Phylogenetic analyses of melanoma reveal complex patterns of metastatic dissemination.

Melanoma is difficult to treat once it becomes metastatic. However, the precise ancestral relationship between primary tumors and their metastases is not well understood. We performed whole-exome sequencing of primary melanomas and multiple matched metastases from eight patients to elucidate their phylogenetic relationships. In six of eight patients, we found that genetically distinct cell populations in the primary tumor metastasized in parallel to different anatomic sites, rather than sequentially from one site to the next. In five of these six patients, the metastasizing cells had themselves arisen from a common parental subpopulation in the primary, indicating that the ability to establish metastases is a late-evolving trait. Interestingly, we discovered that individual metastases were sometimes founded by multiple cell populations of the primary that were genetically distinct. Such establishment of metastases by multiple tumor subpopulations could help explain why identical resistance variants are identified in different sites after initial response to systemic therapy. One primary tumor harbored two subclones with different oncogenic mutations in CTNNB1, which were both propagated to the same metastasis, raising the possibility that activation of wingless-type mouse mammary tumor virus integration site (WNT) signaling may be involved, as has been suggested by experimental models.

Development of Lateral Flow Assay Based on Size-Controlled Gold Nanoparticles for Detection of Hepatitis B Surface Antigen.

In this study, we developed lateral flow assay (LFA) biosensors for the detection of hepatitis B surface antigens using well-controlled gold nanoparticles (AuNPs). To enhance colorimetric signals, a seeded growth method was used for the preparation of size-controlled AuNPs with a narrow size distribution. Different sizes of AuNPs in the range of 342-137.8 nm were conjugated with antibodies and then optimized for the efficient detection of LFA biosensors. The conjugation stability was investigated by UV-vis spectroscopy of AuNP dispersion at various pH values and concentrations of antibody. Based on optimized conjugation conditions, the use of 42.7 ± 0.8 nm AuNPs exhibited superior performance for the detection of LFAs relative to other sizes of AuNPs.

DNAX-activating protein 10 (DAP10) membrane adaptor associates with receptor for advanced glycation end products (RAGE) and modulates the RAGE-triggered signaling pathway in human keratinocytes.

The receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of many inflammatory, degenerative, and hyperproliferative diseases, including cancer. Previously, we revealed mechanisms of downstream signaling from ligand-activated RAGE, which recruits TIRAP/MyD88. Here, we showed that DNAX-activating protein 10 (DAP10), a transmembrane adaptor protein, also binds to RAGE. By artificial oligomerization of RAGE alone or RAGE-DAP10, we found that RAGE-DAP10 heterodimer formation resulted in a marked enhancement of Akt activation, whereas homomultimeric interaction of RAGE led to activation of caspase 8. Normal human epidermal keratinocytes exposed to S100A8/A9, a ligand for RAGE, at a nanomolar concentration mimicked the pro-survival response of RAGE-DAP10 interaction, although at a micromolar concentration, the cells mimicked the pro-apoptotic response of RAGE-RAGE. In transformed epithelial cell lines, A431 and HaCaT, in which endogenous DAP10 was overexpressed, and S100A8/A9, even at a micromolar concentration, led to cell growth and survival due to RAGE-DAP10 interaction. Functional blocking of DAP10 in the cell lines abrogated the Akt phosphorylation from S100A8/A9-activated RAGE, eventually leading to an increase in apoptosis. Finally, S100A8/A9, RAGE, and DAP10 were overexpressed in the psoriatic epidermis. Our findings indicate that the functional interaction between RAGE and DAP10 coordinately regulates S100A8/A9-mediated survival and/or apoptotic response of keratinocytes.

Personalized identification of altered pathways in cancer using accumulated normal tissue data.

