Kisspeptin, a neuropeptide that acts via KISS1R (GPR54) has been reported to regulate the mesenchymal-like transition process in several cancer models. Despite its controversial role, the mesenchymal-like transition in Glioblastoma (GBM) can increase the tumor cell motility, promoting its infiltration that compromises the maximal tumor resection. Preliminary data demonstrated kisspeptin-induced cell scratch closing with mesenchymal-like morphological changes, hypothesizing its role in regulating mesenchymal-like transition in KISS1R-expressing GBM cells. Therefore, this study aims to elucidate the mesenchymal-like transition mechanism in KISS1R-expressing GBM.
The kisspeptin effect on cell proliferation was conducted using a CCK-8 kit while the mesenchymal-like transition was confirmed by rhodamine-phalloidin immunocytochemistry (ICC) analysis. The mechanistic data was initially visualized using liquid chromatography-mass spectrometry (LC-MS/MS), where the molecular markers and mechanisms were further validated using JESS Simple Western.
CCK-8 analysis demonstrated that kisspeptin did not affect the cell viability, indicating the treatment mesenchymal-like effects observed were not due to the cell proliferation and were stress-related. ICC analysis showed that kisspeptin treatment (10 and 100 nM) induced mesenchymal-like transition in KISS1R-expressing GBM, as early as 3 hours and sustained up to 48 hours. While the LC-MS/MS analysis confirmed that the KISS1R-expressing GBMs are not kisspeptin-producing cells, further proteomics profiling revealed the regulation of mesenchymal-related markers (vimentin), cell adhesion molecules, cytoskeletal proteins and matrix proteases, including the upregulation of transgelin-2 and cofilin following kisspeptin treatment. JESS Simple Western confirmed kisspeptin treatment upregulated the KISS1R expression, indicating KiSS/KISS1R system is responsible for mediating mesenchymal-like transition. The data also demonstrated the upregulation of mesenchymal marker (N-cadherin) and transcription factors (ZEB-1, Slug, Snail and β-Catenin).
The current findings illustrated kisspeptin induction of a mesenchymal-like transition process via KiSS/KISS1R with concomitant regulation of classical mesenchymal markers and transcription factors expression. Future comprehensive elucidation of kisspeptin-induced mesenchymal-like transition with recovery assays via respective inhibitors may fill existing knowledge of GBM tumor motility mechanisms.