Aberrant Wnt-1/beta-catenin signaling and WIF-1 deficiency are important events which promote tumor cell invasion and metastasis in salivary gland adenoid cystic carcinoma.

This study investigates whether Wnt components play a role in carcinogenesis, or the invasion and metastasis of salivary glands, also referred to as adenoid cystic carcinoma (sAdCC). Several sAdCC cell lines with low invasive potential (ACC-2), high metastatic potential (ACC-M), and higher invasive potential (T-ACC-M) were examined to determine whether Wnt components correlate with tumors' invasive and metastatic behavior. Immunohistochemistry was performed in a sAdCC tissue array. ACC-M expressed higher levels of Wnt-1, beta-catenin and lower WIF-1 compared to ACC-2 (P<0.05). T-ACC-M exhibited increased mRNA of Wnt-1 and beta-catenin, and decreased WIF-1 compared to ACC-2 and ACC-M. Immuno-histochemistry showed up-regulation of Wnt-1 and down-regulation of WIF-1 in sAdCC compared with normal salivary glands. Beta-catenin was found in the cytoplasm and nuclei of sAdCC. Dislocation of E-cadherin in sAdCC was observed. These results suggest that sAdCC exhibits diverse expressions of Wnt components. It has an important relationship with the invasive phenotype of these cells.

Differences in the developmental origins of the periosteum may influence bone healing.

BACKGROUND AND OBJECTIVE: The jaw bone, unlike most other bones, is derived from neural crest stem cells, so we hypothesized that it may have different characteristics to bones from other parts of the body, especially in the nature of its periosteum. The periosteum exhibits osteogenic potential and has received considerable attention as a grafting material for the repair of bone and joint defects. MATERIAL AND METHODS: Gene expression profiles of jaw bone and periosteum were evaluated by DNA microarray and real-time polymerase chain reaction. Furthermore, we perforated an area 2 mm in diameter on mouse frontal and parietal bones. Bone regeneration of these calvarial defects was evaluated using microcomputed tomography and histological analysis. RESULTS: The DNA microarray data revealed close homology between the gene expression profiles within the ilium and femur. The gene expression of Wnt-1, SOX10, nestin, and musashi-1 were significantly higher in the jaw bone than in other locations. Microcomputed tomography and histological analysis revealed that the jaw bone had superior bone regenerative abilities than other bones. CONCLUSION: Jaw bone periosteum exhibits a unique gene expression profile that is associated with neural crest cells and has a positive influence on bone regeneration when used as a graft material to repair bone defects. A full investigation of the biological and mechanical properties of jaw bone as an alternative graft material for jaw reconstructive surgery is recommended.

Mastication markedly affects mandibular condylar cartilage growth, gene expression, and morphology.

INTRODUCTION: Mandibular growth is believed to be strongly related to mastication. Furthermore, mandibular condylar cartilage is known to be derived from neural crest cells. We examined whether the degree of chewing affects condylar cartilage growth of the mandible. METHODS: Mice were fed diets with varying hardness. Genes specific to neural crest-derived cells were measured by real-time polymerase chain reaction to compare the expression changes between the mandibular and tibia cartilages. The mandibular condylar cartilage was then evaluated histologically, and proliferation was evaluated using proliferating cell nuclear antigen. Immunostaining was conducted for osteopontin, type X collagen, and Musashi1, and real-time polymerase chain reaction was used to assess the expression levels of osteopontin and type X collagen. RESULTS: Markers including P75, Wnt-1, Musashi1, and Nestin were upregulated in the mandibular condylar cartilage as compared with the tibial cartilage. Histologic assessment of the mandibular cartilage showed that the hypertrophic chondrocyte zone was statistically significantly thicker in mice fed a hard diet. Chondrocyte proliferation and Musashi1 expression were lower in mice fed a hard diet. After 4 weeks, numerous osteopontin and type X collagen-positive cells were observed in mice fed a mixed diet. CONCLUSIONS: Mastication affects the balance between differentiation and proliferation in the mandibular condylar cartilage. This phenomenon might be attributed to the presence of neural crest-derived cells.

Wnt/ß-catenin pathway as a potential prognostic and predictive marker in patients with advanced ovarian cancer.

