Noncanonical roles of membranous lysyl-tRNA synthetase in transducing cell-substrate signaling for invasive dissemination of colon cancer spheroids in 3D collagen I gels

Abstract

The adhesion properties of cells are involved in tumor metastasis. Although KRS at the plasma membrane is shown important for cancer metastasis, additionally to canonical roles of cytosolic KRS in protein translation, how KRS and its downstream effectors promote the metastatic migration remains unexplored. Disseminative behaviors (an earlier metastatic process) of colon cancer cell spheroids embedded in 3D collagen gels were studied with regards to cell adhesion properties, and relevance in KRS−/+ knocked-down animal and clinical colon cancer tissues. Time-lapse imaging revealed KRS-dependent cell dissemination from the spheroids, whereas KRS-suppressed spheroids remained static due to the absence of outbound movements supported by cell-extracellular matrix (ECM) adhesion. While keeping E-cadherin at the outward disseminative cells, KRS caused integrin-involved intracellular signaling for ERK/c-Jun, paxillin, and cell-ECM adhesion-mediated signaling to modulate traction force for crawling movement. KRS-suppressed spheroids became disseminative following ERK or paxillin re-expression. The KRS-dependent intracellular signaling activities correlated with the invasiveness in clinical colon tumor tissues and in KRS−/+ knocked-down mice tissues. Collectively, these observations indicate that KRS at the plasma membrane plays new roles in metastatic migration as a signaling inducer, and causes intracellular signaling for cancer dissemination, involving cell-cell and cell-ECM adhesion, during KRS-mediated metastasis.

Dynamic and coordinated single‐molecular interactions at TM4SF5‐enriched microdomains guide invasive behaviors in 2‐ and 3‐dimensional environments

“…Time-lapse images were saved by using an IX81-ZDC microscope (Olympus). Spheroids were immunostained as previously described (15).…”

Lysyl-tRNA synthetase–expressing colon spheroids induce M2 macrophage polarization to promote metastasis

“…HCT116 or SW620 colon cancer cells were processed to form spheroids using stably transfected control cells with control shRNA (shControl), KRS-suppressed cells with shRNA against KRS (shKRS#2 or shKRS#5), or KRSoverexpressing cells with KRS-WT plasmids. The KRS expression levels in the stable cell lines were correlated to ERK activity ( Figure 1B), as previously described (12). The embedded spheroids were treated with control media or CM from THP-1 M1 or M2 macrophages, and time-lapse images were captured for 1 day (1:00:00) or 2 days (2:00:00).…”

“…Moreover, phosphorylation of the T52 residue of KRS by p38 MAPK causes KRS to dissociate from the MSC and translocate to the plasma membrane. At the plasma membrane, KRS associates with and stabilizes 67-kDa laminin receptor (p67LR) and α 6 β 1 integrin, promoting an intracellular signal for migration (6,12). The protein-protein interaction between p67LR and KRS has been the target of anti-KRS reagents to inhibit KRS-mediated cell migration and metastasis (9).…”

Suppression of lysyl-tRNA synthetase, KRS, causes incomplete epithelial-mesenchymal transition and ineffective cell-extracellular matrix adhesion for migration

“…Cells. HCT116 colon cancer cells (american Type Culture Collection, Manassas, VA, USA) were stably transfected with shRNa against KRS, as previously described (18). HCT116 cells overexpressing KRS were established via the stable transfection of a myc-tagged KRS plasmid (7).…”

“…1B). We then checked whether KRS downregulation could affect general protein translation and/or apoptosis, and there were no differences between KRS-expressing and KRS-suppressed cell clones (18). E-cadherin was not clearly observed in cell-cell junctions but N-cadherin was expressed in the junctions with minimal β-catenin when KRS was suppressed, as compared with KRS-expressing cells (Fig.…”