Microarray-based Gene Expression Analysis Research Articles

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.

E-cadherin cytoplasmic domain inhibits cell surface localization of endogenous cadherins and fusion of C2C12 myoblasts.

ABSTRACTMyoblast fusion is a highly regulated process that is essential for skeletal muscle formation during muscle development and regeneration in mammals. Much remains to be elucidated about the molecular mechanism of myoblast fusion although cadherins, which are Ca2+-dependent cell–cell adhesion molecules, are thought to play a critical role in this process. Mouse myoblasts lacking either N-cadherin or M-cadherin can still fuse to form myotubes, indicating that they have no specific function in this process and may be functionally replaced by either M-cadherin or N-cadherin, respectively. In this study, we show that expressing the E-cadherin cytoplasmic domain ectopically in C2C12 myoblasts inhibits cell surface localization of endogenous M-cadherin and N-cadherin, as well as cell–cell fusion. This domain, however, does not inhibit myoblast differentiation according to microarray-based gene expression analysis. In contrast, expressing a dominant-negative β-catenin mutant ectopically, which suppresses Wnt/β-catenin signaling, did not inhibit cell–cell fusion. Therefore, the E-cadherin cytoplasmic domain inhibits cell–cell fusion by inhibiting cell surface localization of endogenous cadherins and not by inhibiting Wnt/β-catenin signaling.

Mechanisms of IKK/NF-κB dependent diabetes development

As a key regulator of inflammatory processes IKK/NF-κB signaling is present and activated in all cell types involved in diabetes development. However, a detailed understanding of the causal relationship between cell-type-specific NF-κB activation and onset and progression of type 1 diabetes needs further investigation. To address this issue, two complementary mouse models (gain- and loss-of-function) for IKK/NF-κB signaling were generated. Both mouse models are based on the conditional expression of mutant IKK2 alleles (constitutive active (IKK2-CA) and dominant negative (IKK2-DN)) via the tetracycline-regulated gene expression system. β-cell-specific IKK/NF-κB activation was already sufficient to initiate a full-blown immune-mediated diabetes including insulitis, hyperglycemia and hypoinsulinemia. Disease development involves a gene expression program mimicking virus-induced diabetes and allergic inflammatory responses as well as increased MHCI/II expression. Potential novel diabetes candidate genes were also identified. Animals of the loss-of-function model also exhibit a diabetes-like phenotype characterized by a complete destruction of the islet architecture including loss of insulin content but without immune cell infiltration. Interestingly, diabetic animals of both models successfully recovered from diabetes upon transgene inactivation. Initial results indicate that this high regeneration capacity might be a consequence of dedifferentiated insulin-negative β-cells, which regain functional β-cell status upon transgene inactivation. Current work addresses the identification of molecular mechanisms involved in this kind of in vivo β-cell regeneration. For this purpose microarray based gene expression analysis was performed using islet samples that were isolated during the recovery process. Preliminary data presented on the poster suggest that autocrine/paracrine mechanisms might be involved.

Uterine Telocytes: A Review of Current Knowledge.

Telocytes (TCs), a novel cell type, are briefly defined as interstitial cells with telopodes (Tps). However, a specific immunocytochemical marker has not yet been found; therefore, electron microscopy is currently the only accurate method for identifying TCs. TCs are considered to have a mesenchymal origin. Recently proteomic analysis, microarray-based gene expression analysis, and the micro-RNA signature clearly showed that TCs are different from fibroblasts, mesenchymal stem cells, and endothelial cells. The dynamics of Tps were also revealed, and some electrophysiological properties of TCs were described (such as membrane capacitance, input resistance, membrane resting potential, and absence of action potentials correlated with different ionic currents characteristics), which can be used to distinguish uterine TCs from smooth muscle cells (SMCs). Here, we briefly present the most recent findings on the characteristics of TCs and their functions in human pregnant and nonpregnant uteri.

Early Gene Expression in Salivary Gland After Isoproterenol Treatment

Isoproterenol, a β-adrenergic agonist, has been shown to induce salivary gland hyperplasia. However, the mechanism involved in this pharmacological phenomenon is not well understood. To gain a better understanding of the underlying changes, including genes, networks and pathways altered by isoproterenol, microarray-based gene expression analysis was conducted on rat parotid glands at 10, 30, and 60 min after isoproterenol injection. After isoproterenol treatment, the number of differentially expressed genes was increased in a time-dependent manner. Pathway analysis showed that cell hyperplasia, p38(MAPK), and IGF-1 were the most altered function, network and pathway, respectively. The balanced regulation of up- and down-expression of genes related to cell proliferation/survival may provide a better understanding of the mechanism of isoproterenol-induced parotid gland enlargement without tumor transformation.

