cDNA Microarray Gene Research Articles

TGFβ inhibition and mesenchymal to epithelial transition initiation by Xenopus egg extract: first steps towards early reprogramming in fish somatic cell

Xenopus egg extract is a powerful material to modify cultured cells fate and to induce cellular reprogramming in mammals. In this study, the response of goldfish fin cells to in vitro exposure to Xenopus egg extract, and subsequent culture, was studied using a cDNA microarray approach, gene ontology and KEGG pathways analyses, and qPCR validation. We observed that several actors of the TGFβ and Wnt/β-catenin signaling pathways, as well as some mesenchymal markers, were inhibited in treated cells, while several epithelial markers were upregulated. This was associated with morphological changes of the cells in culture, suggesting that egg extract drove cultured fin cells towards a mesenchymal-epithelial transition. This indicates that Xenopus egg extract treatment relieved some barriers of somatic reprogramming in fish cells. However, the lack of re-expression of pou2 and nanog pluripotency markers, the absence of DNA methylation remodeling of their promoter region, and the strong decrease in de novo lipid biosynthesis metabolism, indicate that reprogramming was only partial. The observed changes may render these treated cells more suitable for studies on in vivo reprogramming after somatic cell nuclear transfer.

Plumbagin-induced anticancer effects are associated with mitochondrial-encoded respiratory gene downregulation in oral squamous cell carcinoma

Plumbagin (PL) is a known quinoid (5-hydroxyl-2-methyl-1,4-napthoquinone) initially isolated from the roots of Plumbago zeylanica L. It is reported to exert anti-proliferative effects in oral squamous cell carcinoma (OSCC) cells, suggesting that PL would be a promising therapeutic anti-cancer drug for OSCC; however, the molecular basis by which PL suppresses cell survival signaling is poorly understood. In this study, we conducted a comprehensive gene expression analysis to identify the molecular basis by which PL suppresses cell survival signaling. Human OSCC cell lines HSC-3 and SAS were used in this study. Comprehensive gene expression analysis was performed with an Agilent Whole Human Genome DNA microarray 4 × 44 format. Gene Set Enrichment Analysis was conducted to investigate the effect of PL on the oncogenic signaling pathway in the OSCC cells. cDNA microarray and subsequent gene set enrichment analyses showed that PL readily downregulates oxidative phosphorylation, MYC-target genes, and MTORC signaling at 4 h after treatment in HSC-3 and SAS cells. Semi-quantitative PCR analysis showed that gene expression levels of mitochondrial (Mt)-encoded COX-2, COX-3, and ND1 genes are significantly lower in the PL-treated OSCC cells than those in the untreated cells. In addition, an ROS scavenger, NAC, reversed the PL-induced downregulation of expression of Mt-encoded genes. Accordingly, intracellular ATP levels significantly decreased after PL treatment. Collectively, PL downregulates both oxidative phosphorylation and oncogenic signatures and decreases the survival of OSCC cells, thus highlighting its application as a potent treatment for patients with OSCC.

Hypoxia Transcriptomic Modifications Induced by Proton Irradiation in U87 Glioblastoma Multiforme Cell Line.

In Glioblastoma Multiforme (GBM), hypoxia is associated with radioresistance and poor prognosis. Since standard GBM treatments are not always effective, new strategies are needed to overcome resistance to therapeutic treatments, including radiotherapy (RT). Our study aims to shed light on the biomarker network involved in a hypoxic (0.2% oxygen) GBM cell line that is radioresistant after proton therapy (PT). For cultivating cells in acute hypoxia, GSI’s hypoxic chambers were used. Cells were irradiated in the middle of a spread-out Bragg peak with increasing PT doses to verify the greater radioresistance in hypoxic conditions. Whole-genome cDNA microarray gene expression analyses were performed for samples treated with 2 and 10 Gy to highlight biological processes activated in GBM following PT in the hypoxic condition. We describe cell survival response and significant deregulated pathways responsible for the cell death/survival balance and gene signatures linked to the PT/hypoxia configurations assayed. Highlighting the molecular pathways involved in GBM resistance following hypoxia and ionizing radiation (IR), this work could suggest new molecular targets, allowing the development of targeted drugs to be suggested in association with PT.

