt/β-catenin pathway genes in COF, including the upregulation of CTNNB1, TCF7, NKD1, and WNT5 A, and downregulation of CTNNBIP1, FRZB, FZD6, RHOU, SFRP4, WNT10 A, WNT3 A, and WNT4, suggesting activation of the Wnt/β-catenin signaling pathway. NGS revealed 5 single nucleotide variants: TP53 (rs1042522), PIK3 CA (rs2230461), MET (rs33917957), KIT (rs3822214), and APC (rs33974176), but none of them was pathogenic.

Although NGS detected no oncogenic mutation, deregulation of key Wnt/β-catenin signaling pathway genes appears to be relevant to the molecular pathogenesis of COF.

This study was undertaken to determine the frequency, and the clinicopathologic and genetic features, of colon cancers driven by neurotrophic receptor tyrosine kinase (NTRK) gene fusions. Of the 7008 tumors screened for NTRK expression using a pan-Trk antibody, 16 (0.23%) had Trk immunoreactivity. ArcherDx assay detected TPM3-NTRK1 (n=9), LMNA-NTRK1 (n=3), TPR-NTRK1 (n=2) and EML4-NTRK3 (n=1) fusion transcripts in 15 cases with sufficient RNA quality. Patients were predominantly women (median age: 63 y). The tumors involved the right (n=<em>12</em>) and left colon unequally and were either stage T3 (n=<em>12</em>) or T4. Local lymph node and distant metastases were seen at presentation in 6 and 1 patients, respectively. Lymphovascular invasion was present in all cases. Histologically, tumors showed moderate to poor (n=11) differentiation with a partly or entirely solid pattern (n=5) and mucinous component (n=10), including 1 case with sheets of signet ring cells. DNA mismatch repair-deficient phenotype was seen in 13 cases. Tumor-infiltrating CD4/CD8 lymphocytes were prominent in 9 cases. Programmed death-ligand 1 positive tumor-infiltrating immune cells and focal tumor cell positivity were seen in the majority of cases. CDX2 expression and loss of CK20 and MUC2 expression were frequent. CK7 was expressed in 5 cases. No mutations in BRAF, RAS, and PIK3CA were identified. However, other genes of the PI3K-AKT/MTOR pathway were mutated. In several cases, components of <em>Wnt</em>/β-catenin (APC, AMER1, CTNNB1), p53, and TGFβ (ACVR2A, TGFBR2) pathways were mutated. However, no SMAD4 mutations were found. Two tumors harbored FBXW7 tumor suppressor gene mutations. NTRK fusion tumors constitute a distinct but rare subgroup of colorectal carcinomas.

Stimulating bone formation is an important challenge for bone anabolism in osteoporotic patients or to repair bone defects. The osteogenic properties of matrix glycosaminoglycans (GAGs) have been explored; however, the functions of GAGs at the surface of bone-forming cells are less documented. Syndecan-2 is a membrane heparan sulfate proteoglycan that is associated with osteoblastic differentiation. We used a transgenic mouse model with high syndecan-2 expression in osteoblasts to enrich the bone surface with cellular GAGs. Bone mass was increased in these transgenic mice. Syndecan-2 overexpression reduced the expression of receptor activator of NF-kB ligand (RANKL) in bone marrow cells and strongly inhibited bone resorption. Osteoblast activity was not modified in the transgenic mice, but bone formation was decreased in 4-month-old transgenic mice because of reduced osteoblast number. Increased proteoglycan expression at the bone surface resulted in decreased osteoblastic and osteoclastic precursors in bone marrow. Indeed, syndecan-2 overexpression increased apoptosis of mesenchymal precursors within the bone marrow. However, syndecan-2 specifically promoted the vasculature characterized by high expression of CD31 and Endomucin in 6-week-old transgenic mice, but this was reduced in <em>12</em>-week-old transgenic mice. Finally, syndecan-2 functions as an inhibitor of <em>Wnt</em>-β-catenin-T-cell factor signaling pathway, activating glycogen synthase kinase 3 and then decreasing the <em>Wnt</em>-dependent production of <em>Wnt</em> ligands and R-spondin. In conclusion, our results show that GAG supply may improve osteogenesis, but also interfere with the crosstalk between the bone surface and marrow cells, altering the supporting function of osteoblasts.

