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Tumor-associated proteolysis and the urokinase-receptor (u-PAR)

For the accomplishment of almost each step of the metastatic cascade, tumor cells essentially drive the degradation of extracellular matrix components, e.g., collagen type IV, laminin, or vitronectin. To stimulate suchlike activities, tumor cells, and also surrounding stromal cells which interact with them, employ several “tumor-associated proteases”, which are secreted by tumor cells or surrounding stromal cells and can be focused, e.g. at the tumor cell surface, by expression of specific protease receptors. According to the catalytically active site of these proteases they are classified into serine, aspartic, cysteine, threonine, and metalloproteinases. The identity of these proteolytic enzymes is not different from physiologic enzymes which are involved in several tissue remodelling processes such as wound healing, fibrinolysis, inflammation, embryogenesis and angiogenesis. However, the quantity rather than the quality of their expression or activity contributes to the invasive phenotype of malignant cells.

One of the best characterized “tumor-associated proteases” is the urokinase-type plasminogen activator (u-PA), a serine protease of 55 kDa. It converts plasminogen to the active enzyme, plasmin, thus promoting extracellular matrix degradation including fibrin, fibronectin, proteoglycans, laminin and collagen IV (important for a tumor cell’s invasion and intravasation through basement membranes). Its proteolytic activation can occur either in the extracellular space or bound to the specific urokinase-receptor (u-PAR), however, receptor-bound u-PA activates plasminogen much more efficiently, this being reflected by a 40-fold decrease in Km for its substrate. The 55-60 kDa heavily glycosylated, disulfide-linked cell surface receptor (u-PAR) consists of 3 similar repeats approximately 90 residues each, the last of which is anchored to the cell membrane via glycosyl-phosphatidylinositol, this enabling a high intramembrane mobility.

Efficient proteolytic system

Receptor-bound u-PA is inactivated by PAI-1 (-2), the u-PAR/u-PA/PAI complex is internalized into the cell together with α-2-macroglobulin receptor and its ligand, the free u-PAR is recycled to the cell surface, and binding and activation of a second u-PA-molecule can occur. Moreover, the u-PAR is co-localized with integrins, acts as a co-receptor for vitronectin, can be found at cellular focal contacts, lamellipodia or in caveolae, and is able to induce the phosphorylation of focal adhesion kinase (FAK), cytoskeletal proteins and Src-family members. A switch between considerably different signalling cascades brought about by u-PAR-associated molecular interactions can promote such opposing phenomena such as proliferation and dormancy for tumor cells. Thus, the u-PAR/u-PAR system is considered to be one of the most important, dynamic and ubiquitous tumor-associated protease systems, this being reflected by the fact that the u-PAR is overexpressed in the majority of solid carcinoma entities (e.g., breast, gastrointestinal, prostate lung cancers and others), this being associated with a poor clinical prognosis in many instances (for further reading please consider our reviews).

u-PAR system as a novel prognostic factor

Since 1993, our group has been actively contributing to hypothesis generation on how the u-PAR works molecularly, how it is being regulated in malignant cells, and how this could contribute to a better understanding, diagnosis, therapy and classification of tumor diseases. Towards this end, we established the complete u-PAR system as a novel prognostic factor in gastric cancer and demonstrated that the u-PAR is one of the critical molecular characteristics of the metastatically relevant phenotype in minimal residual tumor cells in solid cancer. Moreover, we identified the major transcriptional regulators of u-PAR overexpression in cancer cells and, in a translational approach, demonstrated that the transactivation of u-PAR gene expression via different promoter elements of the u-PAR gene occurs at different levels of tumor specificity, a notion that we currently explore in terms of diagnostic and therapeutic opportunities in a patented approach (Patent-No eu PCT/EPO3/01671. Tissue Specific Exression). Furthermore, we observed that, with specific transcriptional regulators of the u-PAR and further parameters, we were able to modify existing clinical staging concepts toward the identification of completely novel high-risk groups of cancer patients (Maurer). Finally, in recent years we extended translational research projects on u-PAR and identified the EGFR-targeted antibody Cetuximab as an inhibitor of metastasis via blocking u-PAR gene expression, in parallel suggesting the u-PAR as a novel biomarker for Cetuximab sensitivity. Also, we recently implicated the anti-malaria agent Artesunate as a novel metastasis inhibitor in cancer, acting via the u-PAR ligand urokinase (u-PA) and specific matrix-metalloproteinases (MMPs). Furthermore, we established metastasis suppression as a function of the novel tumor suppressor Pdcd4 and showed that this is largely achieved by inhibiting u-PAR gene expression. This was supported by the clinical observation that the loss of Pdcd4, in parallel to an increase of u-PAR gene expression, is an independent prognostic marker in colorectal cancer, Pdcd4 being essentially downregulated in cancer cells by microRNA-21.

