Oral squamous carcinoma cells secrete RANKL directly supporting osteolytic bone loss

Objective: Local invasion of bone is a frequent complication of oral squamous cell carcinoma (OSCC). Development of these osteolytic lesions is mediated by osteoclasts. Receptor activation of NF-kappa B ligand (RANKL) signaling, counteracted by osteoprotegerin (OPG), regulates osteoclastogenesis. Previous studies in rodent models have demonstrated that inhibition of RANKL decreases tumor growth and lesions within bone. However, the contributory role of OSCC cells to this disease process has yet to be defined.Methods: RANKL expression was assessed in a panel of OSCC cell lines by qPCR, flow cytometry, and ELISA. Induction of osteoclastogenesis was assessed by co-culture with macrophages or with OSCC-derived conditioned medium. In an animal model of bone invasion, nude mice were injected intratibially with UMSCC-11B cells expressing a RANKL luciferase promoter to detect tumor-derived RANKL activity. Osteolytic lesions were analyzed by X-ray, micro-CT, and histological methods. RANKL expression was assessed in human OSCC tissues by immunohistochemistry.Results: We demonstrated that OSCCs express varied levels of all RANKL isoforms, both membrane-bound and soluble RANKL. Both co-culture and treatment with OSCC-conditioned media induced osteoclastogenesis. In mice, we demonstrated human RANKL promoter activity during bone invasion. Over the course of the experiment, animals suffered osteolytic lesions as RANKL-driven luciferase expression increased with time. After 8 weeks, human-derived RANKL was detected in areas of bone resorption by immunohistochemistry. Similar epithelial RANKL expression was detected in human OSCC tissues.Conclusion: These data demonstrate the ability of OSCCs to produce RANKL, directly altering the tumor microenvironment to increase osteoclastogenesis and mediate local bone invasion. (C) 2012 Elsevier Ltd. All rights reserved.

Fator de crescimento do endotélio vascular e do transportador de glicose 1 em cães com ceratose actínica e carcinoma de células escamosas

Pós-graduação em Medicina Veterinária - FCAV

PHF21B as a candidate tumor suppressor gene in head and neck squamous cell carcinomas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genome-wide methylation and transcriptome analysis in penile carcinoma: uncovering new molecular markers

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Analysis of the association of an MMP1 promoter polymorphism and transcript levels with chronic periodontitis and end-stage renal disease in a Brazilian population

Chronic periodontitis (CP) and end-stage renal disease (ESRD) are complex inflammatory conditions. Higher levels of MMP-1 were found in fluids and gingival tissues from CP patients and in the blood and tissues from ESRD patients. MMP1-1607 (1G/2G) is a functional polymorphism, as it alters MMP-1 expression. Objective: The aim of this study was to investigate the association of the MMP1-1607 (1G/2G) polymorphism with CP and ESRD and evaluate differences in transcript levels between the groups. Design: A total of 254 individuals were divided into four groups: Group 1, without CP and without chronic kidney disease (CKD) (n = 67); Group 2, with CP and without CKD (n = 60); Group 3, without CP and with CKD stages (ESRD) (n = 52), and Group 4, with CP and with ESRD (n = 75). The MMP1-1607 polymorphism was analysed by PCR-RFLP. MMP1 gene transcripts from gingival tissues were analysed by real-time PCR. Results: No association was found between the MMP1-1607 polymorphism and CP or ESRD. Increased levels of MMP1 transcripts were observed in CP patients with or without ESRD. No differences were observed in the transcript levels according to the genotypes. Conclusion: It was concluded that the MMP1-1607 polymorphism was not associated with either CP or ESRD. However, higher levels of MMP1 gene transcripts were found at gingival sites of CP in patients both with and without ESRD. (C) 2012 Elsevier Ltd. All rights reserved.

