Performed Gene Expression Profile Analysis Research Articles

Reduced expression of alanyl aminopeptidase is a robust biomarker of non-familial adenomatous polyposis and non-hereditary nonpolyposis colorectal cancer syndrome early-onset colorectal cancer.

Early-onset colorectal cancer (EOCRC) has been increasing in incidence worldwide but its genomic pathogenesis is mostly undetermined. This study aimed to identify robust EOCRC-specific gene expression patterns in non-familial adenomatous polyposis (FAP) and non-hereditary nonpolyposis colorectal cancer syndrome (HNPCC) EOCRC. We first performed gene expression profiling analysis using RNA sequencing of discovery cohort comprised of 49 EOCRC (age <50) and 50 late-onset colorectal cancer (LOCRC) (age >70) specimens. To obtain robust gene expression data from this analysis, we validated differentially expressed genes (DEGs) through TCGA cohort (EOCRC:59 samples, LOCRC:229 samples) and our validation cohort (EOCRC:72 samples, LOCRC:43 samples) using real-time RT-PCR. After the validation of DEGs, we validated the selected gene at protein levels using Western blotting. To identify whether genomic methylation regulates the expression of a particular gene, we selected methylation sites using The Cancer Genome Atlas (TCGA) datasets and validated them by pyrosequencing in our validation cohort. The EOCRC patients included in this study had significantly more prominent family history of cancer than the LOCRC patients (23 [46.9%] vs. 13 [26%], p=0.050). Alanyl aminopeptidase (ANPEP) was significantly downregulated in the EOCRC tissues (FC=1.78, p=0.0007) and was also commonly downregulated in the TCGA cohort (FC=-1.08, p=0.0021). Moreover, the ANPEP mRNA and protein expression levels were significantly downregulated in the EOCRC tissues of our validation cohort (p=0.037 and 0.027). In comparisons of the normal and tumor tissues in public datasets, the ANPEP level was significantly lower in the tumor tissue in the TCGA dataset (p < 2.2 × 10-16 ) and GSE196006 dataset (p=0.0005). Furthermore, the ANPEP expression level did not show a decreasing tendency at a young age in the normal colon tissue of the GTEx dataset. Lastly, the hypermethylation of cg26222247 in ANPEP was identified to be weakly associated with reduced ANPEP expression in our EOCRC cohort. The reduced expression of ANPEP was identified as a novel biomarker of non-FAP and non-HNPCC EOCRC.

TLR9 signalling in HCV-associated atypical memory B cells triggers Th1 and rheumatoid factor autoantibody responses

Hepatitis C virus (HCV) infection contributes to the development of autoimmune disorders such as cryoglobulinaemia vasculitis (CV). However, it remains unclear why only some individuals with HCV develop HCV-associated CV (HCV-CV). HCV-CV is characterized by the expansion of anergic CD19+CD27+CD21low/- atypical memory B cells (AtMs). Herein, we report the mechanisms by which AtMs participate in HCV-associated autoimmunity. The phenotype and function of peripheral AtMs were studied by multicolour flow cytometry and co-culture assays with effector T cells and regulatory T cells in 20 patients with HCV-CV, 10 chronicallyHCV-infected patients without CV and 8 healthy donors. We performed gene expression profile analysis of AtMs stimulated or not by TLR9. Immunoglobulin gene repertoire and antibody reactivity profiles of AtM-expressing IgM antibodies were analysed following single B cell FACS sorting and expression-cloning of monoclonal antibodies. The Tbet+CD11c+CD27+CD21- AtM population is expanded in patients with HCV-CV compared to HCV controls without CV. TLR9 activation of AtMs induces a specific transcriptional signature centred on TNFα overexpression, and an enhanced secretion of TNFα and rheumatoid factor-type IgMs in patients with HCV-CV. AtMs stimulated through TLR9 promote type 1 effector T cell activation and reduce the proliferation of CD4+CD25hiCD127-/lowFoxP3+ regulatory T cells. AtM expansions display intraclonal diversity with immunoglobulin features of antigen-driven maturation. AtM-derived IgM monoclonal antibodies do not react against ubiquitous autoantigens or HCV antigens including NS3 and E2 proteins. Rather, AtM-derived antibodies possess rheumatoid factor activity and target unique epitopes on the human IgG-Fc region. Our data strongly suggest a central role for TLR9 activation of AtMs in driving HCV-CV autoimmunity through rheumatoid factor production and type 1 T cell responses. B cells are best known for their capacity to produce antibodies, which often play a deleterious role in the development of autoimmune diseases. During chronic hepatitis C, self-reactive B cells proliferate and can be responsible for autoimmune symptoms (arthritis, purpura, neuropathy, renal disease) and/or lymphoma. Direct-acting antiviral therapy clears the hepatitis C virus and eliminates deleterious B cells.

