Expression analysis of genes associated with human osteosarcoma tumors shows correlation of RUNX2 overexpression with poor response to chemotherapy

Background: Human osteosarcoma is the most common pediatric bone tumor. There is limited understanding of the molecular mechanisms underlying osteosarcoma oncogenesis, and a lack of good diagnostic as well as prognostic clinical markers for this disease. Recent discoveries have highlighted a potential role of a number of genes including: RECQL4, DOCK5, SPP1, RUNX2, RB1, CDKN1A, P53, IBSP, LSAMP, MYC, TNFRSF1B, BMP2, HISTH2BE, FOS, CCNB1, and CDC5L. Methods: Our objective was to assess relative expression levels of these 16 genes as potential biomarkers of osteosarcoma oncogenesis and chemotherapy response in human tumors. We performed quantitative expression analysis in a panel of 22 human osteosarcoma tumors with differential response to chemotherapy, and 5 normal human osteoblasts.Results: RECQL4, SPP1, RUNX2, and IBSP were significantly overexpressed, and DOCK5, CDKN1A, RB1, P53, and LSAMP showed significant loss of expression relative to normal osteoblasts. In addition to being overexpressed in osteosarcoma tumor samples relative to normal osteoblasts, RUNX2 was the only gene of the 16 to show significant overexpression in tumors that had a poor response to chemotherapy relative to good responders. Conclusion: These data underscore the loss of tumor suppressive pathways and activation of specific oncogenic mechanisms associated with osteosarcoma oncogenesis, while drawing attention to the role of RUNX2 expression as a potential biomarker of chemotherapy failure in osteosarcoma. © 2010 Sadikovic et al; licensee BioMed Central Ltd

Hoxa10 controls proliferation, migration and invasion in oral squamous cell carcinoma

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Although HOX genes are best known for acting in the regulation of important events during embryogenesis, including proliferation, differentiation and migration, alterations in their expression patterns have been frequently described in cancers. In previous studies we analyzed the expression profile of the members of the HOX family of homeobox genes in oral samples of normal mucosa and squamous cell carcinoma (OSCC) and identified differently expressed genes such as HOXA10. The present study aimed to validate the increased expression of HOXA10 in OSCCs, and to investigate the effects arising from its knockdown in OSCC cells. The levels of HOXA10 mRNA were determined in human OSCC samples and cell lines by quantitative PCR, and HOXA10- mediated effects on proliferation, apoptosis, adhesion, epithelial-mesenchymal transition (EMT), migration and invasion were studied in HSC-3 tongue carcinoma cells by using retrovirus-mediated RNA interference. Higher expression of HOXA10 mRNA was observed in OSCC cell lines and in tumor tissues compared to normal controls. HOXA10 knockdown significantly reduced the proliferation of the tumor cells which was accompanied by increased levels of p21. HOXA10 silencing also significantly induced the expression of EMT markers and enhanced the adhesion, migration and invasion of HSC-3 cells. No effects on cell death were observed after HOXA10 knockdown. The results of the current study confirm the overexpression of HOXA10 in OSCCs, and further demonstrate that its expression is functionally associated with several important biological processes related to oral tumorigenesis, such as proliferation, migration and invasion.Although HOX genes are best known for acting in the regulation of important events during embryogenesis, including proliferation, differentiation and migration, alterations in their expression patterns have been frequently described in cancers. In previou8436133623FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)sem informaçãosem informaçãoAlthough HOX genes are best known for acting in the regulation of important events during embryogenesis, including proliferation, differentiation and migration, alterations in their expression patterns have been frequently described in cancers. In previo

Optimization and analysis of a quantitative real-time PCR-based technique to determine microRNA expression in formalin-fixed paraffin-embedded samples

