Gene Expression Profiling Reveals a Crucial Role for C/EBPbeta in Proliferation Pathways of ALK+ ALCL Cell Lines

Abstract Introduction: ALK+ anaplastic large cell lymphomas (ALCL) overexpress C/EBPβ, as a consequence of NPM-ALK kinase activity. C/EBPβ is a leucine zipper transcription factor, which plays a major role in cellular differentiation, inflammation, proliferation and metabolism control. To determine the role of C/EBPβ in ALK+ ALCL transformation, and to identify its downstream targets, a highly specific C/EBPβ-shRNA was used to knockdown C/EBPβ. The consequences of C/EBPβ gene-silencing were analyzed by gene expression profiling. Materials and Methods: Four ALK+ ALCL cell lines, SUDHL-1, Kijk, Karpas 299 and SUP-M2 were transfected with lentivirus containing the C/EBPβ shRNA or the vector without shRNA in triplicates. Western Blot analysis and qRT-PCR were performed to quantify the knockdown effect. At day three after infection, RNA was extracted and used for Gene Chip expression analysis (Affymetrix). Using Anova software for statistical analysis, we identified genes, which were regulated in all four cell lines. The effect of C/EBPβ knockdown on proliferation, cell cycle, and viability was analyzed by MTT assay and FACS analysis. Results: In all four ALK+ ALCL, efficient C/EBPβ knockdown resulted in profound growth retardation (up to 84%) compared to control cells after 6 days of infection, and a clear shift from the S phase to the G1 phase in the cell cycle was observed. To identify genes regulated by C/EBPβ in all four cell lines, we performed statistical analysis applying a false discovery rate of 20%, and accepted only genes with a >1,1 and <0,9 fold ratio. We identfied 435 genes regulated after C/EBPβ knockdown (117 upregulated, 318 downregulated). Classification of the differentially expressed genes into biological categories revealed overrepresentation of genes involved in the regulation of kinase activity, cell cycle and proliferation, lymphocyte differentiation, and metabolic processes. In particular, kinases involved in the regulation of JNK activity, which have been shown previously to be involved in proliferation of ALCL, were highly affected by C/EBPβ knockdown. Genomatix Bibliosphere Pathway Analysis revealed C/EBPβ to be connected to pathways involving cell cycle (RUNX3, CCNG1, CDKN2A), apoptosis (FAS, PTPRC, BCL2A1, BIRC3) and MAPK cascades (TRIB1 and several MAP3Ks). Several of the genes identified contain known C/EBPβ binding sites. Conclusions: C/EBPβ silencing induces growth arrest in ALK+ALCL, which correlates with differential expression of genes involved in cell cycle, apoptosis and differentiation. This study reveals C/EBPβ as a master transcription regulator of NPM-ALK induced cellular proliferation, and therefore, an ideal candidate for targeted therapeutic intervention.

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Identification of Genes Which Play a Crucial Role in C/EBPβ Downstream Signalling in ALK+ ALCL Cell Lines.

Blood ◽

10.1182/blood.v114.22.1943.1943 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1943-1943

Author(s):

Irina Bonzheim ◽

Martin Irmler ◽

Natasa Anastasov ◽

Margit Klier-Richter ◽

Sabine Schaefer ◽

...

Keyword(s):

