Comprehensive Microarray Analysis Research Articles

Controlled hydrostatic pressure stress downregulates the expression of ribosomal genes in preimplantation embryos: a possible protection mechanism?

The efficiency of various assisted reproductive techniques can be improved by preconditioning the gametes and embryos with sublethal hydrostatic pressure treatment. However, the underlying molecular mechanism responsible for this protective effect remains unknown and requires further investigation. Here, we studied the effect of optimised hydrostatic pressure treatment on the global gene expression of mouse oocytes after embryonic genome activation. Based on a gene expression microarray analysis, a significant effect of treatment was observed in 4-cell embryos derived from treated oocytes, revealing a transcriptional footprint of hydrostatic pressure-affected genes. Functional analysis identified numerous genes involved in protein synthesis that were downregulated in 4-cell embryos in response to hydrostatic pressure treatment, suggesting that regulation of translation has a major role in optimised hydrostatic pressure-induced stress tolerance. We present a comprehensive microarray analysis and further delineate a potential mechanism responsible for the protective effect of hydrostatic pressure treatment.

PARVB overexpression increases cell migration capability and defines high risk for endophytic growth and metastasis in tongue squamous cell carcinoma.

Background:Tongue squamous cell carcinoma (TSCC) is highly diverse, even in its early stages. This cancer is classified into three subtypes (superficial, exophytic, and endophytic) based on macroscopic appearance. Of these subtypes, the endophytic tumours have the worst prognosis because of their invasiveness and higher frequency of metastasis.Methods:To understand the molecular mechanism underlying the endophytic subtype and to identify biomarkers, we performed a comprehensive gene expression microarray analysis of clinical biopsy samples and also confirmed the clinical relevance of differential gene expression.Results:Expression of the parvin-beta (PARVB) gene and its encoded protein was significantly upregulated in endophytic-type TSCC. PARVB is known to play a critical role in actin reorganization and focal adhesions. Knockdown of PARVB expression in vitro caused apparent decreases in cell migration and wound healing, implying that PARVB has a crucial role in cell motility. Moreover, metastasis-free survival was significantly lower in patients with higher tumour expression of PARVB.Conclusions:These findings suggest that PARVB overexpression is a candidate biomarker for endophytic tumours and metastasis. This protein may be a clinically useful target for adjuvant TSCC therapy.

Abstract A47: Self-renewing cellular compartments in embryonal rhabdomyosarcoma are modulated by hedgehog signaling

Abstract Rhabdomyosarcoma (RMS) is a heterogeneous group of malignancies with features of impaired skeletal muscle differentiation and represents the majority of soft tissue cancers in the pediatric age group. Current treatment regimens show clinical benefit but this has reached a plateau. Since there is significant risk of adverse side effects and secondary tumors later in life, identifying and targeting sub-populations with higher tumorigenic potential and self renewal capacity would not only decrease tumor burden but also lower the probability of relapse. Using in vitro self renewal assays we could identify self-renewing cellular compartments within embryonal RMS (eRMS) cell lines. These compartments showed upregulation of several components of the sonic hedgehog pathway. Here, we now show using both genetic loss- and gain-of-function experiments and clinically relevant small molecule modulators that Hedgehog signalling is important for determining the self-renewal, tumor initiation capability both in vitro as well as in vivo. In addition, the hedgehog pathway can also influence chemoresistance of eRMS cells and altered the differentiation status of the cells by directly modulating the expression of various stem cell associated transcription factors. The pathway was found to be heterogeneously activated within eRMS patient samples in a comprehensive tissue microarray analysis. Importantly, the presence of Hedgehog-active cells identified using effector transcription factors could negatively predict patient survival in a retrospective analysis of this patient cohort. This work elucidates both a novel and central role played by the Hedgehog pathway in eRMS and underscores the importance of studying tumor heterogeneity in pediatric cancer. Citation Format: Sampoorna Satheesha, Amandine Bovay, Gabriele Manzella, Peter Bode, Simone Feuchtgruber, Ewa Koscielniak, Reka Belle, Elisa Casanova, Nurhak Dogan, Beat W. Schäfer. Self-renewing cellular compartments in embryonal rhabdomyosarcoma are modulated by hedgehog signaling. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A47.

Gene expression profiling in juvenile and mature cuttings of Eucalyptus grandis reveals the importance of microtubule remodeling during adventitious root formation.