Low temperature induces von-willebrand factor expression via increased early growth response 1 transcriptional activity in splenic sinusoidal endothelial cells

von Willebrand factor (vWF) is a large plasma glycoprotein that plays an important role in hemostasis by forming molecular bridges with platelets following vascular injury. Previously, we reported that hypothermia enhanced vWF production in the spleen, which resulted in the activation of the platelet pool in a hypothermia-induced murine model. However, the mechanisms that regulate vWF expression under hypothermic conditions remain unclear. In this study, we focused on vWF expression under hypothermic conditions in splenic endothelial cell culture.Human splenic endothelial cells (HSEC) were incubated at 20 °C for 1 h. Total RNA was extracted from the cells, and cDNA microarray gene expression analysis was performed. Genes that may be associated with vWF expression in low temperature culture conditions were then selected for further analysis.Gene expression analysis showed that low temperature conditions increased the expression of FOS and EGR1. We then hypothesized that these factors upregulate vWF mRNA expression in HSEC. The transcriptional inhibitors of EGR1 significantly inhibited vWF mRNA expression in HSEC cultured at a low temperature.Our analysis revealed that low temperatures enhance the gene expression of EGR1, which transcriptionally increases vWF expression. This acute-phase reaction may play an important role in platelet activation in the spleen during hypothermia.

Discovery of novel molecular characteristics and cellular biological properties in ameloblastoma

Ameloblastoma is a rare odontogenic benign tumor accounting for less than 1% of head and neck tumors. Advanced next generation sequencing (NGS) analyses identified high frequency of BRAF V600E and SMO L412F mutations in ameloblastoma. Despite the existence of whole genomic sequence information from patients with ameloblastoma, entire molecular signature of and the characteristics of ameloblastoma cells are still obscure. In this study, we sought to uncover the molecular basis of ameloblastoma and to determine the cellular phenotype of ameloblastoma cells with BRAF mutations. Our comparative cDNA microarray analysis and gene set enrichment analysis (GSEA) showed that ameloblastoma exhibited a distinct gene expression pattern from the normal tissues: KRAS‐responsive gene set is significantly activated in ameloblastoma. Importantly, insulin like growth factor 2 (IGF2), a member of KRAS‐responsive genes, enhances the proliferation of an ameloblastoma cell line AMU‐AM1 with BRAF mutation. In addition, Toll‐like receptor 2 (TLR2) knockdown readily inactivated KRAS‐responsive gene sets as well as increases caspase activities, suggesting that TLR2 signaling may mediate cell survival signaling in ameloblastoma cells. Collectively, the findings may help to further clarify the pathophysiology of ameloblastoma and lead to the development of precision medicine for patients with ameloblastoma.

β-Mangostin suppresses LA-7 cells proliferation in vitro and in vivo: Involvement of antioxidant enzyme modulation; suppression of matrix metalloproteinase and α6β4 integrin signalling pathways

β-mangostin (βM) was isolated from Cratoxylum arborescens to investigate anti-breast cancer effect in vitro and in vivo. βM exhibited an inhibitory effect on the growth of LA-7 cells in vitro with apoptosis formation. In the animal model, βM treatment was found to be effective in improving the tissue antioxidant enzymes such as superoxide dismutase and catalase activity (P < 0.05). βM treatment clearly exhibited apoptosis in mammary tumour tissues, and it was associated with regulation of PCNA and p53. The cDNA microarray gene expression followed by qRT-PCR based validation demonstrated that βM could mediate tumour reduction and prevent metastasis by reduction of MMP-9, MMP-13, and MMP-27. Moreover, the reduction of both 14-3-3β and ITGB4 genes indicated the involvement of α6β4 integrin signalling pathway. These findings showed that β-mangostin is a promising compound candidate as an anti-tumour agent against breast cancer.

Abstract 5257: FGF9 overexpression promotes tumorigenic potential of non-small cell lung cancer (NSCLC) cells, and is associated with poor prognosis in NSCLC patients.