Microarray-based analysis of gene expression in lycopersicon esculentum seedling roots in response to cadmium, chromium, mercury, and lead.

The effects of heavy metals in agricultural soils have received special attention due to their potential for accumulation in crops, which can affect species at all trophic levels. Therefore, there is a critical need for reliable bioassays for assessing risk levels due to heavy metals in agricultural soil. In the present study, we used microarrays to investigate changes in gene expression of Lycopersicon esculentum in response to Cd-, Cr-, Hg-, or Pb-spiked soil. Exposure to (1)/10 median lethal concentrations (LC50) of Cd, Cr, Hg, or Pb for 7 days resulted in expression changes in 29 Cd-specific, 58 Cr-specific, 192 Hg-specific and 864 Pb-specific genes as determined by microarray analysis, whereas conventional morphological and physiological bioassays did not reveal any toxicant stresses. Hierarchical clustering analysis showed that the characteristic gene expression profiles induced by Cd, Cr, Hg, and Pb were distinct from not only the control but also one another. Furthermore, a total of three genes related to "ion transport" for Cd, 14 genes related to "external encapsulating structure organization", "reproductive developmental process", "lipid metabolic process" and "response to stimulus" for Cr, 11 genes related to "cellular metabolic process" and "cellular response to stimulus" for Hg, 78 genes related to 20 biological processes (e.g., DNA metabolic process, monosaccharide catabolic process, cell division) for Pb were identified and selected as their potential biomarkers. These findings demonstrated that microarray-based analysis of Lycopersicon esculentum was a sensitive tool for the early detection of potential toxicity of heavy metals in agricultural soil, as well as an effective tool for identifying the heavy metal-specific genes, which should be useful for assessing risk levels due to heavy metals in agricultural soil.

The Transcription Factor Con7-1 Is a Master Regulator of Morphogenesis and Virulence in Fusarium oxysporum.

Previous studies have demonstrated the essential role of morphogenetic regulation in Fusarium oxysporum pathogenesis, including processes such as cell-wall biogenesis, cell division, and differentiation of infection-like structures. We identified three F. oxysporum genes encoding predicted transcription factors showing significant identities to Magnaporthe oryzae Con7p, Con7-1, plus two identical copies of Con7-2. Targeted deletion of con7-1 produced nonpathogenic mutants with altered morphogenesis, including defects in cell wall structure, polar growth, hyphal branching, and conidiation. By contrast, simultaneous inactivation of both con7-2 copies caused no detectable defects in the resulting mutants. Comparative microarray-based gene expression analysis indicated that Con7-1 modulates the expression of a large number of genes involved in different biological functions, including host-pathogen interactions, morphogenesis and development, signal perception and transduction, transcriptional regulation, and primary and secondary metabolism. Taken together, our results point to Con7-1 as general regulator of morphogenesis and virulence in F. oxysporum.

Improved bone defect healing by a superagonistic GDF5 variant derived from a patient with multiple synostoses syndrome

Multiple synostoses syndrome 2 (SYNS2) is a rare genetic disease characterized by multiple fusions of the joints of the extremities, like phalangeal joints, carpal and tarsal joints or the knee and elbows. SYNS2 is caused by point mutations in the Growth and Differentiation Factor 5 (GDF5), which plays an essential role during skeletal development and regeneration. We selected one of the SYNS2-causing GDF5 mutations, p.N445T, which is known to destabilize the interaction with the Bone Morphogenetic Protein (BMP) antagonist NOGGIN (NOG), in order to generate the superagonistic GDF5 variant GDF5N445T. In this study, we tested its capacity to support regeneration in a rat critical-sized defect model in vivo. MicroCT and histological analyses indicate that GDF5N445T-treated defects show faster and more efficient healing compared to GDF5 wild type (GDF5wt)-treated defects. Microarray-based gene expression and quantitative PCR analyses from callus tissue point to a specific acceleration of the early phases of bone healing, comprising the inflammation and chondrogenesis phase. These results support the concept that disease-deduced growth factor variants are promising lead structures for novel therapeutics with improved clinical activities.