<em>12</em>-O-Tetradecanoylphorbol-13-acetate (TPA) is the most widely used diacylglycerol (DAG) mimetic agent and inducer of protein kinase C (PKC)-mediated cellular response in biomedical studies. TPA has been proposed as a pluripotent cell differentiation factor, but results obtained have been inconsistent. In the present study we show that TPA can be applied as a cardiomyogenesis-promoting factor for the differentiation of mouse embryonic stem (mES) cells in vitro. The mechanism of TPA action is mediated by the induction of extracellular signal-regulated kinase (ERK) activity and the subsequent phosphorylation of GATA4 transcription factor. Interestingly, general mitogens (FGF, EGF, VEGF and serum) or canonical <em>WNT</em> signalling did not mimic the effect of TPA. Moreover, on the basis of our results, we postulate that a TPA-sensitive population of cardiac progenitor cells exists at a certain time point (after days 6-8 of the differentiation protocol) and that the proposed treatment can be used to increase the multiplication of ES cell-derived cardiomyocytes.

<AbstractText>To study the influence of micro ribonucleic acid (miR)-155 on depression-like behaviors of depression mice, and to explore the role of <em>Wnt</em>/b-catenin signaling pathway in behavioral regulation of depression mice.</AbstractText><AbstractText>The mouse model of depression was established via chronic unpredictable mild stress (CUMS). All mice were randomly divided into control group (n=<em>12</em>), model group (n=<em>12</em>), and fluoxetine group (n=<em>12</em>). The expression level of miR-155 in the hippocampus of mice in each group was detected via quantitative Polymerase Chain Reaction (qPCR). The changes in the behaviors of mice in each group were evaluated via behavioral experiments. The apoptosis level in the hippocampus of mice in each group was detected via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Moreover, the content of inflammatory factors in the hippocampus of mice in each group was detected using the enzyme-linked immunosorbent assay (ELISA) kits. The expression levels of <em>Wnt</em>/b-catenin signaling pathway-related proteins in each group were detected via Western blotting.</AbstractText><AbstractText>The expression level of miR-155 in the hippocampus was significantly higher in model group than that in control group (p<0.01). Meanwhile, the expression level of miR-155 was significantly lower in fluoxetine group than that in model group (p<0.01). There were no statistically significant differences in the crossing score and rearing score in the open field test among groups (p>0.05). Compared with those in control group, the immobility time in tail suspension test and forced swimming test were significantly increased (p<0.01), while the sucrose preference degree significantly declined (p<0.01) in model group. Fluoxetine could significantly reduce the immobility time in tail suspension test and forced swimming test (p<0.01) and increase the sucrose preference degree (p<0.01) in model group. The number of TUNEL-positive cells in the hippocampus of mice in model group was significantly larger than that in control group (p<0.01). Fluoxetine could effectively reduce the number of TUNEL-positive cells in the hippocampus (p<0.01). Compared with those in control group, the content of tumor necrosis factor-α (TNF-a), interleukin-1b (IL-1b), and IL-6 in the hippocampus was significantly increased (p<0.01), while the content of IL-10 was significantly decreased (p<0.01) in model group. Fluoxetine could effectively reduce the content of TNF-a, IL-1b, and IL-6 (p<0.01) and increase the content of IL-10 (p<0.01). Besides, in model group, the expression levels of dishevelled-1 (DVL-1) and b-catenin in hippocampus remarkably declined (p<0.01), while the expression levels of glycogen synthase kinase-3b (GSK-3b) and adenomatous polyposis coli (APC) were remarkably increased (p<0.01) compared with those in control group. Fluoxetine could effectively lower the expressions of GSK-3b and APC in the hippocampus (p<0.01) and increase the expressions of DVL-1 and b-catenin (p<0.01) in model group.</AbstractText><AbstractText>MiR-155 is involved in regulating the depression-like behaviors of depression mice through promoting the release of inflammatory factors and the apoptosis of hippocampal neurons. Its mechanism may be related to the inhibition of the <em>Wnt</em>/b-catenin signaling pathway.</AbstractText>

Background: Colorectal cancer (CRC) is a common malignant tumor with unsatisfactory overall prognosis. CircRNAs could be promising prognostic biomarkers in cancers, and play important role in the process of tumorigenesis and progression. Here, we explored the role of hsa_circ_0004831 in blood extracellular vesicles and its prognostic value in CRC.