Abstract Background. Fibroblast growth factor 9 (FGF9) is a member of the FGF family, which modulates cell proliferation, differentiation and motility. Recent studies show that activation of FGF signals including FGF9 is associated with pathogenesis of several cancers. In lung cancer, some reports showed that FGF9 indirectly promoted the growth of lung adenocarcinoma and the intensity of FGF9 staining was positively correlated with the status of disease and the degree of lymph node metastasis in lung adenocarcinoma patients. However, the direct effect of FGF9 on the development and growth of lung cancer has not been clear. Purpose. The purpose of this study is to clarify the role of FGF9 in NSCLC. Method. First, we have performed in vitro analysis to clarify the role of FGF9 in NSCLC. FGF9 was introduced by retroviral infection to make stable cell lines. The cell lines which express no or low FGF9 were selected for the study, namely A549, PC9 and H1975. Overexpression of FGF9 in these cells was confirmed at mRNA and protein levels. Tumorigenic potential was evaluated by soft agar colony formation assay. The effect on proliferation of NSCLC cells was evaluated by MTS proliferation assay. Next, patients survival analysis was also performed to evaluate the effect of FGF9 on the prognosis of NSCLC patients. NSCLC specimens were obtained from 91 patients who underwent surgical resection at Department of Thoracic Surgery, Keio University Hospital from 2001 through 2006 with written informed consent. We have performed cDNA microarray gene expression analysis. Patient survival data was evaluated by genes expression profile. Results. Of the cells studied, A549 with FGF9 overexpression (A549-FGF9) cells significantly increased the anchorage independent colony formation ability compared with A549-empty cells. The numbers of the colonies were significantly higher in A549-FGF9, and the size of the colonies was bigger compared with A549-empty. For patient study, we found 10 out of 91 (11.0%) NSCLC patients overexpressed FGF9. We found FGF9 overexpression was associated with significantly worse prognosis (p=0.001). While none of other FGFs and FGFRs was associated with the prognosis of the patients. Three-year survival rate of FGF9-high patient group and FGF9-low patient group were 40% and 88% respectively. The rate of relapse was significantly higher in FGF9-high patients compared with FGF9-low patients, 60% vs 36.2% (p=0.016). Conclusion. Our in vitro and clinical data indicate that FGF-9 may promote tumorigenic potential, and can be a prognostic indicator in NSCLC patients. Citation Format: Kota Ishioka, Kenzo Soejima, Hiroyuki Yasuda, Keiko Ohgino, Satoshi Yoda, Takashi Sato, Junko Hamamoto, Daisuke Arai, Tetsuo Tani, Aoi Kuroda, Katsuhiko Naoki, Tomoko Betsuyaku. FGF9 overexpression promotes tumorigenic potential of non-small cell lung cancer (NSCLC) cells, and is associated with poor prognosis in NSCLC patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5257. doi:10.1158/1538-7445.AM2013-5257

A novel fumarate hydratase-deficient HLRCC kidney cancer cell line, UOK268: a model of the Warburg effect in cancer

The role of energy deregulation and altered/adapted metabolism in tumor cells is an increasingly important issue in understanding cancer. Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an aggressive form of RCC characterized by germline mutation of fumarate hydratase (FH), followed by somatic loss of the remaining wild-type allele and known to be a highly metastatic and lethal malignancy compared to other RCCs. The intrinsic loss of normal tricarboxylic acid (TCA) cycle presumably aids tumorigenesis due to the necessary metabolic alterations required and the enforced dependence on glycolysis derived energy, mimicking the Warburg effect. Thus, there is considerable utility in establishing a preclinical cell model from these tumors to study energy metabolism deregulation, as well as developing new targeted therapeutic approaches for TCA cycle enzyme-deficient cancers. Here, we describe a new immortalized cell line, UOK268, derived from a patient's primary HLRCC-associated kidney cancer. This represents the first primary renal cell line to model TCA cycle gene loss and provides a perfect partner cell line to our previously described metastasis-derived HLRCC-associated cell line, UOK262. We identified a novel germline FH missense mutation, p.His192Asp, and the subsequent loss of heterozygosity in UOK268. The UOK268 cell line expressed mutant FH protein, which localized to the mitochondria, but with loss of almost all catalytic activity. The UOK268 cells had severely compromised oxidative phosphorylation and increased glycolytic flux. Ingenuity pathways analysis of human mitochondria-focused cDNA microarray (hMitChip3) gene chip data confirmed the altered mRNA expression patterns of genes involved in several important pathways, such as lipid metabolism, apoptosis, and energy production/glycolysis. UOK268 provides a unique model of a primary cell line demonstrating an enforced, irreversible Warburg effect and, combined with UOK262, provides a unique invitro preclinical model for studying the bioenergetics of the Warburg effect in human cancer.

Abstract 3821: Interaction and cross-resistance of cisplatin and pemetrexed in malignant pleural mesothelioma cell lines

