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Thromboxane A2 (TxA2) is a prostanoid formed by thromboxane synthase using the cyclooxygenase product prostaglandin H2 as the substrate. Previously, increased expression of thromboxane synthase was found in prostate tumors, and tumor cell motility was attenuated by inhibitors of thromboxane synthase. This study was undertaken to elucidate how tumor motility is regulated by TxA2. Here, we report that human prostate cancer cells express functional receptors for TxA2 (TP). Ligand binding assay found that PC-3 cells binded to SQ29548, a high-affinity TP antagonist, in a saturable manner with Kd of 3.64 nmol/L and Bmax of 120.4 fmol per million cells. Treatment of PC-3 cells by U46619, a TP agonist, induced PC-3 cell contraction, which was blocked by pretreatment with the TP antagonist SQ29548 or pinane TxA2. The migration of prostate cancer cells was significantly inhibited either by sustained activation of TP or by blockade of TP activation, suggesting that TP activation must be tightly controlled during cell migration. Further studies found that small GTPase RhoA was activated by TP activation, and pretreatment of PC-3 cells with Y27632, a Rho kinase (ROCK) inhibitor, blocked U46619-induced cell contraction. A dominant-negative mutant of RhoA also blocked U46619-induced cell contraction. Taken together, the data suggest that TPs are expressed in prostate cancer and activation of TPs regulates prostate cancer cell motility and cytoskeleton reorganization through activation of Rho. [Cancer Res 2008;68(1):115–21]

Arachidonic acid metabolism leads to the generation of biologically active metabolites that regulate cell growth and proliferation, as well as survival and apoptosis. We have demonstrated previously that platelet-type 12-lipoxygenase (LOX) regulates the growth and survival of a number of cancer cells. In this study, we show that overexpression of platelet-type 12-LOX in prostate cancer PC3 cells or epithelial cancer A431 cells significantly extended their survival and delayed apoptosis when cultured under serum-free conditions. These effects were shown to be a result of enhanced surface integrin expression, resulting in a more spread morphology of the cells in culture. PC3 cells transfected with 12-LOX displayed increased alpha(v)beta(3) and alpha(v)beta(5) integrin expression, whereas other integrins were unaltered. Transfected A431 cells did not express alpha(v)beta(3); however, alpha(v)beta(5) integrin expression was increased. Treatment of both transfected cell lines with monoclonal antibody to alpha(v)beta(5) (and in the case of PC3 cells, anti-alpha(v)beta(3)) resulted in significant apoptosis. In addition, treatment with 100 nM 12(S)-hydroxy-eicosatetraenoic acid, the end product of platelet-type 12-LOX, but not other hydroxy-eicosatetraenoic acids, enhanced the survival of wild-type PC3 and A431 cells and resulted in increased expression of alpha(v)beta(5). Furthermore, Baicalein or N-benzyl-N-hydroxy-5-phenylpentamide, specific 12-LOX inhibitors, significantly decreased alpha(v)beta(5)-mediated adhesion and survival in 12-LOX-overexpressing cells. The results show that 12-LOX regulates cell survival and apoptosis by affecting the expression and localization of the vitronectin receptors, alpha(v)beta(3) and alpha(v)beta(5), in two cancer cell lines.