Abstract 4699: Identification of novel potential biomarkers of response to sunitinib in glioblastoma

Abstract Introduction: Glioblastoma multiforme (GBM) is the most advanced and aggressive form of primary malignant brain tumors in adults. O6-Methylguanine methyltransferase (MGMT) is a DNA repair protein well known for its role in resistance to temozolomide used in standard treatment of patients newly diagnosed with GBM. Sunitinib is an oral multitargeting receptor tyrosine kinase (RTK) inhibitor with antiangiogenic and antiproliferative activities targeting several RTKs. Our group has previously shown the role of MGMT as a negative regulator of angiogenesis and invasion and the differential antiproliferative effect of sunitinib based on expression of MGMT in GBM. Recent gene expression profiling (GEP) studies showed that besides its effects on RTKs, sunitinib affects the expression level of other genes at the transcriptional level in different cancer types. We hypothesized that: (i) the mechanism of action of sunitinib may not be limited to direct inhibition of its known targets (RTKs) in GBM and (ii) expression of MGMT may affect response of GBM tumor cells to sunitinib. We performed GEP analysis to identify genes that could be potentially modulated in response to sunitinib in GBM cell lines isogenic for MGMT. Empty vector (EV)-transfected T98/EV cells with constitutive expression of MGMT and its knockdown counterpart T98/MGMT-shRNA were treated with sunitinib or vehicle control. Treatment of T98/EV and T98/MGMT-shRNA cell lines with sunitinib significantly changed the expression of over 2,000 gene transcripts (analysis performed at Genome Quebec Innovation center, McGill University). Lists of significant genes were fitted into previously known biochemical pathways, available among the canonical pathways from various databases provided by ConsensusPathDB (CPDB). Strikingly, immune system was among the top significantly enriched pathways (p-value&amp;lt;10-3), with some immune pathways not previously reported for response to sunitinib. Conclusion: Our study opens new avenues to further understand the mechanisms of action of sunitinib, investigate its immunomodulatory effects, and identify biomarkers for selection of patients who might benefit from the combination of antiangiogenic and immunebased strategies in GBM. Funding Acknowledgment: Cancer Research Society Operating Grant Competition #22716, and Higher Education Quality Enhancement Project (HEQEP)- Window -4 (Grant Id. CP-4023), University Grant Commission (UGC), Bangladesh. Citation Format: Abu Shadat Mohammod Noman, Bassam Abdulkarim, Siham Sabri. Identification of novel potential biomarkers of response to sunitinib in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4699.

Leveraging increased cytoplasmic nucleoside kinase activity to target mtDNA and oxidative phosphorylation in AML

AbstractMitochondrial DNA (mtDNA) biosynthesis requires replication factors and adequate nucleotide pools from the mitochondria and cytoplasm. We performed gene expression profiling analysis of 542 human acute myeloid leukemia (AML) samples and identified 55% with upregulated mtDNA biosynthesis pathway expression compared with normal hematopoietic cells. Genes that support mitochondrial nucleotide pools, including mitochondrial nucleotide transporters and a subset of cytoplasmic nucleoside kinases, were also increased in AML compared with normal hematopoietic samples. Knockdown of cytoplasmic nucleoside kinases reduced mtDNA levels in AML cells, demonstrating their contribution in maintaining mtDNA. To assess cytoplasmic nucleoside kinase pathway activity, we used a nucleoside analog 2′3′-dideoxycytidine (ddC), which is phosphorylated to the activated antimetabolite, 2′3′-dideoxycytidine triphosphate by cytoplasmic nucleoside kinases. ddC is a selective inhibitor of the mitochondrial DNA polymerase γ. ddC was preferentially activated in AML cells compared with normal hematopoietic progenitor cells. ddC treatment inhibited mtDNA replication, oxidative phosphorylation, and induced cytotoxicity in a panel of AML cell lines. Furthermore, ddC preferentially inhibited mtDNA replication in a subset of primary human leukemia cells and selectively targeted leukemia cells while sparing normal progenitor cells. In animal models of human AML, treatment with ddC decreased mtDNA, electron transport chain proteins, and induced tumor regression without toxicity. ddC also targeted leukemic stem cells in secondary AML xenotransplantation assays. Thus, AML cells have increased cytidine nucleoside kinase activity that regulates mtDNA biogenesis and can be leveraged to selectively target oxidative phosphorylation in AML.

Association of Ribonuclease T2 Gene Polymorphisms With Decreased Expression and Clinical Characteristics of Severity in Crohn’s Disease

Variants in the tumor necrosis factor superfamily member 15 gene (TNFSF15, also called TL1A) have been associated with risk for inflammatory bowel disease (IBD). TL1A affects expression of multiple cytokines to promote mucosal inflammation. Little is known about the TL1A-response pathways that regulate cytokine expression. We investigated T-cell gene expression patterns to determine the mechanisms by which TL1A regulates cytokine production, and whether these associate with outcomes of patients with Crohn's disease (CD). Peripheral T cells isolated from normal donors were cultured with TL1A. We performed gene expression profile analysis by RNA sequencing of subsets of interferon gamma (IFNG)-producing and non-producing cells purified by flow cytometry. Unsupervised hierarchical clustering analysis was used to identify gene expression differences between these subsets. Ribonuclease T2 gene (RNASET2) expression and methylation were assessed by quantitative trait loci analyses. Clinical characteristics of patients (complications, resistance to therapy, and recurrence time) were associated with single nucleotide polymorphisms in RNASET2. We performed motif screening to identify polymorphisms that disrupt transcription factor binding sites. Levels of RNASET2 were knocked down with small interfering RNA in CD4+ T cells and the effect on protein expression was determined by proteomic analysis and cytokine production. Cell aggregation was measured by flow cytometry. We identified 764 genes with at least a 2-fold difference in TL1A-mediated expression between IFNG-secreting and non-secreting T cells (P < 1× 10-5). Many of these genes were located near IBD susceptibility variants. RNASET2 was the only IBD risk-associated gene with >5-fold down-regulation in the IFNG-secreting subset. RNASET2 disease risk variants were associated with decreased expression in peripheral and mucosal tissues and DNA hypermethylation in CD patients requiring surgical intervention. RNASET2 disease risk variants were associated in CD patients with more complicated disease or resistance to therapy, defined in part by failed response to treatment, increased length of intestinal resection, shorter time to repeat surgery, and high Rutgeerts score (>2) in postoperative endoscopy. The RNASET2 variant rs2149092 was predicted to disrupt a consensus binding site for the transcription factor ETS within an enhancer region. Expression of RNASET2 correlated with expression of ETS. RNASET2 knockdown in T cells increased expression of IFNG and intercellular adhesion molecule 1 (ICAM1) and induced T-cell aggregation. A blocking antibody against (ILFA1), disrupting the lymphocyte function-associated antigen 1-intercellular adhesion molecule 1 interaction, reduced T-cell production of IFNG. We identified decreased expression of RNASET2 as a component of TL1A-mediated increase in production of IFNG and as a potential biomarker for patients with severe CD. Further study of the role of RNASET2 in regulating mucosal inflammation may lead to development of novel therapeutic targets.

