Insights from Studies with Oral Cleft Genes Suggest Associations between WNT-pathway Genes and Risk of Oral Cancer

Oral squamous cell carcinoma (OSCC) accounts for more than 90% of the malignant neoplasms that arise in the mucosa of the upper aerodigestive tract. Recent studies of cleft lip/palate have shown the association of genes involved in cancer. WNT pathway genes have been associated with several types of cancer and recently with cleft lip/palate. To investigate if genes associated with cleft lip/palate were also associated with oral cancer, we genotyped 188 individuals with OSCC and 225 control individuals for markers in AXIN2, AXIN1, GSK3 beta, WNT3A, WNT5A, WNT8A, WNT11, WNT3, and WNT9B. Statistical analysis was performed with PLINK 1.06 software to test for differences in allele frequencies of each polymorphism between cases and controls. We found association of SNPs in GSK3B (p = 0.0008) and WNT11 (p = 0.03) with OSCC. We also found overtransmission of GSK3B haplotypes in OSCC cases. Expression analyses showed up-regulation of WNT3A, GSK3B, and AXIN1 and down-regulation of WNT11 in OSCC in comparison with control tissues (P < 0.001). Additional studies should focus on the identification of potentially functional variants in these genes as contributors to human clefting and oral cancer.

Effect of different resistance-training regimens on the WNT-signaling pathway

The purpose of the present study was to evaluate the effects of 8 weeks of strength and power training on the expression of genes related to the canonical WNT pathway and beta-catenin protein levels in physically active men. Twenty-five subjects (27.4 +/- A 4.6 years) were balanced based on their relative maximum strength in the squat exercise (squat 1RM/body mass) and randomly assigned to strength training (ST) (n = 10), power training (PT) (n = 10), and control (C) (n = 5) groups. The ST and the PT groups performed high and low intensity squats, respectively, thrice a week, for 8 weeks. Muscle biopsies from the vastus lateralis muscle were collected before and after the training period. Relative strength and power increased similarly in both ST and PT groups (P < 0.001). Fiber cross-sectional area also increased similarly in both ST and PT groups. Gene expression and beta-catenin protein expression levels were assessed by real-time PCR and Western blot. Certain genes were up-regulated in the ST group (WNT1: 6.4-fold, P < 0.0001; SFRP1: 3.3-fold, P < 0.0001 and LEF1: 7.3-fold, P < 0.0001) and also in the PT group (WNT1: 24.9-fold, P < 0.0001; SFRP1: 2.7-fold, P < 0.0001; LEF1: 34.1-fold, P < 0.0001 and Cyclin D1: 7.7-fold, P < 0.001). In addition, the expression of key WNT pathway genes was substantially more responsive to PT than to ST (WNT1: P < 0.0001; LEF1: P < 0.0001 and Cyclin D1: P < 0.001). Finally, the total beta-catenin protein content increased only in the PT group (P < 0.05). Our data indicate that a PT regimen triggers greater responses in key elements of the WNT pathway.

Molecular profiling of isolated histological components of Wilms tumor implicates a common role for the Wnt signaling pathway in kidney and tumor development

Wilms tumor (WT), a tumor composed of three histological components - blastema (BL), epithelia and stroma - is considered an appropriate model system to study the biological relationship between differentiation and tumorigenesis. To investigate molecular associations between nephrogenesis and WT, the gene expression pattern of individual cellular components was analyzed, using a customized platform containing 4,608 genes. WT gene expression patterns were compared to genes regulated during kidney differentiation. BL had a closer gene expression pattern to the earliest stage of normal renal development. The BL gene expression pattern was compared to that of fetal kidney (FK) and also between FK and mature kidney, identifying 25 common de-regulated genes supposedly involved in the earliest events of WT onset. Quantitative RT-PCR was performed, confirming the difference in expression levels for 13 of 16 genes (81.2%) in the initial set and 8 of 13 (61.5%) in an independent set of samples. An overrepresentation of genes belonging to the Wnt signaling pathway was identified, namely PLCG2, ROCK2 and adenomatous polyposis coli (APC). Activation of the Wnt pathway was confirmed in WT, using APC at protein level and PLCG2 at mRNA and protein level. APC showed positive nuclear immunostaining for an independent set of WT samples, similarly to the FK in week 11. Lack of PLCG2 expression was confirmed in WT and in FK until week 18. Taken together, these results provided molecular evidence of the recapitulation of the embryonic kidney by WT as well as involvement of the Wnt pathway in the earliest events of WT onset. Copyright (C) 2008 S. Karger AG, Basel.