MOTIVATION: Identifying altered pathways in an individual is important for understanding disease mechanisms and for the future application of custom therapeutic decisions. Existing pathway analysis techniques are mainly focused on discovering altered pathways between normal and cancer groups and are not suitable for identifying the pathway aberrance that may occur in an individual sample. A simple way to identify individual's pathway aberrance is to compare normal and tumor data from the same individual. However, the matched normal data from the same individual are often unavailable in clinical situation. Therefore, we suggest a new approach for the personalized identification of altered pathways, making special use of accumulated normal data in cases when a patient's matched normal data are unavailable. The philosophy behind our method is to quantify the aberrance of an individual sample's pathway by comparing it with accumulated normal samples. We propose and examine personalized extensions of pathway statistics, overrepresentation analysis and functional class scoring, to generate individualized pathway aberrance score. RESULTS: Collected microarray data of normal tissue of lung and colon mucosa are served as reference to investigate a number of cancer individuals of lung adenocarcinoma (LUAD) and colon cancer, respectively. Our method concurrently captures known facts of cancer survival pathways and identifies the pathway aberrances that represent cancer differentiation status and survival. It also provides more improved validation rate of survival-related pathways than when a single cancer sample is interpreted in the context of cancer-only cohort. In addition, our method is useful in classifying unknown samples into cancer or normal groups. Particularly, we identified 'amino acid synthesis and interconversion' pathway is a good indicator of LUAD (Area Under the Curve (AUC) 0.982 at independent validation). Clinical importance of the method is providing pathway interpretation of single cancer, even though its matched normal data are unavailable. AVAILABILITY AND IMPLEMENTATION: The method was implemented using the R software, available at our Web site: http://bibs.snu.ac.kr/ipas. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Construction and characterization of Cu²âº, Ni²âº, Zn²âº, and Co²âº modified-DNA crystals.

We studied the physical characteristics of modified-DNA (M-DNA) double crossover crystals fabricated via substrate-assisted growth with various concentrations of four different divalent metallic ions, Cu(2+), Ni(2+), Zn(2+), and Co(2+). Atomic force microscopy (AFM) was used to test the stability of the M-DNA crystals with different metal ion concentrations. The AFM images show that M-DNA crystals formed without deformation at up to the critical concentrations of 6 mM of [Cu(2+)], 1.5 mM of [Ni(2+)], 1 mM of [Zn(2+)], and 1 mM of [Co(2+)]. Above these critical concentrations, the M-DNA crystals exhibited deformed, amorphous structures. Raman spectroscopy was then used to identify the preference of the metal ion coordinate sites. The intensities of the Raman bands gradually decreased as the concentration of the metal ions increased, and when the metal ion concentrations increased beyond the critical values, the Raman band of the amorphous M-DNA was significantly suppressed. The metal ions had a preferential binding order in the DNA molecules with G-C and A-T base pairs followed by the phosphate backbone. A two-probe station was used to measure the electrical current-voltage properties of the crystals which indicated that the maximum currents of the M-DNA complexes could be achieved at around the critical concentration of each ion. We expect that the functionalized ion-doped M-DNA crystals will allow for efficient devices and sensors to be fabricated in the near future.

Practical approach to determine sample size for building logistic prediction models using high-throughput data.

An empirical method of sample size determination for building prediction models was proposed recently. Permutation method which is used in this procedure is a commonly used method to address the problem of overfitting during cross-validation while evaluating the performance of prediction models constructed from microarray data. But major drawback of such methods which include bootstrapping and full permutations is prohibitively high cost of computation required for calculating the sample size. In this paper, we propose that a single representative null distribution can be used instead of a full permutation by using both simulated and real data sets. During simulation, we have used a dataset with zero effect size and confirmed that the empirical type I error approaches to 0.05. Hence this method can be confidently applied to reduce overfitting problem during cross-validation. We have observed that pilot data set generated by random sampling from real data could be successfully used for sample size determination. We present our results using an experiment that was repeated for 300 times while producing results comparable to that of full permutation method. Since we eliminate full permutation, sample size estimation time is not a function of pilot data size. In our experiment we have observed that this process takes around 30min. With the increasing number of clinical studies, developing efficient sample size determination methods for building prediction models is critical. But empirical methods using bootstrap and permutation usually involve high computing costs. In this study, we propose a method that can reduce required computing time drastically by using representative null distribution of permutations. We use data from pilot experiments to apply this method for designing clinical studies efficiently for high throughput data.

Fully automated circulating tumor cell isolation platform with large-volume capacity based on lab-on-a-disc.