MELK is an oncogenic kinase essential for early hepatocellular carcinoma recurrence

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. Many kinases have been found to be intimately involved in oncogenesis and the deregulation of kinase function has emerged as a major mechanism by which cancer cells evade normal physiological constraints on growth and survival. Previously, we have performed gene expression profile analysis on HCC samples and have identified a host of kinases that are remarkably overexpressed in HCC. Among these, the Maternal Embryonic Leucine Zipper Kinase (MELK) is highly overexpressed in HCC and its overexpression strongly correlates with early recurrence and poor patients' survival. Silencing MELK inhibited cell growth, invasion, stemness and tumorigenicity of HCC cells by inducing apoptosis and mitosis. We further showed that the overexpression of MELK in HCC samples strongly correlated with the cell cycle- and mitosis-related genes which are directly regulated as part of the forkhead transcription factor FoxM1-related cell division program. Together, our data establish MELK as an oncogenic kinase involved in the pathogenesis and recurrence of HCC and could provide a promising molecular target to develop therapeutic strategies for patients with advanced HCC.

MiR-338-5p sensitizes glioblastoma cells to radiation through regulation of genes involved in DNA damage response.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor. Despite radical surgery and radiotherapy supported by chemotherapy, the disease still remains incurable with an extremely low median survival rate of 12-15months from the time of initial diagnosis. The main cause of treatment failure is considered to be the presence of cells that are resistant to the treatment. MicroRNAs (miRNAs) as regulators of gene expression are involved in the tumor pathogenesis, including GBM. MiR-338 is a brain-specific miRNA which has been described to target pathways involved in proliferation and differentiation. In our study, miR-338-3p and miR-338-5p were differentially expressed in GBM tissue in comparison to non-tumor brain tissue. Overexpression of miR-338-3p with miRNA mimic did not show any changes in proliferation rates in GBM cell lines (A172, T98G, U87MG). On the other hand, pre-miR-338-5p notably decreased proliferation and caused cell cycle arrest. Since radiation is currently the main treatment modality in GBM, we combined overexpression of pre-miR-338-5p with radiation, which led to significantly decreased cell proliferation, increased cell cycle arrest, and apoptosis in comparison to irradiation-only cells. To better elucidate the mechanism of action, we performed gene expression profiling analysis that revealed targets of miR-338-5p being Ndfip1, Rheb, and ppp2R5a. These genes have been described to be involved in DNA damage response, proliferation, and cell cycle regulation. To our knowledge, this is the first study to describe the role of miR-338-5p in GBM and its potential to improve the sensitivity of GBM to radiation.

Gene Expression Profiling of Primary Vitreoretinal Lymphoma

Background: Primary vitreoretinal lymphoma (PVRL) is a rare form of non-Hodgkin lymphoma, whose original lesions are limited in the intraocular tissues: the vitreous and the retina. Although its morphological and genetic findings show that most cases are diffuse large B-cell lymphoma (DLBCL), the characteristics of the tumor cells of PVRL, including their origin, have not been defined to date. DLBCL is subdivided into 3 groups: activated B-cell (ABC) type, germinal center B-cell (GCB) type, and primary mediastinal B-cell lymphoma on the basis of genes expressed in differentiation or biologic response of B-cell. In order to determine the origin of the tumor cells of PVRL, we performed gene expression profiling analysis.Methods: PVRL was diagnosed using the following criteria: (1) typical eye involvement: a cloudy vitreous body and/or subretinal proliferative lesions, (2) presence of lymphoma cells in the vitreous fluid, and (3) clonality of the infiltrating lymphoma cells in the vitreous fluid using either PCR analysis of IgH gene rearrangements, or flow cytometry analysis. Patients who had (1) accompanied by either (2) or (3) were diagnosed with VRL. VRL confined to the eyes at diagnosis was defined as PVRL. We used the samples from nodal DLBCL whose types were pathologically determined according to Hans classifier (Blood, 103, p 275, 2004) as control. RNA was extracted from the vitreous fluid and the lymph nodes from PVRL and nodal DLBCL patients, respectively. Since the extracted RNA from PVRL was very small in amount, all RNA samples were amplified with WTA reaction. Then, one-color microarray-based gene expression analysis was performed using SurePrint G3 Human GE Microarray 8x60K v2 (Agilent Technology). Data were extracted and the agglomerative hierarchical clustering was performed on the basis of the genes discriminating GCB/ABC signatures that were published initially by Alizadeh et al. (Nature, 403, p503, 2000). Gene ontology (GO) analysis was performed using DAVID data base. We calculated Z-scores and ratios (non-log scaled fold-change) from the normalized signal intensities of each probe for comparison between 2 groups.Results: We performed gene expression profiling analysis for 7 samples from PVRL patients. We also examined 6 samples from nodal DLBCL patients as control; 4 samples were ABC type, and 2 samples were GCB type. Six of 7 PRVL showed different gene expression profiling from ABC type nodal DLBCL. They were close to that of GCB type. Genes regulating cell adhesion were enriched by GO analysis for genes whose expression was different between PRVL and GCB. Next we examined the relation between the gene expression and the outcomes of PVRL. Four GCB-like PRVL samples were obtained from the patients treated with the same strategy: intravitreal administration of methotrexate (MTX) followed by systemic high dose MTX. Among them, 2 patients revealed early relapse in the CNS a year after the treatment whereas the others did not. We compared the gene expression between them. Three genes, SLC35F1, ADAM19, and SMC1A related to neurological disorders, cell migration, and cell division, respectively, were significantly upegulated in the PRVL with early relapse.Conclusion: The majority of PVRL in the present study were different from ABC type and close to GCB type DLBCL. Further studies are required to confirm that and to determine the genes predicting the outcome. DisclosuresNo relevant conflicts of interest to declare.