Activation of the Wnt/Beta-catenin Signaling Pathway during Oral Carcinogenesis Process Is Not Influenced by the Absence of Galectin-3 in Mice

Background/Aim: Galectin-3 has been associated with activated Wnt pathway, translocating beta-catenin into the nucleus. However, it is still unknown whether this lectin drives the Wnt signaling activation in lesions from galectin-3-deficient (Gal3(-/-)) mice. The purpose was to study beta-catenin expression in tongue lesions from Gal3(-/-) and wildtype (Gal3(+/+)) mice and the status of Wnt signaling. Materials and Methods: Twenty Gal3(-/-) and Gal3(+/+) male mice were challenged with 4-nitroquinolin-1-oxide and killed at week 16 and 32. Tongues were processed and stained with H&E to detect dysplasias and carcinomas. An imunohistochemical assay was performed to evaluate beta-catenin expression. Results: Carcinomas were more evident in Gal3(+/+) than Gal3(-/-) mice (55.5% vs. 28.5%, respectively; p>0.05). Elevated expression of non-membranous beta-catenin was observed in dysplasias and carcinomas from both groups (p>0.05). Conclusion: Absence of galectin-3 does not interfere in the pattern of beta-catenin expression and therefore in the mediation of the Wnt signaling pathway.

Glypican-3 regulates migration, adhesion and actin cytoskeleton organization in mammary tumor cells through Wnt signaling modulation

Glypican-3 (GPC3) is a proteoglycan involved in migration, proliferation and cell survival modulation in several tissues. There are many reports demonstrating a downregulation of GPC3 expression in some human tumors, including mesothelioma, ovarian and breast cancer. Previously, we determined that GPC3 reexpression in the murine mammary adenocarcinoma LM3 cells induced an impairment of their in vivo invasive and metastatic capacities together with a higher susceptibility to in vitro apoptosis. Currently, the signaling mechanism of GPC3 is not clear. First, it was speculated that GPC3 regulates the insulin-like growth factor (IGF) signaling system. This hypothesis, however, has been strongly challenged. Recently, several reports indicated that at least in some cell types GPC3 serves as a selective regulator of Wnt signaling. Here we provide new data demonstrating that GPC3 regulates Wnt pathway in the metastatic adenocarcinoma mammary LM3 cell line. We found that GPC3 is able to inhibit canonical Wnt signals involved in cell proliferation and survival, as well as it is able to activate non canonical pathway, which directs cell morphology and migration. This is the first report indicating that breast tumor cell malignant properties can be reverted, at least in part, by GPC3 modulation of Wnt signaling. Our results are consistent with the potential role of GPC3 as a metastasis suppressor.

Altered beta-catenin expression related to cancer progression on actinic cheilitis and squamous cell carcinoma of the lip

beta-Catenin is a bifunctional protein related to cell adhesion and gene transcription when activated by Wnt pathway. Altered expression of beta-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 beta-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 12 cases of SCC of the lip were select and submitted to immunohistochemical staining using beta-catenin antibody. beta-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 beta-catenin, and none of them had nuclear staining. Cytoplasmatic and nuclear staining of beta-catenin on studied cases point to pathway alterations. Results demonstrated that beta-catenin expression is altered on epithelial dysplasia, and it is related to degree of alterations. (C) 2011 Elsevier Inc. All rights reserved.