BACKGROUND: ß-catenin is the key protein in the WNT signalling pathway and it forms adherent junctions together with E-cadherin. In ovarian carcinoma, abnormal expression of ß-catenin, E-cadherin and WNT-1 was observed, but their prognostic and predictive role is unclear. The aim of this study was to clarify the prognostic and predictive role of E-cadherin, ß-catenin and WNT-1 in advanced epithelial ovarian carcinoma (AEOC). METHODS: The expression of E-cadherin, ß-catenin and WNT-1 was determined by immunohistochemistry in AEOC. The correlation between expression of these proteins and progression-free survival (PFS) and overall survival (OS) was evaluated. Statistical analyses included Kaplan-Meier estimation, log-rank test, Spearman correlation and Cox proportional-hazards model. RESULTS: In ovarian cancer, intense expression of E-cadherin, ß-catenin and WNT-1 was found. In multivariate analysis, strong membrane ß-catenin expression was an independent unfavourable predictor for PFS (HR 2.19, 95% CI 1.09-4.39; p = 0.028), while in univariate analysis, strong membrane ß-catenin expression was a prognostic factor for OS in patients with AOC (p = 0.039). In multivariate analysis, only resistance to first-line chemotherapy was an adverse independent prognostic factor for OS (HR 16.84; 95% CI 5.07-55.98; p < 0.0001). Additionally, strong membranous ß-catenin expression was associated with resistance to platinum-based chemotherapy (p = 0.027). CONCLUSIONS: These findings support that WNT/ß-catenin pathway and E-cadherin are important factors in advanced epithelial ovarian cancer.

A pilot trial on the molecular pathophysiology of traumatic temporomandibular joint bony ankylosis in a sheep model. Part I: Expression of Wnt signaling.

OBJECTIVE: To preliminarily investigate the temporal patterns of the endogenous mRNA expression for members of the Wnt signaling and a series of genes regulating bone formation during the development of traumatic temporomandibular joint (TMJ) bony ankylosis in a sheep model. METHODS: Six sheep were used for the induction of bony ankylosis of TMJ. We performed a condylar fracture, excision of the lateral 2/3 disc and serious injury to the glenoid fossa to induce bony ankylosis on the right TMJ. An isolated condylar fracture was performed on the left side. Two sheep were sacrificed at 1 month, 3 months, and 6 months after surgery, respectively. The specimens from the ankylosed joint and the condylar fracture were harvested for RNA extraction respectively. In this report (Part I), only the bony ankylosed samples were used for analysis of gene expressions. The specimens 1 month postoperatively were taken as the control, and the changes of expression of target genes over time were examined by real-time PCR. RESULTS: mRNA expression of Wnt1, Wnt2b, Wnt3a, ß-catenin, Sfrp1, Lrp6, Lef1, CyclinD1, and Runx2 was up-regulated at 3 and 6 months compared with 1 month. The expression of Wnt5a, Sox9, and Osterix was up-regulated with a peak at 3 months, and then fell back to the basal levels at 6 months. The expression of Ocn began to up-regulate until 6 month postoperatively. CONCLUSION: Our findings suggested that Wnt signaling was involved in the formation of traumatic TMJ bony ankylosis and thus may be a potential therapeutic target for the treatment of the disease in the future.

Effect of fibroblasts on breast cancer cell mammosphere formation and regulation of stem cell-related gene expression.

The purpose of this study was to investigate the regulatory effects of breast cancer fibroblasts (BCFs) vs. normal mammary fibroblasts (NMFs) on mammosphere formation and stem cell-related gene expression in breast cancer cells. Breast cancer cells (MCF-7) were cultured in suspension to generate primary and secondary mammospheres. The proportion of CD44+/CD24low/- cells was assessed by flow cytometry (FCM), and Wnt1, Notch1, ß-catenin, CXCR4, SOX2 and ALDH3A1 gene expression was detected by quantitative real-time PCR. The fibroblasts from either breast cancer tissue or normal mammary tissue were purified from tissue specimens and co-cultured with breast cancer cells. The mammosphere formation efficacy was approximately 180/10,000 MCF-7 cells. FCM analysis showed that, compared to the 2.1% positive expression in the MCF-7 monolayer culture cells, the expression of CD44+/CD24low/- in MCF-7 mammosphere cells was significantly elevated to 10.4% (P<0.01). The proportion of the CD44+/CD24low/- subpopulation of the cells in mammospheres was nearly 5-fold higher than that of general MCF-7 cells. Compared with MCF-7 monolayer culture cells, mammosphere cells showed significantly (P<0.01) enhanced expression of Wnt1 [fold-change (FC), 2.25], Notch1 (FC, 2.45), ß-catenin (FC, 1.72), CXCR4 (FC, 4.68), SOX2 (FC, 4.25) and ALDH3A1 (FC, 5.38). When BCFs were co-cultured with MCF-7 cells under mammosphere culture conditions, the length of time of mammosphere formation decreased, the volume of the mammo-spheres increased and the mammosphere-forming efficiency (MFE) was higher than that of NMFs and the control group. Both the BCF and NMF groups showed enhanced gene expression for the following genes: Wnt1 (FC, 3.18 and 1.27, respectively), ß-catenin (FC, 1.75 and 1.22, respectively), Notch1 (FC, 2.09 and 1.31, respectively), CXCR4 (FC, 2.77 and 1.33, respectively), SOX2 (FC, 2.77 and 1.80, respectively) and ALDH3A1 (FC, 5.23 and 1.85, respectively). Cancer fibroblast cells can promote the MFE and up-regulate stem cell-related gene expression in breast cancer cells.