Extensive studies have implicated the role of dietary fatty acids in prostatecancer progression. Platelet-type 12-Lipoxygenase (12-LOX) has beenshown to regulate growth, metastasis, and angiogenesis of prostate cancer. The effect of two 12-LOX inhibitors, Baicalein and N-benzyl-N-hydroxy-5-phenylpentamide (BHPP), on the mechanisms controlling cell cycle progression and apoptosis were examined in two prostate cancer cell lines, PC3 and DU-145. Treatment with Baicalein or BHPP resulted in a dose-dependent decrease in cell proliferation, as measured by BrdUrd incorporation. This growth arrest was shown to be because of cell cycle inhibition at G0/G1, and was associated with suppression of cyclin D1 and D3 protein levels. PC3 cells also showed a strong decrease in phosphorylated retinoblastoma (pRB) protein, whereas the other retinoblastoma-associated proteins, p107 and p130, were inhibited in DU-145 cells. Treatment with 12-hydroxyeicosatetraenoic acid in the presence of Baicalein blocked loss of pRB, whereas 12(S)-HETE alone induced pRB expression. Treatment with either Baicalein or BHPP resulted in significant apoptosis in both cell lines as measured by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. DU-145 cells underwent apoptosis more rapidly than PC-3 cells. The mechanisms involved were decreased phosphorylation of Akt, loss of survivin and subsequent activation of caspase-3 and caspase-7 in each cell line, decreased Bcl-2 and Bcl-X(L) expression in DU-145, and a shift in Bcl-2/Bax levels favoring apoptosis in PC-3 cells. Addition of 12(S)-HETE protected both cell lines from Baicalein-induced apoptosis, whereas other LOX metabolites, 5(S)-HETE, or 15(S)-HETE did not. These results show that the 12-LOX pathway is a critical regulator of prostate cancer progression and apoptosis, by affecting various proteins regulating these processes. Therefore, inhibition of 12-LOX is a potential therapeutic agent in the treatment of prostate cancer.

Integrin alphaIIb beta3 requires its cytoplasmic tails to participate in tumor cell adhesion, spreading, and migration. Using 3' rapid amplification of cDNA ends, we have amplified two alphaIIb cDNAs from human leukemia, prostate adenocarcinoma, and melanoma cells. One of these is the predicted wild-type alphaIIb cDNA, and the other is a novel truncated alphaIIb variant. This variant is unique in that it lacks the transmembrane and cytoplasmic portions of the alphaIIb light chain. The truncated alphaIIb integrin protein is expressed by human leukemia, prostate adenocarcinoma, and melanoma cells but not by platelets or normal prostate epithelial or normal breast epithelial cells. Tumor cells secrete this protein and deposit it on the extracellular matrix. To our knowledge, this is the first report of a naturally occurring variant of an alpha integrin that lacks the transmembrane and cytoplasmic tail.

Previously, we found a positive correlation between the expression of platelet-type 12-lipoxygenase (12-LOX) and the progression of human prostate adenocarcinoma (PCa; Gao et al., Urology, 46: 227-237, 1995). To determine the role of 12-LOX in PCa progression, we generated stable 12-LOX-transfected PC3 cells, which synthesize high levels of 12-LOX protein and 12(S)-hydroxyeicosatetraenoic acid metabolite. In vitro, 12-LOX-transfected PC3 cells demonstrated a proliferation rate similar to neo controls. However, following s.c. injection into athymic nude mice, 12-LOX-transfected PC3 cells formed larger tumors than did the controls. Decreased necrosis and increased vascularization were observed in the tumors from 12-LOX-transfected PC3 cells. Both endothelial cell migration and Matrigel implantation assays indicate that 12-LOX-transfected PC3 cells were more angiogenic than their neo controls. These data indicate that 12-LOX stimulates human PCa tumor growth by a novel angiogenic mechanism.

3p deletion, a common chromosome defect in lung cancer, occurs more frequently in the lung tumor tissues of smoking patients than it does in those of nonsmoking patients. This pilot study evaluated whether 3p aberrations induced by benzo[a]pyrene diol epoxide (BPDE), the metabolic product of benzo[a]pyrene, a constituent of tobacco smoke, were more common in the peripheral blood lymphocytes of 40 lung cancer patients than they were in those of 54 matched controls. Our hypothesis was that 3p sensitivity to BPDE reflects the susceptibility of a specific locus to damage from carcinogens in tobacco smoke. BPDE-induced chromosome 3p21.3 aberrations were significantly more frequent in cases (34.1 per 1000) than they were in controls (22.1 per 1000; P0.0001). However, no such difference was observed for 6q27, a control locus. Using the median value in the controls (20 per 1000) as a cutoff point to classify BPDE-induced sensitivity at 3p21.3 and after adjustment by age, sex, ethnicity, and smoking status, 3p BPDE sensitivity was associated with an elevated risk of 14.1 (95% confidence interval: 3.5, 56.2) for lung cancer. There was also a dose-response relationship between the degree of BPDE sensitivity at 3p21.3 and increased risk for lung cancer. Therefore, 3p may be a molecular target for BPDE damage in lung cancer cases.

