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Colorectal cancer is
now one of the most common causes of death in Australia, with an estimated 1486
new cases in the country in 2010, accounting 12.7% of all cancer deaths (ACIM,
2014). In addition to its significance in Australia,
it is one of the most common global health concerns. At present colorectal
cancer is the third most common cancer worldwide, which cost more than 600,000
lives every year. Most of the colorectal cancer is
diagnosed at a late stage but if
it is diagnosed at an early stage, the five-year survival rate exceeds in 90% cases. This is the reason there is a need to find out
biomarker for early detection and the exact underlying cause for designing a
better treatment for colorectal cancer.

GAEC1 (Gene amplified in esophageal cancer 1)
showed a series of amplifications and
deletions in oesophageal cancer.  The
gene is located at 7q22.1.  GAEC1
has tumorigenic potential approximately equal to the Ras gene family and overexpression
of this gene played a pivotal role in the cancer transformation of oesophageal
squamous cell carcinoma. GAEC1 has
higher amplification in colorectal adenocarcinoma tissues when compared to
non-cancer colorectal tissues. In this study, we focused on finding out the oncogenic properties of GAEC1, correlation with clinical and
pathological features and its underlying mechanism in colorectal cancer
initiation and progression.

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Materials and method:

Human colon cancer cell lines (SW480, SW48, HCT116 cells) and non-neoplastic
colonic epithelium cell (FHC cells) were
purchased from American Type Culture Collection (ATCC). SW480, SW48 and HCT116 cell lines were maintained in Dulbecco’s Modified Eagle Medium
(DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal
bovine serum at 37 ?
in 5% CO2.  FHC cells were maintained in DMEM: F-12 (1:1) with 10%
fetal bovine serum with containing an extra 10 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) (Thermo Fisher Scientific) (for a final
concentration of 25 mM), 10 ng/ml cholera
toxin, 0.005 mg/ml insulin, 0.005 mg/ml
transferrin, 100 ng/ml hydrocortisone.  Fresh frozen human colorectal cancer tissues
and adjacent non-cancer tissues were collected
with no selection bias. Expression levels of mRNA and protein were measured by real-time PCR and western blot analysis
respectively. Immunocytochemistry, immunohistochemistry and immunofluorescence
assay were used to identify the localization
of GAEC1 protein in colon cancer cells and colon cancer tissues.  Flow cytometry was used for the detection of apoptotic cells and cell cycle
alteration. Co-immunoprecipitation
followed by mass spectrometry analysis was used to identify the protein-protein
interaction. Severe combined immunodeficiency (SCID) mice were used for tumour xenograft experiment.

 

Results:

We found differential
expression of GAEC1 protein and mRNA in different pathological stages of colon
cancer cells (SW480-Stage II, SW48-Stage III and HCT116-Stage IV) when compared
to non-neoplastic colon cells (FHC cells). GAEC1 protein was predominantly expressed in the
cytoplasm of colon cancer cells (SW480, SW48, and HCT116) and the nucleus of
non-neoplastic colon epithelial cells (FHC). The transient knockdown of GAEC1
using siRNA induced apoptosis in SW480 and SW48 cells, which was associated
with G2/M phase arrest and decreased expression of Bcl-2 and K-ras proteins and increased
expression of p53.  In addition, down-regulation of GAEC1 significantly inhibited cell proliferation, reduced migration capacity and
decreased clonogenic potentiality of colon cancer cells (SW480 and SW48
cells).  Furthermore, a
xenotransplantation model showed that stable knockdown of GAEC1 using shRNA constructs in colon cancer cells entirely suppressed xenograft tumour growth in
mice.

Approximately 52.5% of patients with colorectal cancers showed high
expression of GAEC1 mRNA whereas 47.5% exhibited low expression compared to
their matched non-neoplastic tissues. Similarly,
~ 66% (53/80) of colorectal cancer tissues showed high GAEC1 protein expression
(positive staining), while the remaining colorectal cancer cases were noted
with no GAEC1 protein (negative) expression. 
 GAEC1 protein was
predominantly located in the cytoplasm and showed low to no expression in normal colon tissues.

High expression
of GAEC1 mRNA was predominantly seen
among patients below 60 years compared to
those patients over 60 years of age (78%,
versus 44%, p=0.008).  Patients with
synchronous colorectal adenocarcinomas mostly exhibited
with low expression of GAEC1 mRNA.  On the other hand, compared to poorly
differentiated colorectal carcinomas (grade III), patients with well and moderately
differentiated colorectal carcinomas 
(grade I+II) colorectal cancers showed a high expression of GAEC1 mRNA.
Similarly, high GAEC1 mRNA expression was
frequently noted among patients presented without any pre-neoplastic
adenomas in their colorectal cancer tissues compared to patients with an
adenoma in their colorectal cancer tissues.

By co-immunoprecipitation followed by mass
spectrometry analysis 31 interacting protein was
identified. The interaction between GAEC1 and four proteins (HIGD1A,
Rhotekin, Granulin and eIF3J) was further confirmed. Western blot analysis detected reduced expression of these proteins
following stable knockdown of GAEC1 in colon cancer cells.

GEAC1
endogenously interacts with p53 in SW480 and SW48 colon cancer cells. In this study, we have noted that overexpression of GAEC1 increased cell proliferation,
migration, and reduced apoptosis in colon cancer cells.  Also, these cells showed cell cycle arrest at
the synthetic phase, activation of Bcl-2, K-ras, pAKT proteins as well as inhibition
of p53, PUMA, p21 and BAX proteins. Furthermore, silencing of GAEC1 reduces the nuclear import of MDM2 and increase the
expression of p53 in the nucleus suggesting that GAEC1 expression is essential for interaction of p53-MDM2 and nuclear translocation of MDM2 in colon cancer
cells.

 

Conclusion:

In summary, the expression analysis, in vitro
and in vivo data indicated that GAEC1 is differentially expressed in
cancer cells and act as an oncogene in colon cancer progression. The
high expression of GAEC1 mRNA/protein, as well as its correlation with multiple
clinical and pathological characteristics in patients with colorectal
carcinoma, strongly, suggests that GAEC1
is a key regulator in the initiation of
colorectal carcinogenesis. In addition,
the protein-protein interaction with a number of
proteins and the effect of GAEC1 modulation on the expression of interacting
proteins indicates the potential role of GAEC1 in the signalling pathway of
colon cancer pathogenesis.  

 

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