BackgroundThe ability to form adventitious roots (AR) is an economically important trait that is lost during the juvenile-to-mature phase change in woody plants. Auxin treatment, which generally promotes rooting in juvenile cuttings, is often ineffective when applied to mature cuttings. The molecular basis for this phenomenon in Eucalyptus grandis was addressed here.ResultsA comprehensive microarray analysis was performed in order to compare gene-expression profiles in juvenile and mature cuttings of E. grandis, with or without auxin treatment on days, 0, 1, 3, 6, 9 and 12 post AR induction. Under these conditions AR primordia were formed only in auxin-treated juvenile cuttings. However, clustering the expression profiles revealed that the time after induction contributed more significantly to the differences in expression than the developmental phase of the cuttings or auxin treatment. Most detected differences which were related to the developmental phase and auxin treatment occurred on day 6, which correlated with the kinetics of AR-primordia formation. Among the functional groups of transcripts that differed between juvenile and mature cuttings was that of microtubules (MT). The expression of 42 transcripts annotated as coding for tubulin, MT-associated proteins and kinesin motor proteins was validated in the same RNA samples. The results suggest a coordinated developmental and auxin dependent regulation of several MT-related transcripts in these cuttings. To determine the relevance of MT remodeling to AR formation, MTs were subjected to subtle perturbations by trifluralin, a MT disrupting drug, applied during auxin induction. Juvenile cuttings were not affected by the treatment, but rooting of mature cuttings increased from 10 to more than 40 percent.ConclusionsThe data suggest that juvenile-specific MT remodeling is involved in AR formation in E. grandis.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-826) contains supplementary material, which is available to authorized users.

MicroRNAs in Barrett's esophagus: future prospects.

MicroRNAs in Barrett's esophagus: future prospects.

Development of Oral Epithelial Cell Line ROE2 with Differentiation Potential from Transgenic Rats Harboring Temperature-Sensitive Simian Virus40 Large T-Antigen Gene

We have developed an immortalized oral epithelial cell line, ROE2, from fetal transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene. The cells grew continuously at either a permissive temperature of 33°C or an intermediate temperature of 37°C. At the nonpermissive temperature of 39°C, on the other hand, growth decreased significantly, and the Sub-G1 phase of the cell cycle increased, indicating that the cells undergo apoptosis at a nonpermissive temperature. Histological and immunocytochemical analyses revealed that ROE2 cells at 37°C had a stratified epithelial-like morphology and expressed cytokeratins Krt4 and Krt13, marker proteins for oral nonkeratinized epithelial cells. Global-scale comprehensive microarray analysis, coupled with bioinformatics tools, demonstrated a significant gene network that was obtained from the upregulated genes. The gene network contained 16 genes, including Cdkn1a, Fos, Krt13, and Prdm1, and was associated mainly with the biological process of skin development in the category of biological functions, organ development. These four genes were validated by quantitative real-time polymerase chain reaction, and the results were nearly consistent with the microarray data. It is therefore anticipated that this cell line will be useful as an in vitro model for studies such as physiological functions, as well as for gene expression in oral epithelial cells.

Cell-Specific Transcriptional Profiling Reveals Candidate Mechanisms Regulating Development and Function of Uterine Epithelia in Mice

All mammalian uteri have luminal (LE) and glandular epithelia (GE) in their endometrium. The LE mediates uterine receptivity and blastocyst attachment for implantation, and the GE synthesize and secrete or transport bioactive substances involved in blastocyst implantation, uterine receptivity, and stromal cell decidualization. However, the mechanisms governing uterine epithelial development after birth and their function in the adult are not fully understood. Here, comprehensive microarray analysis was conducted on LE and GE isolated by laser capture microdissection from uteri on Postnatal Day 10 (PD 10) and day of pseudopregnancy (DOPP) 2.5 and 3.5. This data was integrated with analysis of uteri from gland-containing control and aglandular progesterone-induced uterine gland knockout mice from PD 10 and DOPP 3.5. Many genes were expressed in both epithelia, but there was greater expression of genes in the LE than in the GE. In the neonate, GE-expressed genes were enriched for morphogenesis, development, migration, and retinoic acid signaling. In the adult, LE-expressed genes were enriched for metabolic processes and steroid biosynthesis, whereas retinoid signaling, tight junction, extracellular matrix, and regulation of kinase activity were enriched in the GE. The transcriptome differences in the epithelia support the idea that each cell type has a distinct and complementary function in the uterus. The candidate genes and regulatory networks identified here provide a framework to discover new mechanisms regulating development of epithelia in the postnatal uterus and their functions in early pregnancy.