Mechanisms of Antileukemic Activity of the Multikinase Inhibitors Dasatinib and Ponatinib in Acute Lymphoblastic Leukemia (ALL) Harboring the E2A-PBX1 Fusion Gene

The development of small molecules targeting oncogenic signaling pathways as treatment for leukemias has focused on inhibitors of tyrosine kinases. Subtypes of acute leukemias harboring aberrant fusion genes that involve transcription factors rather than kinases have so far not been amenable to molecularly targeted therapies. The t(1;19) is a recurring translocation found in a small proportion of pediatric (5%) and adult (1-3%) ALL. We observed that leukemic cell growth of E2A-PBX1 positive ALL is inhibited by dasatinib (D) and ponatinib (P) at therapeutically achievable concentrations, but not by the ABL-kinase inhibitor nilotinib (N). The mechanism by which these multikinase inhibitors exert their antiproliferative activity is unclear, as the chimeric E2A-PBX1 fusion gene encodes a protein with transforming properties but has no known link to dysregulated kinase activity.As ALL cells with an E2A-PBX1 gene rearrangement express a functional pre-B cell receptor (pre-BCR), we investigated whether the anti-leukemic effect of D and P is mediated by inhibition of SRC family kinases (SFKs), known key mediators of pre-BCR signaling. In two different cellular models of t(1;19) ALL, i.e. two patient-derived ALL cells (PDLTC) maintained in long-term culture (denoted KR and RL, respectively) and the established t(1;19) cell lines 697 and RCH-ACV, D and P suppress SFK activity in a dose-dependent manner as determined by autophosphorylation of SFKs at Y416. However, 5-fold higher concentrations of D and P were needed to inhibit proliferation than to block SFK phosphorylation. To determine whether cooperation between SFKs and other kinases underlies the anti-proliferative effect of D and P, we examined the role of downstream components of the pre-BCR signaling pathway. Activity of SYK, Btk, PLCgamma2, CD79A and ZAP70 was only moderately reduced in response to high nanomolar concentrations of D and P. Pharmacological inhibition of BTK by ibrutinib, an irreversible Btk inhibitor clinically approved for CLL, dose-dependently inhibited proliferation of all t(1;19) ALL cells evaluated (10-50% at 0.25 µM, max. inhibition of 80% at 5 µM). Inhibition of SYK by the selective ATP-competitive Syk inhibitor fostamatinib had no growth suppressive effects on t(1;19) ALL cells. These results suggest that the antiproliferative effect of D and P involves cooperation between the proximal and distal components of the pre-BCR. To further elucidate the mechanisms underlying responsiveness of t(1;19) ALL cells to D and P and the possible involvement of pre-BCR signaling, we employed a proteomics screen in which the PDLTC KR and RL were exposed to D, N or DMSO, and differentially expressed proteins were identified by SILAC followed by mass spectrometry. With a threshold of 0.5 and 2-fold, 46 proteins were up- and 23 proteins were downregulated in the presence of 0.1 µM D in PDLTC KR cells, 11 proteins were up- and 35 down-regulated in PDLTC RL cells. Analysis of all differentially expressed proteins revealed deregulation of proteins participating in metabolism of proteins, nucleic acids or carbohydrates.To examine whether the dasatinib-induced changes in gene expression in t(1;19) ALL correlated with the proteomic profiles obtained by SILAC/MS, we compared the effects of D, N and DMSO on t(1;19) ALL by differential microarray-based gene expression analysis. In PDLTC KR cells, D exposure was associated with down-regulation of 7 genes associated with cell proliferation (NME1), DNA replication (POLR3G) and cell metabolism (SLC27A2). In PDLTC RL cells, 101 genes were down-regulated, predominantly genes involved in cell cycle progression (CHEK1), cell proliferation (BCAT1), apoptosis (PDCL3) and DNA-replication (ORC6L). Notably, several genes displaying the greatest changes in gene expression (e.g. FABP5) are involved in fatty acid metabolism. In both t(1;19) PDLTC (KR and RL), a significantly smaller number of genes was up-regulated in response to D, including USP18, P2RY14 and F13A1.Unraveling the mechanisms by which dasatinib and ponatinib exert their anti-proliferative activity in t(1;19) ALL may facilitate the rational development of SFK-inhibitor-based therapies in this ALL subtype. DisclosuresNo relevant conflicts of interest to declare.