MP68-15 LYMPH NODE STROMAL CELLS PROMOTE HIGH GRADE UROTHELIAL CELL CARCINOMA CANCER IMPLANTATION, GROWTH, ANGIOGENESIS, AND METASTASIS IN AN ORTHOTOPIC PATIENT-DERIVED XENOGRAFT MODEL

INTRODUCTION AND OBJECTIVES: Extracellular vesicles (EV) are small membrane vesicles secreted by most cell types. They are first found in reticulocytes and play important roles in cell-to-cell communication. Recent studies suggest that EV secreted by tumor cells containing tumor specific proteins and miRNAs. Previous study shows that EV purified from muscle invasive bladder cancer promote cancer progression. This study aims to characterize the genetic contents, such as proteins and nucleic acids and establish the bladder cancer specific EV biomarkers METHODS: EV were purified from bladder cancers, immortalized bladder cells, and urine of healthy volunteers and muscle invasive bladder cancer patients. The protein contents were analyzed by mass spectrometry. To prioritize the proteins of interest, we performed gene expression profile analysis of proteins via public microarray repositories from NCBI Gene Expression Omnibus profile #GDS1479 (carcinoma in situ lesions of the urinary bladder) that consists of 5 categories: normal urothelium, Non-muscle invasive BC with cancer in situ (CIS), superficial TCC without CIS, Muscle invasive bladder cancer, and CIS. In addition to protein, we also performed miRNA expression profiling microarray on EV from high grade bladder cancer cells, and immortalized bladder cells (LC Sciences). RESULTS: More than 400 proteins were found in bladder EV. Among them, we found 9 cancer-associated EV proteins that are particularly interesting because their gene expression levels are significantly elevated in cancer stages as compared to normal urothelium. Those proteins are subjected to further functional characterization via gene knockdown/overexpression strategies. The differences in miRNA expression were compared and differences were determined by a student T test (P<0.05). Results showed significant difference miRNAs between cancer cells vs normal cells. Some of cancer-specific miRNAs which have shown to play critical roles in tumorigenesis and cancer progression will be study priority CONCLUSIONS: Our study enables us to establish a panel of EV protein and miRNA signatures that can be further developed into a urine-based method for rapid detection of stage-specific biomarkers which will be valuable for detecting and managing bladder cancer.

De novo assembly and characterization of Muscovy duck liver transcriptome and analysis of differentially regulated genes in response to heat stress.

High temperature is a major abiotic stress limiting animal growth and productivity worldwide. The Muscovy duck (Cairina moschata), sometimes called the Barbary drake, is a type of duck with a fairly unusual domestication history. In Southeast Asia, duck meat is one of the top meats consumed, and as such, the production of the meat is an important topic of research. The transcriptomic and genomic data presently available are insufficient to understanding the molecular mechanism underlying the heat tolerance of Muscovy ducks. Thus, transcriptome and expression profiling data for this species are required as important resource for identifying genes and developing molecular marker. In this study, de novo transcriptome assembly and gene expression analysis using Illumina sequencing technology were performed. More than 225 million clean reads were generated and assembled into 36,903 unique transcripts with an average length of 1,135bp. A total of 21,221 (57.50%) unigenes were annotated. Gene Ontology (GO) analysis of the annotated unigenes revealed that the majority of sequenced genes were associated with transcription, signal transduction, and apoptosis. We also performed gene expression profiling analysis upon heat treatment in Muscovy ducks and identified 470 heat-response unique transcripts. GO term enrichment showed that protein folding and chaperone binding were significant enrichment, whereas KEGG pathway analyses showed that Ras and MAPKs were activated after heat stress in Muscovy ducks. Our research enriched sequences information of Muscovy duck, provided novel insights into responses to heat stress in these ducks, and serve as candidate genes or markers that can be used to guide future efforts to breed heat-tolerant duck strains.