Tumor microenvironmental genomic alterations in juvenile nasopharyngeal angiofibroma

Background To better characterize the pathophysiology of juvenile nasopharyngeal angiofibroma (JNA), endothelial and stromal cells were evaluated by genomic imbalances in association with transcript expression levels of genes mapped on these altered regions. Methods. High-resolution comparative genomic hybridization (HR-CGH) was used in laser-captured endothelial and stromal cells from 9 JNAs. Ten genes were evaluated by quantitative real-timereverse transcription polymerase chain reaction (qRT-PCR) in 15 cases. Results. Although gains were more frequently detected in endothelial cells, 57% of chromosomal alterations were common by both components. Gene expression analyses revealed a positive correlation between endothelial and stromal components for ASPM, CDH1, CTNNB1, FGF18, and SUPT16H. A significant difference was found for FGF18 and AURKB overexpression in stromal cells and AR down-expression in endothelial cells. Conclusions. A similar pattern of gene expression and chromosomal imbalances in both exponents would suggest a common mechanism of functional regulation. AURKB, FGF18, and SUPT16H were identified as potential molecular markers in JNA. (C) 2011 Wiley Periodicals, Inc. Head Neck 34: 485-492, 2012

Distinct DNA methylation patterns in cirrhotic liver and hepatocellular carcinoma

Abberrant DNA methylation is one of the hallmarks of cancerogenesis. Our study aims to delineate differential DNA methylation in cirrhosis and hepatic cancerogenesis. Patterns of methylation of 27,578 individual CpG loci in 12 hepatocellular carcinomas (HCCs), 15 cirrhotic controls and 12 normal liver samples were investigated using an array-based technology. A supervised principal component analysis (PCA) revealed 167 hypomethylated loci and 100 hypermethylated loci in cirrhosis and HCC as compared to normal controls. Thus, these loci show a "cirrhotic" methylation pattern that is maintained in HCC. In pairwise supervised PCAs between normal liver, cirrhosis and HCC, eight loci were significantly changed in all analyses differentiating the three groups (p < 0.0001). Of these, five loci showed highest methylation levels in HCC and lowest in control tissue (LOC55908, CELSR1, CRMP1, GNRH2, ALOX12 and ANGPTL7), whereas two loci showed the opposite direction of change (SPRR3 and TNFSF15). Genes hypermethylated between normal liver to cirrhosis, which maintain this methylation pattern during the development of HCC, are depleted for CpG islands, high CpG content promoters and polycomb repressive complex 2 (PRC2) targets in embryonic stem cells. In contrast, genes selectively hypermethylated in HCC as compared to nonmalignant samples showed an enrichment of CpG islands, high CpG content promoters and PRC2 target genes (p < 0.0001). Cirrhosis and HCC show distinct patterns of differential methylation with regards to promoter structure, PRC2 targets and CpG islands.

Identification of the p53 family-responsive element in the promoter region of the tumor suppressor gene hypermethylated in cancer 1

The tumor suppressor gene hypermethylated in cancer 1 (HIC1), located on human chromosome 17p13.3, is frequently silenced in cancer by epigenetic mechanisms. Hypermethylated in cancer 1 belongs to the bric à brac/poxviruses and zinc-finger family of transcription factors and acts by repressing target gene expression. It has been shown that enforced p53 expression leads to increased HIC1 mRNA, and recent data suggest that p53 and Hic1 cooperate in tumorigenesis. In order to elucidate the regulation of HIC1 expression, we have analysed the HIC1 promoter region for p53-dependent induction of gene expression. Using progressively truncated luciferase reporter gene constructs, we have identified a p53-responsive element (PRE) 500 bp upstream of the TATA-box containing promoter P0 of HIC1, which is sequence specifically bound by p53 in vitro as assessed by electrophoretic mobility shift assays. We demonstrate that this HIC1 p53-responsive element (HIC1.PRE) is necessary and sufficient to mediate induction of transcription by p53. This result is supported by the observation that abolishing endogenous wild-type p53 function prevents HIC1 mRNA induction in response to UV-induced DNA damage. Other members of the p53 family, notably TAp73beta and DeltaNp63alpha, can also act through this HIC1.PRE to induce transcription of HIC1, and finally, hypermethylation of the HIC1 promoter attenuates inducibility by p53.

