Cleavage of the urokinase receptor (uPAR) on oral cancer cells: regulation by transforming growth factor – β1 (TGF-β1) and potential effects on migration and invasion

Abstract

BackgroundUrokinase plasminogen activator (uPA) receptor (uPAR) is up-regulated at the invasive tumour front of human oral squamous cell carcinoma (OSCC), indicating a role for uPAR in tumour progression. We previously observed elevated expression of uPAR at the tumour-stroma interface in a mouse model for OSCC, which was associated with increased proteolytic activity. The tumour microenvironment regulated uPAR expression, as well as its glycosylation and cleavage. Both full-length- and cleaved uPAR (uPAR (II-III)) are involved in highly regulated processes such as cell signalling, proliferation, migration, stem cell mobilization and invasion. The aim of the current study was to analyse tumour associated factors and their effect on uPAR cleavage, and the potential implications for cell proliferation, migration and invasion.MethodsMouse uPAR was stably overexpressed in the mouse OSCC cell line AT84. The ratio of full-length versus cleaved uPAR as analysed by Western blotting and its regulation was assessed by addition of different protease inhibitors and transforming growth factor - β1 (TGF-β1). The role of uPAR cleavage in cell proliferation and migration was analysed using real-time cell analysis and invasion was assessed using the myoma invasion model.ResultsWe found that when uPAR was overexpressed a proportion of the receptor was cleaved, thus the cells presented both full-length uPAR and uPAR (II-III). Cleavage was mainly performed by serine proteases and urokinase plasminogen activator (uPA) in particular. When the OSCC cells were stimulated with TGF-β1, the production of the uPA inhibitor PAI-1 was increased, resulting in a reduction of uPAR cleavage. By inhibiting cleavage of uPAR, cell migration was reduced, and by inhibiting uPA activity, invasion was reduced. We could also show that medium containing soluble uPAR (suPAR), and cleaved soluble uPAR (suPAR (II-III)), induced migration in OSCC cells with low endogenous levels of uPAR.ConclusionsThese results show that soluble factors in the tumour microenvironment, such as TGF-β1, PAI-1 and uPA, can influence the ratio of full length and uPAR (II-III) and thereby potentially effect cell migration and invasion. Resolving how uPAR cleavage is controlled is therefore vital for understanding how OSCC progresses and potentially provides new targets for therapy.

Effects of transforming growth factor‐β1 on plasminogen activation in stem cells from the apical papilla: role of activating receptor‐like kinase 5/Smad2 and mitogen‐activated protein kinase kinase (MEK)/extracellular signal‐regulated kinase (ERK) signalling

“…TGF‐β1 induced both PAI‐1 and uPAR expression/secretion in retinal pigment epithelial cells (Siren et al ). But TGF‐β1 is shown to stimulate uPA expression in cancer cells (Jaiswal & Yadava ), and induce PAI‐1, but inhibit the cleavage of uPAR of oral squamous carcinoma cells, and thus inhibition of cell migration (Magnussen et al ). For stem cells, Heissig et al () suggests the role of plasmin/fibrinolytic system in stem cell recruitment, cell homing, angiogenesis and tissue regeneration.…”

Blood vitronectin is a major activator of LIF and IL-6 in the brain through integrin–FAK and uPAR signaling

“…This suggests that BC-11 functions through blockade of the enzymatic activity of uPA which may occur in addition to blockade of the binding uPA N-terminal fragments to uPAR. More recently, BC-11 was shown to reduce uPAR cleavage by uPA in a similar fashion to that seen for the serine protease inhibitor aprotinin (Magnussen et al, 2017). Drugs were dissolved in up to 0.1% DMSO of final volume in culture medium (vehicle).…”

Nephronectin promotes breast cancer brain metastatic colonization via its integrin-binding domains

“…RT-qPCR was performed as previously described 47 with minor changes to the protocol as follows; analysis was performed using the LightCycler (Roche, Mannheim, Germany), and target cDNA was amplified through 40 cycles in a 20 μl qPCR mix (FastStart Essential DNA Green Master, Roche Diagnostics GmbH, Mannheim, Germany) containing 10 μM primer mix (primer sequences: see Supplementary file 1). Primer efficiencies were all between 96 and 100%.…”