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Ras proteins regulate signaling pathways that control many cellular responses, such as proliferation, survival, and differentiation. However, there are intriguing questions about the relationship between the developmental timing of specific mutations and the resultant phenotypes in individual cells. In this study, we used the Cre/loxP system for maintaining transgenic zebrafish lines harboring oncogenic Kras(V12) under the nestin promoter, and investigated the developmental effects of Ras activation in neural progenitor cells. Activated human Kras(V12) was induced within pDSNesLCherryLEGFPKRas(V12) transgenic fish by Cre mRNA injection. Cre-mediated gene excision was confirmed by polymerase chain reaction, and the injected embryos were investigated for Kras(V12) effects using the hemotoxylin-eosin staining, terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay, and in situ hybridization. pDSNesLCherryLEGFPKRas(V12) transgenic embryos normally expressed mCherry in their central nervous system throughout the developmental stage. However, Cre mRNA injection efficiently excised the flanking stop sequence, and the injected embryos expressed enhanced green fluorescent protein in their brain with severe edema. Brain histology showed that neuronal cell differentiation could occur in spite of oncogenic Kras(V12) overexpression, but massive apoptosis and brain edema caused early embryonal death. In summary, the overexpression of Kras(V12) induces extensive apoptosis of neural progenitor cells followed by severe edema of the brain. However, some neural progenitor cells are resistant to Kras(V12) and can retain their ability to differentiate into neurons. Finally, our transgenic model demonstrates the inability of Kras(V12) alone to induce brain tumors at the early stage of development.

Bone morphogenetic protein (BMP) 7 counteracts physiological epithelial-to-mesenchymal transition, a process that is indicative of epithelial plasticity in developmental stages. Because epithelial-to-mesenchymal transition and its reversed process mesenchymal-to-epithelial transition (MET) are also involved in cancer progression, we investigated whether BMP7 plays a role in WM-266-4 melanoma cell growth and metastasis. An MTT assay was conducted in WM-266-4 and HEK293T cell lines to show the cell growth inhibition ability of BMP7 and cisplatin. Semiquantitative RT-PCR was used to determine MET in morphologically changed BMP7-treated melanoma cells. MET-induced cells expressed less a basic helix-loop-helix transcription factor (TWIST) in western blot analysis, and we confirm that BMP receptor (Alk2) siRNA transduction could restore TWIST protein expression via blocking of Smad 1, 5 and 8 signaling. Matrigel invasion and cell migration assays were done to investigate the BMP7-induced metastasis inhibition ability. BMP7 treatment only slightly reduced cell growth rate, but induced apparent MET. BMP7 also reduced the invasion and migration ability. Furthermore, BMP7 reduced the resistance of WM-266-4 cells to cisplatin. Collectively, our findings indicate that the metastatis inhibition ability of BMP7 is involved in MET, and that BMP7 could be used as a potential metastasis inhibitor in human melanoma cells.

Sphere-forming abilities in culture condition are considered a hallmark of cancer stem-like cells, which represents tumor cell invasiveness and stem-like characteristics. We aimed to show that the sphere-forming subpopulation of human malignant melanoma cell line WM-266-4 acts differently to zebrafish embryo extracts compared with their bulk counterpart. Spheres were maintained in neural stem cell culture conditions. The embryos of zebrafish at specific developmental stages were collected and the extracts were purified under 100 kDa. Spheres were treated with embyo extracts and proliferation assay and immunocytochemistry were conducted. Spheroid cells expressed nestin and epidermal growth factor receptor (EGFR) but not melanoma antigen recognized by T-cells (MART)1, indicating their stem-like character. Zebrafish embryo extracts at 50% epiboly stage inhibited melanoma bulk cell proliferation in a dose-dependent manner. However, sphere-forming abilities were significantly enhanced under 1 microg/mL concentration of 50% epiboly stage embryo extract treatment. Our findings implicate that we should consider cell subsets of a different character from the tumor origin that can respond differently to exogenous substances or tumor microenvironments. We suggest that cancer research should consider both minor stem-like subpopulations and the other major bulk tumor cells.