Selected publications

  • Heiss MM, Babic R, Allgayer H, Gruetzner U, Jauch KW, Loehrs U, Schildberg FW (1993): Tumor-associated proteolysis and prognosis: New functional risk factors in gastric cancer defined by the urokinase-type plasminogen activator system. Journal of Clinical Oncology, Vol. 13, No. 8, pp. 2084-2093, 1995.
    http://www.ncbi.nlm.nih.gov/pubmed/7636552
  • Allgayer H, Heiss MM, Riesenberg R, Gruetzner KU, Tarabichi A, Babic R, Schildberg FW: Urokinase plasminogen activator receptor (uPA-R) -- a potential characteristic of metastatic phenotypes in minimal residual tumor disease. Cancer Research 57: 1394 - 1399, 1997.
    http://www.ncbi.nlm.nih.gov/pubmed/9102229
  • Allgayer H, Wang H, Wang Y, Heiss MM, Bauer R, Nyormoi O, Boyd D: Transactivation of the urokinase-type plasminogen activator receptor gene through a novel promoter motif bound with an activator-protein-2a-related factor. Journal of Biological Chemistry 274 (8): 4702 - 4714, 1999.
    http://www.ncbi.nlm.nih.gov/pubmed/9988707
  • Allgayer H, Wang H, Gallick GE, Crabtree A, Mazar A, Jones T, Kraker AJ, Boyd DD: Transcriptional induction of the urokinase-receptor (u-PAR) gene by a constitutively active Src: Requirement of an upstream motif (-152/-135) bound with Sp1. Journal of Biological Chemistry 274 (26), 18428 – 18437, 1999.
    http://www.ncbi.nlm.nih.gov/pubmed/10373450
  • Allgayer H, Wang H, Shirasawa S, Sasazuki T, Boyd D: Targeted disruption of the K-Ras oncogene in an invasive colon cancer cell line downregulates urokinase receptor expression and plasminogen-dependent proteolysis. British Journal of Cancer 80, 1884, 1999.
    http://www.ncbi.nlm.nih.gov/pubmed/10471035
  • Dang J, Boyd D, Wang H, Allgayer H, Doe W, Wang Y: A region between -141 and -61 bp containing a proximal AP-1 is essential for constitutive expression of urokinase-type plasminogen activator receptor. European Journal of Biochemistry 264(1):92-9, 1999.
    http://www.ncbi.nlm.nih.gov/pubmed/10447677
  • Wang Y, Dang J, Wang H, Allgayer H, Murrell GA, Boyd D: Identification of a novel nuclear factor-kappaB sequence involved in expression of urokinase-type plasminogen activator receptor. European Journal of Biochemistry 67(11): 3248-3254, 2000.
    http://www.ncbi.nlm.nih.gov/pubmed/10824110
  • Schewe DM, Leupold JH, Boyd DD, Lengyel ER, Wang H, Gruetzner KU, Schildberg FW, Jauch KW, and Allgayer H: Tumor-specific transcription factor binding to an AP-2/Sp1 promoter element of the urokinase-receptor (u-PAR) promoter in a first large series of resected gastrointestinal cancers. Clinical Cancer Research 9(6): 2267-76, 2003.
    http://www.ncbi.nlm.nih.gov/pubmed/12796395
  • Schewe DM, Biller T, Maurer G, Leupold JH, Lengyel ER, Post S, and Allgayer H: Combination analysis of AP-1 family members, Sp1 and an AP-2a-related factor binding to different regions of the urokinase receptor (u-PAR) gene in a large series of resected gastrointestinal cancers. Clin Cancer Res 11(24): 8538-8548, 2005.
    http://www.ncbi.nlm.nih.gov/pubmed/16361535
  • Leupold JH, Yang H-S, Colburn NH, Boyd, DD, Lengyel ER, Post S, Allgayer H: Tumor suppressor Pdcd4 inhibits invasion and regulates urokinase-receptor (uPAR) gene expression via Sp-transcription factors. Oncogene 26(319): 4550-4562, 2007.
    http://www.ncbi.nlm.nih.gov/pubmed/17297470
  • Leupold JH, Asangani I, Lengyel ER, Post S, Allgayer H: Src-induced uPAR gene expression is also mediated via an AP-1 motif and is associated with increased invasive capacity in vivo. Molecular Cancer Research 5(5):485-496, 2007.
    http://www.ncbi.nlm.nih.gov/pubmed/17510314
  • Mudduluru G, Medved F, Grobholz R, Joste C, Gruber A, Leupold JH, Post S, Jansen A, Colburn NH, Allgayer H: Loss of Pdcd4 expression marks adenoma-carcinoma transition, correlates inversely with pAkt, and is a new and independent prognostic factor in resected colorectal cancer. Cancer 110:8, 1697-1707, 2007.
    http://www.ncbi.nlm.nih.gov/pubmed/17849461
  • Nikolova D, Asangani I, Nelson L, Hughes D, Siwak D, Mills G, Harms A, Buchholz E, Pilz L, Manegold C, Allgayer H: Cetuximab attenuates invasion, metastasis, and u-PAR gene expression in non small-cell lung cancer (NSCLC), and u-PAR, besides E-cadherin, is a novel biomarker of Cetuximab sensitivity. Cancer Research 69(6):2461-2670, 2009.
    http://www.ncbi.nlm.nih.gov/pubmed/19276367
  • Rasheed SAK, Efferth T, Asangani IA, Allgayer H: Artesunate - first evidence that an anti-malaria drug inhibits invasion and in vivo metastasis in lung cancer by targeting essential extracellular proteases. Int J Cancer 127(6):1475-85, 2010.
    http://www.ncbi.nlm.nih.gov/pubmed/20232396

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