Low Extracellular Sodium Causes Neuronal Distress Independently of Reduced Osmolality in an Experimental Model of Chronic Hyponatremia

There is evidence that chronic hyponatremia, even when mild, may cause neurological signs and symptoms. These have been traditionally associated with water movement into nervous cells, as a result of the hypotonic state. The aim of the present study was to determine whether low extracellular sodium directly exerts negative effects on human neuronal cells, independently of reduced osmolality. We exposed neuroblastoma SK-N-AS and SH-SY5Y cells to sustained low extracellular sodium, thus mimicking a condition of chronic hyponatremia, both in the presence of reduced and in the presence of unaltered osmolality. We found that very low sodium (i.e., 115 mmol/L in SK-N-AS and 90 mmol/L in SH-SY5Y) significantly reduced cell viability. However, intermediate low sodium was able to cause cell distress, as assessed by the altered expression of anti-apoptotic genes and the reduced ability to differentiate into a mature neuronal phenotype. Noteworthy, these effects were observed also in the presence of unaltered osmolality. Moreover, we performed a comprehensive microarray analysis in cells maintained in normal sodium or in low sodium and unaltered osmolality, and we found that the most altered pathway included genes involved in "cell death and survival." Among the more than 40 differentially expressed genes, the Heme oxygenase gene, which represents a transcriptional response to oxidative stress, showed the highest increase in the expression level. This study demonstrates that low extracellular sodium causes detrimental effects in neuronal cells that are at least in part independent of reduced osmolality. These findings further support the recommendation to effectively correct hyponatremia, even when mild and chronic.

DNA microarray profiling of genes differentially regulated by three heterochromatin protein 1 (HP1) homologs in Drosophila

Heterochromatin protein 1 (HP1) is an epigenetic gene silencing protein that is regulated by lysine 9 methylation of histone H3. Most eukaryotes have at least three HP1 homologs with similar domain structures but with different localization patterns and functions in heterochromatin and euchromatin. However, little is known about the genome-wide effects of the three main HP1 homologs on gene expression. Here, to gain insight into the different gene expression effects of the three HP1 homologs, we performed a comprehensive and comparative microarray analysis of Drosophila HP1 homologs. Bioinformatic analysis of the microarray profiling revealed significant similarity and uniqueness in the genes altered in HP1-knockdown S2 cells in Drosophila. Although global changes of these transcripts were surprisingly subtle (4–6%), there were ∼582 common target genes for the three HP1s that showed transcript levels either reduced or induced >1.5-fold. Depletion of HP1 resulted in up-regulated and down-regulated gene profiles, indicating that HP1 mediates both repression and activation of gene expression. This study is the first to systematically analyze the bioinformatics of HP1 paralogs and provide basic clues to the molecular mechanism by which HP1 might control gene expression in a homolog-specific manner.

Low extracellular sodium causes neuronal distress independently of reduced osmolality in an experimental model of chronic hyponatremia

There is evidence that chronic hyponatremia, even when mild, may cause neurological signs and symptoms. These have been traditionally associated with water movement into nervous cells, as a result of the hypotonic state. The aim of the present study was to determine whether low extracellular sodium directly exerts negative effects on human neuronal cells, independently of reduced osmolality. We exposed neuroblastoma SK-N-AS and SH-SY5Y cells to sustained low extracellular sodium, thus mimicking a condition of chronic hyponatremia, both in the presence of reduced and in the presence of unaltered osmolality. We found that very low sodium (i.e., 115 mmol/L in SK-N-AS and 90 mmol/L in SH-SY5Y) significantly reduced cell viability. However, intermediate low sodium was able to cause cell distress, as assessed by the altered expression of anti-apoptotic genes and the reduced ability to differentiate into a mature neuronal phenotype. Noteworthy, these effects were observed also in the presence of unaltered osmolality. Moreover, we performed a comprehensive microarray analysis in cells maintained in normal sodium or in low sodium and unaltered osmolality, and we found that the most altered pathway included genes involved in “cell death and survival.” Among the more than 40 differentially expressed genes, the Heme oxygenase gene, which represents a transcriptional response to oxidative stress, showed the highest increase in the expression level. This study demonstrates that low extracellular sodium causes detrimental effects in neuronal cells that are at least in part independent of reduced osmolality. These findings further support the recommendation to effectively correct hyponatremia, even when mild and chronic.

