Investigation into Early growth response 1 in colorectal disease: a study of EGR1 expression in colorectal tissue and novel protein interactions in cancer cells
View/ Open
Date
30/11/2012Author
Gernon, Grainne Mary
Metadata
Abstract
Introduction: Early growth response 1 (EGR1) is a zinc-finger transcription factor
involved in the regulation of cell growth. It can act as either a tumour suppressor or a
tumour promoter with a role in the induction of apoptosis in cancer cells by various
pathways and is likely to play a role in colorectal cancer (CRC). EGR1 also appears
to play a significant role in inflammatory pathways, therefore a possible role in
Inflammatory Bowel Disease (IBD) is hypothesised. Patients with IBD have a
greater risk of developing CRC, which is increased with duration of symptoms and
severity of inflammation and dysplasia. The aim of this study is to determine whether
EGR1 is differentially expressed in diseased colon tissue and to investigate novel
EGR1-protein interactions in CRC cell lines.
Methods: The relative EGR1 expression in CRC cell lines and in normal mucosa and
tumours of colorectal cancer patients was determined by qRT-PCR. IBD patient
samples were also examined for differential EGR1 expression levels by qRT-PCR,
before and after stimulation with inflammatory mediators. Statistical analysis of the
data was performed using ‘R’ statistical package, with the mixed-model ANOVA.
Statistical significance was set at < 0.05. The genotype of three EGR1 variants was
determined in the samples using PCR and sequencing, and the methylation status of
regions of the EGR1 promoter was determined using bisulfite sequencing. A yeasttwo
hybrid screen was conducted with EGR1 as bait, and screened against a SW480
CRC cell line library. Interesting novel interactions were investigated using
immunocytochemistry and immunoprecipitation, as was the novel interaction
between EGR1 and NOD2 and between EGR1 and components of the cytoskeleton.
Results: Investigation into the relative EGR1 mRNA expression in CRC has shown
that there is differential expression of EGR1 between matched normal mucosa and
tumour. EGR1 expression is decreased in IBD patients compared with healthy
controls. Induction of EGR1 by inflammatory stimuli also appears to be aberrant in
these patients. The differential expression of EGR1 was not associated with aberrant
methylation of a large region of the EGR1 promoter in either the CRC or IBD
patients or with the genotype of EGR1 variants. EGR1 localises to both the
cytoplasm and the nucleus in CRC cell lines and this study demonstrate interactions
with the IBD susceptibility protein NOD2 and with components of the cyotskeleton.
A yeast-two hybrid screen conducted with EGR1 as bait using a CRC cell line library
has identified several other novel protein interactions of EGR1 in CRC cell lines.
Conclusion: EGR1 is differentially expressed in both CRC and IBD, and in the case
of IBD shows aberrant activity, suggesting that EGR1 may play a role in both
colorectal diseases. EGR1 interacts with the IBD protein NOD2, and components of
the cytoskeleton in CRC cells. Several novel protein interactions with EGR1 have
been identified and warrant further study.