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The membrane-associated prostasin and matriptase belonging to the S1A subfamily of serine proteases, are critical for epithelial development and maintenance. The two proteases are involved in the activation of each other and are both regulated by the protease inhibitors, HAI-1 and HAI-2. The S1A subfamily of serine proteases are generally produced as inactive zymogens requiring a cleavage event to obtain activity. However, contrary to the common case, the zymogen form of matriptase exhibits proteolytic activity, which can be inhibited by HAI-1 and HAI-2, as for the activated counterpart. We provide strong evidence that also prostasin exhibits proteolytic activity in its zymogen form. Furthermore, we show that the activity of zymogen prostasin can be inhibited by HAI-1 and HAI-2. We report that zymogen prostasin is capable of activating zymogen matriptase, but unable to activate its own zymogen form. We propose the existence of an unusual enzyme-enzyme relationship consisting of proteolytically active zymogen forms of both matriptase and prostasin, kept under control by HAI-1 and HAI-2, and located at the pinnacle of an important proteolytic pathway in epithelia. Perturbed balance in this proteolytic system is likely to cause rapid and efficient activation of matriptase by the dual action of zymogen matriptase and zymogen prostasin. Previous studies suggest that the zymogen form of matriptase performs the normal proteolytic functions of the protease, whereas excess matriptase activation likely causes carcinogenesis. HAI-1 and HAI-2 are thus important for the prevention of matriptase activation whether catalysed by zymogen/activated prostasin (this study) or zymogen/activated matriptase (previous studies).

This project had one Specific Aim after the peer and programmatic reviews, to evaluate prostasin, a glycosylphosphatidylinositol (GPI)-anchored extracellular serine protease as a potential metastasis suppressor of breast cancer in nude mice models. Two types of breast cancer metastasis, experimental and spontaneous, were to be used with the cell lines MDA-MB-231 and MDA-MB-435, respectively. We have found that prostasin must be activated by another membrane serine protease, matriptase before becoming functionally active as a proteolytic enzyme. The activating enzyme matriptase, however, is not expressed by either of the two model cell lines, an essential factor previously unrecognized, as well as an underlying reason for our previous inconsistent findings concerning prostasin s impact on breast cancer cell metastasis. In future research on membrane serine proteases in breast cancer cell biology, the newly recognized proteolytic cascade should be addressed with consideration of all of its current and potentially new member proteases., The original document contains color images.

The focus of the project for this report period was the establishment of prostate cancer cell lines with inducible expression of prostasin, for which a role as a tumor suppressor has been implicated. Meanwhile, mapping of prostasin signal transduction pathways was underway using a gene expression knock-down approach in the LNCaP Human prostate cancer cell line. A tetracycline-inducible, prostasin expressing DU-145 cell line has been established, this cell line will allow for proper assessment of prostasin's role as an invasion suppressor and a tumor suppressor. Prostasin serine protease and its serine active-site mutant were able to reduce the motility of DU-145 human prostate cancer cells. Prostasin gene silencing by siRNA in the LNCaP human prostate cancer cells resulted in a down-regulation of the cycln-dependent kinase inhibitor, p21CIP1 at the protein level but not the MRNA level, further suggesting a tumor suppressor role for prostasin serine protease. The protein level change of p21CIP1 is a lead for pathway mapping of prostasin signal transduction.

We have previously shown that membrane-anchored prostasin serine protease was an in vitro suppressor of tumor cell invasion (1-3) . The expression of prostasin is down-regulated in prostate cancers (2), and invasive cancer cell lines of the human prostate and breast (1, 3). We hypothesized that the down-regulation of prostasin is causal to breast cancer invasion and metastasis in vivo. Two highly invasive human breast cancer cell lines, the MDA-MB-231 and MDA-MB-435, were transfected with a prostasin cDNA plasmid to restore prostasin expression. The transfectants of MDA-MB-231 were used in the nude mice model to assess the metastatic potential. The results demonstrated a statistically significant difference of metastasis between cells that express prostasin and the control cells. Prostasin expression in the transfected cells was analyzed by immunocytochemistry and clones that express prostasin more uniformly were obtained for repeat experiments in nude mice for the purpose of maximizing the effect of prostasin., The original document contains color images. All DTIC reproductions will be in black and white.