Methods: The circRNA and mRNA expression level matrix in extracellular vesicles of CRC and normal samples were obtained from the exoRBase database. The corresponding miRNA expression level matrix in extracellular vesicles was downloaded from the BBCancer database. Differentially expressed circRNAs, miRNAs and mRNAs were identified using the limma package of R software at the cut-off criteria of fold change (FC) > 2 and adj. p < 0.05. RT-qPCR assay was conducted to measure hsa_circ_0004831 expression level in CRC blood samples. A circRNA-miRNA-mRNA regulatory network of hsa_circ_0004831 was constructed based on competitive endogenous RNA mechanism and differentially expressed genes. The mRNAs co-expressed with hsa_circ_0004831 were screened at the cut-off criteria of pearson |r| > 0.3 and p < 0.05. Gene set enrichment analysis (GSEA) based on co-expressed mRNAs was used to explore the potential molecular function of hsa_circ_0004831.

<strong class="sub-title"> Results: </strong> Differentially expressed circRNAs, miRNAs and mRNAs were identified and hsa_circ_0004831 had a FC value of 3.92 in CRC blood extracellular vesicles. The RT-qPCR assay showed that the hsa_circ_0004831 was up-regulated in CRC blood samples. The overall survival analysis found that high expression of hsa_circ_0004831 was linked with poorer prognosis. Finally, a circRNA-miRNA-mRNA regulatory network of hsa_circ_0004831 was constructed based on down-regulated miR-4326 and <em>12</em> up-regulated mRNAs. GSEA indicated that mRNAs co-expressed with hsa_circ_0004831 were involved in EMT, <em>WNT</em> and p53 signaling pathways.

Conclusions: The study confirmed the up-regulation of hsa_circ_0004831 in CRC, and it may act as a vital prognostic biomarker. The circRNA-miRNA-mRNA regulatory network of hsa_circ_0004831 could be used to uncover the tumorigenesis and progression of CRC.

Keywords: CircRNA; Colorectal cancer; Competitive endogenous RNA; Extracellular vesicle; Prognosis.

β-Catenin stabilization achieved either via GSK-3β specific inhibition or involving canonical <em>Wnt</em> signalling pathway, contributes to neuroprotection in an oxygen-glucose deprivation (4h OGD) in vitro hypoxia model performed on human cortical neural progenitor cells previously differentiated into neurons and glia. Neuroprotection mechanisms include both acquiring tolerance to injury throughout preconditioning (72 h prior to OGD) or being pro-survival during 24h reoxygenation after the insult. Four hours of OGD induced apoptotic cell death elevation to 73 ± 1% vs. <em>12</em>% measured in control and the LDH level, indicative of necrotic cell injury, elevation by 67 ± 7% (set to 100%). A significant reduction in apoptosis occurred at 24h reoxygenation with indirubin supplement which was 49 ± 6% at 2.5 μM BIO while LDH level was only 47 ± 5% of OGD. Kenpaullone was efficient in reducing both cell deaths at 5 μM (apoptosis 38 ± 1% and necrosis 33 ± 3% less than in OGD). <em>Wnt</em> agonist reduced apoptosis to 45 ± 3% at 0.01 μM, while LDH value was decreased to a level of 53 ± 5% of control. Our findings suggest that GSK-3beta inhibitors/β-catenin stabilizers may ultimately be useful drugs in neuroprotection and neuroregeneration therapies in vivo.

Self-renewal and multipotent differentiation are cardinal properties of intestinal stem cells (ISCs), mediated in part by <em>WNT</em> and NOTCH signaling. Although these pathways are well characterized, the molecular mechanisms that control the 'stemness' of ISCs are still not well defined. Here, we investigated the role of Krüppel-like factor 5 (KLF5) in regulating ISC functions METHODS: We performed studies in adult Lgr5^{EGFP-IRES-creERT2};Rosa26^LSLtdTomato (Lgr5^Ctrl) and Lgr5^{EGFP-IRES-creERT2};Klf5^fl/fl;Rosa26^LSLtdTomato (Lgr5^ΔKlf5) mice. Mice were injected with tamoxifen to activate Cre recombinase, which deletes Klf5 from the intestinal epithelium in Lgr5^ΔKlf5 but not Lgr5^Crtl mice. In experiments involving irradiation, mice were subjected to <em>12</em> Gy total body irradiation (TBI). Tissues were collected for immunofluorescence (IF) analysis and next generation sequencing. Oganoids were derived from fluoresecence activated cell sorted- (FACS-) single cells from tamoxifen-treated Lgr5^ΔKlf5 or Lgr5^Crtl mice and examined by immunofluorescence stain RESULTS: Lgr5⁺ ISCs lacking KLF5 proliferate faster than control ISCs but fail to self-renew, resulting in a depleted ISC compartment. Transcriptome analysis revealed that Klf5-null Lgr5⁺ cells lose ISC identity and prematurely differentiate. Following irradiation injury, which depletes Lgr5⁺ ISCs, reserve Klf5-null progenitor cells fail to dedifferentiate and regenerate the epithelium. Absence of KLF5 inactivates numerous selected enhancer elements and direct transcriptional targets including canonical <em>WNT</em>-and NOTCH-responsive genes. Analysis of human intestinal tissues showed increased levels of KLF5 in the regenerating epithelium as compared to those of healthy controls CONCLUSION: We conclude that ISC self-renewal, lineage specification, and precursor dedifferentiation require KLF5, by its ability to regulate epigenetic and transcriptional activities of ISC-specific gene sets. These findings have the potential for modulating ISC functions by targeting KLF5 in the intestinal epithelium.

