Abstract. miRNA profile deregulation affecting downstream signaling pathways activates endpoints that represent potential biomarkers for prognosis and treatment of tumor patients. In the past 20 years conventional therapy for osteosarcoma (OS) reached a survival plateau, highlighting the need for new therapeutic approaches. In this study, microarray unsupervised and supervised analysis identified, respectively, 100 and 40 differentially expressed miRNAs in OS samples with different grades of malignancy compared to normal bone. When analyzing low-grade and high-grade OS by unsupervised analysis, 12 miRNAs were found to be differentially expressed. Real-time PCR performed on a larger series of OS confirmed a significant lower expression of miR-1, miR-133b and miR-378 * in tumors with respect to control, also showing lower mRNA levels in 31 high-grade OS than in 25 low-grade and in metastatic versus non-metastatic patients. We demonstrated that miR-1 and miR133b were downregulated in OS cell lines compared to normal osteoblasts. Secondly, by transfection with miRNA precursor molecules, we demonstrated that the ectopic expression of miR-1 and miR-133b in U2-OS cell lines significantly reduced cell proliferation and MET protein expression and negatively regulated cell invasiveness and motility in a short-term assay. Cell cycle distribution revealed block in G 1 and delay of cell cycle progression associated with increased apoptosis in miR-1-and miR-133b-transfected cells, respectively. Our data assessed specific miRNA profiling deregulation in OS clinical samples and suggest that the expression of miR-1 and miR-133b may control cell proliferation and cell cycle through MET protein expression modulation. IntroductionOsteosarcoma (OS) is a rare malignant bone neoplasm for which biologic and pathologic information are still largely incomplete. OS has multiple genetic risk factors (1) and is characterized by complex chromosomal abnormalities and genetic cell heterogeneity that in over 70% provide a complex karyotype and drug resistance (2). MicroRNAs (miRNAs) are endogenous non-coding RNAs of 19-24 nucleotides that interacting with the 3' untranslated region (UTR) of mRNA target induce mRNA cleavage when pairing is complete and protein synthesis repression when pairing is incomplete. The relationship between miRNAs and their targets shows combinatorial complication, in terms of both target multiplicity and signal integration (3). MiRNAs are differentially expressed in relation to the developmental state, cell type and tissue (4-6). In literature, there are approximately 1,000 miRNA molecules per cell, with some cells exceeding 50,000 molecules (7). miRNA expression follows a dynamic range and this underscores the regulatory functional importance of miRNAs (3). Recent studies indicate that miRNAs called 'oncomirs' can function either as tumor suppressors or as oncogenes (8).Interconnection between miRNA and cancer is even more evident when analyzing the genomic location of known miRNA genes, more than 50% are in cancer-as...
Enhanced expression levels of NG2 proteoglycan in presurgical original lesions of soft-tissue sarcoma (STS) patients defines with 55% probability the immediate (i.e., within 12 months postsurgery) risk in these individuals to develop postsurgical secondary lesions, independently of any other clinical trait. It, therefore, provides a molecular factor that alone prospects a particularly unfavorable clinical outcome in such patients. Evaluation of the timing of metastasis formation in patients with high and low levels of NG2 in their primitive lesions further stratified the patients in subsets with diverse lag phases in the occurrence of metastatic disease. In our cohort of highgrade STS cases, transcription of NG2 also showed a 81-fold amplification in metastatic lesions, when compared to primitive ones, and this gene overexpression was accompanied by an abundant but nonuniform in situ expression of its product. In a similar manner as seen in primitive lesions, patients with higher levels of metastatic NG2 encountered a significantly more dismal clinical course. Multivariate analysis asserted that in these individuals upregulation of NG2 represented an absolute independent prognostic parameter. Therefore, minimally invasive assessment of the transcription levels of the NG2 gene represents a parameter capable of predicting the arising of metastatic disease within a definite postsurgery time interval, and affords in adjunct in the definition of life expectance in STS patients. ß
Although further studies are needed to evaluate miRNA involvement in osteosarcoma progression, miR-93 overexpression seems to play an important role in osteosarcoma cell growth and invasion.