Differential gene expression in ERα-positive and ERα-negative breast cancer cells upon leptin stimulation

In postmenopausal women, adipositas represents a serious risk factor for cancer development and progression. White adipose tissue secretes the 16 kDa hormone leptin which plays a key role in the regulation of appetite and metabolism. An increasing number of reports indicate that leptin also interferes with signal transduction pathways implicated in the development of breast cancer. In our previous study, we identified the estrogen receptor alpha (ERα) as a relevant enhancer of leptin-induced signal transduction leading to transactivation of signal transducer and activator of transcription 3 (Stat3). The purpose of this study is the investigation of specific target gene expression in response to leptin-mediated Stat3 signaling. We performed a comprehensive microarray analysis of ERα-positive and ERα-negative MDA-MB-231 cells upon leptin treatment and identified 49 genes which showed a significant ERα-dependent regulation in leptin-treated MDA-MB-231 cells. There was no intersection with genes which were merely up- or downregulated by ERα expression and only 9 and 11 genes overlapping targets which were regulated by leptin stimulation either in ERα-expressing or ERα-negative MDA-MB-231 cells, respectively. To demonstrate the specificity, expression of three target genes was validated by quantitative real-time PCR. In conclusion, these data imply that leptin can induce a different set of target genes dependent on ERα expression, which might contribute to the development and progression of cancer diseases.

Multi-tissue microarray analysis identifies a molecular signature of regeneration.

The inability to functionally repair tissues that are lost as a consequence of disease or injury remains a significant challenge for regenerative medicine. The molecular and cellular processes involved in complete restoration of tissue architecture and function are expected to be complex and remain largely unknown. Unlike humans, certain salamanders can completely regenerate injured tissues and lost appendages without scar formation. A parsimonious hypothesis would predict that all of these regenerative activities are regulated, at least in part, by a common set of genes. To test this hypothesis and identify genes that might control conserved regenerative processes, we performed a comprehensive microarray analysis of the early regenerative response in five regeneration-competent tissues from the newt Notophthalmus viridescens. Consistent with this hypothesis, we established a molecular signature for regeneration that consists of common genes or gene family members that exhibit dynamic differential regulation during regeneration in multiple tissue types. These genes include members of the matrix metalloproteinase family and its regulators, extracellular matrix components, genes involved in controlling cytoskeleton dynamics, and a variety of immune response factors. Gene Ontology term enrichment analysis validated and supported their functional activities in conserved regenerative processes. Surprisingly, dendrogram clustering and RadViz classification also revealed that each regenerative tissue had its own unique temporal expression profile, pointing to an inherent tissue-specific regenerative gene program. These new findings demand a reconsideration of how we conceptualize regenerative processes and how we devise new strategies for regenerative medicine.

Identification of Stanniocalcin 2 as a Novel Aryl Hydrocarbon Receptor Target Gene

Proper hepatocyte function is vital for survival; thus, unrepaired destruction of the parenchymal tissue leading to liver decompensation is devastating. Therefore, understanding the homeostatic process regulating liver regeneration is clinically important, and evidence that the aryl hydrocarbon receptor (AhR) can promote cell survival after intrinsic apoptotic stimuli is integral to the regenerative process. The current study uses primary hepatocytes to identify survival mechanisms consistent with normal AhR biology. Taking advantage of the Cre-lox system to manipulate AhR status, we designed a comprehensive microarray analysis to identify immediate and direct changes in the transcriptome concomitant with the loss of the AhR. As a result, we identified a unique data set with minimal overlap, compared with previous array studies, culminating in the identification of Stanniocalcin 2 (Stc2) as a novel receptor target gene previously reported to have a cytoprotective role in endoplasmic reticulum stress. The Stc2 promoter contains multiple putative xenobiotic response elements clustered in a 250-bp region that was shown to recruit the AhR by chromatin immunoprecipitation. Of interest, Stc2 gene expression is refractory to classic exogenous AhR agonists, but responds to cellular stress in an AhR-dependent mechanism consistent with a process promoting cell survival.

