title: Molecular and functional characterization of GAEC1 gene in human colorectal cancer
Creators: name: Wahab, S. M. Riajul
Publisher: name: Griffith University
Year: 2018
Subjects: subject: Molecular characterization
subject: Functional characterization
subject: GAEC1 gene
subject: Colorectal cancer

description: Background: 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. 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.