Stress responses to trichlorophenol in Arabidopsis and integrative analysis of alteration in transcriptional profiling from microarray

Trichlorophenols, also known as TCPs, are one of the most persistent environmental pollutants. It is a matter of concern as they are toxic and cumulative in soil and water bodies which could lead to serious consequences to the biosphere. How plants respond to this compound has rarely been examined previously. In our study, detailed morphological and physiological responses of Arabidopsis to 2,4,6-trichlorophenol, a representative TCP, were investigated. Seed germination and seedling growth were markedly inhibited by 2,4,6-TCP. Furthermore, we performed gene expression profiling analysis upon 2,4,6-TCP treatment in Arabidopsis and identified 34 transcripts induced and 212 repressed more than four folds. Gene Ontology (GO) analysis showed that these TCP-responsive genes are involved in various biological processes, such as secondary metabolism, biological regulation, response to stimulus and other processes related to growth and development. The activities of two reactive oxygen species-related enzymes (peroxidase and superoxide dismutase) and the content of malondialdehyde were increased significantly after 2,4,6-TCP treatment. Our findings have the potential to provide valuable gene resources and theoretical information for more in-depth analyses of TCPs' response in Arabidopsis thaliana and even other organic pollutants.

Abstract 3389: Targeting SALL4 in lung cancer through a novel Cbl-b/IGF1R&amp;EGFR axis

Abstract Lung cancer is the leading cause of cancer deaths in the United States with an estimated &amp;gt;160,000 deaths and &amp;gt;200,000 newly diagnosed cases each year. Over the past 20 years, the survival rate for patients remains extremely poor (∼15% over 5 years). Discovery of novel pathway involved in the pathogenesis of the lung cancer can provide new therapeutic opportunities. Stem cell factor SALL4 has recently merged to be a diagnostic tool and therapeutic target for acute myeloid leukemia and liver cancer. However, very little is known on its role in lung cancer. In order to investigate the functional role of SALL4 in lung cancer tumorigenesis and progression, we first performed gene expression profile analysis and revealed that SALL4 expression level was significant higher in all types of non-small cell lung cancer samples compared to normal lung tissue control, which was validated by IHC. Furthermore, we performed loss of function study using multiple shRNAs. Our data showed that knockdown of SALL4 could induce dramatic cell growth arrest in multiple lung cell lines including large cell carcinoma H661 and adenocarcinoma H522, H292 and PC9, but not in HCC827 cells with none/low SALL4 level. In order to elucidate the downstream pathway of SALL4 in lung cancer, we first conducted gene expression microarray using SALL4 knockdown H661 cells and cells treated with scramble control vectors. Combination of one-way ANOVA analysis and pathway analysis using Pathway Interaction Database (PID) showed that both the EGFR and IGF1R signaling pathways were affected by SALL4 knockdown in H661 lung cancer cells. Using western blot, we further validated that SALL4 depletion could lead to a decrease in both EGFR and IGF1R protein expressions in lung cancer cells. However, we did not observe any changes at the mRNA level for these two receptors upon SALL4 depletion. This leads us to wonder whether SALL4 could affect these two molecules indirectly. It has been well established that receptor tyrosine kinase (RTK) including EGFR and IGF1R can be negatively regulated by Cbl RING ubiquitin ligases. Of interest, our microarray data also showed that loss of SALL4 led to a dramatic upregulation of Cbl-b, a member of Cbl RING ligases, which was further validated by using real-time RT-PCR. In addition, SALL4 downregulation also dramatically increased Cbl-b protein expression in these lung cancer cells. Therefore, we propose that SALL4 can indirectly positively affect the expression of EGFR and IGF1R proteins through Cbl-b. In summary, we have demonstrated that SALL4 expression is aberrantly elevated in non-small cell lung cancer cells. Furthermore our data suggested that SALL4 plays an essential role in lung cancer tumor growth and maintenance by affecting both EGFR and IGFR signaling pathways via regulating Cbl-b expression. Citation Format: Ailing Li, Wenbin Ou, Jonathan Fletcher, Kol Jia Yong, Henry Yang, Ross Soo, Daniel Tenen, Li Chai. Targeting SALL4 in lung cancer through a novel Cbl-b/IGF1R&amp;EGFR axis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3389. doi:10.1158/1538-7445.AM2014-3389

Transcriptome sequencing and whole genome expression profiling of chrysanthemum under dehydration stress

BackgroundChrysanthemum is one of the most important ornamental crops in the world and drought stress seriously limits its production and distribution. In order to generate a functional genomics resource and obtain a deeper understanding of the molecular mechanisms regarding chrysanthemum responses to dehydration stress, we performed large-scale transcriptome sequencing of chrysanthemum plants under dehydration stress using the Illumina sequencing technology.ResultsTwo cDNA libraries constructed from mRNAs of control and dehydration-treated seedlings were sequenced by Illumina technology. A total of more than 100 million reads were generated and de novo assembled into 98,180 unique transcripts which were further extensively annotated by comparing their sequencing to different protein databases. Biochemical pathways were predicted from these transcript sequences. Furthermore, we performed gene expression profiling analysis upon dehydration treatment in chrysanthemum and identified 8,558 dehydration-responsive unique transcripts, including 307 transcription factors and 229 protein kinases and many well-known stress responsive genes. Gene ontology (GO) term enrichment and biochemical pathway analyses showed that dehydration stress caused changes in hormone response, secondary and amino acid metabolism, and light and photoperiod response. These findings suggest that drought tolerance of chrysanthemum plants may be related to the regulation of hormone biosynthesis and signaling, reduction of oxidative damage, stabilization of cell proteins and structures, and maintenance of energy and carbon supply.ConclusionsOur transcriptome sequences can provide a valuable resource for chrysanthemum breeding and research and novel insights into chrysanthemum responses to dehydration stress and offer candidate genes or markers that can be used to guide future studies attempting to breed drought tolerant chrysanthemum cultivars.