High-Throughput MicroRNA (miRNAs) Arrays Unravel the Prognostic Role of MiR-211 in Pancreatic Cancer

BackgroundOnly a subset of radically resected pancreatic ductal adenocarcinoma (PDAC) patients benefit from chemotherapy, and identification of prognostic factors is warranted. Recently miRNAs emerged as diagnostic biomarkers and innovative therapeutic targets, while high-throughput arrays are opening new opportunities to evaluate whether they can predict clinical outcome. The present study evaluated whether comprehensive miRNA expression profiling correlated with overall survival (OS) in resected PDAC patients.Methodology/Principal FindingsHigh-resolution miRNA profiles were obtained with the Toray's 3D-Gene™-miRNA-chip, detecting more than 1200 human miRNAs. RNA was successfully isolated from paraffin-embedded primary tumors of 19 out of 26 stage-pT3N1 homogeneously treated patients (adjuvant gemcitabine 1000 mg/m2/day, days-1/8/15, every 28days), carefully selected according to their outcome (OS<12 (N = 13) vs. OS>30 months (N = 6), i.e. short/long-OS). Highly stringent statistics included t-test, distance matrix with Spearman-ranked correlation, and iterative approaches. Unsupervised hierarchical analysis revealed that PDACs clustered according to their short/long-OS classification, while the feature selection algorithm RELIEF identified the top 4 discriminating miRNAs between the two groups. These miRNAs target more than 1500 transcripts, including 169 targeted by two or more. MiR-211 emerged as the best discriminating miRNA, with significantly higher expression in long- vs. short-OS patients. The expression of this miRNA was subsequently assessed by quantitative-PCR in an independent cohort of laser-microdissected PDACs from 60 resected patients treated with the same gemcitabine regimen. Patients with low miR-211 expression according to median value had a significantly shorter median OS (14.8, 95%CI = 13.1–16.5, vs. 25.7 months, 95%CI = 16.2–35.1, log-rank-P = 0.004). Multivariate analysis demonstrated that low miR-211 expression was an independent factor of poor prognosis (hazard ratio 2.3, P = 0.03) after adjusting for all the factors influencing outcome.Conclusions/SignificanceThrough comprehensive microarray analysis and PCR validation we identified miR-211 as a prognostic factor in resected PDAC. These results prompt further prospective studies and research on the biological role of miR-211 in PDAC.

Silencing of Lipid Metabolism Genes through IRE1α-Mediated mRNA Decay Lowers Plasma Lipids in Mice

XBP1 is a key regulator of the unfolded protein response (UPR), which is involved in a wide range of physiological and pathological processes. XBP1 ablation in liver causes profound hypolipidemia in mice, highlighting its critical role in lipid metabolism. XBP1 deficiency triggers feedback activation of itsupstream enzyme IRE1α, instigating regulated IRE1-dependent decay (RIDD) of cytosolic mRNAs. Here, we identify RIDD as a crucial control mechanism of lipid homeostasis. Suppression of RIDD byRNA interference or genetic ablation of IRE1α reversed hypolipidemia in XBP1-deficient mice. Comprehensive microarray analysis of XBP1 and/or IRE1α-deficient liver identified genes involved in lipogenesis and lipoprotein metabolism as RIDD substrates, which might contribute to the suppression of plasma lipid levels by activated IRE1α. Ablation of XBP1 ameliorated hepatosteatosis, liver damage, and hypercholesterolemia in dyslipidemic animal models, suggesting that direct targeting of either IRE1α or XBP1 might be a feasible strategy to treat dyslipidemias.

PCBP1 is required for maintenance of the transcriptionally silent state in fully grown mouse oocytes

Global transcriptional silencing in fully grown oocytes is a critical event during mammalian oogenesis. However, how this event is regulated remains elusive. Here, we provide evidence that poly(rC)-binding protein 1 (PCBP1), a protein found by us previously to be present in metaphase II (MII) mouse oocytes, participates in maintenance of the transcriptionally silent state in fully grown mouse oocytes. Knocking down Pcbp1 by microinjection of its specific siRNAs into fully grown germinal vesicle (GV) oocytes resulted in remarkable changes in their transcriptional state, including the disequilibrium between the number of oocytes with an NSN (non-surrounded nucleolus) and those with a SN (surrounded nucleolus), and obvious transcriptional reactiviation in oocytes with a SN configuration as evidenced by BrUTP incorporation assay and immunofluorescent labeling of phosphorylated RNA polymerase II CTD and trimethylated H3 lysine 4, markers for active transcription. Furthermore, in a comprehensive microarray analysis of the preovulatory oocyte transcriptome, an incredible number of nearly 4,000 transcripts were upregulated in the Pcbp1 knockdown groups. These data indicate that lack of the function of PCBP1 disrupts the quiescent status of transcription in the fully grown oocytes, and hence supporting a role of this protein in the regulation of global transcriptional silcencing in fully grown mouse oocytes.