Bladder cancer is one of the most common malignant diseases in the urinary system, with poor survival after metastasis. Activation-induced cytidine deaminase (AID), a versatile enzyme involved in antibody diversification, is an oncogenic gene that induces somatic hypermutation and class-switch recombination (CSR). However, the contribution of AID-mediated DNA demethylation to bladder urothelial cell carcinoma (BUCC) remains unclear. Herein, we evaluated the impact on BUCC caused by AID and explored the gene network downstream of AID by using a proteomic approach. Lentiviral vector containing AID-specific shRNA significantly reduced AID expression in T24 and 5637 cells. Silencing AID expression remarkably inhibited tumour malignancies, including cell proliferation, invasion and migration. We used Isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics analysis technology to study the underpinning mechanism in monoclonal T24 cells, with or without AID knockdown. Among the 6452 proteins identified, 99 and 142 proteins in shAICDA-T24 cells were significantly up- or downregulated, respectively (1.2-fold change) compared with the NC-T24 control. After a pipeline of bioinformatics analyses, we identified three tumour-associated factors, namely, matrix metallopeptidase 14 (MMP14), C-X-C motif chemokine ligand <em>12</em> and wntless <em>Wnt</em> ligand secretion mediator, which were further confirmed in human BUCC tissues. Nonetheless, only MMP14 was sensitive to the DNA demethylation molecule 5-aza-2'-deoxycytidine (5-azadC; 5 μM), which reversed the inhibition of carcinogenesis by AID silence in T24 and 5637 cells. Overall, AID is an oncogene that mediates tumourigenesis via DNA demethylation. Our findings provide novel insights into the clinical treatment for BUCC.

Background: The recombinant fusion protein ipafricept (IPA) blocks Wnt signaling, and in combination with gemcitabine (G) and nab-paclitaxel (Nab-P) caused tumor regression in xenografts. This phase 1b study evaluated the combination of IPA with Nab-P+G in untreated metastatic PDAC (mPDAC) patients.

Methods: Dose escalation started with standard dose Nab-P+G and IPA (3.5 mg/kg days 1, 15). Due to fragility fractures seen with different anti-Wnt agents, following cohorts had >=6 patients treated with IPA 3 - 5 mg/kg on day 1, and included bone marker monitoring and prophylactic bisphosphonates as indicated. Based on pre-clinical data sequential dosing was evaluated in cohort 4 (IPA day 1 followed Nab-P+G day 3). Objectives included safety, MTD, RP2D, pharmacokinetics, immunogenicity, pharmacodynamics, and efficacy.

Results: 26 patients were enrolled, 5 in the cohort 1 and 7 each in cohorts 2-4. IPA-related AEs of any grade included fatigue, nausea, vomiting, anorexia and pyrexia. IPA-related AEs >=grade3 included 2 events of AST elevation, and 1 each of nausea, rash, vomiting and leucopenia. No DLTs or fragility fractures were observed. Nine patients (34.6%) had PR, 12 (46.2%) SD as best response, with clinical benefit rate of 81%. Median PFS was 5.9m (95%CI 3.4-18.4), median OS was 9.7m (95%CI: 7.0-14). The study was terminated by the sponsor due to bone-related toxicity within this therapeutic program and concerns for commercial viability. One patient remains on therapy under compassionate use.

