| Colorectal cancer is one of the most frequent occurring cancers and the forth most common cause of cancer deaths in the world. Recurrence and metastasis are common complications after conventional treatments. A subset of cancerous cells known as cancer stem cells(CSCs), characterized by the potential of self-renewal, multiple differentiation, unlimited proliferation, metastasis and chemo-resistance, are the key factor in tumor initiation, progression, recurrence and metastasis. Therefore, studying on the regulation mechanism of tumor stem cells contribute to the design of therapeutic strategies against cancer stem cells.CSCs are located in a niche made up of fibroblasts, immunocytes, endotheliocyte and gliocytes. Genetic mutations of CSCs mediated by this microenvironment is a key component in tumor progression. Inflammatory cytokines within this microenvironment can regulate CSCs through activation of related pathways such as Notch, Hedgehog, STAT3 and NF-κB. Similarly, CSCs can also adjust their niche by secreting cytokines. How CSCs react to inflammatory signals of tumor microenvironment and how CSCs affect tumor microenvironment by autocrine and paracrine is the research direction of our topic.Rab proteins Play an important role in the secretion of cells. Rab proteins are small GTPases belonging to the Ras superfamily and are mainly involved in intracellular vesicle transport. Rab27 is the key protein for intracellular secretion, and contains two isoforms: Rab27 A and Rab27 B. Dysfunction of Rab27 A can lead to disorders such as Griscelli syndrome(GS) and impaired glucose tolerance. Overexpression of Rab27 A promotes growth and metastasis of breast cancer and melanoma. Rab27 A helps provide a suitable microenvironment for cancer progression and metastasis in an exosome-dependent or exosome-independent manner. Our study focuses on the effect of Rab27 A on CSCs and the relationship between Rab27 A and NF-κB signaling pathway.The purpose of our research is to answer whether the Rab27 A affect the function of colon cancer stem cells? Whether Its mechanism is related with the secretion of cytokines?Whether the inflammatory signal NF- κ B affect the expression of Rab27A? 【Aims】1) To determine if correlations exist among Rab27 A, tumor malignant degree,and stemness of CSCs by using clinical samples, cell lines of colon cancers, and sphere culture model of tumor stem cell.2) Whether intervention of Rab27 A affect tumor cell stemness and proliferation?3) Based on the above function changes, to answer whether the Rab27 A effect of CSCs function is related with cytokine secretion.4) Finally, to investigate whether inflammatory microenvironment of CSCs are involved in affecting the expression level of Rab27 A. 【Methods and Results】1) A positive staining rate of 66.7% for Rab27 A was found in clinical samples of colon cancer, through immunohistochemical staining. In addition, we observed the in vitro growth of these six cell lines in tumor sphere culture medium, but with varying sphere shapes. Flow cytometry showed significant difference in the presences of CD44+ cells in these six cell lines. Results from RT-PCR and Western Blot showed that the expression of Rab27 A was significantly different in the six colon cancer cell lines with varying degrees of malignancy.2) Flow cytometry showed a higher percentage of CD44+ and PKH26 high in HT29 sphere compared to normal cancer cells. In addition, we perform HT29 sphere serial passages. RT-PCR indicated that the expression of CSCs markers were higher in passage spheres than normal HT29 cells, while the expression of differentiation markers were lower.3) Results from RT-PCR and Western Blot showed an increase of Rab27 A in HT29 sphere. We screened the HT29 cell line for stable expression of Rab27 A after lentivirus infection. Serum-free culture showed that overexpression of Rab27 A helps make HT29 sphere larger both in number and in size, as well as the growth of passages of sphere. Results of flow cytometry showed that overexpression of Rab27 A caused a higher rate of CD44+ and PKH26 high cells in HT29 cell line. The expression of cyclin D and CDK4 increased, while the expression of p27 decreased with overexpression of Rab27 A, as was shown by RT-PCR and Western Blot. Flow cytometry showed an increase in the proportion of S phase cells in HT29 cells and sphere cells with overexpression of Rab27 A. At the same time, we obtained reverse results after interference of Rab27 A.4) Results indicated that the supernatant from HT29 sphere with overexpression of Rab27 A could promote growth of sphere. Results from ELISA showed an increasing level of VEGF and TGF-β in the supernatant from HT29 sphere with overexpression of Rab27 A. we observed, using laser scanning confocal microscopy, that Rab27 A showed perinuclear unilateral aggregation distribution, and the density of Rab27 A declined from the nucleus to the cell membrane. We also found that the region of high level of Rab27 A cells showed increased proportion of PKH26 high cells. The results indicate that Rab27 A influence CSCs function by promoting the secretion of cytokines.5) In vivo study indicated that 100 cells from HT29 sphere could initiate tumorigenesis in nude mice. With an overexpression of Rab27 A, HT29 cells and spheres grew faster in nude mice. Angiogenesis was faster in overexpression group compared with control, shown by CD31 immunohistochemistry, which may be related with the release of VEGF triggered by Rab27 A.6) The expression level of p65 increased in HT29 sphere according to RT-PCR and Western Blot. Dual luciferase report system showed the activity of NF-κB pathway in HT29 sphere was higher than normal cancer cells. In addition, RT-PCR showed an increase in IL-6 expression, an target gene of NF-κB, in HT29 sphere passages;7) According to RT-PCR and Western Blot, the expression level of Rab27 A in HT29 sphere increased with overexpression of p65, while the NF- κB signal pathway inhibitor PDTC and BAY-11-7082 reduced the Rab27 A expression level. Dual luciferase report system showed that p65 could activate the transcriptional activity of Rab27 A promoter, while PDTC and BAY-11-7082 decreased the transcriptional activity of Rab27 A promoter mediated by p65.8) The growth of HT29 tumor stem-like cells is quicker after treatment with TNFα; Flow cytometry showed increase in percentage of CD44+ cells and PKH26 high cells in HT29 sphere. Dual luciferase report system showed an increase in activity of NF-κB pathway, and RT-PCR and Western Blot showed increase in both p65 and Rab27 A expression levels after treatment with TNFα. 【Conclusion】This study, for the first time, confirmed that Rab27 A promotes the stemness and proliferation of colon cancer stem-like cells, and promotes the growth of colon cancer cells. Activation of NF-κB pathway mediated by TNFα raises the level of p65 in HT29 sphere, which then promotes the expression of Rab27 A, the secretion of VEGF and TGF-β, and finally promotes the stemness and the proliferation rate of HT29 cancer stem cells. This study is the first to demonstrate the mechanism that inflammation enhanced cytokine secretion by increasing the expression of Rab27 A cell secretory protein to participate in the regulation of colon cancer stem cell, which provides a new perspective for further research in CSCs. |
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