Giant cell tumor of bone can be locally aggressive and occasionally can metastasize in the lungs. To identify new markers predictive of aggressive behavior, we analyzed five patients who developed lung metastasis and five who remained disease free for a minimum of 5 years. Using two-dimensional electrophoresis, we detected 28 differentially expressed spots. Fourteen spots were identified using mass spectrometry, including seven up-regulated and seven down-regulated in metastatic samples and classified according to functional categories. We then selected five proteins involved in cell cycle or apoptosis. Thioredoxin peroxidase, allograft inflammatory factor 1, and ubiquitin E2N had more than threefold up-regulation; glutathione peroxidase 1 had 1.9-fold up-regulation; and heat shock protein 27 showed down-regulation in metastatic samples with a very low P value. After validation and analysis of protein levels, evaluation of clinical impact was assessed in a much wider cohort of primary archival specimens. Immunodetection showed a higher frequency of thioredoxin peroxidase, allograft inflammatory factor 1, ubiquitin E2N, and glutathione peroxidase 1 overexpression in primary tumors that developed into lung metastases or that locally relapsed than in the disease-free group, with variable stain intensity and distribution. KaplanMeier analysis showed that high expression of glutathi- Giant cell tumor (GCT) is a benign bone tumor with fairly high local aggressiveness, and development of lung metastases is rare, occurring in 2% to 5% of cases.1 Histologically, the tumor pattern is formed by a network of spindle-shaped mononuclear stroma cells, round mononuclear histiocytic cells, and multinuclear giant cells similar to osteoclasts.2 Cellular components interact with various factors playing a role in osteoclast function regulation. In fact, precursors of osteoclasts express receptor activator of NF-B that in the presence of macrophage colonystimulating factor and its ligand, receptor activator of NF-B ligand, mediates osteoclast formation by increasing the expression of enzymes that dissolve organic and inorganic components of bone.3,4 At the same time, the endogenous osteoprotegerin counteracts these effects by competing for receptor activator of NF-B ligand and neutralizing it. These interactions may provide information to help develop new approaches to biological therapy of this tumor. Drugs that target the osteolytic process lower recurrence rates associated with morbidity and mortality and are considered useful for new clinical treatments. 5,6
Giant cell tumor of bone (GCTb) represents 5% of bone tumors, and although considered benign, 5% metastasize to the lung. The expression of proteins directly or indirectly associated with osteolysis and tumor growth was studied on 163 samples of GCTb. Of these, 33 patients developed lung metastasis during follow-up. The impact of tumor-host interaction on clinical aspects was evaluated with the aim of finding specific markers for new biological therapies, thus improving clinical management of GCTb. Protein expression was evaluated by immunohistochemical analysis on Tissue Microarray. The majority of GCTb samples from patients with metastatic disease were strongly positive to RANKL and its receptor RANK as well as to CAII and MMP-2 and to pro-survival proteins NFIB and c-Fos. Kaplan-Meier analysis indicated a significant difference in metastasis free survival curves based on protein staining. Interestingly, the statistical correlation established a strong association between all variables studied with a higher t coefficient for RANK/RANKL, RANK/NFIB, and RANKL/NFIB pairs. At multivariate analysis co-overexpression of NFIB, RANK and RANKL significantly increased the risk of metastasis with an odds ratio of 13.59 (95%CI 4.12-44.82; p < 0.0005). In conclusion, the interconnection between matrix remodeling and tumor cell activity may identify tumor-host endpoints for new biological treatments. Keywords: giant cell tumor; prognostic biomarkers; bone remodeling; metastasis GCTb is a locally aggressive tumor that occasionally metastasize to the lung. 1,2 It is characterized by a cellular network including multinuclear osteoclastlike giant cells, round cells, and spindle shaped mononuclear cells that represent a proliferative pattern. All cellular components interact with bone matrix mediating the release of soluble growth factors, cytokines, and transcription factors. 3 In a previous study, we selected a panel of proteins involved in cell cycle and apoptosis to identify highrisk patients and suggested the use of proteomic technique to identify new biomarkers associated with a more aggressive tumor behavior. 4 Our recent data demonstrated a significantly different expression of miR-136 between GCTb patients who progressed to lung metastases during follow-up and nonmetastatic patients, identifying the nuclear factor NFIB and the cytokine RANK as target genes involved in proliferation and osteolysis. 5 In GCTb, as well as in bone osteolytic tumors, massive bone resorption is triggered by the RANKL/RANK axis that stimulates osteoclast-dependent and -independent pathways 6 via activation of intracellular mediators such as TNF receptor associated factor (TRAF6), interleukins (IL), carbonic anhydrase II (CAII), metalloproteinases (MMPs), transcription factor c-Fos, and transforming growth factor (TGF-b). In the absence of RANKL inhibitors, a vicious cycle (bone destruction, tumor growth, and further bone destruction) supports tumor expansion. 7 The aim of the study was to find biological prognostic markers of GCTb tu...
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