Differential expression of galectin-3, beta-catenin, and cyclin D1 in adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma of salivary glands

Background: Galectin-3 has been implicated in tumor progression of some malignancies as thyroid, prostate, and salivary gland tumors. Recently, it has been suggested that this protein may be an important mediator of the beta-catenin/Wnt pathway. Moreover, nuclear galectin-3 expression has been implicated in cell proliferation, promoting cyclin D1 activation. Thus, the present study aimed to correlate galectin-3 expression with beta-catenin and cyclin D1 expressions in adenoid cystic carcinoma (ACC) and in polymorphous low-grade adenocarcinoma (PLGA). Methods: Fifteen formalin-fixed paraffin-embedded cases of each tumor were retrieved from the files of the Surgical Oral Pathology Service at the University of Sao Paulo and the proteins were analyzed by immunohistochemistry. Results: Adenoid cystic carcinoma showed galectin-3 immunostaining mainly in the nuclei, while PLGA revealed a positive mostly cytoplasmic reaction to galectin-3 in the largest part of tumor cells. Both tumors showed intense cytoplasmic/nuclear staining for beta-catenin in majority of cases. Cyclin D1 immunoreactivity was not detected in 14/15 PLGA and showed specific nuclear staining in 10/15 cases of ACC in more than 5% of the neoplastic cells. Cyclin D1 expression was correlated with cytoplasmic and nuclear galectin-3 expression in ACC (P < 0.05). Conclusions: These results suggest that in ACC galectin-3 may play a role in cellular proliferation through cyclin D1 activation. In addition, nuclear expression of galectin-3 in ACC may be related to a more aggressive behavior of this lesion. Although beta-catenin seems to play a role in carcinogenesis in both lesions, it seems that it does not bind to galectin-3 for cyclin D1 stimulation.

The Wnt signaling pathway and rheumatoid arthritis

The Wnt signaling pathways play a key role in cell renewal, and there are two such pathways. In patients with rheumatoid arthritis (RA), the synovial membrane expresses genes such as Wnt and Fz at higher levels than those observed in patients without RA. The Wnt proteins are glycoproteins that bind to receptors of the Fz family on the cell surface. The Wnt/Fz complex controls tissue formation during embryogenesis, as well as throughout the process of limb development and joint formation. Recent studies have suggested that this signaling pathway plays a role in the pathophysiology of RA. Greater knowledge of the role of the Writ signaling pathway in RA could improve understanding of the differences in RA clinical presentation and prognosis. Further studies should also focus on Wnt family members as molecular targets in the treatment of RA. (C) 2009 Elsevier B.V. All rights reserved

Ixolaris, a tissue factor inhibitor, blocks primary tumor growth and angiogenesis in a glioblastoma model

Background: The expression levels of the clotting initiator protein Tissue Factor (TF) correlate with vessel density and the histological malignancy grade of glioma patients. Increased procoagulant tonus in high grade tumors (glioblastomas) also indicates a potential role for TF in progression of this disease, and suggests that anticoagulants could be used as adjuvants for its treatment. Objectives: We hypothesized that blocking of TF activity with the tick anticoagulant Ixolaris might interfere with glioblastoma progression. Methods and results: TF was identified in U87-MG cells by flow-cytometric and functional assays (extrinsic tenase). In addition, flow-cytometric analysis demonstrated the exposure of phosphatidylserine in the surface of U87-MG cells, which supported the assembly of intrinsic tenase (FIXa/FVIIIa/FX) and prothrombinase (FVa/FXa/prothrombin) complexes, accounting for the production of FXa and thrombin, respectively. Ixolaris effectively blocked the in vitro TF-dependent procoagulant activity of the U87-MG human glioblastoma cell line and attenuated multimolecular coagulation complexes assembly. Notably, Ixolaris inhibited the in vivo tumorigenic potential of U87-MG cells in nude mice, without observable bleeding. This inhibitory effect of Ixolaris on tumor growth was associated with downregulation of VEGF and reduced tumor vascularization. Conclusion: Our results suggest that Ixolaris might be a promising agent for anti-tumor therapy in humans.