Nectin-2 is a potential target for antibody therapy of breast and ovarian cancers

BackgroundNectin-2 is a Ca2+-independent cell-cell adhesion molecule that is one of the plasma membrane components of adherens junctions. However, little has been reported about the involvement of Nectin-2 in cancer.MethodsTo determine the expression of Nectin-2 in cancer tissues and cancer cell lines, we performed gene expression profile analysis, immunohistochemistry studies, and flow cytometry analysis. We also investigated the potential of this molecule as a target for antibody therapeutics to treat cancers by generating and characterizing an anti-Nectin-2 rabbit polyclonal antibody (poAb) and 256 fully human anti-Nectin-2 monoclonal antibodies (mAbs). In addition, we tested anti-Nectin-2 mAbs in several in vivo tumor growth inhibition models to investigate the primary mechanisms of action of the mAbs.ResultsIn the present study, we found that Nectin-2 was over-expressed in clinical breast and ovarian cancer tissues by using gene expression profile analysis and immunohistochemistry studies. Nectin-2 was over-expressed in various cancer cell lines as well. Furthermore, the polyclonal antibody specific to Nectin-2 suppressed the in vitro proliferation of OV-90 ovarian cancer cells, which express endogenous Nectin-2 on the cell surface. The anti-Nectin-2 mAbs we generated were classified into 7 epitope bins. The anti-Nectin-2 mAbs demonstrated antibody-dependent cellular cytotoxicity (ADCC) and epitope bin-dependent features such as the inhibition of Nectin-2-Nectin-2 interaction, Nectin-2-Nectin-3 interaction, and in vitro cancer cell proliferation. A representative anti-Nectin-2 mAb in epitope bin VII, Y-443, showed anti-tumor effects against OV-90 cells and MDA-MB-231 breast cancer cells in mouse therapeutic models, and its main mechanism of action appeared to be ADCC.ConclusionsWe observed the over-expression of Nectin-2 in breast and ovarian cancers and anti-tumor activity of anti-Nectin-2 mAbs via strong ADCC. These findings suggest that Nectin-2 is a potential target for antibody therapy against breast and ovarian cancers.

Abstract 4441: The ketogenic diet potentiates radiation therapy in a mouse model of glioma: effects on inflammatory pathways and reactive oxygen species.

Abstract Patients with malignant brain tumors have a median survival of approximately one year following diagnosis, regardless of currently available treatments which include surgery, radiation and chemotherapy. Although there have been advances in the development of novel treatments, targeted therapies face the challenge of overcoming phenotypic variability resulting from tumor heterogeneity. One phenotypic trait shared by virtually all cancer cells is dysregulation of metabolism. It has therefore been postulated that one approach to treating brain tumors may be through metabolic alteration such as that which occurs through the use of the ketogenic diet (KD). The KD is high-fat, low-carbohydrate diet that has been utilized for the non-pharmacologic treatment of refractory epilepsy. We and others have shown that this diet enhances survival in mouse models of malignant gliomas. Further, radiation in combination with KD was synergistic, and survival was significantly increased over radiation treatment alone. To gain an understanding of the mechanism(s) by which the KD exerts anti-tumor effects and enhances the efficacy of radiation therapy we performed gene expression profiling analyses on tumor tissue and non-tumor containing brain using the Affymetrix GeneChip® Mouse Genome 430 2.0 array. To identify changes in gene expression representing chronic effects of KD and radiation plus KD we harvested tissue when the animals became symptomatic. We previously reported that tissue from animals maintained on the KD in the absence of radiation treatment showed a reduction in reactive oxygen species (ROS) and cyclooxygenase-2 (COX-2) expression compared to those maintained on a standard diet (Stafford et al, Nutr and Metab (7):74, 2010). We now report that peritumoral edema is significantly reduced early in tumor progression in animals fed the KD when compared to those fed a standard rodent diet. Furthermore, gene expression profiling of animals treated with radiation and KD demonstrated alterations in the chronic expression of crucial genes involved in radiation-induced inflammation including COX-2 and the nuclear transcription factor NF-кB, both of which are implicated in radioresistance. We are repeating these studies using tissues harvested shortly after radiation therapy to identify genes acutely affected by radiation therapy in animals fed a KD. Taken together, our data to date suggests that the KD may be interfering with the radiation-induced inflammatory signaling network implicated in tumor progression. A greater understanding of the effects of the ketogenic diet as an adjuvant therapy will allow for a more rational approach to its clinical use. Citation Format: Eric C. Woolf, Phillip Stafford, Mohammed G. Abdelwahab, Kathryn Fenton, Mark C. Preul, Adrienne C. Scheck. The ketogenic diet potentiates radiation therapy in a mouse model of glioma: effects on inflammatory pathways and reactive oxygen species. [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 4441. doi:10.1158/1538-7445.AM2013-4441

Prognostic Gene Expression Signature for Patients With Hepatitis C–Related Early-Stage Cirrhosis

