| Summary: | 博士 === 國立臺灣大學 === 臨床醫學研究所 === 104 === Background and objective
Colorectal cancer is one of the most common and deadliest malignant disease worldwide. Although current treatment modalities have dramatically improved the survival of patients with stage IV colorectal cancer, colorectal cancers that spread to distant organs are usually not curable. This problem raises questions as to whether current systemic anti-colorectal cancer treatments are targeting molecular mechanisms that are truly critical to cancer growth and metastasis.
Mucin-type O-glycosylation is one of the most common post-translational modifications of proteins and is associated with many important biological functions. Aberrant glycosylation of cell surface molecules is associated with malignant transformation. Tumor-associated Tn and T antigens are oncofetal carbohydrate antigens and present in colon, breast, prostate, and ovarian cancer. They are promising targets for cancer diagnosis, imaging, and therapeutics. However, the glycogenes responsible for regulating Tn and T antigens expression and their biological functions remain largely unknown.
This research is significant in that: (1) it contributes to the understanding of altered glycogene expression in colorectal cancer and the association between their expression level and the patient prognosis; (2) it provides the knowledge about what and how the cancer cell behaviors are regulated by glycogenes; and (3) the gained information will allow us to develop diagnostic and therapeutic reagents for human cancers.
Materials and methods
1. To identify novel glycogenes which can regulate Tn and T antigen expression in colorectal cancer cells, the differential expression of glycogenes in normal colorectal and cancerous tissues from colorectal cancer patients were analyzed by real-time RT-PCR. After the glycogenes with altered expression in colorectal cancer were found, the relation of expression level and the patient prognosis were examined. The glycogenes which have impact on patient’s survival were cloned.
2. To confirm whether these glycogenes can regulate Tn or T antigen expression, colon cancer cell lines were transfected with these genes. Then Tn and T antigen expression on cell surfaces was analyzed by flow cytometry. In addition, we cloned these genes and made their recombinant proteins as well as generated their polyclonal and monoclonal antibodies for subsequent experiments.
3. To investigate the function of these glycogenes in vitro, overexpression and siRNA knockdown techniques were used. Cell-based assays, including cell proliferation, apoptosis, adhesion, migration, invasion, and colony formation assays were performed. Animal study was also conducted to reveal the effect of the glycogenes on cancer metastasis in vivo.
Results
1. Paired tissues from patients of the National Taiwan University Hospital (NTUH) were analyzed by Western blotting. C1GALT1 expression was found to be overexpressed in colorectal cancer tissues compared with adjacent non-tumor tissues. The results from immunohistochemistry of C1GALT1 indicated that 67.8% (59/87) of colorectal tumors showed higher C1GALT1 expression than their normal counterpart tissues. Moreover, a Kaplan-Meier survival analysis showed that the cumulative survival rate of colorectal cancer patients with C1GALT1 overexpression (T > N) was significantly lower than the patients without C1GALT1 overexpression (T ≤ N). In addition, we also observed that C1GALT1 expression is positively associated with T antigen expression as revealed by PNA staining. These findings suggest that C1GALT1 expression is frequently overexpressed in colorectal tumors and its overexpression is associated with poor survival.
2. Overexpression of C1GALT1 slightly increased cell viability in HCT116 and SW480 cells, whereas knockdown of C1GALT1 slightly inhibited cell viability in HCT116 and SW620 cells. We also analyzed migration and invasion by transwell and matrigel invasion assay, respectively. Results showed that overexpression of C1GALT1 significantly enhanced cell migration and invasion in HCT116 and SW480 cells. In contrast, knockdown of C1GALT1 suppressed cell migration and invasion in HCT116 and SW620 cells. These findings suggest that C1GALT1 can regulate malignant behaviors of colon cancer cells in vitro. We then conducted animal experiment and observed that overexpression of C1GALT1 increased lung metastasis of SW480 cells, whereas knockdown of C1GALT1 suppressed lung metastasis of SW620 cells. These results suggest that C1GALT1 expression is able to modulate tumor growth and metastasis in vivo.
3. We found that C1GALT1 can regulate bFGF-induced malignant phenotypes. Since the significance of FGFR2 in colorectal cancer has been clearly demonstrated, we investigated whether C1GALT1 can regulate FGFR2 glycosylation and activity. Only low amounts of endogenous FGFR2 were pulled down by PNA in HCT116, SW480, and SW620 cells, indicating small amounts of T antigens on FGFR2. Interestingly, after neuraminidase treatment, FGFR2 was easily pulled down by PNA, suggesting that FGFR2 carries sialyl T antigens. Moreover, FGFR2 could also be pulled down by VVA, indicating the presence of Tn on FGFR2. Removal of sialic acids with neuraminidase increased VVA binding to FGFR2, indicating the presence of sialyl Tn on FGFR2. These results strongly suggest that FGFR2 is decorated with O-glycans in colon cancer cells.
We next investigated whether C1GALT1 can modify O-glycans on FGFR2. Overexpression of C1GALT1 decreased VVA binding to FGFR2 in HCT116 and SW480 cells, whereas knockdown of C1GALT1 increased VVA binding to FGFR2 in HCT116 and SW620 cells. These results suggest that C1GALT1 is able to modulate O-glycan structures on FGFR2 in colon cancer cells. Furthermore, overexpression of C1GALT1 increased bFGF-induced phosphorylation of FGFR2 and ERK1/2 in HCT116 and SW480 cells. Conversely, knockdown of C1GALT1 suppressed the phosphorylation of FGFR2 and ERK1/2 after bFGF treatment in HCT116 and SW620 cells. These findings suggest that C1GALT1 modifies O-glycans on FGFR2 and regulates bFGF-induced activation of FGFR2 in colon cancer cells.
Conclusion
We found that C1GALT1 is overexpressed in colorectal tumors and its overexpression is associated with poor survival of patients with colorectal tumors. C1GALT1 overexpression enhances the invasive potential and stem-like cell property of colon cancer cells via modifying O-glycosylation and activity of FGFR2. Conversely, C1GALT1 knockdown suppresses these malignant properties in vitro and in vivo. These findings open novel insights into the relevant role of O-glycosylation in colorectal cancer and suggest C1GALT1 as a promising therapeutic target for the treatment of colorectal cancer.
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