Abstract Background: Malignant pleural mesothelioma (MPM) is one of the most lethal cancers with a rapidly increasing incidence worldwide. Although cisplatin and pemetrexed are recognized as key drugs, and their combination chemotherapy is a standard therapy for MPM, their drug-to-drug interactions, cross-resistance, and resistance mechanisms are not well understood. Methods: The interaction between cisplatin and pemetrexed was evaluated by colonogenic assays followed by isobologram analysis in 4 human MPM cell lines. The cell lines were exposed to the agents using a stepwise dose-escalation method to establish drug-resistant sublines. Thymidylate synthase (TS) mRNA expression was evaluated by RT-PCR, and gene expression profiles were assessed by cDNA microarray analysis and gene ontology analysis using Ingenuity Pathway Analisis (IPA) in the drug-resistant sublines. Results: Cisplatin and pemetrexed had synergistic effects in 3 cell lines and an additive effect in the fourth cell line. The former 3 cell lines showed similar sensitivity to pemetrexed in the parental cells and their cisplatin-resistant sublines, whereas the fourth cell line exhibited cross-resistance. In contrast, cisplatin had diverse effects on pemetrexed-resistant sublines. High TS expression did not correlate with natural pemetrexed resistance and its elevatedexpression correlated with acquired pemetrexed resistance in 2 sublines. Microarray analysis disclosed that the genes which related to “cellular development”,” small molecule biochemistry,” “cellular movement,” “cell death, and “cellular growth and proliferation” affected drug-resistant mechanisms deeply, and that this tendency was common to cisplatin-resistant sublines and pemetrexed-resistant sublines. Conclusion: Cisplatin and pemetrexed showed synergistic activity and no cross-resistance in 3 of the 4 MPM cell lines, suggesting the clinical relevance of their combination chemotherapy. TS expression did not necessarily correlate with pemetrexed resistance. Multiple mechanisms of molecular and cellular functions may underlie acquired resistance to cisplatin and pemetrexed in MPM. 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 3821. doi:1538-7445.AM2012-3821

Modulation of gene expression in cold-induced sweetening resistant potato species Solanum berthaultii exposed to low temperature

Cold-induced sweetening (CIS) is a crucial factor influencing the processing quality of potato tubers. To better understand the molecular events of potato CIS and different CIS-sensitivity among various potato species, a suppression subtractive hybridization library and cDNA microarray gene filters were developed. A total of 188 genes were found to be differentially expressed (DE) in Solanum berthaultii (ber) upon cold stimulation. These functional genes were mostly related to cell rescue, defense and virulence, metabolism, energy and protein fate, included in various processes of plant defense against abiotic stresses. Four expression patterns of these DE genes were profiled by qRT-PCR using the cold-stored tubers of both CIS-resistant (ber) and CIS-sensitive (E-potato 3, a variety of S. tuberosum) potatoes. The expression pattern and abundance of many DE genes encoding proteins involved in metabolism were different in these two potato tubers, especially genes associated with amylolysis, sucrose decomposition and glycolysis pathways, indicating distinct regulatory mechanisms between ber and E3 in response to cold stress, which may be crucial for potato CIS. Further investigation of these cold-regulated genes will deepen our understanding of the regulatory mechanisms of potato CIS and direct approaches for the genetic improvement of potato processing quality.

Microarray analysis of global gene regulation in the Cry1Ab‐resistant and Cry1Ab‐susceptible strains of Diatraea saccharalis

Extensive adoption of transgenic Bt corn in recent years for stalk borer control has increased risk of resistance evolution in the target pest populations. A Bt-resistant strain of the sugarcane borer, Diatraea saccharalis, was approximately 100-fold more tolerant to Cry1Ab toxin than the susceptible counterpart. To gain a better understanding of the molecular mechanisms of Bt resistance, the Cry1Ab-susceptible (Cry1Ab-SS) and Cry1Ab-resistant (Cry1Ab-RR) strains of D. saccharalis were subjected to a microarray analysis. Results showed that the expression levels of many genes were significantly different between the Cry1Ab-RR and Cry1Ab-SS strains. Microarray analysis of 7145 cDNAs revealed 384 differentially expressed genes. A total of 273 genes were significantly upregulated 2-51.6-fold, and 111 genes were significantly downregulated 2-22.6-fold in the Cry1Ab-RR strain. The upregulation of three potential resistance-related genes, coding for a glutathione S-transferase (GST), a chymotrypsin-like protease (CHY) and a lipase (LP), was confirmed using real-time PCR, indicating a reproducibility of the microarray data. Ontology analysis revealed that more than twice the number of metabolic-related genes were upregulated compared with downregulated genes with the same biological function. Up to 35.2% of the upregulated genes in the resistant strain were associated with catalytic activity, while only 9.5% of the downregulated genes were related to the same catalytic molecular function. The large portion of metabolic- or catalytic-related genes with significant upregulations indicated a potential large increase in metabolic or catalytic activities in the Cry1Ab-RR strain. This cDNA microarray gene expression data could be used to characterize and identify new genes that may be associated with Bt resistance in D. saccharalis.

P2-05-04: Mapping the Specific Gene Families Activated in the Lymphangiogenesis and Vasculogenic Mimicry Exhibited by Inflammatory Breast Cancer.