Full automation with high purity for circulating tumor cell (CTC) isolation has been regarded as a key goal to make CTC analysis a "bench-to-bedside" technology. Here, we have developed a novel centrifugal microfluidic platform that can isolate the rare cells from a large volume of whole blood. To isolate CTCs from whole blood, we introduce a disc device having the biggest sample capacity as well as manipulating blood cells for the first time. The fully automated disc platform could handle 5 mL of blood by designing the blood chamber having a triangular obstacle structure (TOS) with lateral direction. To guarantee high purity that enables molecular analysis with the rare cells, CTCs were bound to the microbeads covered with anti-EpCAM to discriminate density between CTCs and blood cells and the CTCs being heavier than blood cells were only settled under a density gradient medium (DGM) layer. To understand the movement of CTCs under centrifugal force, we performed computational fluid dynamics simulation and found that their major trajectories were the boundary walls of the DGM chamber, thereby optimizing the chamber design. After whole blood was inserted into the blood chamber of the disc platform, size- and density-amplified cancer cells were isolated within 78 min, with minimal contamination as much as approximately 12 leukocytes per milliliter. As a model of molecular analysis toward personalized cancer treatment, we performed epidermal growth factor receptor (EGFR) mutation analysis with HCC827 lung cancer cells and the isolated cells were then successfully detected for the mutation by PCR clamping and direct sequencing.

Loss-of-function mutations in Notch receptors in cutaneous and lung squamous cell carcinoma.

Squamous cell carcinomas (SCCs) are one of the most frequent forms of human malignancy, but, other than TP53 mutations, few causative somatic aberrations have been identified. We identified NOTCH1 or NOTCH2 mutations in ~75% of cutaneous SCCs and in a lesser fraction of lung SCCs, defining a spectrum for the most prevalent tumor suppressor specific to these epithelial malignancies. Notch receptors normally transduce signals in response to ligands on neighboring cells, regulating metazoan lineage selection and developmental patterning. Our findings therefore illustrate a central role for disruption of microenvironmental communication in cancer progression. NOTCH aberrations include frameshift and nonsense mutations leading to receptor truncations as well as point substitutions in key functional domains that abrogate signaling in cell-based assays. Oncogenic gain-of-function mutations in NOTCH1 commonly occur in human T-cell lymphoblastic leukemia/lymphoma and B-cell chronic lymphocytic leukemia. The bifunctional role of Notch in human cancer thus emphasizes the context dependency of signaling outcomes and suggests that targeted inhibition of the Notch pathway may induce squamous epithelial malignancies.

Design and synthesis of a series of α-benzyl phenylpropanoic acid-type peroxisome proliferator-activated receptor (PPAR) gamma partial agonists with improved aqueous solubility.

In the continuing study directed toward the development of peroxisome proliferator-activated receptor gamma (hPPARγ) agonist, we attempted to improve the water solubility of our previously developed hPPARγ-selective agonist 3, which is insufficiently soluble for practical use, by employing two strategies: introducing substituents to reduce its molecular planarity and decreasing its hydrophobicity via replacement of the adamantyl group with a heteroaromatic ring. The first approach proved ineffective, but the second was productive. Here, we report the design and synthesis of a series of α-benzyl phenylpropanoic acid-type hPPARγ partial agonists with improved aqueous solubility. Among them, we selected (R)-7j, which activates hPPARγ to the extent of about 65% of the maximum observed with a full agonist, for further evaluation. The ligand-binding mode and the reason for the partial-agonistic activity are discussed based on X-ray-determined structure of the complex of hPPARγ ligand-binding domain (LBD) and (R)-7j with previously reported ligand-LDB structures. Preliminal apoptotic effect of (R)-7j against human scirrhous gastric cancer cell line OCUM-2MD3 is also described.

A new cytosolic pathway from a Parkinson disease-associated kinase, BRPK/PINK1: activation of AKT via mTORC2.

Accumulating evidence indicates that dysfunction of mitochondria is a common feature of Parkinson disease. Functional loss of a familial Parkinson disease-linked gene, BRPK/PINK1 (PINK1), results in deterioration of mitochondrial functions and eventual neuronal cell death. A mitochondrial chaperone protein has been shown to be a substrate of PINK1 kinase activity. In this study, we demonstrated that PINK1 has another action point in the cytoplasm. Phosphorylation of Akt at Ser-473 was enhanced by overexpression of PINK1, and the Akt activation was crucial for protection of SH-SY5Y cells from various cytotoxic agents, including oxidative stress. Enhanced Akt phosphorylation was not due to activation of phosphatidylinositol 3-kinase but due to activation of mammalian target of rapamycin complex 2 (mTORC2) by PINK1. Rictor, a specific component of mTORC2, was phosphorylated by overexpression of PINK1. Furthermore, overexpression of PINK1 enhanced cell motility. These results indicate that PINK1 exerts its cytoprotective function not only in mitochondria but also in the cytoplasm through activation of mTORC2.

