Colorectal cancer is now one of the
Colorectal cancer isnow one of the most common causes of death in Australia, with an estimated 1486new 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 colorectalcancer is the third most common cancer worldwide, which cost more than 600,000lives every year. Most of the colorectal cancer isdiagnosed at a late stage but ifit 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 outbiomarker for early detection and the exact underlying cause for designing abetter treatment for colorectal cancer.GAEC1 (Gene amplified in esophageal cancer 1)showed a series of amplifications anddeletions in oesophageal cancer.
Thegene is located at 7q22.1. GAEC1has tumorigenic potential approximately equal to the Ras gene family and overexpressionof this gene played a pivotal role in the cancer transformation of oesophagealsquamous cell carcinoma. GAEC1 hashigher amplification in colorectal adenocarcinoma tissues when compared tonon-cancer colorectal tissues. In this study, we focused on finding out the oncogenic properties of GAEC1, correlation with clinical andpathological features and its underlying mechanism in colorectal cancerinitiation and progression. Materials and method:Human colon cancer cell lines (SW480, SW48, HCT116 cells) and non-neoplasticcolonic epithelium cell (FHC cells) werepurchased 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% fetalbovine 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 finalconcentration of 25 mM), 10 ng/ml choleratoxin, 0.
005 mg/ml insulin, 0.005 mg/mltransferrin, 100 ng/ml hydrocortisone. Fresh frozen human colorectal cancer tissuesand adjacent non-cancer tissues were collectedwith no selection bias. Expression levels of mRNA and protein were measured by real-time PCR and western blot analysisrespectively. Immunocytochemistry, immunohistochemistry and immunofluorescenceassay were used to identify the localizationof GAEC1 protein in colon cancer cells and colon cancer tissues. Flow cytometry was used for the detection of apoptotic cells and cell cyclealteration.
Co-immunoprecipitationfollowed by mass spectrometry analysis was used to identify the protein-proteininteraction. Severe combined immunodeficiency (SCID) mice were used for tumour xenograft experiment. Results:We found differentialexpression of GAEC1 protein and mRNA in different pathological stages of coloncancer cells (SW480-Stage II, SW48-Stage III and HCT116-Stage IV) when comparedto non-neoplastic colon cells (FHC cells). GAEC1 protein was predominantly expressed in thecytoplasm of colon cancer cells (SW480, SW48, and HCT116) and the nucleus ofnon-neoplastic colon epithelial cells (FHC). The transient knockdown of GAEC1using siRNA induced apoptosis in SW480 and SW48 cells, which was associatedwith G2/M phase arrest and decreased expression of Bcl-2 and K-ras proteins and increasedexpression of p53. In addition, down-regulation of GAEC1 significantly inhibited cell proliferation, reduced migration capacity anddecreased clonogenic potentiality of colon cancer cells (SW480 and SW48cells). Furthermore, axenotransplantation model showed that stable knockdown of GAEC1 using shRNA constructs in colon cancer cells entirely suppressed xenograft tumour growth inmice.
Approximately 52.5% of patients with colorectal cancers showed highexpression of GAEC1 mRNA whereas 47.5% exhibited low expression compared totheir 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 notedwith no GAEC1 protein (negative) expression. GAEC1 protein waspredominantly located in the cytoplasm and showed low to no expression in normal colon tissues.High expressionof GAEC1 mRNA was predominantly seenamong patients below 60 years compared tothose patients over 60 years of age (78%,versus 44%, p=0.008). Patients withsynchronous colorectal adenocarcinomas mostly exhibitedwith low expression of GAEC1 mRNA.
On the other hand, compared to poorlydifferentiated colorectal carcinomas (grade III), patients with well and moderatelydifferentiated colorectal carcinomas (grade I+II) colorectal cancers showed a high expression of GAEC1 mRNA.Similarly, high GAEC1 mRNA expression wasfrequently noted among patients presented without any pre-neoplasticadenomas in their colorectal cancer tissues compared to patients with anadenoma in their colorectal cancer tissues. By co-immunoprecipitation followed by massspectrometry analysis 31 interacting protein wasidentified. The interaction between GAEC1 and four proteins (HIGD1A,Rhotekin, Granulin and eIF3J) was further confirmed. Western blot analysis detected reduced expression of these proteinsfollowing stable knockdown of GAEC1 in colon cancer cells.GEAC1endogenously 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 atthe synthetic phase, activation of Bcl-2, K-ras, pAKT proteins as well as inhibitionof p53, PUMA, p21 and BAX proteins. Furthermore, silencing of GAEC1 reduces the nuclear import of MDM2 and increase theexpression of p53 in the nucleus suggesting that GAEC1 expression is essential for interaction of p53-MDM2 and nuclear translocation of MDM2 in colon cancercells. Conclusion:In summary, the expression analysis, in vitroand in vivo data indicated that GAEC1 is differentially expressed incancer cells and act as an oncogene in colon cancer progression. Thehigh expression of GAEC1 mRNA/protein, as well as its correlation with multipleclinical and pathological characteristics in patients with colorectalcarcinoma, strongly, suggests that GAEC1is a key regulator in the initiation ofcolorectal carcinogenesis. In addition,the protein-protein interaction with a number ofproteins and the effect of GAEC1 modulation on the expression of interactingproteins indicates the potential role of GAEC1 in the signalling pathway ofcolon cancer pathogenesis.