Gene Expression ◽

Western Blot ◽

Gene Expression Profiling ◽

Cell Lines ◽

Pathway Analysis ◽

Expression Profiling ◽

Cellular Proliferation ◽

Compensatory Mechanism ◽

Mrna Levels ◽

Qrt Pcr

Abstract Abstract 1943 Poster Board I-966 Introduction: ALK+ anaplastic large cell lymphomas (ALCL) overexpress C/EBPβ, as a consequence of NPM-ALK kinase activity. We recently reported C/EBPβ as a transcription regulator of NPM-ALK induced cellular proliferation. To identify the downstream targets of C/EBPβ that might be responsible for cell proliferation and survival, we performed gene expression profiling and pathway analyses after C/EBPβ gene silencing Materials and Methods: C/EBPβ knockdown was done by lentiviral shRNA-transduction into two ALK+ ALCL cell lines with strong C/EBPβ expression – SUDHL1 and KiJK. At day three after infection, RNA was extracted and used for Gene Chip expression analysis (U133 Plus 2.0 arrays/ Affymetrix). Genes regulated in both cell lines were applied to Genomatix Bibliosphere Pathway analysis. Candidate genes were either strongly influenced by C/EBPβ knockdown, or had promoter binding sites for C/EBPβ, or showed remarkable pathway connections. The influence of C/EBPβ on these genes was validated by qRT-PCR and in part by Western blot. Results: Gene expression profiling analysis resulted in 167 genes being regulated in both cell lines, of which 26 genes were chosen for further analysis. Validation by qRT-PCR confirmed 23/26 genes. Pathway analysis revealed c-Jun, which is a member of the dimeric transcription factor AP-1, as a regulator of C/EBPβ expression. Silencing C/EBPβ led to a clear up-regulation of c-Jun mRNA. Western blot analysis demonstrated that C/EBPβ influenced not only the expression of c-Jun but also its phosphorylation on Ser63 and Ser73. In contrast to what has been reported, we found very low levels of c-Jun expression in ALK+ALCL cells lines and its expression correlated inversely with C/EBPβ mRNA levels. Although it has been shown that c-Jun regulates C/EBPβ expression directly, in ALK+ALCL the expression of C/EBPβ is clearly independent of c-Jun. Our data suggest that c-Jun up-regulation after C/EBPβ knockdown is a compensatory mechanism to maintain C/EBPβ expression. Additionally, of the 26 selected genes, Bibliosphere Analysis identified 12 genes, which might be transcriptionally regulated by C/EBPβ and are primary targets in C/EBPβ downstream signalling. Two of these genes are of particular interest. The anti-apoptotic protein BCL2A1 contains a promoter-binding site for C/EBPβ and has been shown previously to be both strongly regulated in ALK+ALCL and absolutely necessary for its transformation. The second is a DEAD box nucleolar RNA helicase protein involved in ribosomal RNA production and proliferation which we found to be strongly expressed in ALK+ALCL cell lines and primary cases. Conclusions: C/EBPβ silencing in ALK+ALCL cell lines showed 1) an inverse correlation between c-Jun and C/EBPβ mRNA expression levels, 2) the expression of C/EBPβ in ALK+ALCL is independent of c-Jun, 3) genes transcriptionally regulated by C/EBPβ seem to be essential for proliferation and survival in ALK+ALCL. Disclosures: No relevant conflicts of interest to declare.

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Identification of Cyclin D1-Dependent Genes by Gene Expression Profiling in MCL Cell Lines and Primary Cases.

Blood ◽

10.1182/blood.v114.22.3936.3936 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3936-3936

Author(s):

Margit Klier-Richter ◽

Martin Irmler ◽

Irina Bonzheim ◽

Sabine Schaefer ◽

Johannes Beckers ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Cyclin D1 ◽

Gene Expression Profiling ◽

Cell Lines ◽

Expression Profiling ◽

Transcriptional Network ◽

Proliferation Rate ◽

Differentially Expressed ◽

Mrna Levels

Abstract Abstract 3936 Poster Board III-872 Introduction The hallmark of MCL is overexpression of cyclin D1 due to the t(11;14). As cyclin D1 plays a central role in cell cycle regulation and influences the activity of several transcription factors, its overexpression is believed to initiate MCL lymphomagenesis. To dissect the transcriptional network regulated by cyclin D1, and to assess the relationship of cyclin D1 to genes comprising the recently described MCL proliferation signature, the consequences of cyclin D1 knockdown were analyzed by comparative gene expression profiling. Materials and Methods Four MCL cell lines, Granta 519, Jeko-1, Rec-1 and Z-138 were transduced with lentivirus containing the cyclin D1 shRNA or with empty lentivirus in triplicates. At day seven after infection, RNA was extracted and used for Gene Chip expression analysis (U133 Plus 2.0 arrays/ Affymetrix). After robust multi-array average (RMA) preprocessing, genes which were differentially expressed in all four cell lines were identified using BioConductor software. Validation was performed by correlating cyclin D1 levels with candidate gene expression by qRT-PCR in 10 primary MCL cases. Results To identify genes regulated by cyclin D1 in all four cell lines, statistical analysis applying a false discovery rate of 10% was performed, thereby accepting only genes with a >1,1 and <0,9 fold ratio and an average expression >25. We identified 344 genes regulated after cyclin D1 knockdown (93 upregulated, 251 downregulated). Classification of the differentially expressed genes to biological processes revealed overrepresentation of genes involved in replication (23,0%), chromatin packaging and remodelling (12,5%), signal transduction (10,5%), metabolism (9,9%), transcription (9,6%) and cell cycle (6,4%). Of the 20 genes comprising the MCL proliferation signature (Rosenwald et al., Blood 2003), 10 were identified as cyclin D1 dependent. These genes were validated in MCL with high vs. low levels of cyclin D1 mRNA. Although there was a direct correlation between these 10 genes and cyclin D1 levels, it is notable that there was no correlation between the proliferation rate as assessed with MiB1 and the level of cyclin D1 expression Conclusions Comparative gene expression profiling before and after knockdown of cyclin D1 in MCL cell lines reveals a complex transcriptional network influenced by cyclin D1 expression levels. We also show that 10 of the 20 genes comprising the MCL proliferation signature are cyclin D1-dependent. These genes showed good correlation with cyclin D1 mRNA levels in primary cases, however, no correlation with proliferation rate was identified, suggesting cyclin D1-independent mechanisms governing proliferation in MCL. The identification of cyclin D1-dependent genes assists in our understanding of the contribution of cyclin D1 overexpression to the biology and clinical course of MCL. Disclosures: No relevant conflicts of interest to declare.