Abstract Background: Inflammatory breast cancer (IBC) is the most metastatic variant of locally advanced breast cancer. Although IBC is diagnosed less commonly than other types of breast cancer, it is extremely aggressive, and accounts for a disproportionate number of breast cancer related deaths annually. IBC exhibits very specific patterns of lymphangiogenesis and vasculogenic mimicry, however detailed studies of the genes and proteins involved in these angiogenic processes are lacking. This study performed whole unbiased gene transcription studies with validation by protein arrays using all available pre-clinical cell lines and in vivo xenograft models of IBC, including a new model of IBC, FC-IBC01, which exhibits lymphovascular invasion, to identify the specific pathways involved in the distinctive angiogenesis observed in IBC. Materials and Methods: Real-time quantitative RT-PCR, cDNA microarray gene profiling, immunofluorescence with confocal imaging and protein arrays were used to examine differential expression of specific angiogenic gene families including VEGFA,B,C,D, VEGF Receptor genes, and ANG/TIE genes linked to angiogenesis and lymphangiogenesis. Results: Activity of the matrix metalloproteinase, MMP-2, is required for IBC tumor cells to undergo vasculogenic mimicry (VM), which is associated with a loss of TIMP-2, a well known inhibitor of angiogenesis. Therapeutics that target MMP activity can successfully inhibit this VM. Furthermore, pre-clinical models of IBC that form IBC tumor emboli exhibit lymphovascular invasion that is associated with distinct patterns of expression of genes that encode for distinct receptor tyrosine kinases that may represent important therapeutic targets for IBC. Discussion: Identification of the distinct angiogenic pathways that are activated in IBC provides insight into the therapeutic targets that may abrogate the distinct lymphovascular invasion and vasculogenic mimicry that are linked to the aggressive metastasis of IBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-05-04.

The GLI genes as the molecular switch in disrupting Hedgehog signaling in colon cancer

The Hedgehog (HH) signaling pathway leads to activation of GLI, which transcriptionally regulate target genes. Regulated HH signaling activity is critical during embryogenesis while aberrantly activated HH signaling is evident in a variety of human cancers. Canonical HH signaling engages the transmembrane receptor Patched (PTCH) and the signaling intermediate Smoothened (SMO) to activate GLI1 and GLI2. In addition GLI1 and GLI2 are activated by non-canonical oncogenic signaling pathways to further drive HH-dependent survival. We have demonstrated in human colon carcinoma cells that inhibition of the RAS/RAF pathway by U0126 decreases p-ERK protein expression and also inhibits GLI-luciferase activity and GLI1 mRNA and protein levels. Of importance is the demonstration that targeting of SMO (using cyclopamine) has minimal effect on cell survival in comparison to the inhibition of GLI (using GANT61), which induced extensive cell death in 7/7 human colon carcinoma cell lines. Genetic inhibition of the function of GLI1 and GLI2 by transient transfection of the C-terminus deleted repressor GLI3R, reduced proliferation and induced cleavage of caspase-3 and cell death in HT29 cells, similar to the effects of GANT61. Mechanistically, downstream of GLI1 and GLI2 inhibition, γH2AX (a marker of DNA double strand breaks) expression was upregulated, and γH2AX nuclear foci were demonstrated in cells that expressed GLI3R. Activation of the ATM/Chk2 axis with co-localization of γH2AX and p-Chk2 nuclear foci were demonstrated following GLI1/GLI2 inhibition. GANT61 induced cellular accumulation at G1/S and early S with no further progression before cells became subG1, while cDNA microarray gene profiling demonstrated downregulation of genes involved in DNA replication, the DNA damage response, and DNA repair, mechanisms that are currently being pursued. These studies highlight the importance of targeting the GLI genes downstream of SMO for terminating HH-dependent survival, suggesting that GLI may constitute a molecular switch that determines the balance between cell survival and cell death in human colon carcinoma.

Abstract 1656: Termination of hedgehog (HH) survival signaling at the level of the GLI genes induces DNA damage and extensive cell death in human colon carcinoma cells