Conclusions: IPA can be administered with Nab-P+G with reasonable tolerance. Wnt pathway remains a therapeutic target of interest in mPDAC.

Osteosarcoma (OS) is a common malignant primary bone tumor. Its mechanism of development and progression is poorly understood. Currently, there is no effective therapeutic regimens available for the treatment of OS. DEAD-box helicase 5 (DDX5) is involved in oncogenic processes. This study aimed to explore the role of DDX5 in the development and progression of OS and its relationship with transcription factor <em>12</em> (TCF<em>12</em>), which is as an important molecule of <em>Wnt</em> signaling pathway. We found that the expressions of DDX5 and TCF<em>12</em> protein were significantly higher in OS patients tissues and in the MG63 cells than in the corresponding normal tissues and human osteoblast cell hFOB 1.19. Overexpressions of both DDX5 and TCF<em>12</em> were associated with clinicopathological features and poor prognosis of OS patients. siRNA based knockdown of DDX5 inhibited the proliferation of MG63 cells as demonstrated by an <i>in vitro</i> MTS assay and 5-ethynyl-2-deoxyuridine DNA proliferation detection, and promoted apoptosis of MG63 cells measured by flow cytometry. In addition, DDX5 knockdown inhibited the MG63 cell migration and invasion on transwell assays. Further experiments showed that DDX5 knockdown not only inhibited the expression of TCF<em>12</em> but also decreased the mRNA and protein levels of Cyclin E1, an important regulator of G1-S phase progression, suggesting that DDX5 was required for the entry of cells into S phase. Overexpression of TCF<em>12</em> reversed the cell proliferation, migration and invasion in MG63 cells induced by DDX5 knockdown accompanied by the upregulation of Cyclin E1. Additionally, we observed that DDX5 interacted with TCF<em>12</em> in both OS tissues and MG63 cells by Co-immunoprecipitation assays. Taken together, our study revealed that DDX5 interacts with TCF<em>12</em> and promotes the progression of OS by stimulating cell cycle progression. Our results suggest that DDX5 and TCF<em>12</em> could be potential biomarkers for the diagnosis and treatment of OS.

BACKGROUND

The Wnt signaling pathway plays crucial roles in embryonic skeletal development and postnatal bone regeneration. However, mechanisms of Wnt signaling functioning in distraction osteogenesis (DO) haven't been well characterized.

METHODS

We established a DO model using Sprague-Dawley rat tibia. And a Wnt signaling blocking agent, recombinant rat Dickkopf-related protein 1 (rrDkk1), was locally applied in the distracted gap to study the role of Wnt signaling during DO process. Animals in the experimental group received rrDkk1 injections (dose = 25 μg/kg) once daily during distraction period and every third day during consolidation stage (n = 48). Animals in the control group received saline under the same injection strategy (n = 48). Animals at different time points during DO process (1, 3, 6, 12 days after distraction, 10 days and 6 weeks after consolidation) were killed and tissues in the distraction region were harvested for radiography, dual energy X-ray absorptiometry, micro-computed tomography (micro-CT), and histological analyses.

RESULTS

Most Wnt ligands, cofactors, receptors, and antagonists were widely expressed in the distraction callus and were significantly upregulated during DO process. After rrDkk1 administration, the majority of these factors were downregulated at the mRNA level, except sFRP and GSK-3β. At the protein level, both β-catenin and Lef-1 were also suppressed by rrDkk1. In the long term, restricted bone healing was observed in the distracted callus in the rrDkk1 injection group. These findings were confirmed by histological and micro-CT analyses.

CONCLUSIONS

Our findings suggest that Wnt signaling participates in the process of DO, and clinical therapeutic approaches of DO may do well to avoid Wnt pathway suppression.

<em>Wnt</em> signaling regulates multiple aspects of vertebrate central nervous system (CNS) development, including neurogenesis. However, vertebrate genomes can contain up to 25 <em>Wnt</em> genes, the functions of which are poorly characterized partly due to redundancy in their expression. To identify candidate <em>Wnt</em> genes as candidate mediators of pathway activity in specific brain progenitor zones, we have performed a comprehensive expression analysis at three different stages during zebrafish development. Antisense RNA probes for 21 <em>Wnt</em> genes were generated from existing and newly synthesized cDNA clones and used for in situ hybridization on whole embryos and dissected brains. As in other species, we found that <em>Wnt</em> expression patterns in the embryonic zebrafish CNS are complex and often redundant. We observed that progenitor zones in the telencephalon, dorsal diencephalon, hypothalamus, midbrain, midbrain-hindbrain boundary, cerebellum and retina all express multiple <em>Wnt</em> genes. Our data identify <em>12</em> specific ligands that can now be tested using loss-of-function approaches.

