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Myc-Regulated Gene Expression During Tumourigenesis in vivo Sam Robson 1, 2, S. Pelengaris 2, D. Epstein3 and M. Khan 2, 4 1MOAC Doctoral Training Centre, Senate House, University of Warwick, Coventry, CV4 7AL, UK 2Biomedical Research Institute, Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, UK 3Department of Mathematics, University of Warwick, Coventry, CV4 7AL, UK 4Clinical Sciences Research Institute, Warwick Medical School, Walsgrave Hospital, Coventry, UK Results 80 70 60 50 Progression Downregulated Number 40 of Genes 30 Reversal Upregulated 20 Differentially expressed genes were compared during tumour progression and tumour reversal (figure 3). A higher proportion of genes were upregulated during tumourigenesis than downregulated, highlighting Myc’s primary role as a transcriptional activator (though it is unknown how many of these are direct Myc targets). Reversal Downregulated 10 Transcription Protein Kinase Activity Proliferation Insulin Secretion G1-to-S Transition Epigenetic Factors DNA Metabolism Differentiation Chromatin Binding Cell Proliferation (Negative) Cell Proliferation (Positive) Cell Growth and Maintenance Cell Cycle (Positive) Cell Adhesion Apoptosis (Negative) 0 Cell Cycle (Negative) Interestingly, more genes are also upregulated during tumour reversal than are downregulated, suggesting that reversal occurs through alternative pathways to progression. A selection of gene expression profiles can be seen in figure 5. Progression Upregulated Apoptosis (Positive) Microarray data was analysed and filtered to obtain lists of genes with significantly altered gene expression. Progression data corresponds to genes differentially expressed after 1 day of Myc activation; Reversal data corresponds to genes differentially expressed after 7 days of Myc inactivation. Angiogenesis (Negative) Deregulated expression of the c-myc (cellular Myelocytomatosis) protooncogene is seen in a large number of human cancers. [1] The protein product is a transcription factor that forms a heterodimeric complex with Max to promote a variety of tumour related biological functions; cell cycle progression (G1 to S phase), angiogenic growth, inhibition of terminal differentiation, and (perhaps somewhat paradoxically) induction of apoptosis. Control of aberrant Myc expression has been the goal of several therapeutic techniques. [2] However, whilst it is known that Myc directly regulates the expression of a number of genes, [3] the precise genetic pathways involved in Myc induced tumourigenesis are not yet fully understood. It is hoped that a better understanding can be reached through in vivo analysis of changes at the transcriptional level. Mouse models have been created allowing controlled expression of Myc in vivo (figure 2). [4] Transcription of a chimeric c-MycER transgene is targeted to the pancreatic islet β-cells using the rat insulin promoter region. The transcribed protein can be activated through application of the specific ligand 4-Hydroxytamoxifen (4-OHT). Figure 1 shows the effects of Myc activation on islet β-cells. Changes in Gene Expression Angiogenesis (Positive) Introduction Figure 3: Myc regulates the expression of a large number of genes, with a variety of biological functions; particularly those involved with cell growth and differentiation. Similar numbers of genes are upregulated during both progression and reversal of tumour growth. The use of whole pancreas tissue RNA was found to have an effect on the robustness of gene expression data due to the nonuniform nature of the islet proportions in this study. Early and reversed time-points have a much lower islet-to-exocrine proportion than later time-points where islet mass has greatly increased. The technique of laser capture microdissection (LCM) was considered for future work, allowing isolation of pure islet cell populations through the use of a low-power infra-red laser beam. Figure 4: RNA degredation is significantly reduced in pancreatic islets compared to exocrine tissue. This allows for the extraction of intact islet RNA through laser capture microscopy. The LCM protocol allows degradation of RNA by RNAses native to the pancreas tissue. However, we found that the islet tissue RNA was of a higher quality than that of the surrounding exocrine tissue (figure 5), possibly due to their selfcontained nature. This technique allows the extraction of RNA suitable for microarray analysis from homogeneous islet colonies. Figure 1: Activation of c-MycER by administration of 4-OHT leads to an overwhelming apoptotic response in the islet β-cells. Addition of a further anti-apoptotic transgene suppresses angiogenic activity and allows the tumourigenic properties of Myc activation to be analysed. Both phenotypes can be reversed by halting 4OHT administration. [5] Aims To analyse and categorize changes in gene expression at the posttranscriptional level as a result of Myc activation and deactivation, and to compare and contrast these changes to study the differences between tumour progression and regression. Figure 5: Examples of genes showing significant changes in gene expression upon Myc activation. Cell cycle genes, such as Cyclin D2 and Etif2s1, show an initial rise in expression levels as Myc initiates entry of cells into the cell cycle. Reversal sees these gene expression levels drop. Markers for differentiation in β-cells, such as insulin and Pdx1, and genes involved with cell adhesion, such as Mmp9 and E Cadherin, see reduced expression levels upon Myc activation. Many of these changes are indirect targets of Myc, and so changes are not seen for the first day of activation, or during the initial 7 days after inactivation. Methods MycER activated in βcells by daily application of 1mg 4OHT through IP injection for up to 14 days. Reversal data obtained by withholding 4-OHT for 7 days. Total RNA extracted from whole pancreas tissue using Qiagen RNeasy® minikit. RNA integrity confirmed using Agilent 2100 Bioanalyser. RNA hybridised to Affymetrix MOE430 plus 2.0 microarray chips using standard protocols. Expression data was analysed using Genespring® Analysis Platform (Silicon Genetics). Figure 2: Activation of the MycER transgene (by addition of the ligand 4-Hydroxytamoxifen) and subsequent transcriptional activation following dimerization with the protein Max. Conclusions •Activation of Myc leads to differential expression of a large number of genes, particularly those related to cell growth, cell adhesion, and differentiation. •Few genes related to angiogenic growth and cellular proliferation show significant changes in gene expression. •Deactivation of Myc leads to reversal of tumour growth. The changes in gene expression levels indicate that these processes occur through new pathways, as opposed to reversal of those pathways involved in tumour growth. •Results may be compromised by presence of RNA from non-islet cells, leading to non-comparative results. •Microarray studies of laser captured islet tissue will provide improved data on Myc induced gene expression changes allowing further analysis techniques (e.g. Bayesian network analysis, generalised linear modelling, etc.) to be utilised. Acknowledgements References Many thanks to Mike, David and Stella for taking me on for this project, and to Vicky Ifandi, Sylvie Abouna, Göran Mattson and Linda Cheung for their help in the lab. Thanks also to Helen Bird, Sue Davis and Lesley Ward for all their work on the microarray side of things. This project is funded by the Engineering and physical Sciences Research Council and the Association of International Cancer Research through the MOAC Doctoral Training Centre. Mco A 1. C. Nesbit, J. Tersak et al. (1999), “MYC oncogenes and human neoplastic disease”. 2. S. Robson et al. (2006), “c-Myc and downstream targets in the pathogenesis and treatment of cancer”. 3. C. Dang (1999), “c-Myc target genes involved in cell growth, apoptosis, and metabolism”. 4. T. Littlewood et al. (1995), “A modified estrogen receptor ligand-binding domain as an improved switch for the regulation of heterologous proteins”. 5. S. Pelengaris, M. Khan et al. (2002), “Suppression of Myc-induced apoptosis in beta cells exposes multiple oncogenic properties of Myc and triggers carcinogenic progression”.