The expression of cancer stem cell marker, CD133, and its regulatory mechanism in digestive system
Dept. of Medicine/박사
Background: CD133 is a transmembrane glycoprotein that serves as a putative cancer stem cell (CSC) marker in various malignancies. CSCs have been described to have the capacity of progression, metastasis or resistance to chemo-radiotherapy in the malignant tumors. However, the undisputed role of CD133 expression and its regulatory mechanism in tumors of digestive tract is not clear yet. Purpose of the study: To understand the role of CSCs in tumors of digestive system, we evaluated 1) the CD133 expression in various epithelial tumors as well as matched non-neoplastic mucosa of digestive system, 2) the regulatory mechanism of CD133 expression in colorectal cancer, 3) the prognostic significance of CD133 expression in colorectal cancer, and 4) the role of CD133 expression related to chemoresistence in colorectal cancer. Methods: The study includes 480 epithelial tumors of digestive system: 390 adenocarcinomas (271 CRC, 107 gastric (GC), and 12 pancreatic (PC)) and 90 gastroenteropancreatic neuroendocrine tumors (GEP-NETs) (15 gastric, 7 small intestine, 11 colon, 41 rectum, 4 gallbladder, 10 liver, and 2 pancreas) for clinical significance and three CRC cell lines (Caco-2, HT-29, and DLD-1) for in vitro test. Immunohistochemical (IHC) staining for CD133 was performed on the representative paraffin embedded blocks of surgically resected specimens in all 480 cases while real-time RT-PCR was performed on 75 CRC with available fresh frozen tissue. The level of promoter methylation was quantitatively analyzed by pyrosequencing on paraffin-embedded tissue in 171 CRCs. To evaluate the prognostic significance of CD133 expression in CRC and GEP-NETs, clinicopathologic data such as patients’ age, sex, tumor location, differentiation, invasion depth and lymph node metastasis were collected from the pathology reports. Patients who had undergone neo-adjuvant therapy were excluded. Survival data from national cancer registry were used for Kaplan-Meier survival analysis. In in vitro study, CD133 siRNA transfection was performed to Caco-2, HT-29, and DLD-1 cells and we subsequently evaluated the mRNA expression of genes related with chemoresistance (ABCG2 and AKT1), cell proliferation and apoptosis (β-catenin and Survivin) with real-time RT-PCR. Results: In non-neoplastic mucosa of digestive tract, CD133 was expressed on the luminal side of cell membrane in few pyloric type glands in stomach, Brunner’s glands in duodenum, small ducts and centroacinar cells in pancreas, bile ducts in liver and rare cells at crypt base in colon and rectum. Interestingly, CD133 was not expressed in non-neoplastic neuroendocrine cells of digestive tract including pancreatic islets. In neoplastic tissue, the pattern of CD133 expression was mostly luminal membranous and rarely dot-like cytoplasmic in adenocarcinomas in contrast to GEP-NETs that revealed mostly cytoplasmic (diffuse or focal) and rarely luminal expression. CD133 was positive in 35.7% of adenocarcinomas (48% of CRCs, 34% of GCs and 25% of PCs) and 33% of GEP-NETs (30.3% of well-differentiated NET, 26.1% of poorly-differentiated neuroendocrine carcinomas, and 63.6% of mixed adenoneuroendocrine carcinomas). CD133 IHC expression was significantly correlated with CD133 mRNA expression level (P=0.0257). The methylation level of CD133 promoter was inversely correlated with CD133 IHC expression (p<0.0001) and CD133 mRNA expression level (P=0.11). However, no significant correlation between CD133 IHC expression in CRCs and clinicopathologic parameters such as age, sex, tumor histology, stage, invasion depth and lymph node metastasis was found. Similarly, CD133 expression in GEP-NETs was not correlated with tumor grade, site and expression of neuroendocrine markers (chromogranin-A and synaptophysin). Survival analysis in stage II & III CRC patients revealed no significant correlation between CD133 expression and overall survival (OS) (P=0.9689) or disease-free survival (DFS) (P=0.2103). Importantly, CD133+ tumors were significantly associated with longer OS in patients with adjuvant therapy compared to those without adjuvant therapy (p<0.0001, HR 0.125, 95% CI 0.052-0.299). However, patients with CD133- tumors did not show significant difference in survival according to adjuvant therapy (P=0.055, HR 0.500, 95% CI 0.247-1.015). CD133 was also not correlated with OS in neuroendocrine carcinoma patients (P=0.97). In multivariate analysis, CD133 was not an independent prognostic factor in CRC. CD133 was expressed in all three CRC cell lines as evidenced by RT-PCR. The results of qRT-PCR in CRC cell line after CD 133 siRNA transfection indicated that the expression of ABCG2 and AKT1 mRNA was increased while that of β-catenin and Survivin was decreased at 48 and 72 hour. Conclusions: CD133 has a distinctive expression pattern and distribution in adenocarcinomas as well as neuroendocrine neoplasms of digestive system in contrast to its focal or rare expression in non-neoplastic mucosa. CD133 expression seems to be negatively regulated by methylation of its promoter in CRCs. Importantly, this is the first report of CD133 expression in GEP-NETs showing lack of expression in non-neoplastic neuroendocrine cells, indicating an impelling finding that needs further clarification. Although CD133 expression is not an independent prognostic marker, it may prove helpful in predicting the benefit of adjuvant therapy in stage II & III CRCs. This hypothesis was further supported by the in vitro study which showed an increase in the mRNA expression of genes related to chemoresistance (ABCG2 and AKT1) after CD133 knock-down by siRNA transfection in colon cancer cell lines.