Identification of a novel urokinase receptor splice variant and its prognostic relevance in breast cancer.
The cellular receptor for urokinase-type plasminogen activator, uPAR, plays a central role in both cell surface-associated proteolysis and cellular adhesion. In the present study, we systematically searched for splice variants of uPAR mRNA in human cells and tumor tissues by qualitative RT-PCR using specific primers for uPAR exons 1 and 6. Beside the wild-type (wt) uPAR mRNA and the previously described splice variant lacking exon 5 (uPAR-del5), a novel splice variant lacking both exons 4 and 5 (uPAR-del4/5) was found predominantly in various cancer cell lines. To elucidate whether alternatively spliced uPAR mRNA may be translated and post-translationally processed, we generated stably transfected Chinese hamster ovary cells, which harbor expression plasmids of wt uPAR and various uPAR variants including uPAR-del5 and uPAR-del4/5. By ELISA, flow cytofluorometry, and Western blot analysis, we confirmed synthesis and secretion of wt uPAR and the uPAR variants by the use of domain-specific monoclonal antibodies against uPAR. For quantification of uPAR mRNA variants, we established two highly sensitive real-time RT-PCR assays based on LightCycler technology. Study of their expression in a representative set of breast cancer tissues indicated that the novel mRNA variant uPAR-del4/5 was expressed very frequently and independently of uPAR mRNA variants covering exon 4 (uPAR-wt and uPAR-del5). Higher uPAR-del4/5 expression was significantly associated with shorter disease-free survival (p = 0.0004) of breast cancer patients. These results suggest that uPAR-del4/5 mRNA may serve as a novel prognostic marker in breast cancer.
Luther
Thomas
T
Institut für Pathlogie, Technische Universität Dresden, Germany. t.luther@labor-bautzen.de
Kotzsch
Matthias
M
Meye
Axel
A
Langerholc
Thomaz
T
Füssel
Susanne
S
Olbricht
Natalie
N
Albrecht
Sybille
S
Ockert
Detlev
D
Muehlenweg
Bernd
B
Friedrich
Katrin
K
Grosser
Marianne
M
Schmitt
Manfred
M
Baretton
Gustavo
G
Magdolen
Viktor
V
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D017398:Alternative Splicing; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D016466:CHO Cells; D045744:Cell Line, Tumor; D006224:Cricetinae; D018572:Disease-Free Survival; D004797:Enzyme-Linked Immunosorbent Assay; D005091:Exons; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D008875:Middle Aged; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D011379:Prognosis; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000235:genetics*; D055293:Receptors, Urokinase Plasminogen Activator; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection
D000911:Antibodies, Monoclonal; C524898:PLAUR protein, human; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator