An Exosomal miRNA Biomarker for the Detection of Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) remains a difficult tumor to diagnose and treat. To date, PDAC lacks routine screening with no markers available for early detection. Exosomes are 40-150 nm-sized extracellular vesicles that contain DNA, RNA, and proteins. These exosomes are released by all cell types into circulation and thus can be harvested from patient body fluids, thereby facilitating a non-invasive method for PDAC detection. A bioinformatics analysis was conducted utilizing publicly available miRNA pancreatic cancer expression and genome databases. Through this analysis, we identified 18 miRNA with strong potential for PDAC detection. From this analysis, 10 (MIR31, MIR93, MIR133A1, MIR210, MIR330, MIR339, MIR425, MIR429, MIR1208, and MIR3620) were chosen due to high copy number variation as well as their potential to differentiate patients with chronic pancreatitis, neoplasms, and PDAC. These 10 were examined for their mature miRNA expression patterns, giving rise to 18 mature miRs for further analysis. Exosomal RNA from cell culture media was analyzed via RTqPCR and seven mature miRs exhibited statistical significance (miR-31-5p, miR-31-3p, miR-210-3p, miR-339-5p, miR-425-5p, miR-425-3p, and miR-429). These identified biomarkers can potentially be used for early detection of PDAC.

Crystallinity of anodic TiO2 nanotubes and bioactivity.

Anodic TiO2 nanotubes were produced on titanium at 20 V using 1 M Na2SO4 and 0.5 wt% NaF. Oxidation for 3 hours produced amorphous tubes of diameter 100 nm and thicknesses 2 microm. Heat-treatments were done for 3 hours at different temperatures. 300 degrees C treatment converted the amorphous coatings to anatase. 550 and 700 degrees C treatments formed dual anatase and rutile; 850 degrees C treatment crystallized to rutile. The treatment at 700 degrees C produced an oxide surface with higher roughness, lower wetting angle and higher coating adhesion. Bioactivity of the as-oxidized and heated coatings were evaluated by treating them in a simulated body fluid (SBF) to form hydroxyapatite (HA) and the rates of HA formation were compared. Deposits of HA could be seen on the dual oxide structure within 3 days. HA was detected after 7 days in the anatase structure and only after 21 days in the amorphous and rutile structures. In vitro cell culture tests done using mouse osteoblasts indicated that, the 700 degrees C-heated surface showed higher levels of cell activity than the other surfaces. It is concluded that the dual rutile and anatase structure formed by heating the oxide at 700 degrees C is the best of the five surfaces tested.

Ti-9Mn ß-type alloy exhibits better osteogenicity than Ti-15Mn alloy in vitro.

Ti-9Mn and Ti-15Mn were prepared using an arc furnace in order to understand their osteogenic behavior as a biomedical implant. Ti-9Mn surface showed a significantly lower contact angle value (41%) as compared with the Ti-15Mn surface. The higher Ra and lower hydrophilicity values of Ti-9Mn alloy as compared with Ti-15Mn alloy indicates that Ti-9Mn can have better osteoconductive properties. ALP activity of the osteoblast cells on the Ti-9Mn alloy was elevated by 45% on day 7 and 20% on day 14 as compared to the Ti-15Mn alloy that reflects faster induction of osteoblast phenotypes of MG63 cells. Filopodia and lamellipodia structures were spread more on the Ti-9Mn specimens as compared to the Ti-15Mn alloy. Cell viability on Ti-9Mn alloy increased by 25% and 32%, respectively after 7 and 14 days of culture as compared to Ti-15Mn alloy. On day 14 of culture, the relative expression of RUNX2, COL1, and OC on Ti-9Mn alloy were elevated by 35%, 21%, and 30% respectively than the Ti-15Mn alloy. Ti-9Mn alloy also exhibited an inductive effect on the cell proliferation, and upregulation in the expression of ALP, RUNX2, and OC that is, the genes related to osteoblastic differentiation. Hence, the present in vitro results suggest that Ti-9Mn can be a preferred implant material than the Ti-15Mn alloy.

Mesenchymal Stem Cell-Secreted Extracellular Vesicles Instruct Stepwise Dedifferentiation of Breast Cancer Cells into Dormancy at the Bone Marrow Perivascular Region.

In the bone marrow (BM), breast cancer cells (BCC) can survive in dormancy for decades as cancer stem cells (CSC), resurging as tertiary metastasis. The endosteal region where BCCs exist as CSCs poses a challenge to target them, mostly due to the coexistence of endogenous hematopoietic stem cells. This study addresses the early period of dormancy when BCCs enter BM at the perivascular region to begin the transition into CSCs, which we propose as the final step in dormancy. A two-step process comprises the Wnt-ß-catenin pathway mediating BCC dedifferentiation into CSCs at the BM perivascular niche. At this site, BCCs responded to two types of mesenchymal stem cell (MSC)-released extracellular vesicles (EV) that may include exosomes. Early released EVs began the transition into cycling quiescence, DNA repair, and reorganization into distinct BCC subsets. After contact with breast cancer, the content of EVs changed (primed) to complete dedifferentiation into a more homogeneous population with CSC properties. BCC progenitors (Oct4alo), which are distant from CSCs in a hierarchical stratification, were sensitive to MSC EVs. Despite CSC function, Oct4alo BCCs expressed multipotent pathways similar to CSCs. Oct4alo BCCs dedifferentiated and colocalized with MSCs (murine and human BM) in vivo. Overall, these findings elucidate a mechanism of early dormancy at the BM perivascular region and provide evidence of epigenome reorganization as a potential new therapy for breast cancer. SIGNIFICANCE: These findings describe how the initial process of dormancy and dedifferentiation of breast cancer cells at the bone marrow perivascular niche requires mesenchymal stem cell-derived exosomes, indicating a potential target for therapeutic intervention.

Bioinformatics approaches to cancer gene discovery.

The Cancer Gene Anatomy Project (CGAP) database of the National Cancer Institute has thousands of known and novel expressed sequence tags (ESTs). These ESTs, derived from diverse normal and tumor cDNA libraries, offer an attractive starting point for cancer gene discovery. Data-mining the CGAP database led to the identification of ESTs that were predicted to be specific to select solid tumors. Two genes from these efforts were taken to proof of concept for diagnostic and therapeutics indications of cancer. Microarray technology was used in conjunction with bioinformatics to understand the mechanism of one of the targets discovered. These efforts provide an example of gene discovery by using bioinformatics approaches. The strengths and weaknesses of this approach are discussed in this review.

Mining Exosomal Genes for Pancreatic Cancer Targets.

BACKGROUND: Exosomes, cell-derived vesicles encompassing lipids, DNA, proteins coding genes and noncoding RNAs (ncRNAs) are present in diverse body fluids. They offer novel biomarker and drug therapy potential for diverse diseases, including cancer. MATERIALS AND METHODS: Using gene ontology, exosomal genes database and GeneCards metadata analysis tools, a database of cancer-associated protein coding genes and ncRNAs (n=2,777) was established. Variant analysis, expression profiling and pathway mapping were used to identify putative pancreatic cancer exosomal gene candidates. RESULTS: Five hundred and seventy-five protein-coding genes, 26 RNA genes and one pseudogene directly associated with pancreatic cancer were identified in the study. Nine open reading frames (ORFs) encompassing enzymes, apoptosis and transcriptional regulators, and secreted factors and five cDNAs (enzymes) emerged from the analysis. Among the ncRNA class, 26 microRNAs (miRs), one pseudogene, one long noncoding RNA (LNC) and one antisense gene were identified. Furthermore, 22 exosome-associated protein-coding targets (a cytokine, enzymes, membrane glycoproteins, receptors, and a transporter) emerged as putative leads for pancreatic cancer therapy. Seven of these protein-coding targets are FDA-approved and the drugs-based on these could provide repurposing opportunities for pancreatic cancer. CONCLUSION: The database of exosomal genes established in this study provides a framework for developing novel biomarkers and drug therapy targets for pancreatic cancer.

Druggable cancer secretome: neoplasm-associated traits.

BACKGROUND: The genome association databases provide valuable clues to identify novel targets for cancer diagnosis and therapy. Genes harboring phenotype-associated polymorphisms for neoplasm traits can be identified using diverse bioinformatics tools. The recent availability of various protein expression datasets from normal human tissues, including the body fluids, enables for baseline expression profiling of the cancer secretome. Chemoinformatics approaches can help identify drug-like compounds from the protein 3D structures. MATERIALS AND METHODS: The National Center for Biotechnology Information (NCBI) Phenome Genome Integrator (PheGenI) tool was enriched for neoplasm-associated traits. The neoplasm genes were characterized using diverse bioinformatics tools for pathways, gene ontology, genome-wide association, protein expression and functional class. Chemogenomics analysis was performed using the canSAR protein annotation tool. RESULTS: The neoplasm-associated traits segregated into 1,305 genes harboring 2,837 single nucleotide polymorphisms (SNPs). Also identified were 65 open reading frames (ORFs) encompassing 137 SNPs. The neoplasm genes and the associated SNPs were classified into distinct tumor types. Protein expression in the secretome was seen for 913 of the neoplasm-associated genes, including 17 novel uncharacterized ORFs. Druggable proteins, including enzymes, transporters, channel proteins and receptors, were detected. Thirty-four novel druggable lead genes emerged from these studies, including seven cancer lead targets. Chemogenomics analysis using the canSAR protein annotation tool identified 168 active compounds (<1 µM) for the neoplasm genes in the body fluids. Among these, 7 most active lead compounds with drug-like properties (1-600 nM) were identified for the cancer lead targets, encompassing enzymes and receptors. CONCLUSION: Over seventy percent of the neoplasm trait-associated genes were detected in the body fluids, such as ascites, blood, tear, milk, semen, urine, etc. Ligand-based druggabililty analysis helped establish lead prioritization. The association of these proteins with diverse cancer types and other diseases provides a framework to develop novel diagnosis and therapy targets.

Phenome-genome association studies of pancreatic cancer: new targets for therapy and diagnosis.

BACKGROUND: Pancreatic cancer, has a very high mortality rate and requires novel molecular targets for diagnosis and therapy. Genetic association studies over databases offer an attractive starting point for gene discovery. MATERIALS AND METHODS: The National Center for Biotechnology Information (NCBI) Phenome Genome Integrator (PheGenI) tool was enriched for pancreatic cancer-associated traits. The genes associated with the trait were characterized using diverse bioinformatics tools for Genome-Wide Association (GWA), transcriptome and proteome profile and protein classes for motif and domain. RESULTS: Two hundred twenty-six genes were identified that had a genetic association with pancreatic cancer in the human genome. This included 25 uncharacterized open reading frames (ORFs). Bioinformatics analysis of these ORFs identified putative druggable proteins and biomarkers including enzymes, transporters and G-protein-coupled receptor signaling proteins. Secreted proteins including a neuroendocrine factor and a chemokine were identified. Five out of these ORFs encompassed non coding RNAs. The ORF protein expression was detected in numerous body fluids, such as ascites, bile, pancreatic juice, milk, plasma, serum and saliva. Transcriptome and proteome analyses showed a correlation of mRNA and protein expression for nine ORFs. Analysis of the Catalogue of Somatic Mutations in Cancer (COSMIC) database revealed a strong correlation across copy number variations and mRNA over-expression for four ORFs. Mining of the International Cancer Gene Consortium (ICGC) database identified somatic mutations in a significant number of pancreatic patients' tumors for most of these ORFs. The pancreatic cancer-associated ORFs were also found to be genetically associated with other neoplasms, including leukemia, malignant melanoma, neuroblastoma and prostate carcinomas, as well as other unrelated diseases and disorders, such as Alzheimer's disease, Crohn's disease, coronary diseases, attention deficit disorder and addiction. CONCLUSION: Based on Genome-Wide Association Studies (GWAS), copy number variations, somatic mutational status and correlation of gene expression in pancreatic tumors at the mRNA and protein level, expression specificity in normal tissues and detection in body fluids, six ORFs emerged as putative leads for pancreatic cancer. These six targets provide a basis for accelerated drug discovery and diagnostic marker development for pancreatic cancer.

Down's syndrome-associated Single Minded 2 gene as a pancreatic cancer drug therapy target.

