Cell softening in malignant progression of human lung cancer cells by activation of receptor tyrosine kinase AXL

Abstract

To study the role of cell softening in malignant progression, Transwell assay and atomic force microscope were used to classify six human non-small cell lung cancer cell lines into two groups: a high motility-low stiffness (HMLS) group and a low motility-high stiffness (LMHS) group. We found a significant role of activity of the AXL receptor tyrosine kinase, which belongs to the TAM (Tyro3, AXL, Mer) family, in the stimulation of motility and cell softening. HMLS cells expressed higher AXL levels than LMHS cells and contained phosphorylated AXL. H1703 LMHS cells transfected with exogenous AXL exhibited increased motility and decreased stiffness, with low levels of actin stress fibre formation. Conversely, the AXL-specific inhibitor R428 and AXL-targeting siRNA reduced motility and increased stiffness in H1299 HMLS cells. Knockdown of AXL stimulated actin stress fibre formation, which inhibited tumour formation in a mouse xenograft model. The Ras/Rac inhibitor SCH 51344, which blocks disruption of actin stress fibres, exerted similar effects to AXL inactivation. We therefore propose that the Ras/Rac pathway operates downstream of AXL. Thus, AXL activation-induced cell softening promotes malignant progression in non-small cell lung cancer and represents a key biophysical property of cancer cells.

(−)-Epigallocatechin gallate inhibits stemness and tumourigenicity stimulated by AXL receptor tyrosine kinase in human lung cancer cells

“…A control siRNA (siControl) was obtained from OriGene Technologies, Inc. (Rockville, MD, USA). siRNA was transfected into H1299 cells using Lipofectamine RNAiMAX (Invitrogen, CA, USA) as described previously 11 . Cell stiffness, tumour sphere formation, and western blot analyses were conducted after 2 days.…”

“…Western blot analysis. Cells or tumours were lysed in strong lysis buffer containing 20 mM Tris-HCl at pH 8.0, 150 mM sodium chloride, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 0.5 mM sodium pyrophosphate, and 5 mM sodium fluoride, as described previously 11 . The lysates were subjected to SDS-PAGE, and then transferred onto a nitrocellulose membrane.…”

“…AXL is a receptor tyrosine kinase. We previously reported that AXL stimulates H1299 cell softening, and treatment with the R428 AXL inhibitor reversed it 11 . As aforementioned, H1299 cells contain both high amounts of AXL protein and p-AXL at Y702.…”

“…Our experiments showed that a human NSCLC cell line, H1299, expressed high amounts of both AXL and phosphorylated AXL (p-AXL, an active form) and revealed that treating H1299 cells with AXL-targeted siRNA increased the average Young's modulus value. These findings indicated that the stiffness of the H1299 cells was reversed and that motility was inhibited to the levels of the low-motile high-stiffness H1703 NSCLC cell line, as if the cells had been treated with EGCG 11 . Thus, AXL appears to play a significant role in the stemness of H1299-sdCSCs.…”

“…We recently reported that AXL receptor tyrosine kinase (AXL) stimulates cell softening and motility in H1299 and Lu99 human lung cancer cells 11 . AXL was isolated as a novel transforming gene from patients with chronic myelogenous leukemia; the name 'AXL' was derived from the Greek word for 'non-controlled' 12,13 .…”