Dual induction of PKR with E2F-1 and IFN-alpha to enhance gene therapy against hepatocellular carcinoma

Overexpression of the transcription factor E2F-1 induces apoptosis in tumor cells. This apoptotic effect is partly mediated through the induction of the double-stranded RNA-activated protein kinase (PKR). Here, we investigate if agents that upregulate PKR could enhance the apoptotic effect of E2F-1 overexpression in liver tumors. In human hepatocellular carcinoma (HCC) cells (Hep3B, HepG2, Huh7), adenovirus-mediated overexpression of E2F-1 (AdCMV-E2F) transcriptionally increased PKR mRNA. The subsequent increase of total and phosphorylated PKR protein was followed by induction of apoptosis. When AdCMV-E2F was combined with the PKR modifier interferon alpha (IFNalpha), PKR was additionally upregulated and both PKR activation and apoptosis were increased. Subcutaneous xenograft tumors were selectively targeted using an adenoviral vector expressing E2F-1 under the control of the human telomerase reverse transcriptase (hTERT) promoter (AdhTERT-E2F). Weekly systemic administration of AdhTERT-E2F inhibited tumor growth. The tumor suppressive effect of AdhTERT-E2F therapy was further enhanced in combination with IFNalpha.Our results demonstrate that PKR activating agents enhance the anti-tumor effect of E2F-1 overexpression in HCC in-vitro and in-vivo. Hence, modulation of PKR is a potential strategy to increase the efficacy of PKR-dependent anti-tumor therapies.

Analysis of galectin expression and identification of endogenous ligands in a human colon carcinoma cell line

The 14.5 kDa (galectin-1) and 31 kDa (galectin-3) lectins are the most well characterized members of a family of vertebrate carbohydrate-binding proteins known as the galectins. Evidence has been obtained implicating these galectins in events as diverse as cell-cell and cell-extracellular matrix interactions, growth regulation, transformation, differentiation, and programmed cell death. In the present study, sodium butyrate was found to be a potent inducer of galectin-1 in the KM12 human colon carcinoma cell line. Prior to treatment with butyrate this cell line expresses only galectin-3. These cells were utilized as an in vitro model system to study galectin expression as well as that of their endogenous ligands. The initial phase of this project involved the examination of the induction of galectin-1 by butyrate at the protein level. These studies indicated that galectin-1 induction by butyrate was relatively rapid reaching nearly maximal levels after only 24 hours. Additionally, the induction was found to be reversible upon the removal of butyrate and to precede the increase in expression of the well characterized differentiation marker, carcinoembryonic antigen (CEA). The second phase of this project involved the characterization of potential glycoprotein ligands for galectin-1 and galectin-3. This work demonstrated that the polylactosaminoglycan-containing glycoproteins laminin, CEA, and the lysosome-associated glycoproteins-1 and -2 (LAMPs-1 and -2) are capable of serving as ligands for both galectin-1 and -3. The third phase of this project involved the analysis of the induction of the galectin-1 promoter by butyrate. Through the analysis of deletion constructs transiently transfected into KM12 cells, the region of the galectin-1 promoter mediating a high level of induction by butyrate was localized primarily within a proximal portion of the promoter containing a CCAAT element and an Sp1 binding site. The CCAAT-binding activity in the KM12 nuclear extracts was subsequently dentified as NF-Y by gel shift analysis. These studies suggest that: (1) the galectins may be involved in modulating adhesive interactions in human colon carcinoma cells through the binding of several polylactosaminoglycans shown to play a role in adhesion and (2) high level induction of the galectin-1 promoter by butyrate can proceed through a discreet, proximal element containing an NF-Y-binding CCAAT box and an Sp1 site. ^

TWIST1 and TWIST2 promoter methylation and protein expression in tumor stroma influence the epithelial-mesenchymal transition-like tumor budding phenotype in colorectal cancer.

Tumor budding in colorectal cancer is likened to an epithelial-mesenchymal transition (EMT) characterized predominantly by loss of E-cadherin and up-regulation of E-cadherin repressors like TWIST1 and TWIST2. Here we investigate a possible epigenetic link between TWIST proteins and the tumor budding phenotype. TWIST1 and TWIST2 promoter methylation and protein expression were investigated in six cell lines and further correlated with tumor budding in patient cohort 1 (n = 185). Patient cohort 2 (n = 112) was used to assess prognostic effects. Laser capture microdissection (LCM) of tumor epithelium and stroma from low- and high-grade budding cancers was performed. In colorectal cancers, TWIST1 and TWIST2 expression was essentially restricted to stromal cells. LCM results of a high-grade budding case show positive TWIST1 and TWIST2 stroma and no methylation, while the low-grade budding case was characterized by negative stroma and strong hypermethylation. TWIST1 stromal cell staining was associated with adverse features like more advanced pT (p = 0.0044), lymph node metastasis (p = 0.0301), lymphatic vessel invasion (p = 0.0373), perineural invasion (p = 0.0109) and worse overall survival time (p = 0.0226). Stromal cells may influence tumor budding in colorectal cancers through expression of TWIST1. Hypermethylation of the tumor stroma may represent an alternative mechanism for regulation of TWIST1.