Background: Normal-weight polycystic ovary syndrome (PCOS) women exhibit adipose resistance in vivo accompanied by enhanced subcutaneous (SC) abdominal adipose stem cell (ASC) development to adipocytes with accelerated lipid accumulation per cell in vitro. The present study examines chromatin accessibility, RNA expression and fatty acid (FA) synthesis during SC abdominal ASC differentiation into adipocytes in vitro of normal-weight PCOS versus age- and body mass index-matched normoandrogenic ovulatory (control) women to study epigenetic/genetic characteristics as well as functional alterations of PCOS and control ASCs during adipogenesis.

<strong class="sub-title"> Results: </strong> SC abdominal ASCs from PCOS women versus controls exhibited dynamic chromatin accessibility during adipogenesis, from significantly less chromatin accessibility at day 0 to greater chromatin accessibility by day <em>12</em>, with enrichment of binding motifs for transcription factors (TFs) of the AP-1 subfamily at days 0, 3, and <em>12</em>. In PCOS versus control cells, expression of genes governing adipocyte differentiation (PPARγ, CEBPα, AGPAT2) and function (ADIPOQ, FABP4, LPL, PLIN1, SLC2A4) was increased two-sixfold at days 3, 7, and <em>12</em>, while that involving <em>Wnt</em> signaling (FZD1, SFRP1, and WNT10B) was decreased. Differential gene expression in PCOS cells at these time points involved triacylglycerol synthesis, lipid oxidation, free fatty acid beta-oxidation, and oxidative phosphorylation of the TCA cycle, with TGFB1 as a significant upstream regulator. There was a broad correspondence between increased chromatin accessibility and increased RNA expression of those <em>12</em> genes involved in adipocyte differentiation and function, <em>Wnt</em> signaling, as well as genes involved in the triacylglycerol synthesis functional group at day <em>12</em> of adipogenesis. Total content and de novo synthesis of myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1), and oleic (C18:1) acid increased from day 7 to day <em>12</em> in all cells, with total content and de novo synthesis of FAs significantly greater in PCOS than controls cells at day <em>12</em>.

Conclusions: In normal-weight PCOS women, dynamic chromatin remodeling of SC abdominal ASCs during adipogenesis may enhance adipogenic gene expression as a programmed mechanism to promote greater fat storage.

Keywords: Adipogenesis; Adipose stem cells; Cellular programming; Chromatin accessibility; De novo synthesis of fatty acids; Fat storage; Gene expression; Polycystic ovary syndrome; Total content; Transcriptional factors.

To explore the role of retinoic acid (RA) in liver, we developed transgenic mice expressing retinoic acid receptor alpha dominant negative form (RARE) in hepatocytes using by albumin promoter and enhancer. At 4 months of age, the RARE transgenic mice developed microvesicular steatosis and spotty focal necrosis. Mitochondrial beta-oxidation activity of fatty acids and expression of its related enzymes including VLCAD, LCAD and HCD were down-regulated. On the other hand, peroxisomal beta-oxidation and its related enzymes including AOX and BFE were up-regulated. Expression of CYP4a10, CYP4a<em>12</em> and CYP4a14 was increased, suggesting that omega-oxidation of fatty acids in microsome was accelerated. In addition, formation of H(2)O(2) and 8-hydroxy-2'-deoxyguanosine was increased. After <em>12</em> months of age, these mice developed liver tumors which are hepatocellular carcinoma or adenoma. The incidence of tumor formation was increased with age. Expression of beta-catenin and cyclin D1 was enhanced, and TCF-4/beta-catenin complex was increased whereas RARalpha/beta-catenin complex was decreased. Feeding on high RA diet reversed histological and biochemical abnormalities, and inhibited occurrence of liver tumor. Taken together, hepatic loss of RA function leads to development of steatohepatitis and liver tumor and RA plays an important role in preventing hepatocarcinogenesis in association with fatty acid metabolism and <em>Wnt</em> signaling.