137 SPLITTING OF IVP BOVINE BLASTOCYST AFFECTS MORPHOLOGY AND GLOBAL GENE EXPRESSION OF RESULTING DEMI-EMBRYOS DURING ELONGATION

Embryo splitting has been used since the early 1980s to produce identical twins and increase the pregnancy rate per available embryo. However, very little is known about the effect of splitting on embryo development and competence. Indeed splitting could provoke a negative effect on embryo survival and it can be presumed that each demi-embryo might respond differently to the injury. In this sense, even when embryos are genetically and morphologically identical at the moment of splitting, their developmental potential and molecular characteristics might change as a consequence of the intense manipulation or epigenetic differences due to the interaction with the environment. We have proposed an approach to evaluate the effect of blastocyst splitting on the morphological and gene expression in in vivo development up to the filamentous stage. For that, the effect of splitting on bovine embryo development was evaluated during the elongation period by transferring split and nonsplit IVF-derived blastocysts to cattle recipients and collecting them at Day 17 of development. The number of collected embryos, embryo size, and global gene expression was compared between both groups. Collected elongated embryos derived from split blastocyst were compared with time matched collected control embryos. From 14 transferred hemi-embryos, 5 (35.7%) were collected while 9 elongated from 17 controls were recovered (52.9%). Neither the recovery rate nor the average length of the elongated embryos was significantly different between the two treatments. However, when embryos were rated depending on their size, more than 50% of embryos from the control group had a length surpassing 100 mm, while only 33% of the split embryos reached that size. Global gene expression was performed using 2-colour microarray-based gene expression analysis. This was a whole-genome microarray study comparing 10 individual elongated embryos derived from split and nonsplit IVF blastocysts. Genes were considered differentially expressed if the fold change is greater than 2 (up or down-regulation) with P ≤ 0.05. A total of 29 585 transcripts were detected in all embryos. From those, 449 (1.5%) were differentially expressed between elongated embryos derived from split and nonsplit IVF blastocysts, among them, 248 (0.83%) genes were down-regulated and 201 (0.67%) genes were up-regulated in split embryos. Gene ontology analysis identified deregulated genes related with intrinsic component of membrane (ELOVL7, GJA1, LAPTM4B, LDLR, SLC18A2, SLC1A3, SLC38A5, TSPAN13), lipid transporter activity (RBP4, APOA1, MTTP), and organophosphate ester transport (GJA1, GJB1, ATP9B). In conclusion, we showed that splitting affect the in vivo developmental capability and gene expression profile during the elongation period of bovine embryos. However, further studies are needed to determine the long-term effect of this technique to produce viable offspring. This work was partially supported by Fondecyt No. 11100082 and Fondequip No. EQM12113 from the Ministry of Education of Chile.

Whole genome response in guinea pigs infected with the high virulence strain Mycobacterium tuberculosis TT372

In this study we conducted a microarray-based whole genomic analysis of gene expression in the lungs after exposure of guinea pigs to a low dose aerosol of the Atypical Beijing Western Cape TT372 strain of Mycobacterium tuberculosis, after harvesting lung tissues three weeks after infection at a time that effector immunity is starting to peak. The infection resulted in a very large up-regulation of multiple genes at this time, particularly in the context of a "chemokine storm" in the lungs. Overall gene expression was considerably reduced in animals that had been vaccinated with BCG two months earlier, but in both cases strong signatures featuring gamma interferon [IFNγ] and tumor necrosis factor [TNFα] were observed indicating the potent TH1 response in these animals. Even though their effects are not seen until later in the infection, even at this early time point gene expression patterns associated with the potential emergence of regulatory T cells were observed. Genes involving lung repair, response to oxidative stress, and cell trafficking were strongly expressed, but interesting these gene patterns differed substantially between the infected and vaccinated/infected groups of animals. Given the importance of this species as a relevant and cost-effective small animal model of tuberculosis, this approach has the potential to provide new information regarding the effects of vaccination on control of the disease process.

Abstract 543: Identification of lincRNAs in the HOX domain in adult medulloblastoma by microarray analysis