Change and Aging Senescence as an Adaptation

Understanding why we age is a long-lived open problem in evolutionary biology. Aging is prejudicial to the individual, and evolutionary forces should prevent it, but many species show signs of senescence as individuals age. Here, I will propose a model for aging based on assumptions that are compatible with evolutionary theory: i) competition is between individuals; ii) there is some degree of locality, so quite often competition will be between parents and their progeny; iii) optimal conditions are not stationary, and mutation helps each species to keep competitive. When conditions change, a senescent species can drive immortal competitors to extinction. This counter-intuitive result arises from the pruning caused by the death of elder individuals. When there is change and mutation, each generation is slightly better adapted to the new conditions, but some older individuals survive by chance. Senescence can eliminate those from the genetic pool. Even though individual selection forces can sometimes win over group selection ones, it is not exactly the individual that is selected but its lineage. While senescence damages the individuals and has an evolutionary cost, it has a benefit of its own. It allows each lineage to adapt faster to changing conditions. We age because the world changes.

A role for mammalian target of rapamycin (mTOR) pathway in non alcoholic steatohepatitis related-cirrhosis

Non-alcoholic fatty liver disease (NAFLD) encompasses the whole spectrum of steatosis, nonalcoholic steatohepatitis (NASH), and NASH-related cirrhosis (NASH/Cir). Although molecular advances have been made in this field, the pathogenesis of NAFLD is not completely understood. The gene expression profiling associated to NASH/Cir was assessed, in an attempt to better characterize the pathways involved in its etiopathogenesis. Methods: In the first step, we used cDNA microarray to evaluate the gene expression profiles in normal liver (n=3) and NASH/Cir samples (n=3) by GeneSifter (TM) analysis to identify differentially expressed genes and biological pathways. Second, tissue microarray was used to determine immunohistochemical expression of phosphorylated mTOR and 4E-BP1 in 11 normal liver samples, 10 NASH/Cir samples and in 37 samples of cirrhosis of other etiologies to further explore the involvement of the mTOR pathway evidenced by the gene expression analysis. Results: 138 and 106 genes were, respectively, up and down regulated in NASH/Cir in comparison to normal liver. Among the 9 pathways identified as significantly modulated in NASH/Cir, the participation of the mTOR pathway was confirmed, since expression of cytoplasmic and membrane phospho-mTOR were higher in NASH/Cir in comparison to cirrhosis of other etiologies and to normal liver. Conclusions: Recent findings have suggested a role for the cellular ""nutrient sensor"" mTOR in NAFLD and the present study corroborates the participation of this pathway in NASH/Cir. Phospho-mTOR evaluation might be of clinical utility as a potential marker for identification of NASH/Cir in cases mistakenly considered as cryptogenic cirrhosis owing to paucity of clinical data.

Novel Primate-Specific Genes, RMEL 1, 2 and 3, with Highly Restricted Expression in Melanoma, Assessed by New Data Mining Tool

Melanoma is a highly aggressive and therapy resistant tumor for which the identification of specific markers and therapeutic targets is highly desirable. We describe here the development and use of a bioinformatic pipeline tool, made publicly available under the name of EST2TSE, for the in silico detection of candidate genes with tissue-specific expression. Using this tool we mined the human EST (Expressed Sequence Tag) database for sequences derived exclusively from melanoma. We found 29 UniGene clusters of multiple ESTs with the potential to predict novel genes with melanoma-specific expression. Using a diverse panel of human tissues and cell lines, we validated the expression of a subset of three previously uncharacterized genes (clusters Hs.295012, Hs.518391, and Hs.559350) to be highly restricted to melanoma/melanocytes and named them RMEL1, 2 and 3, respectively. Expression analysis in nevi, primary melanomas, and metastatic melanomas revealed RMEL1 as a novel melanocytic lineage-specific gene up-regulated during melanoma development. RMEL2 expression was restricted to melanoma tissues and glioblastoma. RMEL3 showed strong up-regulation in nevi and was lost in metastatic tumors. Interestingly, we found correlations of RMEL2 and RMEL3 expression with improved patient outcome, suggesting tumor and/or metastasis suppressor functions for these genes. The three genes are composed of multiple exons and map to 2q12.2, 1q25.3, and 5q11.2, respectively. They are well conserved throughout primates, but not other genomes, and were predicted as having no coding potential, although primate-conserved and human-specific short ORFs could be found. Hairpin RNA secondary structures were also predicted. Concluding, this work offers new melanoma-specific genes for future validation as prognostic markers or as targets for the development of therapeutic strategies to treat melanoma.

