Role Of Wnt/β-catenin Signaling Pathway In Nasopharyngeal Carcinoma SP Cells With A "Seed" Characteristic

BackgruoundNasopharyngeal carcinoma is a common highly invasive malignant tumor in the Southeast Asia and South China, which has serious impact on people’s health. In China, its incidence is especially high in Guangzhou. At present, the causes and pathogenesis of nasopharyngeal carcinoma are still not completely clear, but its occurrence is associated with multiple factors, such as heredity, dietary habits, infection of EB virus, and environment. The risk of nasopharyngeal carcinoma is high in males than in females, and the sex ratio of male to female is about 2:1. With the deepening of studies on nasopharyngeal carcinoma, the main treatment methods at present are radiotherapy or comprehensive therapy based on radiotherapy, which have good short-term therapeutic effects on most of nasopharyngeal carcinoma patients. Although intensity-modulated radiotherapy is available at present, which has improved the 5-year survival rate of nasopharyngeal carcinoma to about 65%-70%, but distant metastasis and local recurrence are still main reasons for treatment failure. It is reported that recurrence and metastasis occurred within 5 years after treatment in about 55% of nasopharyngeal carcinoma patients. A large amount of clinical practice suggests that the resistance of nasopharyngeal carcinoma to radiotherapy or chemotherapy is one of the main factors leading to treatment failure. Therefore, in order to improve the 5-year survival rate of nasopharyngeal carcinoma patients, it is a problem demanding prompt solution to explore new drugs or treatment methods. Because targeted drugs have advantages of tumor specificity as well as less toxic and side effects, there are increasing developments and studies on molecular targeted drugs, the key factor of which lies in the difference of targets, so specific tumor markers and signaling pathways should be found.In recent years, the cancer stem cell (CSC) theory holds an opinion that the tumor recurrence, metastasis and resistance to treatment are all associated with the CSC. This theory provides a reasonable explanation for the mechanisms behind treatment failure of nasopharyngeal carcinoma from a new perspective. CSC refers to a cell in tumor tissues, which has self-renewal ability and can produce a series of heterogeneous tumor cells in the tumor. It has the potential of unlimited proliferation and multi-directional differentiation, and it only makes up a small proportion in the tumor cells, but it has the potential of self-renewal and multi-directional differentiation, drug resistance and colony formation ability, as well as tumor formation ability, and thus is associated with tumor metastasis and recurrence. CSC and normal stem cells have similar self-renewal mechanism, which is achieved by asymmetric division:the division of a CSC forms a new CSC and another daughter cell which can ultimately differentiate into various cells including tumor cells, and the result of such division is to maintain the stability of CSC number and form tumor. How to study the characteristics of pharyngeal carcinoma CSC such as specific surface markers and signaling pathways influencing CSC on the basis of traditional therapy is important in developing new drugs and treatment methods to kill CSC or CSC-like cells of nasopharyngeal carcinoma, so as to eradicate the basis of tumor recurrence at the source, which has great significance on improving the 5-year survival rate of nasopharyngeal carcinoma patients. Studies have shown that the side population cells (SP cells) can be isolated from nasopharyngeal carcinoma cell lines, which are a special cell subset, found by DNA fluorescent dye Hoechst 33342 and flow cytometry during the isolation of bone marrow hematopoietic stem cells, with a series of biological characteristics of stem cells such as self-renewal and multi-directional differentiation. SP cells are considered as a kind of more original stem cells, or they are greatly correlated with stem cells, which can be reflected in the biological behavior characteristics of SP cells such as self-renewal, unlimited proliferation and strong tumorigenicity. SP phenotype is a common mark of stem cells from various sources, and thus the SP cells have many common characteristics with CSC. They can enrich CSC and also have resistance to radiotherapy and chemotherapy. Up to now, studies on the SP cells of nasopharyngeal carcinoma are rare. The present study sorted SP cells from nasopharyngeal carcinoma cells and investigated the importance of