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRs) are non-coding RNA molecules involved in post-transcriptional regulation, with diverse functions in tissue development, differentiation, cell proliferation and apoptosis. miRs may be less prone to degradation during formalin fixation, facilitating miR expression studies in formalin-fixed paraffin-embedded (FFPE) tissue.</p> <p>Results</p> <p>Our study demonstrates that the TaqMan Human MicroRNA Array v1.0 (Early Access) platform is suitable for miR expression analysis in FFPE tissue with a high reproducibility (correlation coefficients of 0.95 between duplicates, p < 0.00001) and outlines the optimal performance conditions of this platform using clinical FFPE samples. We also outline a method of data analysis looking at differences in miR abundance between FFPE and fresh-frozen samples. By dividing the profiled miR into abundance strata of high (Ct<30), medium (30≤Ct≤35), and low (Ct>35), we show that reproducibility between technical replicates, equivalent dilutions, and FFPE <it>vs</it>. frozen samples is best in the high abundance stratum. We also demonstrate that the miR expression profiles of FFPE samples are comparable to those of fresh-frozen samples, with a correlation of up to 0.87 (p < 0.001), when examining all miRs, regardless of RNA extraction method used. Examining correlation coefficients between FFPE and fresh-frozen samples in terms of miR abundance reveals correlation coefficients of up to 0.32 (low abundance), 0.70 (medium abundance) and up to 0.97 (high abundance).</p> <p>Conclusion</p> <p>Our study thus demonstrates the utility, reproducibility, and optimization steps needed in miR expression studies using FFPE samples on a high-throughput quantitative PCR-based miR platform, opening up a realm of research possibilities for retrospective studies.</p

The interplay of matrix metalloproteinase-8, transforming growth factor-beta 1 and vascular endothelial growth factor-C cooperatively contributes to the aggressiveness of oral tongue squamous cell carcinoma

Background: Matrix metalloproteinase-8 (MMP-8) has oncosuppressive properties in various cancers. We attempted to assess MMP-8 function in oral tongue squamous cell carcinoma (OTSCC). Methods: MMP-8 overexpressing OTSCC cells were used to study the effect of MMP-8 on proliferation, apoptosis, migration, invasion and gene and protein expression. Moreover, MMP-8 functions were assessed in the orthotopic mouse tongue cancer model and by immunohistochemistry in patient samples. Results: MMP-8 reduced the invasion and migration of OTSCC cells and decreased the expression of MMP-1, cathepsin-K and vascular endothelial growth factor-C (VEGF-C). VEGF-C was induced by transforming growth factor-beta 1 (TGF-beta 1) in control cells, but not in MMP-8 overexpressing cells. In human OTSCC samples, low MMP-8 in combination with high VEGF-C was an independent predictor of poor cancer-specific survival. TGF-beta 1 treatment also restored the migration of MMP-8 overexpressing cells to the level of control cells. In mouse tongue cancer, MMP-8 did not inhibit metastasis, possibly because it was eliminated in the peripheral carcinoma cells. Conclusions: The suppressive effects of MMP-8 in OTSCC may be mediated through interference of TGF-beta 1 and VEGF-C function and altered proteinase expression. Together, low MMP-8 and high VEGF-C expression have strong independent prognostic value in OTSCC.Peer reviewe

Design and protocol of the multimorbidity and mental health cohort study in frailty and aging (MiMiCS-FRAIL):unraveling the clinical and molecular associations between frailty, somatic disease burden and late life depression

BACKGROUND: To explore the mutual relationship between multimorbidity, mental illness and frailty, we have set-up the Multimorbidity and Mental health Cohort Study in FRAILty and Aging (MiMiCS-FRAIL) cohort. At the population level, multimorbidity, frailty and late-life depression are associated with similar adverse outcomes (i.e. falls, disability, hospitalization, death), share the same risk factors, and partly overlap in their clinical presentation. Moreover, these three variables may share a common underlying pathophysiological mechanism like immune-metabolic dysregulation. The overall objectives of MiMiCS-FRAIL are 1) to explore (determinants of) the cross-sectional and longitudinal relationship between multimorbidity, depression, and frailty among non-demented geriatric outpatients; 2) to evaluate molecular levels of senoinflammation as a broad pathophysiological process underlying these conditions; and 3) to examine adverse outcomes of multimorbidity, frailty and depression and their interconnectedness. METHODS: MiMiCS-FRAIL is an ongoing observational cohort study of geriatric outpatients in Brazil, with an extensive baseline assessment and yearly follow-up assessments. Each assessment includes a comprehensive geriatric assessment to identify multimorbidity and geriatric syndromes, a structured psychiatric diagnostic interview and administration of the PHQ-9 to measure depression, and several frailty measures (FRAIL, Physical Phenotype criteria, 36-item Frailty Index). Fasten blood samples are collected at baseline to assess circulating inflammatory and anti-inflammatory cytokines, leukocytes' subpopulations, and to perform immune-metabolic-paired miRome analyses. The primary outcome is death and secondary outcomes are the number of falls, hospital admissions, functional ability, well-being, and dementia. Assuming a 5-year mortality rate between 25 and 40% and a hazard rate varying between 1.6 and 2.3 for the primary determinants require a sample size between 136 and 711 patients to detect a statistically significant effect with a power of 80% (beta = 0.2), an alpha of 5% (0.05), and an R2 between the predictor (death) and all covariates of 0.20. Local ethical board approved this study. DISCUSSION: Frailty might be hypothesized as a final common pathway by which many clinical conditions like depression and chronic diseases (multimorbidity) culminate in many adverse effects. The MiMiCS-FRAIL cohort will help us to understand the interrelationship between these variables, from a clinical perspective as well as their underlying molecular signature