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Gene Expression Profiling Uncovers Molecular Classifiers for the Recognition of Anaplastic Large-Cell Lymphoma Within Peripheral T-Cell Neoplasms

Journal of Clinical Oncology ◽

10.1200/jco.2008.20.9759 ◽

2010 ◽

Vol 28 (9) ◽

pp. 1583-1590 ◽

Cited By ~ 116

Author(s):

Roberto Piva ◽

Luca Agnelli ◽

Elisa Pellegrino ◽

Katia Todoerti ◽

Valentina Grosso ◽

...

Keyword(s):

Gene Expression ◽

T Cell ◽

Gene Expression Profiling ◽

Cell Lines ◽

Anaplastic Large Cell Lymphoma ◽

Expression Profiling ◽

Cell Lymphoma ◽

Large Cell ◽

Large Cell Lymphoma ◽

Alk Positive

Purpose To unravel the regulatory network underlying nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) –mediated lymphomagenesis of anaplastic large-cell lymphoma (ALCL) and to discover diagnostic genomic classifiers for the recognition of patients with ALK-positive and ALK-negative ALCL among T-cell non-Hodgkin's lymphoma (T-NHL). Patients and Methods The transcriptome of NPM-ALK–positive ALCL cell lines was characterized by silencing the expression of ALK or STAT3, a major effector of ALK oncogenic activity. Gene expression profiling (GEP) was performed in a series of systemic primary T-NHL (n = 70), including a set of ALK-positive and ALK-negative ALCL (n = 36). Genomic classifiers for ALK-positive and ALK-negative ALCL were generated by prediction analyses and validated by quantitative reverse-transcriptase polymerase chain reaction and/or immunohistochemistry. Results In ALCL cell lines, two thirds of ALK-regulated genes were concordantly dependent on STAT3 expression. GEP of systemic primary T-NHL significantly clustered ALK-positive ALCL samples in a separate subgroup, underscoring the relevance of in vitro ALK/STAT3 signatures. A set of genomic classifiers for ALK-positive ALCL and for ALCL were identified by prediction analyses. These gene clusters were instrumental for the distinction of ALK-negative ALCL from peripheral T-cell lymphomas not otherwise specified (PTCLs-NOS) and angioimmunoblastic lymphomas. Conclusion We proved that experimentally controlled GEP in ALCL cell lines represents a powerful tool to identify meaningful signaling networks for the recognition of systemic primary T-NHL. The identification of a molecular signature specific for ALCL suggests that these T-NHLs may represent a unique entity discernible from other PTCLs, and that a restricted number of genes can be instrumental for clinical stratification and, possibly, therapy of T-NHL.

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Assessing Various Control Samples for Microarray Gene Expression Profiling of Laryngeal Squamous Cell Carcinoma

Biomolecules ◽

10.3390/biom11040588 ◽

2021 ◽

Vol 11 (4) ◽

pp. 588

Author(s):

Adam Ustaszewski ◽

Magdalena Kostrzewska-Poczekaj ◽

Joanna Janiszewska ◽

Malgorzata Jarmuz-Szymczak ◽

Malgorzata Wierzbicka ◽

...

Keyword(s):

Gene Expression ◽

Squamous Cell Carcinoma ◽

Epithelial Cells ◽

Cell Carcinoma ◽

Gene Expression Profiling ◽

Cell Lines ◽

Squamous Cell ◽

Expression Profiling ◽

Laryngeal Squamous Cell Carcinoma ◽

Control Samples

Selection of optimal control samples is crucial in expression profiling tumor samples. To address this issue, we performed microarray expression profiling of control samples routinely used in head and neck squamous cell carcinoma studies: human bronchial and tracheal epithelial cells, squamous cells obtained by laser uvulopalatoplasty and tumor surgical margins. We compared the results using multidimensional scaling and hierarchical clustering versus tumor samples and laryngeal squamous cell carcinoma cell lines. A general observation from our study is that the analyzed cohorts separated according to two dominant factors: “malignancy”, which separated controls from malignant samples and “cell culture-microenvironment” which reflected the differences between cultured and non-cultured samples. In conclusion, we advocate the use of cultured epithelial cells as controls for gene expression profiling of cancer cell lines. In contrast, comparisons of gene expression profiles of cancer cell lines versus surgical margin controls should be treated with caution, whereas fresh frozen surgical margins seem to be appropriate for gene expression profiling of tumor samples.