Solid phase DNA extraction with a flexible bead-packed microfluidic device to detect methicillin-resistant Staphylococcus aureus in nasal swabs.

We have developed a bead-packed microfluidic device with a built-in flexible wall to automate extraction of nucleic acids from methicillin-resistant Staphylococcus aureus (MRSA) in nasal swabs. The flexible polydimethylsiloxane (PDMS) membrane was designed to manipulate the surface-to-volume ratio (SVR) of bead-packed chambers in the range of 0.05 to 0.15 (µm(-1)) for a typical solid phase extraction protocol composed of binding, washing, and eluting. In particular, the pneumatically assisted close packing of beads led to an invariant SVR (0.15 µm(-1)) even with different bead amounts (10-16 mg), which allowed for consistent operation of the device and improved capture efficiency for bacteria cells. Furthermore, vigorous mixing by asynchronous membrane vibration enabled ca. 90% DNA recovery with ca. 10 µL of liquid solution from the captured cells on the bead surfaces. The full processes to detect MRSA in nasal swabs, i.e., nasal swab collection, prefiltration, on-chip DNA extraction, and real-time polymerase chain reaction (PCR) amplification, were successfully constructed and carried out to validate the capability to detect MRSA in nasal swab samples. This flexible microdevice provided an excellent analytical PCR detection sensitivity of ca. 61 CFU/swab with 95% confidence interval, which turned out to be higher than or similar to that of the commercial DNA-based MRSA detection techniques. This excellent performance would be attributed to the capability of the flexible bead-packed microdevice to enrich the analyte from a large initial sample (e.g., 1 mL) into a microscale volume of eluate (e.g., 10 µL). The proposed microdevice will find many applications as a solid phase extraction method toward various sample-to-answer systems.

Highly efficient assay of circulating tumor cells by selective sedimentation with a density gradient medium and microfiltration from whole blood.

Isolation of circulating tumor cells (CTCs) by size exclusion can yield poor purity and low recovery rates, due to large variations in size of CTCs, which may overlap with leukocytes and render size-based filtration methods unreliable. This report presents a very sensitive, selective, fast, and novel method for isolation and detection of CTCs. Our assay platform consists of three steps: (i) capturing CTCs with anti-EpCAM conjugated microbeads, (ii) removal of unwanted hematologic cells (e.g., leukocytes, erythrocytes, etc.) by selective sedimentation of CTCs within a density gradient medium, and (iii) simple microfiltration to collect these cells. To demonstrate the efficacy of this assay, MCF-7 breast cancer cells (average diameter, 24 µm) and DMS-79 small cell lung cancer cells (average diameter, 10 µm) were used to model CTCs. We investigated the relative sedimentation rates for various cells and/or particles, such as CTCs conjugated with different types of microbeads, leukocytes, and erythrocytes, in order to maximize differences in the physical properties. We observed that greater than 99% of leukocytes in whole blood were effectively removed at an optimal centrifugal force, due to differences in their sedimentation rates, yielding a much purer sample compared to other filter-based methods. We also investigated not only the effect of filtration conditions on recovery rates and sample purity but also the sensitivity of our assay platform. Our results showed a near perfect recovery rate (~99%) for MCF-7 cells and very high recovery rate (~89%) for DMS-79 cells, with minimal amounts of leukocytes present.

Noble polymeric surface conjugated with zwitterionic moieties and antibodies for the isolation of exosomes from human serum.

