Transcript No Slide Title - University of Warwick

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”.