Gene expression profiles in promoted-growth rice seedlings that germinated from the seeds implanted by low-energy N+ beam

The stimulation effect that some beneficial agronomic qualities have exhibited in present-generation plants have also been observed due to ion implantation on plants. However, there is relatively little knowledge regarding the molecular mechanism of the stimulation effects of ion-beam implantation. In order to extend our current knowledge about the functional genes related to this stimulation effect, we have reported a comprehensive microarray analysis of the transcriptome features of the promoted-growth rice seedlings germinating from seeds implanted by a low-energy N+ beam. The results showed that 351 up-regulated transcripts and 470 down-regulated transcripts, including signaling proteins, kinases, plant hormones, transposable elements, transcription factors, non-coding protein RNA (including miRNA), secondary metabolites, resistance proteins, peroxidase and chromatin modification, are all involved in the stimulating effects of ion-beam implantation. The divergences of the functional catalog between the vacuum and ion implantation suggest that ion implantation is the principle cause of the ion-beam implantation biological effects, and revealed the complex molecular networks required to adapt to ion-beam implantation stress in plants, including enhanced transposition of transposable elements, promoted ABA biosynthesis and changes in chromatin modification. Our data will extend the current understanding of the molecular mechanisms and gene regulation of stimulation effects. Further research on the candidates reported in this study should provide new insights into the molecular mechanisms of biological effects induced by ion-beam implantation.

Molecular profiling and comparative genomic hybridization analysis in human melanoma cell lines and identification of BRAF amplification in a PLX4032 acquired resistance cell line.

8592 Background: Melanoma is the most aggressive form of skin cancer. Its management is evolving rapidly due to an improved understanding of the molecular heterogeneity and the development of effective, personalized targeted therapies. PLX4032 is a BRAF inhibitor that induces tumor response in patients with BRAF mutation whose response is limited due to acquired resistance. We used comprehensive microarray and genomic analysis to molecularly characterize a panel of melanoma cell lines with the goal of identifying new molecular subgroups. To better understand the mechanisms of acquired resistance to PLX4032, we included two cell lines that were conditioned in vitro to acquire resistance. Methods: Using microarray, we studied 52 melanoma cell lines for mRNA expression and performed array CGH using genome microarrays. Data analysis was done using Rosetta Resolver and Agilent Analytics 4.0 software. Results: Microarray analysis suggests melanoma is comprised of at least two distinct molecular groups. One group has a differentiated melanocyte phenotype and the other has an expression signature characteristic of progenitor-like cells. The progenitor-like group expresses SOX9, WNT5A and WNT5B and also has the lowest relative expression of MITF. The most differentiated group had the highest MITF and SOX5 levels, while the progenitor-like cell lines have decreased expression. MITF appears to be a strong candidate marker for this group. Positive correlation exists between MITF expression levels and gene copy number, as almost all of the MITF samples amplified by CHG analysis are also overexpressed. A third group shows strong expression of SOX5, SOX10 and Nestin. One of the most important findings is M14-R cell line, conditioned to acquire PLX4032 resistance, has a focal, high level amplification of BRAF (Log2 ratio = 3, 8-fold amplification) when compared with the parental cell line, which is extremely sensitive to PLX4032. Conclusions: These results afford new insight into the potential pathogenesis and classification and provide a greater understanding of melanoma. BRAF amplification could be a novel mechanism of resistance to PLX4032.

Investigatory study of biomarkers for gastric cancer based on a cDNA bank.