their "stem" mechanism. Because nasopharyngeal carcinoma is mostly undifferentiated squamous cell carcinoma, we selected poorly differentiated CNE-2 cell line as the main subject of the present study.Wnt/β-catenin signaling pathway not only exists in normal stem cells to play important roles in the maintenance of self-renewal, differentiation inhibition, proliferation, migration and apoptosis of cells, but may also play a key role in the control of immature CSC. In the CSC of many tumors, Wnt/β-catenin signaling pathway has been found to have a role of mediating drug resistance and tumor reccurrence. Moreover, it is now generally considered that the maintenance of CSC’s "stem property" relies on Wnt/β-catenin signaling pathway. The key factor for the abnormal change of Wnt/β-catenin signaling pathway in tumor cells is that the disordered degradation of P-catenin leads to the aggregation of free β-catenin in the cytoplasm, which then combines with TCF/LEF and enters the nucleus to initiate the transcription of downstream genes. Abnormal p-catenin is a common event in nasopharyngeal carcinoma, and studies have shown that β-catenin plays a crucial rule in maintaining the CSC property of nasopharyngeal carcinoma. Based on previous studies, we have found that the Wnt/β-catenin signaling pathway is of great importance in the proliferation and differentiation of SP cells, but the action mechanisms of Wnt/β-catenin signaling pathway in SP cells of nasopharyngeal carcinoma are rarely reported.The metastasis of malignant tumors mainly involves three routes:implantation metastasis, lymphatic metastasis and hematogenous metastasis, of which lymphatic metastasis and hematogenous metastasis are main routes of metastasis. Metastasis is a very complex process, with multiple stages but also continuity. The metastasis of tumor cells involves the following steps:the invasion and growth of tumor cells, the adhesion of tumor cells to basement membrane and the erosion of basement membrane, the penetration of tumor cells through endothelial cells to enter blood circulation, the attachment of tumor cells to the microvessels of target organ, finally the outward penetration of tumor cells through endothelial cells and the formation of metastasis in target tissues. In the process of invasion and metastasis, the adhesion of tumor cells to vascular endothelial cells is one of the decisive steps for metastasis, which requires the activation and expression of multiple genes. Studies have shown that the glycoprotein formed by CD44 gene expression participates in the specific adhesion between tumor cells and basement membrane as well as among different cells, and it is closely associated with the growth, invasion and metastasis of tumors. It is one of the specific surface markers on CSC, and has been well documented as a specific surface marker on CSC in breast cancer, pancreatic cancer, intestinal cancer, and head and neck squamous cell carcinoma. Particularly, the nasopharyngeal carcinoma has the same pathological type and close location with other squamous cell carcinomas on the head and neck, so we have reason to speculate that CD44 may be also a specific surface marker for nasopharyngeal carcinoma. Because CD44 is a target gene at the downstream of Wnt/β-catenin signaling pathway, and the maintenance of CSC’s "stem property" is now considered to mainly rely on the Wnt/β-catenin signaling pathway, investigating how the changes in the expression may alter the number and biological characteristics, especially the expression of downstream target gene CD44 and the characteristics of CD44+ SP cells, will be of great importance in finding the CSC of nasopharyngeal carcinoma and inhibiting the abnormality of Wnt/β-catenin signaling pathway to change the characteristics of CSC or kill the CSC, so that a premise will be provided to the creation of new drugs or treatment methods for nasopharyngeal carcinoma. Studies on the interference of Wnt/β-catenin signaling pathway to change or kill the CSC are rare. Generally, it is considered that small molecular inhibitors (siRNA) or biological inhibitors can be used to block the abnormality of Wnt/p-catenin signaling pathway, so as to change the characteristics of CSC and achieve the purpose of treatment. At present, no reports related to the influence of Wnt/β-catenin signaling pathway interference on the SP cells of nasopharyngeal carcinoma are available. Therefore, the present study sorted SP cells from nasopharyngeal carcinoma CNE-2 cells and then sorted CD44+ cells from SP cells to investigate the Wnt/β-catenin signaling pathway in SP