An approach for the identification of targets specific to bone metastasis using cancer genes interactome and gene ontology analysis

Metastasis is one of the most enigmatic aspects of cancer pathogenesis and is a major cause of cancer-associated mortality. Secondary bone cancer (SBC) is a complex disease caused by metastasis of tumor cells from their primary site and is characterized by intricate interplay of molecular interactions. Identification of targets for multifactorial diseases such as SBC, the most frequent complication of breast and prostate cancers, is a challenge. Towards achieving our aim of identification of targets specific to SBC, we constructed a 'Cancer Genes Network', a representative protein interactome of cancer genes. Using graph theoretical methods, we obtained a set of key genes that are relevant for generic mechanisms of cancers and have a role in biological essentiality. We also compiled a curated dataset of 391 SBC genes from published literature which serves as a basis of ontological correlates of secondary bone cancer. Building on these results, we implement a strategy based on generic cancer genes, SBC genes and gene ontology enrichment method, to obtain a set of targets that are specific to bone metastasis. Through this study, we present an approach for probing one of the major complications in cancers, namely, metastasis. The results on genes that play generic roles in cancer phenotype, obtained by network analysis of 'Cancer Genes Network', have broader implications in understanding the role of molecular regulators in mechanisms of cancers. Specifically, our study provides a set of potential targets that are of ontological and regulatory relevance to secondary bone cancer.Comment: 54 pages (19 pages main text; 11 Figures; 26 pages of supplementary information). Revised after critical reviews. Accepted for Publication in PLoS ON

Prognostic Biomarkers for Esophageal Adenocarcinoma Identified by Analysis of Tumor Transcriptome

Despite many attempts to establish pre-treatment prognostic markers to understand the clinical biology of esophageal adenocarcinoma (EAC), validated clinical biomarkers or parameters remain elusive. We generated and analyzed tumor transcriptome to develop a practical biomarker prognostic signature in EAC.Untreated esophageal endoscopic biopsy specimens were obtained from 64 patients undergoing surgery and chemoradiation. Using DNA microarray technology, genome-wide gene expression profiling was performed on 75 untreated cancer specimens from 64 EAC patients. By applying various statistical and informatical methods to gene expression data, we discovered distinct subgroups of EAC with differences in overall gene expression patterns and identified potential biomarkers significantly associated with prognosis. The candidate marker genes were further explored in formalin-fixed, paraffin-embedded tissues from an independent cohort (52 patients) using quantitative RT-PCR to measure gene expression. We identified two genes whose expression was associated with overall survival in 52 EAC patients and the combined 2-gene expression signature was independently associated with poor outcome (P<0.024) in the multivariate Cox hazard regression analysis.Our findings suggest that the molecular gene expression signatures are associated with prognosis of EAC patients and can be assessed prior to any therapy. This signature could provide important improvement for the management of EAC patients

OPN/CD44v6 overexpression in laryngeal dysplasia and correlation with clinical outcome

Laryngeal dysplasia is a common clinical concern. Despite major advancements, a significant number of patients with this condition progress to invasive squamous cell carcinoma. Osteopontin (OPN) is a secreted glycoprotein, whose expression is markedly elevated in several types of cancers. We explored OPN as a candidate biomarker for laryngeal dysplasia. To this aim, we examined OPN expression in 82 cases of dysplasia and in hyperplastic and normal tissue samples. OPN expression was elevated in all severe dysplasia samples, but not hyperplastic samples, with respect to matched normal mucosa. OPN expression levels correlated positively with degree of dysplasia (P=0.0094) and negatively with disease-free survival (P<0.0001). OPN expression was paralleled by cell surface reactivity for CD44v6, an OPN functional receptor. CD44v6 expression correlated negatively with disease-free survival, as well (P=0.0007). Taken as a whole, our finding identify OPN and CD44v6 as predictive markers of recurrence or aggressiveness in laryngeal intraepithelial neoplasia, and overall, point out an important signalling complex in the evolution of laryngeal dysplasia

Degradation, Bioactivity, and Osteogenic Potential of Composites Made of PLGA and Two Different Sol–Gel Bioactive Glasses