4070 Background: We have constructed a cDNA bank using mRNA extracted from frozen specimens of gastric cancer tissue and adjacent normal gastric mucosa and studied biomarkers for the individualized therapy of gastric cancer and the identification of new treatment targets. We report currently available results. Methods: We studied 227 patients in whom at least 5 years had elapsed since surgery for gastric cancer. The disease stage was IB in 29 patients, II in 66, III in 103, and IV in 29. Among the 139 patients who postoperatively received fluoropyrimidine anticancer agents, 82 with stage II or III disease were given adjuvant chemotherapy with S-1. A total 116 genes were selected as candidate biomarkers on the basis of the results of comprehensive DNA microarray analysis, extraction from a serial analysis of gene expression (SAGE) database, and other studies. The relative expression levels of these 116 genes in gastric cancer tissue and adjacent normal mucosa were measured in each case by a quantitative polymerase chain reaction assay using the cDNA databank described above, and the relations between clinical histopathological factors and treatment outcomes were examined. Results: In patients who underwent gastrectomy, high expression levels of the secreted protein acidic and rich in cysteine (SPARC), sulfatase 1 (SULF1), and inhibin, beta A (INHBA) genes were significantly associated with poor outcomes. In patients with stage II or III disease who received adjuvant chemotherapy with S-1, high expression levels of the insulin-like growth factor receptor 1 (IGF-1R), KIAA1199, thymidylate synthase (TS), and regenerating IV (Reg IV) genes were significantly associated with poor survival. Conclusions: Investigatory studies using a cDNA bank of biomarkers for gastric cancer suggested that expression levels of the SPARC, SULFI, and INHBA genes are useful prognostic factors in patients who undergo gastrectomy for gastric cancer. Expression levels of the IGF-1R, KIAA1199, TS, Reg IV, and INHBA genes may be useful biomarkers in patients who receive adjuvant chemotherapy with S-1.

Abstract 186: High-throughput microRNA (miRNAs) arrays unravel the prognostic role of miR211 in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the most lethal among solid tumors. Only a subset of patients benefit from chemotherapy, and identification of novel prognostic factors is warranted. Recently, miRNAs emerged as a pivotal class of regulators for multiple genes involved in cancer, including PDAC (Giovannetti et al, Cancer Res 2010), and miRNA expression pattern classified tumors better than mRNA data (Lu et al, Nature 2005), suggesting their use for diagnostic purposes. However, high-throughput arrays detecting more than 1000 miRNAs are opening new opportunities to evaluate whether their profiles can also predict clinical outcome. Therefore, the present study evaluated whether comprehensive miRNA expression profiling by microarray technology can predict overall survival (OS) in resected PDAC patients. High-resolution miRNA profiles were obtained with the Toray's 3D-GeneTM miRNA chip, detecting 1200 types of human miRNA. RNA was isolated from formaldehyde fixed paraffin embedded primary tumors of 26 stage-pT3N1Mx homogeneously treated patients (gemcitabine 1000 mg/m2/day on days 1/8/15, every 28 days), selected according to their outcome (OS &amp;lt;12 months vs. OS &amp;gt;30 months). Microarray slides of the 19 samples that passed RNA quality check were scanned using 3D-GeneTM Scanner 3000 with appropriate photomultiplier settings for red channel. Each microarray was scanned three times, and then merged into one dataset analyzed. Highly stringent statistics included t-test, Spearman ranked correlation to generate a distance matrix, and iterative approaches. Unsupervised hierarchical analysis revealed that the PDAC specimens clustered according to their long/short OS classification, while the feature selection algorithm RELIEF identified the top 10 discriminating miRNAs between the two groups. MiR211 emerged as the best discriminating miRNA, with significantly higher expression in long vs. short OS patients. The expression of this miRNA was subsequently assessed by quantitative RT-PCR in an independent cohort of laser microdissected tumors from 60 resected stage-pT3N1Mx PDAC patients treated with the same gemcitabine regimen. Patients with low miR211 expression, according to median value (12.8 a.u. calculated as ΔCt vs. RNU6), had a significantly shorter median OS (14.8, 95%CI=13.1-16.5, vs. 25.7 months, 95%CI=16.2-35.1, log-rank P=0.004). Multivariate analysis demonstrated that low miR211 expression was an independent factor of poor prognosis for OS (hazard ratio 2.3, P=0.03) after adjusting for all the factors influencing clinical outcome. In conclusion, through comprehensive microarray analysis and PCR validation we identified miR211 as prognostic factor in resected PDAC. This miRNA was already associated with melanoma invasiveness, but our results prompt further prospective studies as well as research on miR211 biological role in PDAC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 186. doi:1538-7445.AM2012-186