No abstract

SummaryBreakdowno ft he extracellular matrix is crucial forc ancer invasion and metastasis.Iti sa ccomplishedb yt he concerted action of several proteases, including the serine protease plasmin and anumberofmatrix metalloproteases.Theactivity of each of these proteasesisregulated by an arrayofactivators, inhibitors and cellular receptors.Thus,the generation of plasmininvolves the pro-enzyme plasminogen,the urokinaset ype plasminogen activator uPAa nd its pro-enzyme pro-uPA, the uPAi nhibitor PAI-1, the cell surfaceu PA receptor uPAR, and thep lasmin inhibitor α 2 -antiplasmin. Furthermore, the regulation of extracellular proteolysis in cancerinvolves acomplex interplaybetween cancercells and non-malignant stromalcells in the expression of themolecular components involved.For some types of cancer, this cellular interplaymimicsthat observedinthe tissueofori- KeywordsCancer invasion, plasminogen activation, matrix degradation, tissueremodelling, cancertherapy gin duringnon-neoplastic tissue remodellingprocesses.We proposethat cancer invasion can be considered as uncontrolled tissue remodelling. Inhibitiono fe xtracellular proteases is an attractivea pproach to cancer therapy.B ecause proteasesh ave manyd ifferentf unctions in the normal organism, efficienti nhibitionwill have toxic sideeffects.In cancerinvasion,like in normal tissue remodellingp rocesses, therea ppearst ob eafunctional overlapbetween differentextracellular proteases.This redundancymeans thatcombinationsofproteaseinhibitorsmust be used.Such combination therapy, however, is alsolikelytoincrease toxicity.Therefore foreach typeofcancer,acombination of proteasei nhibitorst hat is optimised with respectt ob oth maximal therapeutic effect and minimal toxic side effects need to be identified.

We used linker insertion-deletion mutagenesis to study the catalytic domain of the Harvey murine sarcoma virus v-rasH transforming protein, which is closely related to the cellular rasH protein. The mutants displayed a wide range of in vitro biological activity, from those that induced focal transformation of NIH 3T3 cells with approximately the same efficiency as the wild-type v-rasH gene to those that failed to induce any detectable morphologic changes. Correlation of transforming activity with the location of the mutations enabled us to identify three nonoverlapping segments within the catalytic domain that were dispensable for transformation and six other segments that were required for transformation. Segments that were necessary for guanosine nucleotide (GDP) binding corresponded to three of the segments that were essential for transformation; two of the three segments share strong sequence homology with other purine nucleotide-binding proteins. Loss of GDP binding was associated with apparent instability of the protein. Lesions in two of the three other required regions significantly reduced GDP binding, while small lesions in the last required region did not impair GDP binding or membrane localization. We speculate that this latter region interacts with the putative cellular target of ras. The results suggest that transforming ras proteins require membrane localization, guanosine nucleotide binding, and an additional undefined function that may represent interaction with their target.

We have characterized a transcriptional enhancer of the human urokinase‐type plasminogen activator (uPA) gene and found a regulatory element required for co‐operation between a PEA3‐‐AP‐1 element and an AP‐1 site in the enhancer. We designated this regulatory element co‐operation mediator (COM). Both the PEA3‐‐AP‐1 element, the AP‐1 site and the COM are required for efficient phorbol ester induction of transcription from the uPA promoter in the HepG2 hepatoma cell line. We show that the COM is also required for co‐operation between the PEA3‐‐AP‐1 element and a glucocorticoid response element, both in the presence or absence of TPA, indicating that the COM is generally capable of mediating synergism between inducible enhancer elements. The COM contains multiple overlapping binding sites for nuclear proteins, designated uPA enhancer factors 1–4 (UEF‐1‐4). We have identified putative binding sites for UEF‐1, −2 and −3. The UEF‐1 and −3 sites in the uPA enhancer are highly conserved between species. We demonstrate the binding of UEF‐3 to the NIP element, a previously characterized regulatory element in the human interleukin‐3 and stromelysin promoters, suggesting that this factor plays a role in regulation of a variety of genes.

A prominent phenotype of plasmin deficiency in mice is reduced metastasis in the MMTV-PymT transgenic breast cancer model. Proteolytically active plasmin is generated from inactive plasminogen by one of 2 activators, uPA or tPA. We now find that uPA deficiency alone significantly reduces metastasis >7-fold in the MMTV-PymT model. We studied a cohort of 55 MMTV-PymT transgenic mice, either uPA-deficient or wild-type controls. Tumor incidence, latency, growth rate and final primary tumor burden were not significantly affected by uPA deficiency. In contrast, average lung metastasis volume was reduced from 1.58 mm 3 in wild-type controls to 0.21 mm 3 in uPA-deficient mice (p ‫؍‬ 0.023). Tumor cell dissemination to brachial lymph nodes was also reduced from 53% (28/53) in wild-type controls to 31% (17/54) in uPA-deficient mice (p ‫؍‬ 0.032). Mice without plasminogen display a severe pleiotropic phenotype. By comparison, spontaneous phenotypes are modest in uPA-deficient mice, probably because they still have active tPA. We show that metastasis is strongly and selectively decreased in uPA-deficient mice, suggesting that uPAdirected antimetastatic therapy would be efficacious and have limited side effects.