Abstract Temporally and spatially regulated activity of the HH signaling pathway is important for normal embryonic development and organ patterning while aberrant activation of this pathway is involved in the development of genomic instability and dysregulated proliferation in several human cancers. There is emerging evidence that HH signaling progresses during colon carcinogenesis and in metastatic disease, however little is known functionally about this signaling pathway and how it affects the survival and pathogenesis of colon cancer. Canonical HH signaling occurs via Ptch1, Smo, and activation of Gli1 and Gli2, which transcriptionally regulate HH target genes. We demonstrate that a small molecule inhibitor of Gli1 and Gli2, GANT61, in contrast to cyclopamine (Smo inhibitor), induced extensive cell death in six human colon carcinoma cell lines. Detailed mechanistic studies in HT29 cells demonstrated that GANT61 induced transient accumulation of cells at the G1/S boundary and in early S at 24 hr and 32 hr, respectively, along with upregulation of p21Cip1, cyclin E and cyclin A in G1- and S- phase cells. By 40 hr, GANT61-treated cells (but not cells treated with cyclopamine) became subG1. shRNA-mediated knockdown of p21Cip1 failed to alter the cytotoxicity of GANT61. cDNA microarray gene profiling in GANT61-treated cells identified significant changes in expression of genes involved in DNA damage signaling, DNA replication and DNA repair. In single cell assays (Comet), GANT61 induced DNA damage by 24 hr with increased Tail Moment and in particular Tail Length; within 4 hr, γH2AX foci were detected at sites of DNA breaks (confocal microscopy). Upregulation of activated forms of ATM and Chk2 but not ATR or Chk1 at 4 hr, that were sustained (24 hr), was observed by western analysis following GANT61 exposure. Further, by 4 hr, p-Chk2 foci co-localized with γH2AX foci in nuclei. Partial knockdown of GLI1 and GLI2 using shRNA partially protected HT29 cells from GANT61-induced cell death, and reduced nuclear γH2AX foci. Studies of the extrinsic and intrinsic pathways of apoptosis demonstrated increased expression of Fas and DR5 receptors, decreased expression of PDGFRα (GLI1 target which regulates Fas), and decreased Bcl-2 expression (GLI2 target) following GANT61 treatment. Expression of DNFADD (to abrogate death receptor signaling) and/or over-expression of Bcl-2 (to block mitochondria-mediated apoptosis) partially rescued from GANT61-induced cell death. Data demonstrate the critical role of HH signaling in colon cancer cell survival, and that inhibition of this pathway at the level of the GLI genes in contrast to Smo induces 1) DNA damage, 2) transient accumulation of cells at G1/S and early S, 3) ineffective checkpoint activation, and 4) extensive cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1656. doi:10.1158/1538-7445.AM2011-1656

CDNA Microarray Gene Expression Profiling of Hedgehog Signaling Pathway Inhibition in Human Colon Cancer Cells

BackgroundHedgehog (HH) signaling plays a critical role in normal cellular processes, in normal mammalian gastrointestinal development and differentiation, and in oncogenesis and maintenance of the malignant phenotype in a variety of human cancers. Increasing evidence further implicates the involvement of HH signaling in oncogenesis and metastatic behavior of colon cancers. However, genomic approaches to elucidate the role of HH signaling in cancers in general are lacking, and data derived on HH signaling in colon cancer is extremely limited.Methodology/Principal FindingsTo identify unique downstream targets of the GLI genes, the transcriptional regulators of HH signaling, in the context of colon carcinoma, we employed a small molecule inhibitor of both GLI1 and GLI2, GANT61, in two human colon cancer cell lines, HT29 and GC3/c1. Cell cycle analysis demonstrated accumulation of GANT61-treated cells at the G1/S boundary. cDNA microarray gene expression profiling of 18,401 genes identified Differentially Expressed Genes (DEGs) both common and unique to HT29 and GC3/c1. Analyses using GenomeStudio (statistics), Matlab (heat map), Ingenuity (canonical pathway analysis), or by qRT-PCR, identified p21Cip1 (CDKN1A) and p15Ink4b (CDKN2B), which play a role in the G1/S checkpoint, as up-regulated genes at the G1/S boundary. Genes that determine further cell cycle progression at G1/S including E2F2, CYCLIN E2 (CCNE2), CDC25A and CDK2, and genes that regulate passage of cells through G2/M (CYCLIN A2 [CCNA2], CDC25C, CYCLIN B2 [CCNB2], CDC20 and CDC2 [CDK1], were down-regulated. In addition, novel genes involved in stress response, DNA damage response, DNA replication and DNA repair were identified following inhibition of HH signaling.Conclusions/SignificanceThis study identifies genes that are involved in HH-dependent cellular proliferation in colon cancer cells, and following its inhibition, genes that regulate cell cycle progression and events downstream of the G1/S boundary.