The original purpose was to identify a receptor for maspin, a serpin tumor suppressor of breast and prostate cancers. Prostasin and hepsin serine proteases were examined as candidates. Prostasin was found to be down-regulated in high-grade prostate cancers and absent in invasive prostate and breast cancer cells, in which prostasin promoter hypermethylation was found. Prostasin was shown to be an invasion suppressor, via its GPI-anchored membrane form. Prostasin is involved in epithelial sodium channel activation and is regulated by the serpin protease nexin-1 (PN-1). Other cellular protein changes elicited by prostasin expression were also observed. A new project has been initiated to investigate the mechanisms of prostasin's anti-invasion function. A second new project has been initiated to evaluate the potential of using prostasin as a metastasis suppressor of breast cancer. A new hypothesis is proposed for a potential indirect interaction between maspin and prostasin based on their individually confirmed roles in sodium transport regulation. We demonstrated an up-regulation of hepsin in prostate cancer, but no direct interaction was established between maspin and hepsin at the biochemical or cellular level. Drosophila genetics was and will continue to be employed to investigate hepsin's role in cancer., The original document contains color images. All DTIC reproductions will be in black and white.

The purpose was to identify a maspin receptor. Prostasin and hepsin serine proteases were examined as candidates. Prostasin down-regulation observed in high-grade prostate cancer eliminated this protease as a candidate. Prostasin expression is absent in highly invasive DU-145 and PC-3 cells, while expressed in normal human prostate epithelial cells and the non-invasive LNCaP cells. A forced re-expression of prostasin in DU-145 and Pc-3 cells reduced the invasiveness by 68% and 42%, respectively, while showing no effect on cell proliferation. The anti-invasion property was associated with cellular prostasin, which exists on cell membrane as an active protease via a GPI-anchor. In DU-145 and PC-3 cells expressing recombinant prostasin, a protein band at 120-130 kDa range in SDS-PAGE was found to be reduced in tyrosine phosphorylation while a reduction of protein kinase C alpha expression was also observed. These cellular protein changes elicited by prostasin expression provide leads for investigation of prostasin anti-invasion signal transduction. We demonstrated an up-regulation of hepsin in prostate cancer and are in pursuit of maspin-hepsin interaction by yeast genetics. Drosophila genetics will also be employed to investigate hepsin's role in cancer. Our findings may lead to the development of diagnostics or drugs leads., Original contains color plates: All DTIC reproductions will be in black and white.

The purpose of this project was to identify a maspin receptor, and to investigate the signal transduction in maspin-induced cancer suppression. Human prostasin was examined as a candidate for maspin receptor. Our major progress is in the following areas. First, prostasin expression is found down-regulated in prostate cancer. Second, PC-3 cells transfected with a human prostasin cDNA displayed a significantly reduced level of invasiveness (50-80% inhibition). Third, the prostasin-transfected PC-3 cells have an increased maspin expression, suggesting a concerted tumor suppression mechanism between these two proteins. Fourth, prostasin expression in LNCaP cells can be up-regulated by dihydrotestosterone, suggesting a possibility that prostasin's down-regulation in prostate cancer is not due to gene damage. Fifth, a human maspin-expressing fruit-fly strain has been established, as well as a strain that expresses human hepsin, a second candidate for maspin receptor. Overall, we have tested the initial hypothesis at or exceeding the pace intended. Several unexpected findings were made regarding the behaviors of human prostasin, and the stage is set for the follow-through investigation on how maspin and prostasin work synergistically to suppress prostate cancer. A prostate cancer therapy and methods of diagnosis are being developed as part of the extended goal of our research., Original contains color plates: All DTIC reproductions will be in black and white.