<strong class="sub-title"> Objective: </strong> To study whether vitamin D (VitD) inhibits cell proliferation and <em>Wnt</em>/β-catenin and transforming growth factor-β (TGFβ) signaling pathways in uterine leiomyomas independent of mediator complex subunit <em>12</em> (MED<em>12</em>) mutation status.

<strong class="sub-title"> Design: </strong> Prospective study comparing leiomyoma vs. myometrial tissues and human uterine leiomyoma primary (HULP) cells treated with or without VitD and analyzed by MED<em>12</em> mutation status.

Setting: Hospital and university laboratories.

Patient(s): Women with uterine leiomyoma without any treatment (n = 37).

Intervention(s): Uterine leiomyoma and myometrium samples were collected from women undergoing surgery because of symptomatic leiomyoma pathology.

Main outcome measure(s): Analysis of Wnt/β-catenin and TGFβ pathways and proliferation by quantitative real-time polymerase chain reaction in leiomyoma and myometrial tissue as well as in VitD-treated HULP cells analyzed by Sanger sequencing.

<strong class="sub-title"> Results: </strong> Sequencing data showed that 46% of leiomyomas presented MED<em>12</em> mutation, whereas no mutations were detected in adjacent myometrium. Expression of <em>Wnt</em>/β-catenin and TGFβ pathway genes was significantly increased in MED<em>12</em>-mutated leiomyomas compared to matched myometrium; no significant differences were found in wild-type (WT) leiomyomas. In HULP cells, VitD significantly decreased PCNA expression of both MED<em>12</em>-mutated and WT groups. VitD treatment decreased WNT4 and β-catenin expression in both groups compared to controls, with significance for WNT4 expression in MED<em>12</em>-mutated samples. Similarly, VitD significantly inhibited TGFβ3 expression in cells from both groups. MMP9 expression also decreased.

<strong class="sub-title"> Conclusion: </strong> Despite molecular differences between MED<em>12</em>-mutated and WT leiomyomas, VitD inhibited <em>Wnt</em>/β-catenin and TGFβ pathways in HULP cells, suggesting VitD as an effective treatment to reduce proliferation and extracellular matrix formation in different molecular subtypes of uterine leiomyomas.

<strong class="sub-title"> Keywords: </strong> MED<em>12</em> mutation; TGFβ signaling pathway; Uterine leiomyoma; <em>Wnt</em>/β-catenin pathway; cell proliferation.

<AbstractText>To unravel the primary open angle glaucoma (POAG) related proteomic changes in aqueous humour (AH).</AbstractText><p><div><b>METHODS</b></div>Totally 35 patients listed for cataract surgery (controls: <i>n</i>=<em>12</em>, age: 67.4±13.6y) or trabeculectomy for POAG (<i>n</i>=23, age: 72.5±8.3y) were included. AH samples of those patients were obtained during cataract surgery or trabeculectomy. AH samples were subsequently pooled into the experimental groups under equal contribution in terms of protein amount of each individual patient. Protein samples were analyzed by a linear trap quadrupol Orbitrap Mass Spectrometry device with an upstream liquid chromatography system. The obtained raw data were analyzed using the Maxquant proteome software and compared. Proteins with a fold-change ratio higher than a cut-off of 2 were considered as noticeably altered.</p><AbstractText>A total number of 175 proteins could be identified out of the AH from POAG and cataract by means of quantitative mass spectrometric analysis. Apolipoprotein D (fold change, 3.16 times), complement C3 (2.96), pigment epithelium-derived factor (2.86), dickkopf-related protein 3 (2.18) and wingless-related integration (<em>Wnt</em>) inhibitory factor 1 (2.35) were significantly upregulated within the AH of glaucoma compared to cataract serving as controls.</AbstractText><AbstractText>AH provides a tool to analyze changes in glaucoma and shows striking changes in <em>Wnt</em> signaling inhibitory molecules and other proteins.</AbstractText>

