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p06/0412(S)-hydroxyeicosatetraenoic [12(S)-HETE], a lipoxygenase metabolite of arachidonic acid, has been demonstrated to enhance metastatic capacity during tumor progression via its receptor. Using cloning and binding strategy, we have identified a G-protein coupled receptor, GPR31, as a 12(S)-HETE receptor. In this project, we aimed to characterize the biochemical properties of GPR31 and study the role of GPR31 in tumor survival and metastasis. We have constructed a secondary structure model for GPR31 and using the structural characteristics, we designed two peptides to produce antibodies that against both sides of this membrane protein. We have analyzed the expression of GPR31 in cell lines and human prostate specimens. The GPR31 protein was expressed in various cancer cells and stained in prostate cancer tissue. In order to further characterize this receptor, we designed a series experiments to determine its biochemical properties. Binding experiments with or without GPR31 expressed in Chinese hamster ovary cells demonstrated that this receptor has a Kd value of 4.8 for [3H]12(S)-HETE specific binding with Bmax value of 38.3 pmol/mg protein. We also purified the two antibodies from immunized rabbit antiserums. This project will provide significant insights into the process of prostate cancer progression and metastasis., The original document contains color images.

A significant achievement in the current reporting period is our ability to immunostain both the 12-LOX protein and Beta4 integrin in paraffin- embedded prostate tumor tissues circumventing the problems described in the previous report. With the new protocol we have stained about 20 cases so far and the remaining cases are in progress. We have generated several stable transfectants of PC-3 cells expressing mutant forms of the Beta4 integrin and studied their interaction with 12-LOX. During this study we have identified that the peptide spanning between amino acids 1126 and 1315 of the cytoplasmic tail of the Beta4 integrin shows strong interaction with 12-LOX. An important observation is that the full-length cytoplasmic tail of Beta4 integrin when expressed ectopically disrupts the interaction with 12-LOX with Beta4 integrin in a dominant negative manner. This interaction also resulted in a decrease in the biosynthesis of the enzymatic product 12-HETE as well as reduction in the tumor growth rate from subcutaneously injected PC-3 cells in athymic nu/nu mice., The original document contains color images.

A significant achievement in the current reporting period is our ability to immunostain both the 12-LOX protein and 4 integrin in paraffin-embedded prostate tumor tissues circumventing the problems described in the previous report. With the new protocol we have stained about 20 cases so far and the remaining cases are in progress. We have generated several stable transfectants of PC-3 cells expressing mutant forms of the 4 integrin and studied their interaction with 12-LOX. During this study, we have identified that the peptide spanning between amino acids 1126 and 1315 of the cytoplasmic tail of the 4 integrin shows strong interaction with 12-LOX. An important observation is that the full-length cytoplasmic tail of 4 integrin, when expressed ectopically, disrupts the interaction with 12-LOX with 4 integrin in a dominant negative manner. This interaction also resulted in a decrease in the biosynthesis of the enzymatic product, 12-HETE, as well as reduction in the tumor growth rate from subcutaneously injected PC-3 cells in athymic nu/nu mice., The original document contains color images.

During the progression of human PCa, hemidesmosomes are lost. Hemidesmosomes are adhesion structures that anchor epithelial cells to basement membrane and function as a tumor suppressor. We found that 12-lipoxygenase directly interacts with beta4 integrin. We hypothesize that an increase in 12-LOX activity can cause the disassembly of hemidesmosomes, mobilization of alpha6beta4 integrin from hemidesmosomes to other parts of the cell membrane, and stimulate tumor invasive growth. We proposed to conduct a correlation study using clinical tumor specimens. We will study whether 12(S)-HETE can disrupt hemidesmosomes and whether 12-LOX inhibitors promote the formation of hemidesmosomes. Then we will study the underlying signaling pathway, especially PKCalpha, initiated by 12(S)-HETE, in the disassembly of hemidesmosomes. Next we will overexpress beta4 integrin and study the role of the interaction between 12-LOX and beta4 integrin in the adhesion, proliferation, migration, and survival, in response to HGF/SF. Finally we will xenograft these transfected cells into mice, to evaluate whether any phenotypic changes of tumor cells in vitro can be recapitulated in vivo. The work will significantly advance our understanding about the complex process of prostate cancer progression as well as the possible role played by dietary fat in the progression of prostate cancer.