We have developed poly(l-lactide-co-glycolide) (PLGA) based composites using sol–gel derived bioactive glasses (S-BG), previously described by our group, as composite components. Two different composite types were manufactured that contained either S2—high content silica S-BG, or A2—high content lime S-BG. The composites were evaluated in the form of sheets and 3D scaffolds. Sheets containing 12, 21, and 33 vol.% of each bioactive glass were characterized for mechanical properties, wettability, hydrolytic degradation, and surface bioactivity. Sheets containing A2 S-BG rapidly formed a hydroxyapatite surface layer after incubation in simulated body fluid. The incorporation of either S-BG increased the tensile strength and Young’s modulus of the composites and tailored their degradation rates compared to starting compounds. Sheets and 3D scaffolds were evaluated for their ability to support growth of human bone marrow cells (BMC) and MG-63 cells, respectively. Cells were grown in non-differentiating, osteogenic or osteoclast-inducing conditions. Osteogenesis was induced with either recombinant human BMP-2 or dexamethasone, and osteoclast formation with M-CSF. BMC viability was lower at higher S-BG content, though specific ALP/cell was significantly higher on PLGA/A2-33 composites. Composites containing S2 S-BG enhanced calcification of extracellular matrix by BMC, whereas incorporation of A2 S-BG in the composites promoted osteoclast formation from BMC. MG-63 osteoblast-like cells seeded in porous scaffolds containing S2 maintained viability and secreted collagen and calcium throughout the scaffolds. Overall, the presented data show functional versatility of the composites studied and indicate their potential to design a wide variety of implant materials differing in physico-chemical properties and biological applications. We propose these sol–gel derived bioactive glass–PLGA composites may prove excellent potential orthopedic and dental biomaterials supporting bone formation and remodeling

Hepatitis B Virus X Protein Drives Multiple Cross-Talk Cascade Loops Involving NF-κB, 5-LOX, OPN and Capn4 to Promote Cell Migration

Hepatitis B virus X protein (HBx) plays an important role in the development of hepatocellular carcinoma (HCC). However, the mechanism remains unclear. Recently, we have reported that HBx promotes hepatoma cell migration through the upregulation of calpain small subunit 1 (Capn4). In addition, several reports have revealed that osteopontin (OPN) plays important roles in tumor cell migration. In this study, we investigated the signaling pathways involving the promotion of cell migration mediated by HBx. We report that HBx stimulates several factors in a network manner to promote hepatoma cell migration. We showed that HBx was able to upregulate the expression of osteopontin (OPN) through 5-lipoxygenase (5-LOX) in HepG2-X/H7402-X (stable HBx-transfected cells) cells. Furthermore, we identified that HBx could increase the expression of 5-LOX through nuclear factor-κB (NF-κB). We also found that OPN could upregulate Capn4 through NF-κB. Interestingly, we showed that Capn4 was able to upregulate OPN through NF-κB in a positive feedback manner, suggesting that the OPN and Capn4 proteins involving cell migration affect each other in a network through NF-κB. Importantly, NF-κB plays a crucial role in the regulation of 5-LOX, OPN and Capn4. Thus, we conclude that HBx drives multiple cross-talk cascade loops involving NF-κB, 5-LOX, OPN and Capn4 to promote cell migration. This finding provides new insight into the mechanism involving the promotion of cell migration by HBx

Molecular analysis of metastasis in a polyomavirus middle T mouse model: the role of osteopontin

INTRODUCTION: In order to study metastatic disease, we employed the use of two related polyomavirus middle T transgenic mouse tumor transplant models of mammary carcinoma (termed Met and Db) that display significant differences in metastatic potential. METHODS: Through suppression subtractive hybridization coupled to the microarray, we found osteopontin (OPN) to be a highly expressed gene in the tumors of the metastatic mouse model, and a lowly expressed gene in the tumors of the lowly metastatic mouse model. We further analyzed the role of OPN in this model by examining sense and antisense constructs using in vitro and in vivo methods. RESULTS: With in vivo metastasis assays, the antisense Met cells showed no metastatic tumor formation to the lungs of recipient mice, while wild-type Met cells, with higher levels of OPN, showed significant amounts of metastasis. The Db cells showed a significantly reduced metastasis rate in the in vivo metastasis assay as compared with the Met cells. Db cells with enforced overexpression of OPN showed elevated levels of OPN but did not demonstrate an increase in the rate of metastasis compared with the wild-type Db cells. CONCLUSIONS: We conclude that OPN is an essential regulator of the metastatic phenotype seen in polyomavirus middle T-induced mammary tumors. Yet OPN expression alone is not sufficient to cause metastasis. These data suggest a link between metastasis and phosphatidylinositol-3-kinase-mediated transcriptional upregulation of OPN, but additional phosphatidylinositol-3-kinase-regulated genes may be essential in precipitating the metastasis phenotype in the polyomavirus middle T model