Krajewski Adrian, Gagat Maciej, Mikołajczyk Klaudia, Izdebska Magdalena, Żuryń Agnieszka, Grzanka Alina
Department of Histology and Embryology, Nicolaus Copernicus University in Toruń, Collegium Medicum in Bydgoszcz, Bydgoszcz, Poland.
Cancer Manag Res. 2020 Dec 22;12:13085-13097. doi: 10.2147/CMAR.S279169. eCollection 2020.
Cyclins are well-known cell cycle regulators. The activation of cyclin-dependent kinases by cyclins allows orchestration of the complicated cell cycle machinery and drives the cell from the G1 phase to the end of the mitotic phase. In recent years, it has become evident that cyclins are involved in processes beyond the cell cycle. Cyclin F does not activate CDKs but forms part of the Skp1-Cul1-F-box (SCF) complex where it is responsible for protein target recognition and subsequent degradation in a proteasome-dependent manner.
Here, we report that the downregulation of cyclin F in the A-375 melanoma cell line increases cell viability and colony formation in a cell cycle independent manner. Lower levels of cyclin F do not appear to affect the cell cycle, based on flow cytometry measuring BrdU incorporation and propidium iodide staining. By means of immunofluorescence staining and Western blot analysis, we observed changes in cell morphology-related markers which suggested ongoing epithelial-mesenchymal transition (EMT) in response to cyclin F downregulation. Increases in vimentin and N-cadherin protein levels, decreases in levels of epithelial markers such as ZO-1, along with changes in morphology to a spindle-like shape with the appearance of actin stress fibers, are all hallmarks of EMT. These changes are associated with increased invasive and migratory potential, based on 2D migration assays. Moreover, we observe an increase in RhoABC, talin and paxillin levels, the proteins involved in controlling cell signaling and motility. Lastly, upon knocking down cyclin F expression, we observed a decrease in thrombospondin-1 expression, suggesting a role of cyclin F in angiogenesis.
Cyclin F depletion induces proliferation and EMT processes in the A-375 melanoma model.
细胞周期蛋白是著名的细胞周期调节因子。细胞周期蛋白激活细胞周期蛋白依赖性激酶，从而协调复杂的细胞周期机制，并驱动细胞从G1期进入有丝分裂期末期。近年来，越来越明显的是，细胞周期蛋白参与了细胞周期以外的过程。细胞周期蛋白F不激活细胞周期蛋白依赖性激酶，而是构成Skp1-Cul1-F-box（SCF）复合物的一部分，在该复合物中，它负责蛋白质靶标的识别，并随后以蛋白酶体依赖性方式进行降解。
在此，我们报告，A-375黑色素瘤细胞系中细胞周期蛋白F的下调以细胞周期非依赖性方式增加细胞活力和集落形成。基于流式细胞术测量BrdU掺入和碘化丙啶染色，较低水平的细胞周期蛋白F似乎不影响细胞周期。通过免疫荧光染色和蛋白质印迹分析，我们观察到细胞形态相关标志物的变化，这表明响应细胞周期蛋白F下调而发生了上皮-间质转化（EMT）。波形蛋白和N-钙黏蛋白水平增加，ZO-1等上皮标志物水平降低，以及形态变为纺锤状并出现肌动蛋白应力纤维，这些都是EMT的标志。基于二维迁移试验，这些变化与侵袭和迁移潜能增加有关。此外，我们观察到RhoABC、踝蛋白和桩蛋白水平增加，这些蛋白质参与控制细胞信号传导和运动。最后，在敲低细胞周期蛋白F表达后，我们观察到血小板反应蛋白-1表达降低，这表明细胞周期蛋白F在血管生成中发挥作用。
在A-375黑色素瘤模型中，细胞周期蛋白F的缺失诱导增殖和EMT过程。