Integrins play an important role in mediating tumor cell-extracellular matrix (ECM) and tumor cell-endothelial cell interactions. The integrin alphaIIb beta3 (GPIIb-IIIa) is expressed on the surface of platelets in an inactive state and requires a conformational change to recognize extracellular matrix proteins such as fibrinogen, fibronectin, vitronectin, and others. In this study, we questioned whether human melanoma cells express the alphaIIb beta3 integrin. Reverse transcription-PCR/Southern blotting, Northern blotting, and dot blotting demonstrated the presence of the platelet-type alphaIIb beta3 integrin in human melanoma WM 983B, WM 983A, and WM 35 cells. AP-2, a monoclonal antibody (mAb) to alphaIIb beta3, positively stained two human melanoma specimens, indicating expression of this integrin in vivo. Phorbol 12-myristate 13-acetate and 12(S)-hydroxyeicosatetraenoic acid, two activators of protein kinase C, stimulated adhesion of melanoma cells to immobilized fibronectin and PAC-1, a mAb to alphaIIb beta3. PAC-1 specifically recognizes the conformationally active form of platelet alphaIIb beta3. Phorbol 12-myristate 13-acetate-stimulated adhesion of WM 983B cells to PAC-1 was completely blocked by an RGD peptide, thus providing evidence that tumor cell adhesion to PAC-1 is mediated via the alphaIIb beta3 integrin but not the Fc receptor. Confocal immunofluorescent studies demonstrated that fibronectin-adherent melanoma cells possess an intracellularly localized pool of high-affinity alphaIIb beta3. Invasion of WM 983B cells through fibronectin was stimulated by 12(S)-hydroxyeicosatetraenoic acid, and this stimulated invasion was blocked by the mAb PAC-1. The data suggest that melanoma cells express the high-affinity alphaIIb beta3 integrin, which is involved in tumor invasion.

The underlying mechanism by which elevated omega-6 fatty acids, like arachidonic acid, lead to increased prostate cancer (PCa) risk is unclear. However, it is known that the 12-Lipoxygenase (12-LOX) enzyme that metabolizes arachidonic acid, as well as the 12(S)-HETE metabolic end product together with its cognate receptor, 12-HETER1, are strongly associated with PCa. 12(S)-HETE affects many cancer phenotypes including cell migration and metastasis. It causes the disassembly of complex cellular anchors to the substrata called hemidesmosomes that are comprised of numerous proteins, including ITGB4, which we identified as an interacting partner of 12-LOX. ITGB4 also physically interacts with c-Met at the cell membrane in multiple cell lines, in a cell line and stimulus-specific manner. PCa metastases have increased levels of both 12-LOX and c-Met. Therefore we sought to determine if ITGB4, c-MET and 12-LOX comprise a signaling axis. Our findings revealed that HGF-c-Met-induced invasion could be attenuated by enzymatic inhibitors of 12LOX, but that addition of exogenous 12(S)-HETE could not overcome the block. Furthermore, the physical interaction between β4 and c-Met remained unaffected by enzymatic inhibitors in PCa cells. Likewise, LC-MS analyses, showed that HGF stimulation did not lead to increased 12(S)-HETE production, and knockdown of 12-HETER1, by shRNA had no effect on HGF-induced invasion. Therefore, we hypothesize that 12LOX has dual functions whereby its enzymatic activity leads to hemidesmosome dissociation and cell release from the substrata, and its capacity to modulate c-MET/ITGB4 signaling may be attributed to a scaffolding function. Citation Format: Elizabeth Tovar, Stephanie Tucker, Kenneth V. Honn. 12-Lipoxygenase regulation of prostate cancer progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5124. doi:10.1158/1538-7445.AM2013-5124