New zwitterionic polymer-coated immunoaffinity beads were developed to resist nonspecific protein adsorption from undiluted human serum for diagnostic applications of exosomes. A zwitterionic sulfobetaine monomer with an amine functional group was employed for simple surface chemistry and antifouling properties. An exosomal biomarker protein, epithelial cell adhesion molecule (EpCAM), was selected as a target molecule in this work. The beads were coated with polyacrylic acids (PAA) for increasing biorecognition sites, and protein G was then conjugated with carboxylic acid groups on the surfaces for controlling EpCAM antibody orientation. The remaining free carboxylic acid groups were modified with sulfobetaine moieties, and anti-EpCAM antibody was finally introduced. The amount of anti-EpCAM on the beads was increased by 40% when compared with PAA-uncoated beads. The surfaces of the beads exhibited near-net-zero charge, and nonspecific protein adsorption was effectively suppressed by sulfobetaine moieties. EpCAM was captured from undiluted human serum with almost the same degree of efficiency as from PBS buffer solution using the newly developed immunoaffinity beads.

A direct extraction method for microRNAs from exosomes captured by immunoaffinity beads.

A direct extraction method was developed for exosomal microRNAs. After isolation of exosomes from human serum by immunoaffinity magnetic beads, microRNAs were extracted by just mixing beads with a lysis solution and heating without further purification. The lysis solution was composed of a nonionic detergent and salt (NaCl). The concentration of each component was optimized to maximize lysis efficiency and to inhibit adsorption of extracted microRNAs on beads. MicroRNAs extracted by this method could be quantitatively analyzed by qRT-PCR, indicating that the method could replace conventional methods for extracting microRNAs from immunobead-captured exosomes.

Preclinical safety and efficacy of in situ REIC/Dkk-3 gene therapy for prostate cancer.

The preclinical safety and therapeutic efficacy of adenoviral vectors that express the REIC/Dkk-3 tumor suppressor gene (Ad-REIC) was examined for use in prostate cancer gene therapy. The Ad-human (h) and mouse (m) REIC were previously demonstrated to induce strong anti-cancer effects in vitro and in vivo, and we herein report the results of two in vivo studies. First, intra-tumor Ad-hREIC administration was examined for toxicity and therapeutic effects in a subcutaneous tumor model using the PC3 prostate cancer cell line. Second, intra-prostatic Ad-mREIC administration was tested for toxicity in normal mice. The whole-body and spleen weights, hematological and serum chemistry parameters, and histological evaluation of tissues from throughout the body were analyzed. Both experiments indicated that there was no significant difference in the examined parameters between the Ad-REIC-treated group and the control (PBS- or Ad-LacZ-treated) group. In the in vitro analysis using PC3 cells, a significant apoptotic effect was observed after Ad-hREIC treatment. Confirming this observation, the robust anti-tumor efficacy of Ad-hREIC was demonstrated in the in vivo subcutaneous prostate cancer model. Based on the results of these preclinical experiments, we consider the adenovirus-mediated REIC/Dkk-3 in situ gene therapy to be safe and useful for the clinical treatment of prostate cancer.

Tumor suppressor REIC/Dkk-3 interacts with the dynein light chain, Tctex-1.

REIC/Dkk-3 is a member of the Dickkopf family proteins known as Wnt-antagonists, and REIC/Dkk-3 expression is downregulated in a broad range of cancer types. REIC/Dkk-3 acts as a tumor suppressor in multiple cancer cell lines by inducing apoptosis through endoplasmic reticulum (ER) stress signaling. However, the intracellular interaction partners of REIC/Dkk-3 have not been fully elucidated. By employing yeast two-hybrid screening, we identified the human dynein light chain, Tctex-1, as a novel interaction partner of REIC/Dkk-3. We further disclosed that the interaction involves the 136-157 amino acid region of REIC/Dkk-3 by using the mammalian two-hybrid system. Interestingly, this binding region of REIC/Dkk-3 with Tctex-1 contains an amino acid sequence motif [-E-X-G-R-R-X-H-] which was previously reported as the Tctex-1 binding domain of dynein intermediate chain (DIC). Immunocytochemistry demonstrated that both REIC/Dkk-3 and Tctex-1 were localized around the ER of human fibroblasts, and the similar distribution pattern of the proteins suggests that their interaction occurs around the ER. This is the first study showing the interaction of a Dickkopf family protein with a dynein motor complex protein. The link between REIC/Dkk-3 and Tctex-1 may be of significance for understanding the molecular functions of the proteins in ER stress signaling and intracellular dynein motor dynamics, respectively.

Mutational hotspots in the mitochondrial genome of lung cancer.

