6502

SKP2

Both SKP2 (S-phase kinase-associated protein 2) and transforming growth factor-beta1 (TGF-beta1) play important roles in cancer metastasis through different mechanisms: TGF-beta1 via induction of epithelial-mesenchymal transition (EMT) and SKP2 via downregulating p27(kip1). Recent studies indicated that c-Myc and Akt1 were active players in metastasis. In this study we demonstrated a crosstalk between these pathways. Specifically, we found that TGF-beta1 treatment increased SKP2 expression accompanied with increased phosphorylation of Akt1 and c-Myc protein accumulation during EMT. We demonstrated that Akt1 was required for TGF-beta1-mediated SKP2 upregulation and that c-Myc transcription factor specifically bound to the promoter of SKP2 for its enhanced transcription. Analysis of 25 samples of normal human skin, nevi, and melanomas revealed a positive correlation between c-Myc and SKP2 accumulation. Furthermore, accumulation of SKP2 and c-Myc proteins was significantly higher in metastatic melanoma samples as compared with that in primary melanomas, which again was higher than that in normal skin or nevi. In summary, our results integrated TGF-beta1 signals to SKP2 via Akt1 and c-Myc during EMT, and provided, to our knowledge, a previously unreported mechanistic molecular event for TGF-beta1-induced metastasis in human melanoma.

Osteosarcoma (OS), a common worldwide primary aggressive bone malignancy, arises from primitive transformed cells of mesenchymal origin and usually attacks adolescents and young adults. Methotrexate (MTX) is the anti-folate drug used as a pivotal chemotherapeutic agent in the treatment of OS. However, patients with OS often develop drug resistance, leading to poor treatment outcomes. In the present study, in order to explore the underlying mechanisms responsible for MTX resistance, we established MTXresistant OS cells using the U2OS and MG63 cell lines and examined whether MTXresistant OS cells underwent epithelial-mesenchymal transition (EMT) by Transwell assay, wound healing assay, MTT assay, RT-PCR and western blot analysis. We found that the viability of the MTXresistant cells remained relatively unaltered following further treatment with MTX compared to the parental cells. The resistant cells appeared to possess a mesenchymal phenotype, with an elongated and more spindlelike shape, and acquired enhanced invasive, migratory and attachment abilities. The measurement of EMT markers also supported EMT transition in the MTXresistant OS cells. Our result further demonstrated that the overexpression of S-phase kinase-associated protein 2 (Skp2) was closely involved in the resistance of OS cells to MTX and in the acquirement of EMT properties. Thus, the pharmacological inhibition of Skp2 may prove to be a novel therapeutic strategy with which to overcome drug resistance in OS.