Abstract Introduction: Medulloblastoma (MB) is defined at molecular level into at least four major subtypes: WNT, SHH, Group C and Group D. Previously, we have demonstrated that levels of HOX transcripts in medulloblastoma specimens correlate with their malignant phenotype. HOX gene regulation involves long noncoding RNAs (lncRNAs), the epigenetic activator of the Trithorax group (TrxG) and the epigenetic repressors of the polycomb group (PcG). Therefore, we performed transcriptoma profiling in adult MBs and normal cerebellum samples to elucidate potential epigenetic regulatory mechanisms involved in tumor progression. Methodology: We analyzed frozen tumor samples from 5 MB adult patients with different tumor subtypes and with ages ranging from 18-33 years and 3 control cerebellar tissues obtained from infants with ages from 23 weeks to six months. RNA was isolated and integrity was assessed using the Agilent 2100 Bioanalyzer. Two colors microarray-based gene expression analysis was performed according to the manufacture's instructions. Quality control and quantile normalization were done using R. Hierarchical clustering was performed using subsets of genes identified in previous studies analysis to classify the tumor samples. There was also considerable variability among the control samples which was separately studied using bootstrap analysis and rank based tests. Results The hierarchical clustering of samples considering the 200 most variable genes, followed by molecular analyses suggests the existence of at least three groups among the tumor specimens analyzed. Hierarchical clustering of samples using the Hox genes demonstrates that the HOX pattern expression might be related to defined medulloblastoma molecular subgroups. For example, in SHH, group C and group D, HOXA3, HOXA6 and HOXB4, are upregulated while HOXC4 is increased in the Wnt group. Interestingly, in control samples these HOX genes showed little variation. Hierarchical clustering of tumor samples using 1414 long intergenic noncoding RNAs (lincRNAs) from the chromosomal (7, 17, 12 and 2) of HOX genes shows similar grouping between control and tumor samples. Moreover five lincRNAs, whose distance from HOX is comparable with the distance from HOTAIR to HOX, were identified: three of these five are in HOX C, one in HOX B9 and another close to HOX D3 genes with the distance varying from 3,800 - 11,819 bp from HOX genes. In our analysis, HOTAIR showed little expression across the samples and HOX regulators (TrxG and PcG complexes) displayed variation in expression level among samples. We found that there is considerable heterogeneity among the control samples used in this study. Conclusion: This study suggests that lncRNAs might affect the expression of HOX gene products in adult medulloblastoma. Validation analyses and genetic studies are undergoing to elucidate the possible mechanisms of action of this class of ncRNAs during MB progression. Note: This abstract was not presented at the meeting. Citation Format: Julia B. Veiga, Ricardo Bonfim Silva, Kuruvilla Joseph Abraham, Daniela B. Pretti da Cunha Tirapelli, Fernando Silva Ramalho, Gerson Chadi, Jéssica Ruivo Maximino, Dimas B. Tadeu Covas, Angelo A. Cardoso, Carlos Gilberto B. Carlotti, Aparecida M. Fontes. Identification of lincRNAs in the HOX domain in adult medulloblastoma by microarray analysis. [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 543. doi:10.1158/1538-7445.AM2014-543

Abstract 204: Understanding breast cancer resistance to chemotherapy: Characterization of cancer cell sub-populations in residual and relapsed tumors

Abstract Tumor recurrence fueled by residual tumor cells having survived chemotherapy represents the principal cause of breast cancer treatment failure. Triple-negative breast cancer (TNBC) is a heterogeneous disease at both molecular and cellular level, and the presence of different tumor cell sub-populations is likely the reason for this heterogeneity and for the incomplete response to neoadjuvant chemotherapy observed for most TNBC. To identify and isolate tumor cell sub-populations that resist to chemotherapy, we used a panel of 45 antibody fluorochrome conjugates in combination with multi-parameter flow cytometry to screen for the expression of a set of cell surface markers during the course of tumor chemotherapy. This set of markers represented both proteins involved in stem cell function and proteins known to be over-expressed in stem cells or cancer stem cell sub-populations. As a source of tumor samples, we used a panel of TNBC patient-derived xenografts (PDXs). These tumor models are known to preserve the morphology, molecular characteristics and drug response profile of the original patient tumors. We used TNBC PDX models to reproduce in vivo chemotherapy-induced tumor regression and relapse. Tumor dissociation, depletion of mouse cells and multi-parameter flow cytometry allowed us to measure the percentage of marker expression only in the fraction of human tumor cells. We found that the expression of most of markers was very heterogeneous among the different TNBC models analyzed. In residual tumors, we observed enrichment or depletion of several cell populations expressing stem and cancer stem cell markers such as CD44+ and CD133/1+. Interestingly, in the majority of relapsed tumors, the percentage of these marker-positive cells shifted back to pre-treatment levels. Moreover, the percentage of certain tumor cell sub-populations was higher in untreated chemotherapy-resistant tumors compared to untreated chemotherapy-sensitive ones. To study the properties of enriched tumor cells sub-populations, we coupled cell separation of marker-positive tumor cells to large scale molecular analysis. Microarray-based gene expression analysis of sorted tumor cells revealed cell sub-populations with up-regulated signaling pathways related to stem cell function, cell migration or EMT. Further in vivo and in vitro assays with sorted sub-populations are currently being performed to search for possible functional role of selected markers during tumor resistance to chemotherapy and initiation of tumor relapse. In summary, by coupling PDX and cell surface marker screening technologies, we have identified distinct tumor cell sub-populations which are associated with tumor resistance to chemotherapy. We believe that this approach will help gaining knowledge of tumor cell adaptation and selection in response to chemotherapy, which will ultimately lead to design tailored treatments for TNBCs. Citation Format: Andrea Aloia, Evgeniya Petrova, Olivier Deas, Sophie Banis, Enora Le Ven, Andreas Bosio, Olaf Hardt, Stefano Cairo, Jean-Gabriel Judde. Understanding breast cancer resistance to chemotherapy: Characterization of cancer cell sub-populations in residual and relapsed tumors. [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 204. doi:10.1158/1538-7445.AM2014-204