Expression of eight genes of nuclear factor-kappa B pathway in multiple myeloma using bone marrow aspirates obtained at diagnosis

Purpose: To evaluate the expression of NF-kappa B pathway genes in total bone marrow samples obtained from MM at diagnosis using real-time quantitative PCR and to evaluate its possible correlation with disease clinical features and survival. Material and methods: Expression of eight genes related to NF-kappa B pathway (NFKB1, IKB, RANK, RANKL, OPG, IL6, VCAM1 and ICAM1) were studied in 53 bone marrow samples from newly diagnosed MM patients and in seven normal controls, using the Taqman system. Genes were considered overexpressed when tumor expression level was at least four times higher than that observed in normal samples. Results: The percentages of overexpression of the eight genes were: NFKB1 0%, IKB 22.6%, RANK 15.1%, RANKL 31.3%, OPG 7.5%, IL6 39.6%, VCAM1 10% and ICAM1 26%. We found association between IL6 expression level and International Staging System (ISS) (p = 0.01), meaning that MM patients with high ISS scores have more chance of overexpression of IL6. The mean value of ICAM1 relative expression was also associated with the ISS score (p = 0.02). Regarding OS, cases with IL6 overexpression present worse evolution than cases with IL6 normal expression (p = 0.04). Conclusion: We demonstrated that total bone marrow aspirates can be used as a source of material for gene expression studies in MM. In this context, we confirmed that IL6 overexpression was significantly associated with worse survival and we described that it is associated with high ISS scores. Also, ICAM1 was overexpressed in 26% of cases and its level was associated with ISS scores.

Selection of suitable housekeeping genes for expression analysis in glioblastoma using quantitative RT-PCR

Background: Considering the broad variation in the expression of housekeeping genes among tissues and experimental situations, studies using quantitative RT-PCR require strict definition of adequate endogenous controls. For glioblastoma, the most common type of tumor in the central nervous system, there was no previous report regarding this issue. Results: Here we show that amongst seven frequently used housekeeping genes TBP and HPRT1 are adequate references for glioblastoma gene expression analysis. Evaluation of the expression levels of 12 target genes utilizing different endogenous controls revealed that the normalization method applied might introduce errors in the estimation of relative quantities. Genes presenting expression levels which do not significantly differ between tumor and normal tissues can be considered either increased or decreased if unsuitable reference genes are applied. Most importantly, genes showing significant differences in expression levels between tumor and normal tissues can be missed. We also demonstrated that the Holliday Junction Recognizing Protein, a novel DNA repair protein over expressed in lung cancer, is extremely over-expressed in glioblastoma, with a median change of about 134 fold. Conclusion: Altogether, our data show the relevance of previous validation of candidate control genes for each experimental model and indicate TBP plus HPRT1 as suitable references for studies on glioblastoma gene expression.

beta-Hydroxy-beta-methylbutyrate (HM beta) supplementation stimulates skeletal muscle hypertrophy in rats via the mTOR pathway

beta-Hydroxy-beta-methylbutyrate (HM beta) supplementation is used to treat cancer, sepsis and exercise-induced muscle damage. However, its effects on animal and human health and the consequences of this treatment in other tissues (e. g., fat and liver) have not been examined. The purpose of this study was to evaluate the effects of HM beta supplementation on skeletal muscle hypertrophy and the expression of proteins involved in insulin signalling. Rats were treated with HM beta (320 mg/kg body weight) or saline for one month. The skeletal muscle hypertrophy and insulin signalling were evaluated by western blotting, and hormonal concentrations were evaluated using ELISAs. HM beta supplementation induced muscle hypertrophy in the extensor digitorum longus (EDL) and soleus muscles and increased serum insulin levels, the expression of the mammalian target of rapamycin (mTOR) and phosphorylation of p70S6K in the EDL muscle. Expression of the insulin receptor was increased only in liver. Thus, our results suggest that HM beta supplementation can be used to increase muscle mass without adverse health effects.