Cirrhosis affects 1% to 2% of the world population and is the major risk factor for hepatocellular carcinoma (HCC). Hepatitis C cirrhosis-related HCC is the most rapidly increasing cause of cancer death in the United States. Noninvasive methods have been developed to identify patients with asymptomatic early-stage cirrhosis, increasing the burden of HCC surveillance, but biomarkers are needed to identify patients with cirrhosis who are most in need of surveillance. We investigated whether a liver-derived 186-gene signature previously associated with outcomes of patients with HCC is prognostic for patients with newly diagnosed cirrhosis but without HCC. We performed gene expression profile analysis of formalin-fixed needle biopsy specimens from the livers of 216 patients with hepatitis C-related early-stage (Child-Pugh class A) cirrhosis who were prospectively followed up for a median of 10 years at an Italian center. We evaluated whether the 186-gene signature was associated with death, progression of cirrhosis, and development of HCC. Fifty-five (25%), 101 (47%), and 60 (28%) patients were classified as having poor-, intermediate-, and good-prognosis signatures, respectively. In multivariable Cox regression modeling, the poor-prognosis signature was significantly associated with death (P = .004), progression to advanced cirrhosis (P < .001), and development of HCC (P = .009). The 10-year rates of survival were 63%, 74%, and 85% and the annual incidence of HCC was 5.8%, 2.2%, and 1.5% for patients with poor-, intermediate-, and good-prognosis signatures, respectively. A 186-gene signature used to predict outcomes of patients with HCC is also associated with outcomes of patients with hepatitis C-related early-stage cirrhosis. This signature might be used to identify patients with cirrhosis in most need of surveillance and strategies to prevent the development of HCC.

Gene Expression Signatures That Delineate Biologic and Prognostic Subgroups in Peripheral T-Cell Lymphoma

AbstractAbstract 679 Background:Peripheral T-cell lymphoma (PTCL) represents approximately 10–12% of all non-Hodgkin lymphoma (NHL) in the Western world, with a higher incidence in Asian populations. The World Health Organization classification recognizes a number of distinctive subtypes of PTCL including angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), adult T-cell leukemia/lymphoma (ATLL), extranodal NK/T-cell lymphoma of nasal type (ENKTCL), and many other rare entities that present mainly as extranodal PTCL. However, with current immunophenotypic and molecular markers, about 30–50% of PTCL cases are not classifiable and are categorized as PTCL-not otherwise specified (PTCL-NOS). With the exception of ALK(+)ALCL, the PTCLs generally have a poor outcome and, thus a better understanding of the biology of these diseases is greatly needed to improve the long-term survival of these patients. Methods:In the current study, we performed gene expression profiling analysis on a large and well- characterized series of PTCL and ENKTCL cases (n=372) from the Lymphoma Leukemia Molecular Profiling Project (LLMPP), the International Peripheral T-cell Lymphoma Project (IPTCL) and other major institutions to define robust molecular classifiers, oncogenic pathways and prognosticators for the more common PTCL entities, as well as unique molecular and prognostic subgroups within PTCL-NOS. Molecular signatures for diagnosis and prognosis were generated in training data sets and validated in separate cohorts. Results:Robust molecular classifiers for AITL, two types of systemic ALCL (ALK(+) and ALK(-)), ATLL and ENKTCL were identified (Figure 1). These classifiers reflect the pathobiology of the tumor cells, as well as their microenvironment, and represent a refinement of what we reported previously (Iqbal et.al Blood, 2010; Iqbal et.al Leukemia. 2011). Importantly, ALK(-)ALCL can be differentiated from ALK(+)ALCL and PTCL-NOS with a unique gene expression signature. Approximately 14% of PTCL-NOS were re-classified as ALK(-)ALCL and showed expression of CD30 protein, TIA-1 or granzyme B by immunohistochemistry. ENKTCL can be separated molecularly into NK-cell lymphoma and gd-PTCL, the latter of which was also identified in 9% of PTCL-NOS. The remaining PTCL-NOS cases could be separated into two major subgroups related to T-cell differentiation and characterized by either high expression of GATA3 (30%) or TBX21(T-BET) (45%) and many of the corresponding target genes (Figure 2). Cases with high expression of GATA3 had poor overall survival and showed enriched Wnt and mTOR pathways, but no prominent microenvironment signature. The high TBX21 subgroup had a remarkably good outcome for patients with a high plasma cell-like gene expression signature, but poor overall survival when expressing a high cytotoxic signature (Figure 3). The molecular prognosticator for AITL largely reflected the role of the tumor microenvironment, with the presence of a high B-cell signature correlating with favorable outcome, whereas high dendritic cell/monocyte signatures were associated with inferior survival. Conclusion:We have organized the most comprehensive molecular profiling study of PTCL, and have not only refined the molecular diagnostic and prognostic signatures for the common subtypes of PTCL, but also segregated PTCL-NOS in meaningful biological and prognostic subtypes. Molecular diagnostic and prognostic signatures of PTCL frequently include components of the tumor-host interactions, highlighting the importance of the microenvironment in PTCL biology. This study provides an important framework for additional analysis to identify novel therapeutic targets to improve the outcome of patients with PTCL. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

H05 Gene expression profile in fibroblasts of Huntington's disease patients and controls

Huntington's disease (HD), MIM 143100, is a dominantly inherited progressive neurodegenerative disorder characterised by motor, behavioural and cognitive dysfunction, caused by an expanded CAG repeat region in exon 1 of...

