RESUMO
The limited availability of melanoma stem cells is a major challenge for therapeutic reagent screening and study of molecular mechanisms. It has been shown that induced expression of four stem cell factors (Oct4, Sox2, Klf4, and c-Myc) changes the phenotype of osteosarcoma and breast cancer cells to osteosarcoma stem cells and breast cancer stem cells, respectively. The present study aimed to explore whether these four factors might change the phenotype of melanoma cells to melanoma stem cells and, if so, to examine the possible molecular signal involved. Melanoma B16-F10 cells were transfected with the plasmid TetO-FUW-OSKM which contains cDNA expressing four factors, driven by the Tet-On element. We found that expression of the four transcription factors was highly induced by DOX in the stable melanoma cell clones. Further studies confirmed that induced expression of these factors remodeled the phenotype of the melanoma cells to melanoma stem cells (MSCs). This conclusion was supported by the evidence that induced expression of these factors increased the numbers of tumor-initiating cells, (namely MSCs), both in an in vitro cell culture system and in a mouse in vivo model. The conclusion was further supported by the observation that the induction of these factors exclusively increased the mRNA of signal transducer and activator of transcription 3 which has been reported to play a crucial role in stem cell maintenance. Thus, phenotypic remodeling of melanoma cells following the induction of these four factors provided a simple and optimal means to constantly obtain MSCs for screening new therapeutic reagents. The result also reveals that Stat3 may be a crucial link between the induction of the four factors and the cell remodeling, suggesting its potential role as a target to fight melanoma.
Assuntos
Reprogramação Celular/genética , Melanoma Experimental/genética , Melanoma/genética , Células-Tronco Neoplásicas/metabolismo , Fator de Transcrição STAT3/genética , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Melanoma/patologia , Melanoma Experimental/patologia , Camundongos , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição SOXB1/genética , Transdução de Sinais/genética , TransfecçãoRESUMO
Background: The crotonylation of histones is discovered of late as one of the post-translational modifications (PTMs) that can regulate gene expression. However, the function of crotonylation on nonhistone proteins in vascular smooth muscle cells (VSMCs) is unclear. Here, we aim to find the cellular characteristics of crotonylated nonhistone proteins and the cross talk with ubiquitinated proteins in VSMC phenotypic remodeling using the modified omics and proteomic analysis. Methods: We performed the modified omics and proteomic analysis of VSMCs before and after the stimulation with platelet-derived growth factor-BB (PDGF-BB). The crotonylated and ubiquitinated pan-antibody was used to enrich proteins and then subjected to a high-throughput mass spectrometry analysis. The enrichment analysis was performed within differentially modified proteins in regard to GO terms, KEGG, and protein domains. Results: As a result, there were 2,138 crotonylation sites in 534 proteins and 1,359 ubiquitination sites corresponding to 657 proteins. These crotonylated proteins detected after PDGF-BB stimulation might be involved in various vital cellular pathways and carry out important functions in VSMCs. Some of them closely took part in significant physiological processes of VSMC phenotypic remodeling, including glycolysis/gluconeogenesis, vascular smooth muscle contraction, and the PI3K-Akt signaling pathway. Furthermore, the KEGG pathway enrichment analysis showed the involvement of ubiquitinated proteins in the physiological processes of VSMC phenotypic remodeling, including glycolysis/gluconeogenesis, vascular smooth muscle contraction, RAS signaling pathway, or the PI3K-Akt signaling pathway. A cross talk analysis showed that there were 199 sites within the 177 proteins modified by crotonylation and ubiquitination simultaneously. Protein-protein interaction (PPI) network analysis indicated that crotonylated and ubiquitinated proteins play an important role in cellular bioprocess commonly and possibly have a synergistic effect. Conclusion: In summary, our bioinformatics analysis shows that the crotonylation and ubiquitination of nonhistone proteins play an essential role in VSMC phenotypic transformation induced by PDGF-BB stimulation. The cross talk between crotonylation and ubiquitination in glycolysis is possibly a novel mechanism during VSMC phenotypic remodeling.