Tall cell papillary thyroid carcinoma: new diagnostic criteria and mutations in BRAF and TERT

The tall cell (TC) variant of papillary thyroid carcinoma (PTC) has an unfavorable prognosis. The diagnostic criteria remain inconsistent, and the role of a minor TC component is unclear. Molecular diagnostic markers are not available; however, there are two potential candidates: BRAF V600E and telomerase reverse transcriptase (TERT) promoter mutations. Using a novel approach, we enriched a collective with PTCs that harbored an adverse outcome, which overcame the limited statistical power of most studies. This enabled us to review 125 PTC patients, 57 of which had an adverse outcome. The proportion of TCs that constituted a poor prognosis was assessed. All of the tumors underwent sequencing for TERT promoter and BRAF V600E mutational status and were stained with an antibody to detect the BRAF V600E mutation. A 10% cutoff for TCs was significantly associated with advanced tumor stage and lymph node metastasis. Multivariate analysis showed that TCs above 10% were the only significant factor for overall, tumor-specific, and relapse-free survival. Seven percent of the cases had a TERT promoter mutation, whereas 61% demonstrated a BRAF mutation. The presence of TC was significantly associated with TERT promoter and BRAF mutations. TERT predicted highly significant tumor relapse (P<0.001). PTCs comprised of at least 10% TCs are associated with an adverse clinical outcome and should be reported accordingly. BRAF did not influence patient outcome. Nevertheless, a positive status should encourage the search for TCs. TERT promoter mutations are a strong predictor of tumor relapse, but their role as a surrogate marker for TCs is limited.

Chemopreventive function of retinoid X receptors in human squamous cell carcinoma of the skin

Retinoid therapy has been successful for the treatment of skin squamous cell carcinoma (SCC). A suppression of the predominant retinoid X receptor expressed in skin, retinoid X receptor α (RXRα), has been reported in skin SCC. These observations have led to the hypothesis that retinoid receptor loss contributes to the tumorigenic phenotype of epithelial cancers. To test this hypothesis, the RXRα gene was mapped in order to generate a targeting construct. Additionally the transcriptional regulation of the human RXRα a gene in keratinocytes was characterized after identifying the transcription initiation sites, the promoter, and enhancer regions of this gene. The structure is highly conserved between human and mouse. A nontumorigenic human skin-derived cell line called near diploid immortalized keratinocytes (NIKS) has the advantage of growing as organotypic raft cultures, under physiological conditions closely resembling in-vivo squamous stratification. We have exploited the raft culture technique to develop an in-vitro model for skin SCC progression that includes the NIKS cells, HaCaT cells, a premalignant cell line, and SRB 12-p9 cells, a tumorigenic SCC skin cell line. The differentiation, proliferation and nuclear receptor ligand response characteristics of this system were studied and significant and novel results were obtained. RXRs are obligate heterodimerization partners with many of the nuclear hormone receptors, including retinoic acid receptors (RARs), vitamin D3 receptors (VDR), thyroid hormone receptors (T3 R) and peroxisome proliferator activate receptors (PPARs), which are all known to be active in skin. Treatment of the three cell lines in raft culture with the RXR specific ligand BMS649, BMS961 (RARγ-specific), vitamin D3 (VDR ligand), thryoid hormone (T3R ligand) and clofibrate (PPARa ligand), and the combination of BMS649 with each of the 4 receptor partner ligands, resulted in distinct effects on differentiation, proliferation and apoptosis. The effects of activation of RXRs in each of the four-receptor pathways; in the context of skin SCC progression, with an emphasis on the VDR/RXR pathway, are discussed. These studies will lead to a better understanding of RXRα action in human skin and will help determine its role in SCC tumorigenesis, as well as its potential as a target for the prevention, treatment, and control of skin cancer. ^

An Sp1/Sp3 site polymorphism associated with hypermethylation of the candidate tumor suppressor gene RIL in cancer

