RAP80 regulates epithelial-mesenchymal transition related with metastasis and malignancy of cancer.
Bottom Line: The downregulation of RAP80 increases ZEB1 protein and decreases miR200c expression to activate EMT signaling in the form of drastic inhibitions of E-cadherin, p16 and p21 expression.Using in vivo metastasis analysis, RAP80 knockdown cells are shown to dramatically metastasize into the lung and generate more malignant phenotype compared to controls.Interestingly, the expression level of RAP80 was positively correlated with the survival rate in lung adenocarcinoma and breast cancer patients.
Affiliation: Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, Korea.Show MeSH
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Mentions: To investigate the role of RAP80 on EMT, RAP80 knockdown cells were generated by the stable expression of RAP80 shRNA in HeLa cells using shRAP80 lentivirus to test if EMT signal is induced. In RAP80 knockdowned cells, vimentin and c‐myc (mesenchymal specific proteins) are increased; in contrast, E‐cadherin (a epithelial specific protein) is markedly decreased (Fig. 1a). We also show that vimentin mRNA transcript is increased by RAP80 knockdown (Fig. 1b). However, N‐cadherin is barely detected and not changed by Rap80 knockdown (Fig. 1a). Among four independent Rap80 knockdown cells, we selected two independent Rap80 knockdown cells (shRap80‐1 and shRap80‐2) for further analysis. First, RAP80 knockdown cell lines were tested for morphological changes induced by EMT. As shown in Figure 2(a), RAP80 knockdown cells tend to shrink and detach from surfaces to become globular, indicating that loss of RAP80 induces EMT‐like morphological changes. To find further evidence for the induction of EMT in RAP80 knockdown cells, we tested anchorage independent growth and tumor sphere formation, another characteristic change of EMT.23 Anchorage independent growth and tumor sphere formation were tested with cells grown in poly‐HEMA (poly‐hydroxyethyl methacrylate)‐treated surfaces to prevent cell adhesion. Under non‐adhesive culture conditions, the number and morphology of tumor spheres with control and RAP80 knockdown cells were analyzed. As shown in Figure 2(b,c), the number of tumor spheres is greatly increased in the shRAP80‐1 and the shRAP80‐2 cell lines, indicating EMT induction in RAP80 knockdown cells. In addition, the morphology of spheres is different in control compared to RAP80 knockdown cells. The control vector‐infected cells formed small spheres that were round with tightly connected cells (Fig. 2b, left panel). In contrast, RAP80 knockdown cells (shRAP80‐1 and shRAP80‐2) formed large spheres that were perfectly round and, instead, were loosely connected and easily dissociated (Fig. 2b middle and right panel). To confirm cell viability under non‐adhesive culture conditions, MTT assay was performed. As shown in Figure 2(d), RAP80 knockdown cells showed significant increase of cell viability under non‐adhesive culture conditions, indicating that loss of RAP80 induces anchorage‐independent cell growth. Together, the loss of RAP80 expression induces EMT‐related cellular phenotypes, such as tumor sphere formation and anchorage‐independent growth.
Affiliation: Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, Korea.