Polypodium leucotomos targets multiple aspects of oral carcinogenesis and it is a potential antitumor phytotherapy against tongue cancer growth

Introduction: Oral cancer refers to malignant tumors, of which 90% are squamous cell carcinomas (OSCCs). These malignancies exhibit rapid progression, poor prognosis, and often mutilating therapeutical approaches. The determination of a prophylactic and/or therapeutic antitumor role of the polyphenolic extract Polypodium leucotomos(PL) would be relevant in developing new tools for prevention and treatment.Methods: We aimed to determine the antitumor effect of PL by treating OSCC cell lines with PL metabolites and evaluating its action during OSCC progression in vivo.Results: PL treatment successfully impaired cell cycling and proliferation, migration, and invasion, enhanced apoptosis, and modulated macrophage polarization associated with the tumoral immune-inflammatory response of tongue cancer cell lines (TSCC). PL treatment significantly decreased the expression of MMP1 (p &lt; 0.01) and MMP2 (p &lt; 0.001), and increased the expression of TIMP1 (p &lt; 0.001) and TIMP2 (p &lt; 0.0001) in these cells. The mesenchymal-epithelial transition phenotype was promoted in cells treated with PL, through upregulation of E-CAD (p &lt; 0.001) and reduction of N-CAD (p &lt; 0.05). PL restrained OSCC progression in vivo by inhibiting tumor volume growth and decreasing the number of severe dysplasia lesions and squamous cell carcinomas. Ki-67 was significantly higher expressed in tongue tissues of animals not treated with PL(p &lt; 0.05), and a notable reduction in Bcl2 (p &lt; 0.05) and Pcna (p &lt; 0.05) cell proliferation-associated genes was found in dysplastic lesions and TSCCs of PL-treated mice. Finally, N-cad(Cdh2), Vim, and Twist were significantly reduced in tongue tissues treated with PL.Conclusion: PL significantly decreased OSCC carcinogenic processes in vitro and inhibited tumor progression in vivo. PL also appears to contribute to the modulation of immune-inflammatory oral tumor-associated responses. Taken together, these results suggest that PL plays an important antitumor role in processes associated with oral carcinogenesis and may be a potential phytotherapeutic target for the prevention and/or adjuvant treatment of TSCC

MicroRNA and protein profiles in invasive versus non-invasive oral tongue squamous cell carcinoma cells in vitro

Complex molecular pathways regulate cancer invasion. This study overviewed proteins and microRNAs (miRNAs) involved in oral tongue squamous cell carcinoma (OTSCC) invasion. The human highly aggressive OTSCC cell line HSC-3 was examined in a 3D organotypic human leiomyoma model. Non-invasive and invasive cells were laser-captured and protein expression was analyzed using mass spectrometry-based proteomics and miRNA expression by microarray. In functional studies the 3D invasion assay was replicated after silencing candidate miRNAs, miR-498 and miR-940, in invasive OTSCC cell lines (HSC-3 and SCC-15). Cell migration, proliferation and viability were also studied in the silenced cells. In HSC-3 cells, 67 proteins and 53 miRNAs showed significant fold-changes between non-invasive vs. invasive cells. Pathway enrichment analyses allocated "Focal adhesion" and "ECM-receptor interaction" as most important for invasion. Significantly, in HSC-3 cells, miR-498 silencing decreased the invasion area and miR-940 silencing reduced invasion area and depth. Viability, proliferation and migration weren't significantly affected. In SCC-15 cells, down-regulation of miR-498 significantly reduced invasion and migration. This study shows HSC-3 specific miRNA and protein expression in invasion, and suggests that miR-498 and miR-940 affect invasion in vitro, the process being more influenced by mir-940 silencing in aggressive HSC-3 cells than in the less invasive SCC-15.Peer reviewe