cells, the expression of its downstream target gene CD44 and the in vitro biological characteristics of CD44+ SP cells, so as to learn about the mechanism of Wnt/β-catenin signaling pathway in maintaining the "stem" property of SP cells in nasopharyngeal carcinoma, which will be of great significance in developing new drugs and treatment methods to kill nasopharyngeal carcinoma CSC and CSC-like cells and finally eradicate the recurrence of nasopharyngeal carcinoma at the source.The main content of the present study was as follows:(1) sorting SP cells from poorly differentiated human nasopharyngeal carcinoma cell line (CNE-2) for culture; (2) sorting CD44+SP cells from CNE-2 SP cells for culture; (3) detecting the expression of CD44 in the SP cells and NSP cells of nasopharyngeal carcinoma; (4) testing the different biological behaviors of CNE-2 cells, CD44+SP cells and CD44-SP cells, including experiments such as cell growth rate calculation, migration assay, plate colony formation assay, cell cycle analysis and chemotherapeutic drug sensitivity test of cells cultured in vitro; (5) blocking the Wnt/β-catenin signaling pathway through transfection with P-catenin siRNA, detecting the number of CNE-2 cells and CD44+SP cells in the cell line transfected with β-catenin siRNA, and observing the biological behaviors of these cells, including experiments such as cell growth rate calculation, migration assay, plate colony formation assay, cell cycle analysis and chemotherapeutic drug sensitivity test of cells cultured in vitro.Chaper I Expression and its significance of CD44 in SP cells of nasopharyngeal carcinomaObjectiveDiscussion of expression and its biological significance of CD44 in SP cells of nasopharyngeal carcinoma.MethodsFlow cytometry was used to sort cultured CNE-2 cells of nasopharyngeal carcinoma for obtaining CD44-SP and CD44+SP cells. Biological differences of CNE-2, CNE-2 SP, CNE-2 NSP, CNE-2 CD44+SP and CNE-2 CD44-SP cells were statistically analyzed by experiments such as cell migration experiments, plate clone formation assay, cell cycle analysis and sensitivity tests to chemotherapeutics.Results2.3% of SP cells were extracted from CNE-2 cells of nasopharyngeal carcinoma, among which 36.5% was CD44+SP cells. Abilities of proliferation, cell migration and plate clone of CD44+SP cells were significantly higher than other cells (P<0.05), and its tolerance to chemotherapeutics was significantly higher too (P<0.05).ConclusionThe proportion of SP cells in nasopharyngeal carcinoma cells was small, but SP cells had strong activeness in the aspect of cell proliferation with a "seed" characteristic of tumor cells. As CD44+SP cells played an important role in proliferation and chemotherapy resistance of nasopharyngeal carcinoma, it indicated that CD44 may be can be one of the surface markers of SP cells of nasopharyngeal carcinoma.Chapter II The Function and Meaning of Wnt/p-catenin Signaling Pathway by controling Tumor marker CD44 in Nasopharyngeal Carcinoma SP CellsObjectiveThe Wnt/β-catenin signaling pathway is abnormally expressed in various types of tumors, CD44 is also the pathway downstream target gene, and has been shown to be one of the specific surface markers of various malignant stem cells. This study aims to explore the molecular mechanism and expression of Wnt/β-catenin signaling pathway and tumor marker CD44 in nasopharyngeal carcinoma cells after transfection with β-Catenin when the Wnt/β-catenin signaling pathway was blocked.MethodsSP cells and CD44+SP cells isolated from the nasopharyngeal carcinoma cell line CNE-2 were cultured and identified. Changes in the number and biological characteristics of CNE-2 and CD44+SP cells in vitro were investigated after the Wnt/β-catenin signaling pathway was blocked.ResultsSP cells accounted for 2.3% of nasopharyngeal carcinoma CNE-2 cells, and CD44+SP cells accounted for 36.5% of the SP cells. CD44+SP cells showed significantly higher in vitro proliferation and resistance to chemotherapy transfection with β-Catenin siRNA, for proliferation, cloning efficiency, and tolerance to chemotherapeutic drugs,CNE-2 cells and CD44+SPcellstransfected withβ-catenin had significantly lower proliferation and cloning efficiency,had significantly higher toleranceto chemotherapeutic drugs,and statistical differences were found in all three characteristics (P<0.05)ConclusionWnt/β-catenin signaling pathway abnormalities are closely related to the biological significance of nasopharyngeal carcinoma CD44+SP cells. Interference of this pathway can change the characteristics of nasopharyngeal carcinoma stem cells.