Book of abstract 2008

Clinical use of cleaved forms of the urokinase receptor
Gunilla Høyer Hansen, Charlotte Almasi, Helle Pappot, Ib Jarle Christensen
Finsen Laboratory, Copenhagen Biocenter, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark
It is well established that the urokinase plasminogen activator, uPA and its cellular
receptor uPAR are involved in cancer invasion and metastasis and the tumor tissue and
circulating levels correlate with cancer patient survival. In addition to the cellular binding,
uPA cleaves uPAR in the linker region between domains I and II and thereby inactivates
uPAR´s binding potential. Cleavage of uPAR thus reflects the activity of uPA and possibly
the aggressiveness of the tumor. uPA in physiological relevant concentrations cleaves
glycolipid-anchored uPAR but not soluble uPAR (suPAR). To elucidate the mechanism
of uPA-mediated uPAR cleavage and identify the different uPAR forms we have used
PMA–stimulated U937 cells. On the cell surface as well as inside the cell, both intact
uPAR(I-III) and cleaved uPAR(II-III) are present, whereas in the media only intact suPAR
and uPAR(I) are detected. Treatment of the cells with Pi-PLC releases both uPAR(I-III)
and uPAR(II-III) from the cell surface. Biotinylation of cell surface proteins showed that
when uPAR cleavage was inhibited by incubation with a neutralizing anti-uPA antibody,
uPAR(II-III) was not found on the cell surface. The intracellular pattern of uPAR(I-III)
and uPAR(II-III) was not affected by inhibiting the cell surface cleavage. However, in
the media from cells grown in the presence of the neutralizing anti-uPA antibody, both
suPAR(I-III) and suPAR(II-III) were present. This indicates that uPAR(II-III) is shed
from the cell surface when uPA-mediated cleavage of uPAR is blocked.
In tumor tissue and body fluids both intact and cleaved forms of uPAR have been
identified using Western blotting and immunoassays quantifying the individual uPAR
forms. In tumor extracts from patients with non-small cell lung cancer uPAR(I) is
a stronger prognostic marker then the total amount of all uPAR forms. Also in serum
from patients with non-small cell lung cancer the level of uPAR(I) correlates with overall
survival. The concentrations of uPAR(I) as well as suPAR(II-III) are significantly elevated
in serum samples
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from patients with prostate cancer compared to the concentrations in
serum from men with benign prostatic conditions. Furthermore, specific measurements
of uPAR(I) were found to improve specificity of prostate cancer detection. Similarly high
concentration of plasma uPAR(I) is an independent preoperative marker of poor prognosis
in patients with ovarian cancer. The combination of plasma suPAR(I-III)+suPAR(II-III)
and CA125 discriminates between malignant and benign ovarian tumors with an AUC
of 0.94. We will verify our findings in a larger collection of plasma/serum samples from
prostate, ovarian and non-small cell lung cancer patients and investigate the prognostic
significance and possible diagnostic utility of cleaved uPAR variants in other forms of
cancer.
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