Programmed cell death 4 loss increases tumor cell invasion and is regulated by miR-21 in oral squamous cell carcinoma

<p>Abstract</p> <p>Background</p> <p>The tumor suppressor Programmed Cell Death 4 (<it>PDCD4</it>) has been found to be under-expressed in several cancers and associated with disease progression and metastasis. There are no current studies characterizing PDCD4 expression and its clinical relevance in Oral Squamous Cell Carcinoma (OSCC). Since nodal metastasis is a major prognostic factor in OSCC, we focused on determining whether PDCD4 under-expression was associated with patient nodal status and had functional relevance in OSCC invasion. We also examined <it>PDCD4 </it>regulation by microRNA 21 (miR-21) in OSCC.</p> <p>Results</p> <p><it>PDCD4 </it>mRNA expression levels were assessed in 50 OSCCs and 25 normal oral tissues. <it>PDCD4 </it>was under-expressed in 43/50 (86%) OSCCs, with significantly reduced mRNA levels in patients with nodal metastasis (<it>p = 0.0027</it>), and marginally associated with T3-T4 tumor stage (<it>p = 0.054</it>). PDCD4 protein expression was assessed, by immunohistochemistry (IHC), in 28/50 OSCCs and adjacent normal tissues; PDCD4 protein was absent/under-expressed in 25/28 (89%) OSCCs, and marginally associated with nodal metastasis (<it>p = 0.059</it>). A matrigel invasion assay showed that PDCD4 expression suppressed invasion, and siRNA-mediated PDCD4 loss was associated with increased invasive potential of oral carcinoma cells. Furthermore, we showed that miR-21 levels were increased in PDCD4-negative tumors, and that <it>PDCD4 </it>expression may be down-regulated in OSCC by direct binding of miR-21 to the 3'UTR <it>PDCD4 </it>mRNA.</p> <p>Conclusions</p> <p>Our data show an association between the loss of PDCD4 expression, tumorigenesis and invasion in OSCC, and also identify a mechanism of PDCD4 down-regulation by microRNA-21 in oral carcinoma. PDCD4 association with nodal metastasis and invasion suggests that PDCD4 may be a clinically relevant biomarker with prognostic value in OSCC.</p

Integrated proteomics identified up-regulated focal adhesion-mediated proteins in human squamous cell carcinoma in an orthotopic murine model

Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by combining an orthotopic murine model of oral squamous cell carcinoma (OSCC), mass spectrometry-based proteomics and biological network analysis. Our results indicated the up-regulation of proteins involved in actin cytoskeleton organization and cell-cell junction assembly events and their expression was validated in human OSCC tissues. In addition, the functional relevance of talin-1 in OSCC adhesion, migration and invasion was demonstrated. Taken together, this study identified specific processes deregulated in oral cancer and provided novel refined OSCC-targeting molecules.Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by co95e98208FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO2009/54067-3; 2010/19278-0; 2011/22421-2; 2009/53839-2; 2011/02267-9470567/2009-0; 470549/2011-4; 301702/2011-0; 470268/2013-1; 505413/2013-

Extracellular vesicles derived from cancer-associated fibroblasts induce the migration and invasion of oral squamous cell carcinoma

As one of the most abundant constituents of the tumour microenvironment (TME), cancer-associated fibroblasts (CAF) display critical roles during tumour progression and metastasis. Multiple classes of molecules including growth factors, cytokines, proteases and extracellular matrix proteins, are produced by CAF to act as mediators of the stroma-tumour interactions. One of the main channels for this communication is associated with extracellular vesicles (EV), which are secreted particles loaded with protein and genetic information. In this study, we evaluated the effects of EV derived from CAF primary human cell lines (n = 5) on proliferation, survival, migration, and invasion of oral squamous cell carcinoma (OSCC) cells. As controls, EV from human primary-established normal oral fibroblasts (NOF, n = 5) were used. Our in vitro assays showed that CAF-EV significantly induces migration and invasion of OSCC cells and promote a disseminated pattern of HSC-3 cell invasion in the 3D organotypic assay. Furthermore, gene expression analysis of EV-treated cancer cells revealed changes in the pathways associated with tumour metabolism and up-regulation of tumour invasion genes. Our findings suggest a significant role of CAF-EV in promoting the migration and invasion of OSCC cells, which are related to the activation of cancer-related pathways.Peer reviewe