Magnussen Synnove Norvoll, Hadler-Olsen Elin, Costea Daniela Elena, Berg Eli, Jacobsen Cristiane Cavalcanti, Mortensen Bente, Salo Tuula, Martinez-Zubiaurre Inigo, Winberg Jan-Olof, Uhlin-Hansen Lars, Svineng Gunbjorg
Department of Medical Biology, Faculty of Health Sciences, UiT - The Arctic University of Norway, N-9037, Tromsø, Norway.
Diagnostic Clinic - Clinical Pathology, University Hospital of North Norway, Tromsø, Norway.
BMC Cancer. 2017 May 19;17(1):350. doi: 10.1186/s12885-017-3349-7.
Urokinase 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.
Mouse 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.
We 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.
These 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.
尿激酶型纤溶酶原激活物（uPA）受体（uPAR）在人类口腔鳞状细胞癌（OSCC）的侵袭性肿瘤前沿上调，表明uPAR在肿瘤进展中发挥作用。我们之前在OSCC小鼠模型中观察到肿瘤-基质界面处uPAR表达升高，这与蛋白水解活性增加有关。肿瘤微环境调节uPAR的表达及其糖基化和裂解。全长和裂解的uPAR（uPAR（II-III））都参与细胞信号传导、增殖、迁移、干细胞动员和侵袭等高度调控的过程。本研究的目的是分析肿瘤相关因子及其对uPAR裂解的影响，以及对细胞增殖、迁移和侵袭的潜在影响。
在小鼠OSCC细胞系AT84中稳定过表达小鼠uPAR。通过蛋白质印迹分析全长与裂解uPAR的比例，并通过添加不同的蛋白酶抑制剂和转化生长因子-β1（TGF-β1）评估其调控。使用实时细胞分析分析uPAR裂解在细胞增殖和迁移中的作用，并使用肌瘤侵袭模型评估侵袭。
我们发现，当uPAR过表达时，一部分受体会被裂解，因此细胞同时呈现全长uPAR和uPAR（II-III）。裂解主要由丝氨酸蛋白酶特别是尿激酶型纤溶酶原激活物（uPA）进行。当用TGF-β1刺激OSCC细胞时，uPA抑制剂PAI-1的产生增加，导致uPAR裂解减少。通过抑制uPAR的裂解，细胞迁移减少，通过抑制uPA活性，侵袭减少。我们还可以表明，含有可溶性uPAR（suPAR）和裂解的可溶性uPAR（suPAR（II-III））的培养基可诱导内源性uPAR水平低的OSCC细胞迁移。
这些结果表明，肿瘤微环境中的可溶性因子，如TGF-β1、PAI-1和uPA，可影响全长和uPAR（II-III）的比例，从而可能影响细胞迁移和侵袭。因此，解决uPAR裂解如何被控制对于理解OSCC的进展至关重要，并可能为治疗提供新的靶点。
