Biological Characteristics And Molecular Basis Of Cancer Stem Cell Subpopulations In Human Gastric Cancer

Gastric cancer (GC) is the second leading cause of cancer related mortality, despite the development of better diagnostic tools, including endoscopy the five year survival rate remains low. The 5-year survival rate in gastric cancer patients is still less than 30%, and this low survival rate is mainly due to relapse, metastasis and drug resistance. Therefore, it is important to identify highly specific therapeutic targets to improve the curative effect of gastric cancer and even radical cure effect. It opens up a new way to study the mechanism of the occurrence and development of gastric cancer.Recently, emerging of cancer stem cells (CSCs) hypothesis provides us a new way insight into the mechanism underlining gastric cancer initiation and progression. To demonstrate the existence of gastric cancer stem cells (GCSCs) and explore their development, invasion, metastasis and recurrence, the study to find molecular markers and identify of GCSCs marker and the regulation mechanism of related molecules.In the present study, we established two methods of serum-free spheroid formation and fluorescence-activated cell sorting (FACS) to isolate and enrich GCSCs from gastric cancer cell lines, real-time quantitative PCR showed the high expression of stemness related genes Sox 2, Oct-4 and Nanog in the positive cells of CD44 and CD90 in SNU-5 and SNU-16 compared to the negative cells. Serum-free spheroid formation showed higher spheroid formation ability in the positive cells of CD44 and CD90 in SNU-5 and SNU-16 compared to the negative cells; Xenograft assay showed the positive cells of CD44 and CD90 in SNU-5 and SNU-16 were highly tumorigenic. Those data suggesting that the positive cells of CD44 and CD90 in SNU-5 and SNU-16 were possess stem cell properties.Invasive ability of the positive cells of CD44 and CD90 in gastric cancer cell lines SNU-5 and SNU-16 were measured by transwell invasion assay, and we found that the positive cells of CD44 with higher invasive ability in vitro compared to the positive cells of CD90; CCK8 assay showed that the positive cells of CD90 were higher proliferation in vitro compared to the positive cells of CD44, we found xenograft tumors formed by the positive cells of CD44 in gastric cancer cell lines SNU-5 and SNU-16 invaded into the capsule and metastasized to lymph nodes. In contrast, there were compact capsules surrounding the positive cells of CD90 in gastric cancer cell lines SNU-5 and SNU-16 without any invasion or metastasis. We further studied the expression of CD44 related to epithelial-mesenchymal transition (EMT) and the expression of CD90 in relation to the cell cycle progression. The presence of CD44 cells is associated with metastasis, whereas the presence of CD90 cells is associated with high tumorigenic capacity. A series of genes differential expression between CD44 and CD90 cells from gastric cancer stem cell SNU-5 were screened by gene microarray. Among the differential expression molecular, ENO1 was particularly over-expressed in GCSCs (CD44). Then, roles of ENO1 in gastric cancer sternness, invasion, metastasis and its relationship with clinicopathological features were explored.High ENO1 expression and low ENO1 expression was found in SNU-5 and BGC-823 by Western blot; ENO1 was co-localized with stem cell marker CD44 in SNU-5. Silenced ENO1 expression in GCSCs (CD44) by shRNA, the ability of spheroid formation, invasion and metastasis was impaired; Over-expressing ENO1 in GCSCs (CD44) from BGC-823, the spheroid formation, invasion and metastasis of GCSCs was increased. In addition, the function anti-ENO1 monoclonal antibody of 21 A3 in inhibiting CD44 positive cells in vitro showed that 21 A3 functional antibody could significantly inhibit the spheroid formation and invasion ability of CD44 positive cells in SNU-5. ENO1 plays important roles in gastric cancer sternness maintenance, invasion and metastasis. Gene array analysis showed that ENO1 expression mainly resulted in change of PI3K/Akt signaling pathway. Silencing ENO1 expression from GCSCs (CD44) from SNU-5 showed vimentin, cyclin D1 and C-myc was down-regulated, meanwhile, the expression of E-cadherin and p21 was up-regulated, which resulted in decreased phosphorylation of PI3K and Akt, accompanied by muPA and mENO1 down-regulated expression. Over-expressing ENO1 in GCSCs (CD44) from BGC-823, demonstrated that increased phosphorylation of PI3K and Akt, accompanied by muPA and mENO1 up-regulated expression, indicating that the function of ENO1 may be dependent on the PI3K/Akt signaling pathway.In summary, Serum-free spheroid formation and FACS is an effective method for isolation/enrichment of GCSCs. GCSCs (CD44 and CD90) possesses highly invasive, metastasis and tumorigenicity properties. Multi-signaling pathways, such as PI3K/Akt, Wnt, TGF-beta and anti-apoptosis signaling pathway, may be involved in regulation of the GCSCs (CD44) behaviors; Our results also demonstrated that ENO1 play important roles in stemness maintenance, invasion and metastasis of GCSCs (CD44). Expression of ENO1 is positively correlated with clinic pathologicparameters and negatively correlated with survival time, indicating that it can be used as anindicator of poor prognosis of patients with gastric cancer.