Download Cancer-inducing genes

Cancer-inducing genes
CRGs (cooperation response genes)
Paper Presentation
01.12.2008
Nadine Sündermann
Cancer-inducing genes
• Oncogenes and/or the loss of tumour suppressor genes
generally cooperate with each other in transforming cells
into cancer cells
• A new study identifies a list of genes synergistically
regulated by the Ras oncogene and loss of the p53
tumour suppressor gene
• Many of these genes are essential for tumour formation
in vivo, whereas few of the genes regulated by either Ras
or p53 alone are important
Ras/p53 signalling
Oncogenic mutations in p53 and Ras
•
•
Oncogenic mutations in transcription
factor p53 and in the small GTPase
protein Ras can transform normal cells
into cancer cells
Have individually limited effects on
promoting cancer:
 p53 mutation affects the
expression of group A genes
 Ras mutation modifies the
expression of group B genes
 both p53 and Ras are mutated in the
same cell, they synergistically regulate a
subset of genes (AB) known as
cooperation response genes (CRGs),
which turn out to be crucial mediators
of tumour formation
The mutation spectrum of p53
• The mutation spectrum of p53 in human cancer can help identify
particular carcinogens and define the biochemical mechanisms
responsible for the genetic lesions in DNA that cause human cancer
• Establishment of a new approach to find
genes important in tumorigenesis
• may also represent novel targets for tumour
therapy
The strategy
• Identification of genes regulated synergistically by
cooperating oncogenic mutations at genomic scale,
mRNA expression profiles were used
• Comparison of young adult murine colon (YAMC) cells
with YAMC cells expressing
- mutant p53175H (mp53)
- activated H-RasV12 (Ras)
- both mutant proteins together (mp53/Ras)17
• Method: Affymetrix microarrays and TaqMan low density
array qPCR
Identification and characterization of CRGs
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Raw expression values (log2) of 538
differentially expressed genes (represented
by 657 probe sets) for:
 mp53 cells
 Ras cells
compared to YAMC ctrls
 mp53/Ras cells
rank-ordered according to synergy score
Red and green indicate relative gene
expression in the cells vs. YAMC cells
purple or blue indicate the synergy score for
each gene plotted
synergy score of 0.9 or less defines CRGs.
The cutoff is indicated by arrowheads or the
threshold line
CRG expression in mp53/Ras
cells as compared with YAMC
cells, mp53 cells and Ras cells
- data for 76 CRGs
- Plots indicating fold change in CRG
expression (log2)
CRG analysis
• CRGs encode proteins involved in regulation of cell
signalling, transcription, apoptosis, metabolism,
transport or adhesion
• a large proportion are misexpressed in human cancer
• for 47 out of 75 CRGs tested, co-regulation is found
in primary human colon cancer and our murine colon
cancer cell model
• altered expression of 29 CRGs has been reported in a
variety of human cancer types
Differential expression and synergy scores
of CRGs in mp53/Ras cells
Co-regulation of CRGs
- in mp53/Ras cells and human cancer on the basis of a literature
survey for a variety of human cancers
- two independent expression analyses of primary human colon
cancers
Modulation of CRG expression has common features in
malignant cell transformation of both murine and human cells
Is tumour formation reduced after CRG
perturbations?
• Genetic perturbation experiments were carried out using
retrovirus-mediated reexpression of corresponding complementary
DNAs (cDNAs) for downregulated genes and shRNA-dependent
stable knockdown using multiple independent targets for
overexpressed genes
• Injection of murine and human cancer cells with indicated
perturbations into nude mice
• Measurement of Tumour volume weekly for 4 weeks after injection
• Number of injections (n) and significance levels as compared to
matched controls are indicated
Reversal of the changes in CRG expression
• significantly reduced tumour formation by mp53/Ras
cells in 14 out of 24 cases
 indicates a critical role in malignant transformation for a
surprisingly large fraction of these genes
Synergistic response of downstream genes
to oncogenic mutations
• Distribution of gene perturbations over the set of genes
differentially expressed in mp53/Ras cells, rank-ordered by synergy
score
• Colourcoded bars indicate perturbed genes;
• CRG cutoff synergy score (0.9) is indicated by a horizontal line
 malignant transformation strongly relies on the class of genes
synergistically regulated by cooperating oncogenic mutations
CRG perturbations reduce tumour formation of
both mp53/Ras and human cancer cells
• Plac8 knockdown is functionally rescued by expression of
shRNA-resistant Plac8
CRG perturbations reduce tumour formation of
both mp53/Ras and human cancer cells
CRG perturbations reduce tumour formation of
both mp53/Ras and human cancer cells
CRG perturbation - conclusion
• CRG perturbations reduce tumour formation of both
mp53/Ras and human cancer cells
• Perturbations of CRGs in human cancer cells (Fig. 4b, d,
f,) had similarly strong tumour inhibitory effects to those
in the genetically tractable murine mp53/Ras cells
• genetic perturbations disrupt tumour formation
downstream of the initiating oncogenic mutations
Summary
• efficacy of CRG perturbations performed in human colon
cancer cells comparable to murine colon cell
transformation model  suggesting dependence of the
malignant state on a similar set of genes in both
backgrounds
• human cancer cells carry oncogenic mutations in genes in
addition to Ras or Raf and p53 suggesting that CRGs may
have a large involvement in the generation and
maintenance of the cancer cell phenotype in a variety of
contexts
 CRGs may provide a valuable source for identification by
rational means of the much sought ‘Achilles’ heel’ in human
cancer
Thank you
for your attention!!