[The correlation between the expression of Wnt1 and prognosis in resected non-small cell lung cancer].

BACKGROUND AND OBJECTIVE: Wnt1 protein is the first factor in Wnt signaling pathway. It has been proven that high expression of Wnt1 protein was associated with malignant proliferation in many tumors. The aim of this study is to investigate the correlation between Wnt1 protein overexpression, clinicopathologic features and survival in resected non-small-cell lung cancer (NSCLC). METHODS: Immunohistochemical staining of Envision was applied to investigate the expression of Wnt1 protein in specimens of 115 NSCLC and 19 benign pulmonary diseases. The correlation between the Wnt1 protein in specimens of 115 NSCLC and clinicopathologic features was analysed with chi2 test, and the correlation between the Wnt1 protein expression and the patient survival was evaluated with Kaplan-Meier survival curve and Cox regression. RESULTS: The positive rate ofWnt1 protein in specimens of NSCLC was 62.6%, which was significantly higher than 31.6% of benign pulmonary diseases (chi2 = 4.474, P = 0.034). But it was not correlated with clinicopathologic features. Kaplan-Meier survival analysis and Log-rank test suggested that patients with Wnt1 protein overexpression had poor prognosis (P = 0.003). And Cox regression suggested Wnt1 protein expression was an independent prognostic factor of NSCLC. CONCLUSION: The expression of Wnt1 was remarkably higher in specimens of resected NSCLC than that in benign pulmonary diseases. Overexpression of Wnt1 protein in NSCLC was correlated with prognosis and can be served as an independent prognostic factor of NSCLC.

Molecular events involved in the morphogenesis of multicystic dysplastic kidney.

INTRODUCTION: Wnt-1 is capable of inducing metanephric mesenchyme to undergo tubulogenesis. A relationship between the degree of cystogenesis and reduced E-cadherin (E-cad) expression was described. Syndecan-1 (Sdc-1) has a critical role in kidney development. MATERIALS AND METHODS: Ten multicystic dysplastic kidneys (MCDKs) were stained with hematoxylin and eosin and immunohistochemistry was performed using Wnt-1, E-cad and Sdc-1 antibodies. Eight unaffected kidneys were used as controls. RESULTS: Strong Wnt-1 immunostaining occurred inside cystic/tubular epithelial cells and in blastematous foci. An immunoreaction was observed in glomerular epithelial cells. In controls, just weak cytoplasmic Wnt-1 positivity was seen in tubular epithelial cells. E-cad reaction was negative in MCDKs while strong immunostaining was common in tubular cells of controls. A strong Sdc-1 immunoreaction depicted cystic, tubular and glomerular epithelial cells in MCDKs while Sdc-1 expression documented weak positivity in tubular epithelium alone. CONCLUSIONS: Our data are in accordance with an involvement of Wnt-1 in normal nephrogenesis and with its role in altered epithelial differentiation of metanephric mesenchyme in MCDKs. Wnt-1 signal may function to suppress E-cad expression, a predisposing event for cystogenesis. High expression of Sdc-1 in tubular/cystic epithelial cells of MCDKs might alter the normal transition of stages of the developmental process and modify the anion charge of the glomerular barrier.

Expression and clinical significance of Wnt-1 and beta-catenin in nasopharyngeal carcinoma.