Abstract 3993: A microarray-based gene expression analysis identified diagnostic biomarkers for unknown primary cancer

Abstract Background: Patients with cancer of unknown primary (CUP) present with metastatic disease for which the primary site cannot be found. Clinically, CUPs exhibit common characteristics, such as rapid progression and early dissemination, with a silent primary tumor that may become involuted and undetectable. Existence of such common properties prompts us to hypothesize that there may be potential biological markers that elucidate CUP as a whole. Methods: Tumor mRNA samples from 60 patients with CUP were measured for the expression of ∼22,000 genes using the Affymetrix U133A Plus 2.0 GeneChip and analyzed by applying normalization and classification algorithms to the gene expression data. The similarity of each tumor specimen's gene expression pattern is then compared to the gene-expression profiles specific to non-CUP groups (containing tumors from 24 known primary sites) that were constructed using publicly available raw microarray datasets. The t-tests were performed to compare the CUP with non-CUP groups and the top 59 CUP specific genes with the highest fold change were selected (p-value < 0.001). Results: This study enabled the identification of genes that exhibited a unique expression pattern in CUP. As a high metastasis potential and vulnerability to apoptosis would explain the properties of CUP well, we first searched for genes related to metastasis and apoptosis and found 14 genes among 44 that were up-regulated by more than 2.5-fold in the CUP samples. Some of these genes were associated with the epithelial-to-mesenchymal transition (EMT), a function that has been increasingly recognized as a key step in cancer metastasis. We also identified 6 genes for ribosomal proteins among 44 up-regulated genes, two of which (RPS7 and RPL11) were known to be involved in the Mdm2 - p53 pathway. Conclusions: The protein products of the up-regulated and down-regulated genes identified in this study suggest a biological attribute of CUP and, therefore, may become clinically useful biomarkers for CUP. Citation Format: Yoshihiko Fujita, Issei Kurahashi, Takayasu Kurata, Yasuhiro Koh, Kazuko Sakai, Kazuhiko Nakagawa, Kazuto Nishio. A microarray-based gene expression analysis identified diagnostic biomarkers for unknown primary cancer. [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 3993. doi:10.1158/1538-7445.AM2014-3993

The oncogene EVI1 enhances transcriptional and biological responses of human myeloid cells to all-trans retinoic acid

The product of the ecotropic virus integration site 1 (EVI1) gene, whose overexpression is associated with a poor prognosis in myeloid leukemias and some epithelial tumors, regulates gene transcription both through direct DNA binding and through modulation of the activity of other sequence specific transcription factors. Previous results from our laboratory have shown that EVI1 influenced transcription regulation in response to the myeloid differentiation inducing agent, all-trans retinoic acid (ATRA), in a dual manner: it enhanced ATRA induced transcription of the RARβ gene, but repressed the ATRA induction of the EVI1 gene itself. In the present study, we asked whether EVI1 would modulate the ATRA regulation of a larger number of genes, as well as biological responses to this agent, in human myeloid cells. U937 and HL-60 cells ectopically expressing EVI1 through retroviral transduction were subjected to microarray based gene expression analysis, and to assays measuring cellular proliferation, differentiation, and apoptosis. These experiments showed that EVI1 modulated the ATRA response of several dozens of genes, and in fact reinforced it in the vast majority of cases. A particularly strong synergy between EVI1 and ATRA was observed for GDF15, which codes for a member of the TGF-β superfamily of cytokines. In line with the gene expression results, EVI1 enhanced cell cycle arrest, differentiation, and apoptosis in response to ATRA, and knockdown of GDF15 counteracted some of these effects. The potential clinical implications of these findings are discussed.