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Erratum to: Gene expression profiling of breast tumor cell lines to predict for therapeutic response to microtubule-stabilizing agents

Breast Cancer Research and Treatment ◽

10.1007/s10549-012-1974-z ◽

2012 ◽

Vol 134 (1) ◽

pp. 449-451

Author(s):

Gais Kadra ◽

Pascal Finetti ◽

Yves Toiron ◽

Patrice Viens ◽

Daniel Birnbaum ◽

...

Keyword(s):

Gene Expression ◽

Tumor Cell ◽

Gene Expression Profiling ◽

Cell Lines ◽

Expression Profiling ◽

Breast Tumor ◽

Therapeutic Response ◽

Tumor Cell Lines ◽

Breast Tumor Cell ◽

Stabilizing Agents

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Microarray gene expression profiling in colorectal (HCT116) and hepatocellular (HepG2) carcinoma cell lines treated withMelicope ptelefolialeaf extract reveals transcriptome profiles exhibiting anticancer activity

PeerJ ◽

10.7717/peerj.5203 ◽

2018 ◽

Vol 6 ◽

pp. e5203 ◽

Cited By ~ 6

Author(s):

Mohammad Faujul Kabir ◽

Johari Mohd Ali ◽

Onn Haji Hashim

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Dna Replication ◽

Cell Lines ◽

Microarray Data ◽

Expression Profiling ◽

Cell Cycle Progression ◽

Anticancer Activities ◽

Microarray Gene Expression ◽

Cycle Progression

BackgroundWe have previously reported anticancer activities ofMelicope ptelefolia(MP) leaf extracts on four different cancer cell lines. However, the underlying mechanisms of actions have yet to be deciphered. In the present study, the anticancer activity of MP hexane extract (MP-HX) on colorectal (HCT116) and hepatocellular carcinoma (HepG2) cell lines was characterized through microarray gene expression profiling.MethodsHCT116 and HepG2 cells were treated with MP-HX for 24 hr. Total RNA was extracted from the cells and used for transcriptome profiling using Applied Biosystem GeneChip™ Human Gene 2.0 ST Array. Gene expression data was analysed using an Applied Biosystems Expression Console and Transcriptome Analysis Console software. Pathway enrichment analyses was performed using Ingenuity Pathway Analysis (IPA) software. The microarray data was validated by profiling the expression of 17 genes through quantitative reverse transcription PCR (RT-qPCR).ResultsMP-HX induced differential expression of 1,290 and 1,325 genes in HCT116 and HepG2 cells, respectively (microarray data fold change, MA_FC ≥ ±2.0). The direction of gene expression change for the 17 genes assayed through RT-qPCR agree with the microarray data. In both cell lines, MP-HX modulated the expression of many genes in directions that support antiproliferative activity. IPA software analyses revealed MP-HX modulated canonical pathways, networks and biological processes that are associated with cell cycle, DNA replication, cellular growth and cell proliferation. In both cell lines, upregulation of genes which promote apoptosis, cell cycle arrest and growth inhibition were observed, while genes that are typically overexpressed in diverse human cancers or those that promoted cell cycle progression, DNA replication and cellular proliferation were downregulated. Some of the genes upregulated by MP-HX include pro-apoptotic genes (DDIT3, BBC3, JUN), cell cycle arresting (CDKN1A, CDKN2B), growth arrest/repair (TP53, GADD45A) and metastasis suppression (NDRG1). MP-HX downregulated the expression of genes that could promote anti-apoptotic effect, cell cycle progression, tumor development and progression, which include BIRC5, CCNA2, CCNB1, CCNB2, CCNE2, CDK1/2/6, GINS2, HELLS, MCM2/10 PLK1, RRM2 and SKP2. It is interesting to note that all six top-ranked genes proposed to be cancer-associated (PLK1, MCM2, MCM3, MCM7, MCM10 and SKP2) were downregulated by MP-HX in both cell lines.DiscussionThe present study showed that the anticancer activities of MP-HX are exerted through its actions on genes regulating apoptosis, cell proliferation, DNA replication and cell cycle progression. These findings further project the potential use of MP as a nutraceutical agent for cancer therapeutics.

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