BACKGROUND AND OBJECTIVE: As signaling molecule and key component of Wnt/beta-catenin signaling pathway respectively, Wnt-1 and beta-catenin are abnormally expressed in several malignancies and correlate with poor prognosis. This study was to investigate the expression and clinical significance of Wnt-1 and beta-catenin in nasopharyngeal carcinoma (NPC). METHODS: The expression of Wnt-1 and beta-catenin in 111 specimens of NPC was detected by SP immunohistochemistry. Their correlations to relapse-free survival (RFS), metastasis-free survival (MFS) and progression-free survival (PFS) were analyzed. RESULTS: The high expression of beta-catenin was observed in 64 (57.7%) of the 111 cases. Its high expression rate was significantly higher in advanced NPC than in early stage NPC (63.1% vs. 40.7%, p = 0.041). The RFS, MFS and PFS were lower in high beta-catenin expression group than in low beta-catenin expression group (p < 0.05). Cox regression analysis demonstrated that beta-catenin was related to poor prognosis of NPC patients. The high expression of Wnt-1 was observed in 68 (61.3%) of the 111 cases, but its expression had no effect on RFS, MFS and PFS (p > 0.05). CONCLUSIONS: Wnt/beta-catenin signaling pathway may be activated abnormally in some NPC patients. beta-catenin may be a prognostic factor of NPC.

The precerebellar linear nucleus in the mouse defined by connections, immunohistochemistry, and gene expression.

The linear nucleus (Li) is a prominent cell group in the caudal hindbrain, which was first described in a study of cerebellar afferents in the rat by [Watson, C.R.R., Switzer, R.C. III, 1978. Trigeminal projections to cerebellar tactile areas in the rat origin mainly from N. interpolaris and N. principalis. Neurosci. Lett. 10, 77-82.]. It was named for its elongated appearance in transverse sections. Since this original description in the rat, reference to the nucleus seems to have been largely absent from experimental studies of mammalian precerebellar nuclei. We therefore set out to define the cytoarchitecture, cerebellar connections, and molecular characteristics of Li in the mouse. In coronal Nissl sections at the level of the rostral inferior olive, it consists of two parallel bands of cells joined at their dorsal apex by a further band of cells, making the shape of the Greek capital letter pi. Our three-dimensional reconstruction demonstrated that the nucleus is continuous with the lateral reticular nucleus (LRt) and that the ambiguus nucleus sits inside the arch of Li. Cerebellar horseradish peroxidase injections confirmed that the cells of Li project to cerebellum. We have shown that Li cells express Atoh1 and Wnt1 lineage markers that are known to label the rhombic lip derived precerebellar nuclei. We have examined the relationship of Li cells to a number of molecular markers, and have found that many of the cells express a nonphosphorylated epitope in neurofilament H (SMI 32), a feature they share with the LRt. The mouse Li therefore appears to be a rostrodorsal extension of the LRt.

Partial biological characterization of cancer stem-like cell line (WJ(2)) of human glioblastoma multiforme.

To provide suitable models for human GBM cancer stem cells in vitro and in vivo, and investigate their biological characteristics, a new human GBM cancer stem-like cell line, WJ2, was established in this experiment through serial passages from adherent monolayer culture to nonadherent tumor sphere culture in turns; Its partial biological characteristics were studied through cell proliferation and tumor sphere assay; cell cycle distribution, side population, and CD133 phenotype were analyzed with FCM. The expressions of CD133, Nestin, and GFAP of cancer stem-like cells and xenograft tumor cells were detected with RT-PCR and immunohistochemistry. Biological characterization, side population, CD133 phenotype and CD133 Nestin, BCRP-1, Wnt-1 gene expression revealed the stemness of this cancer stem-like cell line. Tumorigenicity heterotransplanted in nude mice; histopathological characteristics of xenograft tumor, and expressions of CD133, Nestin, and GFAP of xenograft tumor cells indicated that xenograft tumors recapitulated the phenotype and biological characterization of human primary GBM. All findings of this experimental study suggested that WJ(2) cancer stem-like cell line could accurately mimic human GBM cancer stem cell in vitro and in vivo; it would be useful in the cellular and molecular studies as well as in testing novel therapies of CSC-based anti-cancer therapies for human GBM.

Porcupine-mediated lipid-modification regulates the activity and distribution of Wnt proteins in the chick neural tube.