Therapeutic concentration of lithium stimulates complement C3 production in dendritic cells and microglia via GSK-3 inhibition.

Evidence indicates that widely prescribed mood stabilizer, lithium (Li), mediates cellular functions of differentiated monocytic cells, including microglial migration, monocyte-derived dendritic cell (MoDC) differentiation, and amelioration of monocytic malfunctions observed in neuropsychiatric diseases. Here, we surveyed molecules which take major roles in regulating these monocytic cellular functions. MoDCs treated with 1 and 5 mM Li, and microglia separated from Li-treated mice were subjected to microarray-based comprehensive gene expression analyses. Findings were validated using multiple experiments, including quantitative PCR, ELISA and immunostaining studies. Differing effects of Li on the two cell types were observed. Inflammation- and chemotaxis-relevant genes were significantly over-represented among Li-induced genes in MoDCs, whereas no specific category of genes was over-represented in microglia. The third component of complement (C3) was the only gene which was significantly induced by a therapeutic concentration of Li in both MoDCs and microglia. C3 production was increased by Li via GSK-3 inhibition. Li-induced C3 production was seen only in differentiated monocytic cells, but not in circulating monocytes. Our findings highlight a link between Li treatment and C3 production in differentiated monocytic cells, and reveal a regulatory role of GSK-3 in C3 production. Induction of microglial C3 production might be a novel neuroprotective mechanism of Li via regulating interactions between microglia and neurons. GLIA 2015;63:257-270.

EIF3a is over-expressed in urinary bladder cancer and influences its phenotype independent of translation initiation.

The eukaryotic translation initiation factor (eIF) 3a, the largest subunit of the eIF3 complex, is a key functional entity in ribosome establishment and translation initiation. In the past, aberrant eIF3a expression has been linked to the pathology of various cancer types but, so far, its expression has not been investigated in transitional cell carcinomas. Here, we investigated the impact of eIF3 expression on urinary bladder cancer (UBC) cell characteristics and UBC patient survival. eIF3a expression was reduced through inducible knockdown in the UBC-derived cell lines RT112, T24, 5637 and HT1197. As a consequence of eIF3a down-regulation, UBC cell proliferation, clonogenic potential and motility were found to be decreased and, concordantly, UBC tumour cell growth rates were found to be impaired in xenotransplanted mice. Polysomal profiling revealed that reduced eIF3a levels increased the abundance of 80S ribosomes, rather than impairing translation initiation. Microarray-based gene expression and ontology analyses revealed broad effects of eIF3a knockdown on the transcriptome. Analysis of eIF3a expression in primary formalin-fixed paraffin embedded UBC samples of 198 patients revealed that eIF3a up-regulation corresponds to tumour grade and that high eIF3a expression corresponds to longer overall survival rates of patients with low grade tumours. From our results we conclude that eIF3a expression may have a profound effect on the UBC phenotype and, in addition, may serve as a prognostic marker for low grade UBCs.

Cancer stem cell differentiation: TGFβ1 and versican may trigger molecules for the organization of tumor spheroids.

Cancer stem cells (CSCs) have the ability to self-renew similar to normal stem cells. This process is linked with metastasis and resistance to chemotherapy and radiotherapy. In the present study, we constructed an in vitro differentiation model for CSCs. CSCs isolated and proliferated for one passage were maintained as monolayers or spheroid-forming cells with serum included media for differentiation process. Differentiation of adhesion molecules and cellular ultrastructural properties were investigated and compared in both monolayer and spheroid cultures. CD133+/CD44+ cancer-initiating cells were isolated from DU-145 human prostate cancer cell line monolayer cultures and propagated as tumor spheroids and compared with the remaining heterogeneous cancer cell bulk population. Microarray-based gene expression analysis was applied to determine genes with differential expression and protein expression levels of candidates were analyzed by immunohistochemistry. Electron microscopy showed detailed analysis of morphology. TGFβ1 was found to be significantly upregulated in monolayer CSCs. High expression levels of VCAN, COL7A1, ITGβ3, MMP16, RPL13A, COL4A2 and TIMP1 and low expression levels of THBS1, MMP1 and MMP14 were detected when CSCs were maintained as serum-grown prostate CSC spheroids. Immunohistochemistry supported increased immunoreactivity of TGFβ1 in monolayer cultures and VCAN in spheroids. CSCs were found to possess multipotential differentiation capabilities through upregulation and/or downregulation of their markers. TGFβ1 is a triggering molecule, it stimulates versican, Col7A1, ITGβ3 and, most importantly, the upregulation of versican was only detected in CSCs. Our data support a model where CSCs must be engaged by one or more signaling cascades to differentiate and initiate tumor formation. This mechanism occurs with intracellular and extracellular signals and it is possible that CSCc themselves may be a source for extracellular signaling. These molecules functioning in tumor progression and differentiation may help develop targeted therapy.