We determined the somatic mutations in the mitochondrial genomes of 70 lung cancer patients by pair-wise comparative analyses of the normal- and tumor-genome sequences acquired using Affymetrix Mitochondrial Resequencing Array 2.0. The overall mutation rates in lung cancers were Approximately 100 fold higher than those in normal cells, with significant statistical correlation with smoking (p=0.00088). Total of 532 somatic mutations were evenly distributed in 499 positions with very low overall frequency (1.07/bp), but the non-synonymous mutations causing amino acid substitution occurred more frequently (1.83/bp), particularly at two positions, 8701 and 10398 (10.5/bp) that code for ATPase6 and NADH dehydrogenase 3, respectively. Despite the randomness or even distribution of the mutations, these two mutations occurred together in 86% of the cases. The linkage between the two most frequent mutations suggests that they were selected together, possibly due to their cooperative role during cancer development. Indeed, the mutation at 10398 was shown by Canter, Pezzotti, and their colleagues in 2009, as a risk factor for breast cancer. In this study, we identified two potential biomarkers that might be functionally linked together during the development of cancer.

Expression of REIC/Dkk-3 in normal and hyperproliferative epidermis.

Dickkopf (Dkk) family members are known as Wnt modulators involved in the development, cell growth/differentiation and cancer. REIC/Dkk-3, which does not interfere with Wnt signalling, has been proposed to be a tumor suppressor gene, but its physiological function has remained unclear. In this study, we analysed the expression of REIC/Dkk-3 in normal interfollicular epidermis (IFE) and hyperproliferative epidermis. REIC/Dkk-3 was expressed in human and mouse IFE, being localized at the interface of upper spinous layer and granular layer. Skin cancer cell lines lost REIC/Dkk-3 expression as reported previously. When we analysed patient samples, REIC/Dkk-3 expression was down-regulated in the hyperproliferative epidermis including skin cancers and non-cancerous proliferative diseases. REIC/Dkk-3 expression was also suppressed in the regenerative and inflammative epidermis of model mice. These findings will certainly contribute to the extension of studies on REIC/Dkk-3.

S100A11, a dual growth regulator of epidermal keratinocytes.

S100A11, a member of the family of S100 proteins, is a dimmer, each monomer of which has two EF-hands. Expression of S100A11 is ubiquitous in various tissues at different levels, with a high expression level in the skin. We have analyzed functions of S100A11 mainly in normal human keratinocytes (NHK) as a model cell system of human epithelial cells. High Ca(2+) and transforming growth factor-ß (TGF-ß), two representative growth suppressors for NHK, need a common S100A11-mediated pathway in addition to unique pathways (NFAT1-mediated pathway for high Ca(2+) and Smad-mediated pathway for TGF-ß) for exhibiting a growth inhibitory effect. S100A11 has another action point for growth suppression in NHK. Annexin A1 (ANXA1) complexed with S100A11 efficiently binds to and inhibits cytosolic phospholipase A2 (cPLA2), the activity of which is needed for the growth of NHK. On exposure of NHK to epidermal growth factor (EGF), ANXA1 is cleaved at 12Trp, and this truncated ANXA1 loses binding capacity to S100A11, resulting in maintenance of an active state of cPLA2. On the other hand, we found that S100A11 is actively secreted by NHK. Extracellular S100A11 acts on NHK to enhance the production of EGF family proteins, resulting in growth stimulation. These findings indicate that S100A11 plays a dual role in growth regulation, being suppressive in cells and being promotive from outside of cells.

A novel tumor suppressor, REIC/Dkk-3 gene identified by our in vitro transformation model of normal human fibroblasts works as a potent therapeutic anti-tumor agent.

Reduced Expression in Immortalized Cell (REIC) was cloned by subtractive hybridization method as a gene whose expression is reduced in many human immortalized and neoplastic tumor cells. The REIC, when over-expressed by an adenovirus (Ad-REIC), exhibited a dramatic therapeutic effect on a wide variety of human cancers through a mechanism triggered by ER-stress-mediated JNK activation. In addition to this direct effect on cancer cells, Ad-REIC exerted another cytotoxicity on human cancers, an indirect host-mediated effect due to overproduction of IL-7 by mis-targeted normal cells. This "one-bullet two-arms" finding may lead to a powerful new therapeutic approach to the treatment of human cancers.