A long-term goal of developmental biology is to understand how morphogens establish gradients that promote proper tissue patterning. A number of reports describe the formation of the Wg (Wnt1) gradient in Drosophila and have shown that Porcupine, a predicted membrane-bound O-acyl transferase, is required for the correct distribution of Wg protein. The discovery that Wnts are palmitoylated on a conserved cysteine residue suggests that porcupine activity and Wnt palmitoylation are important for the generation of Wnt gradients. To establish the role of porcupine in Wnt gradient formation in vertebrates, we tested the role of porcupine/Wnt palmitoylation in human embryonic kidney 293T cells and in the chick neural tube. Our results lead us to conclude that: (1) vertebrate Wnt1 and Wnt3a possess at least one additional site for porcupine-mediated lipid-modification; (2) porcupine-mediated lipid-modification of Wnt proteins promotes their activity in 293T cells and in the chick neural tube; and (3) porcupine-mediated lipid-modification reduces the range of activity of Wnt1 and Wnt3a in the chick neural tube. These findings highlight the importance of porcupine-mediated lipid modifications in the formation of vertebrate Wnt activity gradients.

Syndecan-1 and Wingless-type protein-1 in human ameloblastomas.

BACKGROUND: Aberrant Wingless type 1 glycoprotein (Wnt) pathway in ameloblastomas and a role of syndecan-1 (SDC1) in activating Wnt signalling were perspected. SDC1 shifting from epithelium to stroma was reported in invasive non-odontogenic neoplasms. The aim of this study was to reveal the role of SDC1 and Wnt1 in intraosseous ameloblastomas (IA(s)). METHODS: SDC1 and Wnt1 expressions were investigated in 29 ameloblastoma subtypes and seven tooth buds. RESULTS: SDC1 immunostaining strongly depicted stromal cells, extracellular matrix (ECM) and basement membranes of ameloblastomas. It also showed epithelial tumour cells in the acanthomatous and plexiform subtypes, and it often occurred in stellate reticulum cells and basal ameloblasts of tooth buds. Parallel Wnt1 expression occurred in ameloblastomatous epithelial cells, but it was common in basal cells of tooth buds too. Statistically, a significant correlation was found between the percentage of IA(s)-bearing SDC1-positive stromal cells and ECM and the percentage of IA(s)-bearing Wnt1-positive epithelial cells. CONCLUSIONS: A role of SDC1 in stromal cells and ECM can be hypothesized as a critical factor for carcinogenesis and local invasiveness of IA(s).

Development of leiomyosarcoma of the uterus in MMTV-CR-1 transgenic mice.

Overexpression of Cripto-1 (CR-1) in FVB/N mice using the MMTV-LTR promoter results in increased mammary tumourigenesis in these female transgenic mice (MMTV-CR-1). Here, we characterize uterine tumours that developed in 15/76 (19.7%) of MMTV-CR-1 female nulliparous or multiparous mice during 24 months of observation compared with 0/33 (0%) of FVB/N normal control mice observed during the same time period (p < 0.01). The uterine tumours collected from the MMTV-CR-1 mice were classified as leiomyosarcomas and found to express the CR-1 transgene by polymerase chain reaction analysis and immunohistochemistry. Detection by western blot analysis showed higher levels of phosphorylated (P) forms of c-src, Akt, GSK-3beta, and dephosphorylated (DP)-beta-catenin in lysates from MMTV-CR-1 uterine leiomyosarcomas in comparison with lysates from normal control FVB/N uteri. Immunostaining showed increased nuclear localization of beta-catenin in the MMTV-CR-1 uterine leiomyosarcomas. Increased immunostaining for CR-1 was detected in 9/13 (69.2%) cases of human leiomyosarcoma compared with staining in 3/15 (20%) human leiomyoma sections. Stronger immunostaining for P-src, P-Akt, P-GSK-3beta and increased nuclear localization of beta-catenin was also seen in human leiomyosarcomas in comparison with leiomyomas. Normal human uterine smooth muscle (UtSM) cells treated with exogenous soluble rhCR-1 showed increased levels of P-src, P-Akt, P-GSK-3beta and dephosphorylated (DP)-beta-catenin and increased proliferation (p < 0.05) and migration (p < 0.01) in comparison with untreated control UtSM cells. Inhibitors against c-src, Akt or beta-catenin, individually or in combination, significantly reduced CR-1-induced migration. These results suggest a role for CR-1 during uterine tumourigenesis either directly by activating c-src and Akt and/or via cross-talk with the canonical Wnt signalling pathway, as suggested by the increased expression of P-GSK-3beta, DP-beta-catenin, and increased nuclear localization of beta-catenin in human and MMTV-CR-1 mice leiomyosarcomas.