The initiation and progression of prostate cancer remain not well understood to enable development of interventional therapy. Thromboxane synthase is an enzyme downstream of cyclooxygenase, utilizing prostaglandin H to form thromboxane A2. Using immunohistochemistry analysis, we found that 25% of clinical prostate tumor specimens had strong expression of thromboxane synthase; 33% of cases had medium expression and 42% of cases had weak expression of thromboxane synthase. Prostate cancer cells isolated from lymph node metastasis had higher levels of thromboxane synthase expression and activity than those isolated from the primary tumor sites in an animal model. We cloned and sequenced full-length thromboxane synthase cDNA from PC-3 cells and constructed an expression vector. Increased expression of thromboxane synthase in DU145 cells was found to stimulate cell migration. Inhibition of thromboxane synthase or blockade of thromboxane A2 function inhibited prostate cancer cell migration. Further we found that PCa cells express receptors for TXA2 and stimulation with TXA2 mimetic led to the activation of RhoA and cell retraction. Our study suggest that thromboxane synthase and its eicosanoid product play a contributory role in prostate cancer progression.

Angiogenesis is an integral part of human prostate cancer progression. In the Phase I of this grant, we have identified arachidonate 12-lipoxygenase as a promoter of angiogenesis in human prostate carcinoma by stimulating VECF expression and via the pro-angiogenic activity of 12(S)-HETE. The first objective of phase II of this grant is to delineate the regulation of VEGF gene expression by 12-LOX in PCa cells, specifically the involvement of Spl and possibly AP2. The second objective of this proposal is to describe the signal transduction pathway by which 12-LOX stimulates VEGF expression. These two objectives are based upon our observations in the Phase I of this grant that 12-LOX, when overexpressed in human PCa cells, stimulates tumor angiogenesis and growth. The third objective to evaluate the therapeutic potential of 12-LOX inhibitors for PCa treatment., The original document contains color images. All DTIC reproductions will be in black and white.

The initiation and progression of prostate cancer remain not well understood to enable rational development of interventional therapy. Thromboxane synthase is an enzyme downstream cyclooxygenase, utilizing prostaglandin H to form thromboxane A2. Using immunohistochemistry analysis, we found that 25% of clinical prostate tumor specimens had strong expression of thromboxane synthase; 33% of cases had medium expression and 42% of cases had weak expression of thromboxane synthase. Prostate cancer cells isolated from lymph node metastasis had higher levels of thromboxane synthase expression and activity than those isolated from the primary tumor sites in an animal model. We cloned and sequenced full-length thromboxane synthase cDNA from PC-3 cells and constructed an expression vector. Increased expression of thromboxane synthase in DUl45 cells was found to stimulate cell migration. Inhibition of thromboxane synthase or blockade of thromboxane A2 function inhibited prostate cancer cell migration. Our study suggest that thomboxane synthase and its eicosanoid product play a contributory role in prostate cancer progression.

The initiation and progression of prostate cancer remain not well understood to enable rational development of interventional therapy. Thromboxane synthase is an enzyme downstream cyclooxygenase, utilizing prostaglandin H to form thromboxane A2. Using immunohistochemistry analysis, we found that 25% of clinical prostate tumor specimens had strong expression of thromboxane synthase; 33% of cases had medium expression and 42% of cases had weak expression of thromboxane synthase. Prostate cancer cells isolated from lymph node metastasis had higher levels of thromboxane synthase expression and activity than those isolated from the primary tumor sites in an animal model. We cloned and sequenced full-length thromboxane synthase cDNA from PC-3 cells and constructed an expression vector. Increased expression of thromboxane synthase in DUl45 cells was found to stimulate cell migration. Inhibition of thromboxane synthase or blockade of thromboxane A2 function inhibited prostate cancer cell migration. Our study suggest that thomboxane synthase and its eicosanoid product play a contributory role in prostate cancer progression.