Gene silencing due to promoter methylation is an alternative to mutations and deletions, which inactivate tumor suppressor genes (TSG) in cancer. We identified RIL by Methylated CpG Island Amplification technique as a novel aberrantly methylated gene. RIL is expressed in normal tissues and maps to the 5q31 region, frequently deleted in leukemias. We found methylation of RIL in 55/80 (69%) cancer cell lines, with highest methylation in leukemia and colon. We also observed methylation in 46/80 (58%) primary tumors, whereas normal tissues showed substantially lower degrees of methylation. RIL expression was lost in 13/16 cancer cell lines and was restored by demethylating agent. Screening of 38 cell lines and 13 primary cancers by SSCP revealed no mutations in RIL, suggesting that methylation and LOH are the primary inactivation mechanisms. Stable transfection of RIL into colorectal cancer cells resulted in reduction in cell growth, clonogenicity, and increased apoptosis upon UVC treatment, suggesting that RIL is a good candidate TSG. ^ In searching for a cause of RIL hypermethylation, we identified a 12-bp polymorphic sequence around the transcription start site of the gene that creates a long allele containing 3CTC repeat. Evolutionary studies suggested that the long allele appeared late in evolution due to insertion. Using bisulfite sequencing, in cancers heterozygous for RIL, we found that the short allele is 4.4-fold more methylated than the long allele (P = 0.003). EMSA results suggested binding of factor(s) to the inserted region of the long allele, but not to the short. EMSA mutagenesis and competition studies, as well as supershifts using nuclear extracts or recombinant Sp1 strongly indicated that those DNA binding proteins are Sp1 and Sp3. Transient transfections of RIL allele-specific expression constructs showed less than 2-fold differences in luciferase activity, suggesting no major effects of the additional Sp1 site on transcription. However, stable transfection resulted in 3-fold lower levels of transcription from the short allele 60 days post-transfection, consistent with the concept that the polymorphic Sp1 site protects against time-dependent silencing. Thus, an insertional polymorphism in the RIL promoter creates an additional Sp1/Sp3 site, which appears to protect it from silencing and methylation in cancer. ^

Molecular epidemiological studies on genetic predispositions to squamous cell carcinoma of the head and neck

Two molecular epidemiological studies were conducted to examine associations between genetic variation and risk of squamous cell carcinoma of the head and neck (SCCHN). In the first study, we hypothesized that genetic variation in p53 response elements (REs) may play roles in the etiology of SCCHN. We selected and genotyped five polymorphic p53 REs as well as a most frequently studied p53 codon 72 (Arg72Pro, rs1042522) polymorphism in 1,100 non-Hispanic White SCCHN patients and 1,122 age-and sex-matched cancer-free controls recruited at The University of Texas M. D. Anderson Cancer Center. In multivariate logistic regression analysis with adjustment for age, sex, smoking and drinking status, marital status and education level, we observed that the EOMES rs3806624 CC genotype had a significant effect of protection against SCCHN risk (adjusted odds ratio= 0.79, 95% confidence interval =0.64–0.98), compared with the -838TT+CT genotypes. Moreover, a significantly increased risk associated with the combined genotypes of p53 codon 72CC and EOMES -838TT+CT was observed, especially in the subgroup of non-oropharyneal cancer patients. The values of false-positive report probability were also calculated for significant findings. In the second study, we assessed the association between SCCHN risk and four potential regulatory single nucleotide polymorphisms (SNPs) of DEC1 (deleted in esophageal cancer 1) gene, a candidate tumor suppressor gene for esophageal cancer. After adjustment for age, sex, and smoking and drinking status, the variant -606CC (i.e., -249CC) homozygotes had a significantly reduced SCCHN risk (adjusted odds ratio = 0.71, 95% confidence interval = 0.52–0.99), compared with the -606TT homozygotes. Stratification analyses showed that a reduced risk associated with the -606CC genotype was more pronounced in subgroups of non-smokers, non-drinkers, younger subjects (defined as ≤ 57 years), carriers of TP53 Arg/Arg (rs1042522) genotype, patients with oropharyngeal cancer or late-stage SCCHN. Further in silico analysis revealed that the -249 T-to-C change led to a gain of a transcription factor binding site. Additional functional analysis showed that the -249T-to-C change significantly enhanced transcriptional activity of the DEC1 promoter and the DNA-protein binding activity. We conclude that the DEC1 promoter -249 T>C (rs2012775) polymorphism is functional, modulating susceptibility to SCCHN among non-Hispanic Whites. Additional large-scale, preferably population-based studies are needed to validate our findings.^