Abstract 785: Gene expression profiling identifies two stable clusters of lobular carcinoma in situ

Abstract INTRODUCTION: Historical data suggests that lobular carcinoma in situ (LCIS) is not a precursor to invasive cancer but rather is a marker of increased risk, conferred equally to both breasts. Emerging evidence of genomic similarities between some LCIS and invasive cancers suggests that as subset of LCIS may exhibit precursor potential. The aim of this study was to look for evidence of distinct molecular subtypes of LCIS using cDNA microarray gene expression analysis. METHODS: Mastectomy specimens from 9 patients with classical LCIS only, 74 patients with classical LCIS &amp; synchronous invasive cancer (51 ILC, 16 IDC, 7 mixed) and 5 patients with LCIS + DCIS were selectively sampled using laser capture microdissection. Representative patient matched normal (NL), LCIS and invasive cells were microdissected from fresh frozen (FF) tissue samples. RNA was extracted and hybridized to Affy. HG-U133A 2.0 oligonucleotide arrays. Unsupervised hierarchical clustering of all LCIS samples and matched paired analysis of NL vs LCIS and LCIS vs ILC were performed using the R software (bioconductor package) and data were analyzed with Ingenuity Pathway Analysis filtered for human genes; p 2. RESULTS: Suitable RNA was obtained from 48 FF LCIS samples. Patient matched NL and ILC samples were available for 42 and 17 cases, respectively. Hierarchical cluster analysis of all LCIS (n=48) demonstrated two stable clusters with median co-cluster frequency of 79% and 83%. The NL vs LCIS paired comparison (n=42) showed 306 differentially expressed genes (DEG) in Cluster 1 (FC &amp;gt;2: 3.9% DEG) and 2012 DEG in Cluster 2 (FC &amp;gt;2: 2.5% DEG). There were no common DEGs with FC &amp;gt; 2 between clusters 1 and 2 for the NL vs LCIS comparison. The LCIS vs ILC paired comparison (n=17) showed 87 DEG in Cluster 1 (FC &amp;gt;2: 58.6%) and 339 DEG in Cluster 2 (FC &amp;gt;2: 27.7% DEG). There were 18/87 (21%) shared DEG between Cluster 1 and Cluster 2 for the LCIS vs ILC comparison. The functions of these 18 genes include biologically relevant genes involved in cell adhesion, cell-matrix interaction, cell movement, subcellular organization and cell proliferation. CONCLUSION: Unsupervised hierarchical analysis demonstrated two stable distinct clusters of LCIS. Matched paired analysis of NL vs LCIS suggests that LCIS in cluster 1 is more similar to NL epithelium than LCIS in cluster 2. The absence of overlap in DEG with FC &amp;gt; 2 for this comparison suggests that the initiation of lobular neoplasia may be driven by alternate genes in these two clusters. Matched paired analysis of LCIS vs ILC demonstrates 3.89 times more DEG in cluster 2 than 1, however the proportion of genes with FC &amp;gt;2 was 2.2 times higher in cluster 1, suggesting both quantitative and qualitative differences in these groups. These preliminary analyses support the hypothesis that LCIS is a heterogeneous lesion, further analysis to determine if there is a subtype of LCIS with true precursor potential is underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 785.

Microarray Analysis of Papillary Thyroid Cancers in Korean

Background/AimsPapillary thyroid cancer (PTC) is the most common malignancy of the thyroid gland. It involves several molecular mechanisms. The BRAF V600E mutation has been identified as the most common genetic abnormality in PTC. Moreover, it is known to be more prevalent in Korean PTC patients than in patients from other countries. We investigated distinct genetic profiles in Korean PTC through cDNA microarray analysis.MethodsTranscriptional profiles of five PTC samples and five paired normal thyroid tissue samples were generated using cDNA microarrays. The tumors were genotyped for BRAF mutations. The results of the cDNA microarray gene expression analysis were confirmed by real-time PCR and immunohistochemistry analysis of 35 PTC patients.ResultsFour of the five patients whose PTC tissues were subjected to microarray analysis were found to carry the BRAF V600E mutation. Microarrays analysis of the five PTC tissue samples showed the expression of 96 genes to be increased and that of 16 genes decreased. Real-time reverse transcription-polymerase chain reaction (RT-PCR) confirmed increased expression of SLC34A2, TM7SF4, COMP, KLK7, and KCNJ2 and decreased expression of FOXA2, SLC4A4, LYVE-1, and TFCP2L1 in PTC compared with normal tissue. Of these genes, TFCP2L1, LYVE-1, and KLK7 were previously unidentified in PTC microarray analysis. Notably, Foxa2 activity in PTC was reduced, as shown by its cytoplasmic localization, in immunohistochemical analyses.ConclusionsThese findings demonstrate both similarities and differences between our results and previous reports. In Korean cases of PTC, Foxa2 activity was reduced with its cytoplasmic accumulation. Further studies are needed to confirm the relationship between FOXA2 and BRAF mutations in Korean cases of PTC.