We report here a pancreatic cancer drug therapy utility of a gene involved in Down's syndrome. Single Minded 2 gene (SIM2) from Down's Syndrome Critical Region was expressed in pancreatic cancer-derived cell lines as well as in tumor tissues, but not in the normal pancreas. A related member of the SIM family, SIM1, did not show similar specificity. Inhibition by antisense technology of one of the isoforms of SIM2, the short-form (SIM2-s) expression in the CAPAN-1 pancreatic cancer cell line, caused a pronounced growth inhibition and induced cell death through apoptosis. The specificity of antisense was inferred from inhibition of SIM2-s mRNA but not the related members of SIM family. In view of the high mortality rate of pancreatic cancer patients, these findings have important implications for the future of pancreatic cancer treatment.

Down's syndrome-associated single minded gene as a novel tumor marker.

The Cancer Genome Anatomy Project (CGAP) database has thousands of Expressed Sequence Tags encompassing both known and novel genes. Bioinformatics of the CGAP database led to the prediction that Single Minded Gene (sim2) could be specific to colon tumors. The sim2 gene is located in a minimum region of the chromosome 21 often implicated in trisomia called Down's Syndrome Critical Region. To date, the sim proteins have not been shown to be involved in cancer. Intrigued by the possible association of a Down's syndrome-related gene to solid tumors, efforts were undertaken to validate the expression specificity. The sim2 isoform (sim2-short-form, sim2-s) expression was seen in carcinomas of colon, pancreas and prostate, but not in corresponding normal tissues. Stage-specific expression of the sim2-s protein was seen in normal matched paraffin sections of the colon tumors. In a matched set of tissues of Benign Prostatic Hyperplasia (BPH) and prostate carcinomas, sim2-s expression was detected in the BPH. The expression specificity of sim2-s in select solid tumors offers both diagnostic and therapeutic potential and warrants additional study.

Bioinformatics-based discovery of a novel factor with apparent specificity to colon cancer.

In a previous study, a data mining tool called Digital Differential Display (DDD) from the Cancer Genome Anatomy Project (CGAP) was used to predict solid tumor- and organ-specific genes from the expressed sequence tag (EST) database. To validate the use of bioinformatics approaches in gene discovery, one of the ESTs, which was predicted to be colon tumor-specific, was chosen for further study. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis of matched sets of cDNAs from normal and colon tumor tissues indicated that the EST was specifically expressed in the majority of colon tumors. Expression was also detected in early adenomas. Among other normal tissues, EST expression was detected only in the small intestine. The colon tumor specificity of this EST was inferred from the lack of expression in carcinomas of the breast, lung, ovary, pancreas and prostate. To validate the computational prediction of specificity, a full-length cDNA encompassing the entire open reading frame was cloned and, in view of its apparent specificity to the colon tumors, this gene was termed Colon Carcinoma Related Gene (CCRG). CCRG encodes a novel cysteine-rich motif and a putative signal peptide sequence. Supernatant from COS cells transfected with the CCRG expression vector stimulated proliferation of colon cancer cells. Immunoreactive CCRG was also detected in the paraffin sections of colon tumor samples. CCRG belongs to a new class of growth factors and may be important in the diagnosis and treatment of colon cancers. Identification of CCRG using bioinformatics approaches validates gene discovery using computational approaches.

A novel transmembrane glycoprotein cancer biomarker present in the X chromosome.

BACKGROUND: The uncharacterized proteins of the human proteome offer an untapped potential for cancer biomarker discovery. Numerous predicted open reading frames (ORFs) are present in diverse chromosomes. The mRNA and protein expression data, as well as the mutational and variant information for these ORF proteins are available in the cancer-related bioinformatics databases. MATERIALS AND METHODS: ORF proteins were mined using bioinformatics and proteomic tools to predict motifs and domains, and cancer relevance was established using cancer genome, transcriptome and proteome analysis tools. RESULTS: A novel testis-restricted ORF protein present in chromosome X called CXorf66 was detected in the serum, plasma and neutrophils. This gene is termed secreted glycoprotein in chromosome X (SGPX). The SGPX gene is up-regulated in cancer of the brain, lung and in leukemia, and down-regulated in liver and prostate cancer. Brain cancer in female patients exhibited elevated copy numbers of the SGPX gene. CONCLUSION: The SGPX gene is a putative novel cancer biomarker. Our results demonstrate the feasibility of mining the 'dark matter' of the cancer proteome for rapid cancer biomarker discovery.

