| Colorectal carcinoma is one of the most common cancers whose incidence increased rapidly in China. It is the fourth leading cause of cancer deaths, with a 5-year survival about 50%-60%. The malignant behaviors including invasion and metastasis are the major reasons of treatment failure in colorectal carcinoma. Recently, the mechanisms of invasion, metastasis and recurrence in colorectal carcinoma are still uncertain. A better understanding of the factors involved in the initiation, progression and metastasis of colon cancer is base of developing novel and more efficient diagnostic and therapeutic strategies. It plays an important role in increasing the survival of colorectal carcinoma patients.With the development of stem cell and cancer biology research, people found a small population of cells that have the ability of self-renewing and giving rise to a new tumor, termed Cancer stem cell (CSC). CSCs have been isolated from several types of solid tumor, including breast cancer, colorectal cancer, prostate cancer, lung cancer, brain tumor and pancreatic cancer. CSCs play an important role in carcinogenesis, resistance to chemotherapy and may lead to cancer recurrence and metastasis. Research on the malignant behavior and deregulated signaling pathways of CSCs is helpful to understand the mechanism of carcinogenesis and to find the new therapy target. There is little report about the molecular mechanism of CSC involved in these events. Our study cultured primary cells derived from liver metastasis of colon cancer patient and isolated CD 133+cells using Magnetic cell sorting. We compared the colony formation ability in vitro and the tumor initiating capacity in vivo of the two types of cells. CD133+colon cancer cells showed a higher ability of colony formation in soft agar than CD 133-cell. When cultured in the serum free medium, there were more colon cancer spheres that generated from CD133+cells than CD133-cells. However, the growth curves of the two types of cells are similar when cultured in complete medium. Furthermore, tumorigenicity assay was performed by injecting both the CD 133+and CD 133-cells into NOD/SCID mice. The CD 133+cells showed significantly higher tumorigenicity than CD 133-cells. All these results indicated that there are caner stem cells in CD 133+subpopulation in colon cancer and CD 133 is an effective surface marker to enrich colon cancer stem cells.Therefore, a search was undertaken for genes that were differentially expressed between the two cell types by using cDNA GeneChip analysis. In total,321 genes were upregulated and 65 genes were downregulated in CD 133+colon cancer cells by>2 fold change. The expression of 11 representative genes was examined by real-time PCR to verify the results of the cDNA GeneChip analysis. Pathway analysis showed the changed genes mainly distributed with PI3K/Akt, Notch, JAK/STAT, MAPK and TGF-βpathways.PI3K/Akt and MAPK signaling pathways were dysregulation in many types of cancers. They played an important role in the cell proliferation, differentiation, and apoptosis, and the activation of their components have closely related to the invasion and metastasis of tumor. We tested the kinase activities of Akt, Erkl/2, p38, and JNK kinase of the two cell fractions using Akt and MAPK kinases assay. Different activities of Akt and Erk1/2 were found between CD133+and C133-cells. Akt was significantly activated in CD133+cell and Erkl/2 activity was up-regulated by 2.38 fold. These results suggested the Akt and MAPK signaling pathways may be important for the CD133+cell. Next, we examined whether the activation of Erkl/2 and Akt was required for the colony fomation of CD133+cell in soft agar. Inhibiting the Akt and Erk1/2 using special pathway inhibitors, the colonies formed by CD 133+cells decreased significantly. Furthermore, we observed that CD133+cells transduced with Akt and Erk shRNA also showed reduction in colony formation. The activation of PI3K/Akt and MAPK may contribute to the tumorigicity of CD 133+colon cancer cell. Moreover, we found the migration ability of Akt knockdown CD 133+cell showed significantly decreased comparing with the control.We proved that the activation of Akt and MAPK pathways were vital for the colon CSCs'tumourigenicity. Inhibitors of Akt and MAPK can reduce the colony formation ability of the colon CSCs, which might provide a target for inhibiting CSC in the treatment of cancer. Further work is necessary to investigate the mechanism of Akt and Erkl/2 activation in the biological events of CSCs. Combination therapies that apply CSCs targeting agents together with those that kill non-CSC populations need to be developed. |
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