Leukamenin E Induces K8/18 Phosphorylation and Blocks the Assembly of Keratin Filament Networks Through ERK Activation

Abstract

Leukamenin E is a natural ent-kaurane diterpenoid isolated from Isodon racemosa (Hemsl) Hara that has been found to be a novel and potential keratin filament inhibitor, but its underlying mechanisms remain largely unknown. Here, we show that leukamenin E induces keratin filaments (KFs) depolymerization, largely independently of microfilament (MFs) and microtubules (MTs) in well-spread cells and inhibition of KFs assembly in spreading cells. These effects are accompanied by keratin phosphorylation at K8-Ser73/Ser431 and K18-Ser52 via the by extracellular signal-regulated kinases (ERK) pathway in primary liver carcinoma cells (PLC) and human umbilical vein endothelial cells (HUVECs). Moreover, leukamenin E increases soluble pK8-Ser73/Ser431, pK18-Ser52, and pan-keratin in the cytoplasmic supernatant by immunofluorescence imaging and Western blotting assay. Accordingly, leukamenin E inhibits the spreading and migration of cells. We propose that leukamenin E-induced keratin phosphorylation may interfere with the initiation of KFs assembly and block the formation of a new KFs network, leading to the inhibition of cell spreading. Leukamenin E is a potential target drug for inhibition of KFs assembly.

Cell State and Cell Type: Deconvoluting Circulating Tumor Cell Populations in Liquid Biopsies by Multi-Omics

“…In readdressing blood draws from patient PC1, using a combination of WGS, morphometrics and targeted proteomics, we found that copy number-neutral CK+ cells were morphologically distinct from clonal CTCs, had a high expression of the endothelial marker CD31, indicating an endothelial cell lineage (Figures 4 and 5) and, further, trended toward a lower CK signal (CKdim) than the clonal cells. Endothelial cells, despite their mesodermal origin, have been reported to express certain CKs such as CK7, CK1, CK8 and CK18, explaining why they can be found using a pan-CK-based rare-cell detection assay [40][41][42][43][44]. Consistent with that result, we have shown that both spiked endothelial cell lines as well as MI CECs were robustly detected using CK and Vim as inclusion and CD45 as exclusion markers.…”

“…Our earlier results on the prostate cancer index patient (PC 1) showed that single-cell copy-number profiling of rare CK+ cells could distinguish clonally altered tumor cells (CTCs) from cells of an uncertain origin displaying neutral genomes, and, further, that both populations varied according to the state of the disease [40][41][42][43][44]. The data presented here take this observation further and demonstrate that through a combination of epithelial, mesenchymal, endothelial, and leukocyte markers, it is possible to further discriminate multiple cell types within the population of rare CK+ circulating cells.…”