Genomic deletions at 1p and 14q are associated with an abnormal cDNA microarray gene expression pattern in meningiomas but not in schwannomas

The molecular pathology of meningiomas and shwannomas involve the inactivation of the NF2 gene to generate grade I tumors. Genomic losses at 1p and 14q are observed in both neoplasms, although more frequently in meningiomas. The inactivation of unidentified genes located in these regions appears associated with tumor progression in meningiomas, but no clues to its molecular/clinical meaning are available in schwannomas. Recent microarray gene expression studies have demonstrated the existence of molecular subgroups in both entities. In the present study, we correlated the presence of genomic deletions at 1p, 14q, and 22q with the expression patterns of 96 tumor-related genes obtained by cDNA low-density microarrays in a series of 65 tumors including 42 meningiomas and 23 schwannomas. Two expression pattern groups were identified by cDNA mycroarray analysis when compared to the expression pattern in normal control RNA in both meningiomas and schwannomas, each one with patterns similar and different from the normal control. Meningioma and schwannoma subgroups differed in the expression of 38 and 16 genes, respectively. Using MLPA and microsatellites, we identified genomic losses at 1p, 14q, and 22q at nonrandom frequencies (12.5–69%) in meningiomas and schwannomas. Losses at 22q were almost equally frequent in both molecular expression subgroups in both neoplasms. However, deletions at 1p and 14q accumulated in meningiomas with a gene expression pattern different from the normal pattern, whereas the inverse situation occurred in schwannomas. Those anomalies characterized the schwannomas with expression pattern similar to the normal control. These findings suggest that deletions at 1p and 14q enhance the development of an abnormal tumor-related gene expression pattern in meningiomas, but this fact is not corroborated in schwannomas.

Pancreatic Expression of DOG1

The cDNA microarray gene profile of gastrointestinal stromal tumors (GISTs) revealed that DOG1 (TMEM16A) gene was mostly expressed in these neoplasms. Immunohistochemically, DOG1 protein was found positive in a significant proportion of GISTs. However, normal tissues' expression of DOG1 is not yet completely studied. Our study intended to identify the DOG1 protein expression in normal adult and fetal tissues, in comparison with that of GISTs, using an anti-DOG1 polyclonal serum. Fourteen CD117/CD34-positive GIST cases were tested for DOG1. Tissue samples from autopsies of 15 human fetuses and 11 adults were tested immunohistochemically on simple or double staining with antibodies raised against: DOG1, insulin, glucagon, somatostatin, NK1, PGP9.5, chromogranin A, and synaptophysin. All the tested GISTs were positive for DOG1, with a membranous and cytoplasmic location. The normal tissues showed a distinct positivity for DOG1 only in the endocrine pancreas, in both fetal and adult ones. The other tissues tested showed a weak or negative reaction. The DOG1 staining pattern in the pancreas islets was granular, like that of neuroendocrine markers. The location of DOG1 expression in pancreatic islets was partly similar to neuroendocrine markers chromogranin A, PGP9.5, and synaptophysin. The positive cells were situated centrally, in the vicinity of insulin-bearing cells as seen on double staining. DOG1 positivity in fetal and adult pancreatic islets suggests the strong antibody affinity for neuroendocrine cells. Before making a final conclusion regarding the suitability of DOG1 as a new neuroendocrine marker, a large survey of neuroendocrine lesions must be undertaken, including carcinoid tumors of various sites and pancreatic endocrine tumors. To the best of our knowledge, this particular localization has not been reported yet for DOG1.

The MAGi RNA extraction method: a highly efficient and simple procedure for fresh and dry plant tissues

AbstractBACKGROUND: Samples from different plant species, different organs or tissues at different times of the year, usually show great differences in their cell compositions, pH, and the endogenous RNase activities, decreasing the RNA yield and quality.RESULTS: In this study we describe a reagent and a simple total RNA isolation method for plant organs, tissues and dry seeds. The RNA extraction reagent (MAGi) is non‐toxic and can be stored at room temperature for several months to years. The principle of the total RNA extraction is that tissues are lysed in extraction solution with the aid of mortar homogenization–maceration, and cellular proteins, polysaccharides and DNA are removed from the RNA. We tested the reported method on more than 16 different types of plant seed and 15 different tissues and organs of pepper.CONCLUSION: The RNA extraction procedure reported in the present study greatly reduces the time required to isolate dry seed total RNA and other tissues by more than half as compared with the previously reported methods. The range of typical RNA yield and quality represents a significant improvement over existing protocols. The quality is high enough to be considered as suitable method for RT‐PCR, cDNA library construction and microarray gene expression studies. Copyright © 2008 Society of Chemical Industry