Antipsychotic drug-associated gene–miRNA interaction in T-lymphocytes

Antipsychotic drugs (APDs) can have a profound effect on the human body that extends well beyond our understanding of their neuropsychopharmacology. Some of these effects manifest themselves in peripheral blood lymphocytes, and in some cases, particularly in clozapine treatment, result in serious complications. To better understand the molecular biology of APD action in lymphocytes, we investigated the influence of chlorpromazine, haloperidol and clozapine in vitro, by microarray-based gene and microRNA (miRNA) expression analysis. JM-Jurkat T-lymphocytes were cultured in the presence of the APDs or vehicle alone over 2 wk to model the early effects of APDs on expression. Interestingly both haloperidol and clozapine appear to regulate the expression of a large number of genes. Functional analysis of APD-associated differential expression revealed changes in genes related to oxidative stress, metabolic disease and surprisingly also implicated pathways and biological processes associated with neurological disease consistent with current understanding of the activity of APDs. We also identified miRNA-mRNA interaction associated with metabolic pathways and cell death/survival, all which could have relevance to known side effects of APDs. These results indicate that APDs have a significant effect on expression in peripheral tissue that relate to both known mechanisms as well as poorly characterized side effects.

Bortezomib effect on E2F and cyclin family members in human hepatocellular carcinoma cell lines

To evaluate the effects of the proteasome inhibitor bortezomib (BZB) on E2Fs and related genes in hepatocellular carcinoma (HCC) cells. The mRNA levels of the E2F family members (pro-proliferative: E2F1-3 and anti-proliferative: E2F4-8) and of their related genes cyclins and cyclin-dependent kinases (cdks) were evaluated in two HCC cell lines following a single BZB administration. mRNA levels of the epithelial-mesenchymal transition (EMT) genes were also measured in both cell lines after BZB treatment. The BZB concentration (40 nmol/L) used was chosen to stay well below the maximal amount/cm² recommended for in vivo application, and 2 d incubation was chosen as this time point has been found optimal to detect BZB effects in our previous studies. The HCC cell lines, HepG2 and JHH6, were chosen as they display different phenotypes, hepatocyte-like for HepG2 and undifferentiated for JHH6, thus representing an in vitro model of low and high aggressive forms of HCC, respectively. The mRNA levels of the target genes were measured by two-color microarray-based gene expression analysis, performed according to Agilent Technologies protocol and using an Agilent Scan B. For the E2F family members, mRNA levels were quantified by real-time reverse transcription polymerase chain reaction (RT-PCR). Using small interfering RNA's, the effects of E2F8 depletion on cell number was also evaluated. After BZB treatment, microarray analysis of the undifferentiated JHH6 revealed a significant decrease in the expression of the pro-proliferative E2F member E2F2. Quantitative RT-PCR data were in keeping with the microarray analysis, and showed a significant increase and decrease in E2F8 and E2F2 mRNA levels, respectively. In contrast, BZB treatment of the hepatocyte-like HCC cell line HepG2 had a significant impact on mRNA levels of 5 of the 8 E2F members. In particular, mRNA levels of the pro-proliferative E2F members E2F1, E2F2, and of the anti-proliferative member E2F8, decreased over 80%. Notably, a reduction in E2F8 expression in HepG2 and JHH6 cells following siRNA treatment had no impact on cell proliferation. As observed with JHH6, BZB treatment of HepG2 cells induced a significant increase in mRNA levels of an anti-proliferative E2F member, E2F6 in this case. As was observed with E2F's, more dramatic changes in mRNA levels of the E2F related genes cyclins and Cdks and EMT genes were observed after BZB treatment of HepG2 compared to JHH6. The differential expression of E2Fs and related genes induced by BZB in diverse HCC cell phenotypes contribute to bortezomib's mechanism of action in hepatocellular carcinoma.