Rehmannia glutinosa Libosch (RG), is officially listed in the Chinese Pharmacopoeia and is widely used in China. The leaves of RG (LR) is an important vegetative organ of the plant. At present, the total glycosides of RG (TLR) were extracted from RG, and developed a national second class of new drugs to the Dihuangye total glycoside capsule (DTG). Additionally, DTG has the effect of nourishing yin and tonifying kidney, promoting blood circulation and blood cooling, and applicable to chronic glomerulonephritis mild to Qi and Yin Deficiency. Moreover, diabetic nephropathy (DN) rats model was induced by intraperitoneal injection of a small dose of streptozotocin (45 mg/kg) and high-fat diet and plus 5% glucose drinking water. Over 15 days, after oral administration TLR and DTG in DN rats, samples from serum, urine and kidney were collected for biochemical indicators measurements, pathological analysis, western blotting and metabolomics. Therefore, the analytical results of biochemical indicators, histopathological observations and western blotting showed that TLR and DTG exhibited a significant effect in renal protection. And 27 endogenous metabolites (<em>12</em> in serum and 15 in urine) could be tentatively identified in the process of DN in rats using metabolomics method. Those endogenous metabolites were chiefly involved in sphingolipid metabolism; pentose, glucuronate interconversion; terpenoid backbone biosynthesis; purine metabolism and retinol metabolism. After drug intervention, these endogenous metabolites turned back to normal level some extent (P < 0.05). Furthermore, TLR and DTG prevent high glucose-induced glomerular mesangial cells (GMCs) by inhibiting TGF-β1 and <em>Wnt</em>/β-catenin signaling pathway, providing a powerful supports to develop a new therapeutic agent for DN. This study paved the way for further exploration of the pathogenesis of DN, early diagnosis and the evaluation of curative effect.

Background: Mesenchymal stem cells (MSCs) exhibit differential effect (augmentation or inhibition) on cancer cells depending on the tissue of origin. Given the increasing demand to use MSCs in regenerative medicine, it is vital to ensure that the MSCs being employed are not pro-carcinogenic.

Objective: To assess the effect of human MSC derived conditioned media (CM) on human cancer cell lines.

Materials and methods: PubMed, SCOPUS, and Web of Science were searched using the keyword combination 'human mesenchymal stem cell and conditioned media and human cancer cell line and in-vitro'.

<strong class="sub-title"> Results: </strong> MSC-CM pro-carcinogenic molecules were IL-6, IL-8, FGF10, VEGF, PDGF, TGF-b1, IGF-1, GRO-a, OSP, MMPs, TNFα, IL-4, IL-10, IL-13, IL-17, IL-1 β, G-CSF, MCP‑1, MIP‑1α, MIP‑1β, RANTES, MIG, IP‑10, HGFa, ETX, DKK1; anti-carcinogenic molecules were IFN-β, OST, LIGHT, FRTK3, INF-γ, IP-10, LAP, IL‑1RA, IL‑2, IL-5, IL-7, IL-<em>12</em>, IL-15, IFN-α, IFN‑γ. Effector pathways were STAT 1, JAK2/STAT3, Ras-Raf-MEK-ERK, <em>Wnt</em>/β-catenin, NF-κB, ERK1/2, PI3K/ Akt/mTOR, MAPK/ERK. BMSC, ADMSC, UCMSC, WJMSC DPMSC, AMSC, and UTCMSC had a differential effect on carcinogenesis. GMSC, LMSC, FDMSC were anti-carcinogenic. OMSC was pro-carcinogenic.

Conclusion: Use of MSC-CM with a pro-carcinogenic effect must be restricted in cancer patients irrespective of the nature of the application.

Keywords: Cancer; Conditioned media; Culture; Mesenchymal stem cells.

BACKGROUND

Medulloblastoma is the most common malignant brain tumor in children, currently treated uniformly based on histopathology and clinico-radiological risk stratification leading to unpredictable relapses and therapeutic failures. Identification of molecular subgroups have thrown light on the reasons for these and now reveals clues to profile molecularly based personalized therapy against these tumors.

METHODS

Research and online contents were evaluated for pediatric medulloblastoma which included latest information on the molecular subgroups and their clinical relevance and update on efforts to translate them into clinics.

RESULTS

Scientific endeavors over the last decade have clearly identified four molecular variants (<em>WNT</em>, SHH, Group 3, and Group 4) and their demographic, genomic, and epigenetic profile. Latest revelations include significant heterogeneity within these subgroups and <em>12</em> different subtypes of MB are now identified with disparate outcomes and biology. These findings have important implications for stratification and profiling future clinical trials against these formidable tumors.

CONCLUSIONS

With the continued outpouring of genomic/epigenomic data of these molecular subgroups and evolution of further subtypes in each subgroup, the challenge lies in comprehensive evaluation of these informations. Current and future endeavors are now needed to profile personalized therapy for each child based on the molecular risk stratification of medulloblastoma, with a hope to improve survival outcome and reduce relapses.