Differential expression of NF-κB target genes in MALT lymphoma with and without chromosome translocation: insights into molecular mechanism

Mucosa-associated lymphoid tissue (MALT) lymphoma is characterized by t(11;18)(q21;q21)/API2-MALT1, t(1;14)(p22;q32)/BCL10-IGH and t(14;18)(q32;q21)/IGH-MALT1, which commonly activate the nuclear factor (NF)-kappaB pathway. Gastric MALT lymphomas harboring such translocations usually do not respond to Helicobacter pylori eradication, while most of those without translocation can be cured by antibiotics. To understand the molecular mechanism of these different MALT lymphoma subgroups, we performed gene expression profiling analysis of 21 MALT lymphomas (13 translocation-positive, 8 translocation-negative). Gene set enrichment analysis (GSEA) of the NF-kappaB target genes and 4394 additional gene sets covering various cellular pathways, biological processes and molecular functions have shown that translocation-positive MALT lymphomas are characterized by an enhanced expression of NF-kappaB target genes, particularly toll like receptor (TLR)6, chemokine, CC motif, receptor (CCR)2, cluster of differentiation (CD)69 and B-cell CLL/lymphoma (BCL)2, while translocation-negative cases were featured by active inflammatory and immune responses, such as interleukin-8, CD86, CD28 and inducible T-cell costimulator (ICOS). Separate analyses of the genes differentially expressed between translocation-positive and -negative cases and measurement of gene ontology term in these differentially expressed genes by hypergeometric test reinforced the above findings by GSEA. Finally, expression of TLR6, in the presence of TLR2, enhanced both API2-MALT1 and BCL10-mediated NF-kappaB activation in vitro. Our findings provide novel insights into the molecular mechanism of MALT lymphomas with and without translocation, potentially explaining their different clinical behaviors.

The Functional Role of Microrna 15a/16-1 as Tumor Suppressor Genes in Multiple Myeloma.

Abstract 1963Poster Board I-986 Background:Multiple Myeloma (MM), a cancer of plasma cells is characterized by frequent chromosomal alterations. Deletion of chromosome 13, especially band 13q14, is commonly observed in early stages of MM, suggesting the presence of tumor suppressor genes within this region. When studied in the context of CLL, the miR 15a and 16-1 cluster was associated with a distinct miR signature and clinical outcome. Over-expression of miR16 caused induction of apoptosis and downregulation of the anti apoptotic gene BCL2 in a megakaryocytic leukemia cell line and induced growth arrest in MM cells. Nonetheless, being lost in CLL, MM, MCL and LPL, their functional role has not been studied using a loss-of-function approach in any of these lymphoid malignancies. Here, we describe the generation of an in vivo system for the long term, stable knockdown of miR 15a/ 16-1 expression in myeloma cells to recapitulate the conditions seen in chromosome 13q14 deleted MM. Methods:Using lentiviral vectors to stably express a competitive sponge miR16 inhibitor we set up a system to functionally validate the role of microRNA 15a/16-1 cluster. Pure populations of lentivirally transduced MM cell lines were sorted by flow cytometry using GFP marker. Decreased miRs 15a and 16 expression levels were assessed by Northern blot and R-luciferase reporter system. Cell growth rate was measured using trypan blue counting, and thymidine incorporation. Cell cycle analysis was measured by flow cytometry after staining with PI. Intracellular signal modulation was demonstrated by Western blotting. RNA from MM cell lines expressing the control sponge or sponge16 were hybridized on an Affymetrix U133A 2.0 array chip, and validated using quantitative real time PCR. Xenograft murine models were performed using the stable MM cell lines injected into 6-week old NOD.CB17-PrkdcSCID/J irradiated mice. Results:Selected stable miR knockdown MM cell lines exhibited significantly reduced expression of miRs15a/16-1 as assessd by both by mRNA levels and miR luciferase reporter assays. The knockdown cells showed a significant increase in growth rates (1.5-2 fold) compared to control cells, as measured by viable cell counts and proliferation by thymidine incorporation in vitro. Importantly, miR16 inhibition decreased animal survival in a xenograft model of MM by increasing tumor load, invasiveness and host angiogenesis. To further delineate the role of miR15a/16 in MM and to gain additional insight into the possible target genes regulated by this cluster, we performed gene expression-profiling analysis in controls and miR16 deficient MMS1 and RPMI cell lines. Since our sponge system produces downregulation of the miRs, we focused on the upregulated probes. Expression profiling analysis of miR16 deficient cells identified a surprisingly large number of downstream target-genes such as FGFR1, PI3KCa, MDM4, VEGFa, as well as secondary affected genes such as JUN and Jag1. These results were verified both at the mRNA level and the protein level, as well as in other MM cell lines. Moreover, we were able to show that these knockdown cells were partially addicted to some of these pathways using specific drug inhibitors. We further validated designated genes as direct miR16 targets by showing binding sites within the conserved 3' UTR and also within the mRNA coding region, thus indicating that the miRs may have many more possible targets than anticipated by conventional prediction methods. Conclusions:Using this loss-of-function system, which mimics the pleiotropic chronic effects of microRNA loss at the 13q chromosomal deletion, provides a valuable tool to investigate their function as tumor suppressor genes in MM pathogenesis, affecting multiple targets, and may represent a novel potential for therapeutic targeting in MM and other lymphoid malignancies. Disclosures:Munshi:Seattle Genetics, Inc.: Research Funding.