Open reading frames associated with cancer in the dark matter of the human genome.

BACKGROUND: The uncharacterized proteins (open reading frames, ORFs) in the human genome offer an opportunity to discover novel targets for cancer. A systematic analysis of the dark matter of the human proteome for druggability and biomarker discovery is crucial to mining the genome. Numerous data mining tools are available to mine these ORFs to develop a comprehensive knowledge base for future target discovery and validation. MATERIALS AND METHODS: Using the Genetic Association Database, the ORFs of the human dark matter proteome were screened for evidence of association with neoplasms. The Phenome-Genome Integrator tool was used to establish phenotypic association with disease traits including cancer. Batch analysis of the tools for protein expression analysis, gene ontology and motifs and domains was used to characterize the ORFs. RESULTS: Sixty-two ORFs were identified for neoplasm association. The expression Quantitative Trait Loci (eQTL) analysis identified thirteen ORFs related to cancer traits. Protein expression, motifs and domain analysis and genome-wide association studies verified the relevance of these OncoORFs in diverse tumors. The OncoORFs are also associated with a wide variety of human diseases and disorders. CONCLUSIONS: Our results link the OncoORFs to diverse diseases and disorders. This suggests a complex landscape of the uncharacterized proteome in human diseases. These results open the dark matter of the proteome to novel cancer target research.

Inhibition of Single Minded 2 gene expression mediates tumor-selective apoptosis and differentiation in human colon cancer cells.

A Down's syndrome associated gene, Single Minded 2 gene short form (SIM2-s), is specifically expressed in colon tumors but not in the normal colon. Antisense inhibition of SIM2-s in a RKO-derived colon carcinoma cell line causes growth inhibition, apoptosis, and inhibition of tumor growth in a nude mouse tumoriginicity model. The mechanism of cell death in tumor cells is unclear. In the present study, we investigated the pathways underlying apoptosis. Apoptosis was seen in a tumor cell-specific manner in RKO cells but not in normal renal epithelial cells, despite inhibition of SIM2-s expression in both of these cells by the antisense. Apoptosis was depended on WT p53 status and was caspase-dependent; it was inhibited by a pharmacological inhibitor of mitogen-activated protein kinase activity. Expression of a key stress response gene, growth arrest and DNA damage gene (GADD)45alpha, was up-regulated in antisense-treated tumor cells but not in normal cells. In an isogenic RKO cell line expressing stable antisense RNA to GADD45alpha, a significant protection of the antisense-induced apoptosis was seen. Whereas antisense-treated RKO cells did not undergo cell cycle arrest, several markers of differentiation were deregulated, including alkaline phosphatase activity, a marker of terminal differentiation. Protection of apoptosis and block of differentiation showed a correlation in the RKO model. Our results support the tumor cell-selective nature of SIM2-s gene function, provide a direct link between SIM2-s and differentiation, and may provide a model to identify SIM2-s targets.

Identification of Down's syndrome critical locus gene SIM2-s as a drug therapy target for solid tumors.

We report here a cancer drug therapy use of a gene involved in Down's syndrome. Using bioinformatics approaches, we recently predicted Single Minded 2 gene (SIM2) from Down's syndrome critical region to be specific to certain solid tumors. Involvement of SIM2 in solid tumors has not previously been reported. Intrigued by a possible association between a Down's syndrome gene and solid tumors, we monitored SIM2 expression in solid tumors. Isoform-specific expression of SIM2 short-form (SIM2-s) was seen selectively in colon, prostate, and pancreatic carcinomas but not in breast, lung, or ovarian carcinomas nor in most normal tissues. In colon tumors, SIM2-s expression was seen in early stages. Antisense inhibition of SIM2-s expression in a colon cancer cell line caused inhibition of gene expression, growth inhibition, and apoptosis. The administration of the antisense, but not the control, oligonucleotides caused a pronounced inhibition of tumor growth in nude mice with no major toxicity. Our findings provide a strong rationale for the genes-to-drugs paradigm, establish SIM2-s as a molecular target for cancer therapeutics, and may further understanding of the cancer risk of Down's syndrome patients.