The integrin alphaIIb(beta)3 was initially believed to be expressed only in cells from the megakaryocytic lineage, such as platelets or HEL cells. In this study, we report for the first time that human prostate carcinoma PC-3 and DU-145 cells express alphaIIb(beta)3. Reverse transcription-PCR from HEL (positive control), PC-3, and DU-145 cells amplified a predicted alphaIIb fragment that hybridized to the full-length alphaIIb cDNA probe. DNA sequencing of the PCR fragments revealed 100% sequence homology to the corresponding extracellular domain of platelet alphaIIb but minimal sequence homology to integrins (alpha)v or a5. An RNase protection assay was used to confirm the results from reverse transcription-PCR. An antisense riboprobe to alphaIIb mRNA hybridized to total RNA from HEL, PC-3, and DU-145 cells, suggesting that alphaIIb mRNA is transcribed in these tumor cells. In situ hybridization on surgical specimens from human prostate tumor tissue stained positive with an antisense riboprobe to alphaIIb mRNA. The expression of alphaIIb(beta)3 protein in PC-3 and DU-145 cells was demonstrated by Western and dot blotting and flow cytometry with monoclonal antibodies (mAbs) to alphaIIb (MAB 1990), beta3, and alphaIIb(beta)3 (AP-2). A protein kinase C activator, phorbol 12-myristate 13-acetate, increased the adhesion of PC-3 cells to PAC-1, a mAb specific to the high-affinity state of alphaIIb(beta)3, by more than 80-fold. The invasion of DU-145 cells through a reconstituted basement membrane was blocked 40-50% by mAbs AP-2 or PAC-1. These data collectively suggest that: (a) prostate tumor cells express alphaIIb(beta)3; (b) surface expression of alphaIIb(beta)3 integrin is regulated by protein kinase C; and (c) mAbs to this receptor inhibit invasion of prostate cancer cells through a reconstituted basement membrane.

The binding of autocrine motility factor (AMF) to its cell surface receptor, gp78, stimulates tumor cell motility. In this report, we provide evidence that stimulation of gp78 by either AMF or a monoclonal antibody to gp78 (3F3A) increases adhesion and spreading of metastatic murine melanoma (B16a) cells on fibronectin. This gp78-regulated increase is mediated by up-regulation of surface alphaIIbbeta3++ and alpha5beta1 integrin receptors. In addition, AMF treatment of B16a cells increased translocation of alphaIIbbeta3 and alpha5beta1 from the cytoplasm to the cell surface. However, alphaIIbbeta3 and alpha5beta1 demonstrate separate and unique staining patterns at the surface of B16a cells in response to stimulation of gp78. Furthermore, stimulation of B16a cells with AMF increased their invasion through Matrigel. This stimulated invasion was inhibited by antibodies to alphaIIbbeta3 but not by antibodies to alpha5beta1. The increased integrin surface expression and function in response to AMF was blocked by N-benzyl-N-hydroxy-5-phenylpentanamide, an inhibitor of 12-lipoxygenase, and calphostin C, an inhibitor of protein kinase C. The results demonstrate that AMF stimulates integrin-mediated B16a cell adhesion, spreading, and invasion, and these events are regulated by a signaling pathway involving 12-lipoxygenases and protein kinase C.

A putative tumor suppressor gene, the BRCA1 gene, on chromosome 17q21 has recently been identified and shown to be mutated in breast and ovarian cancers. We have undertaken the present study to explore the possible involvement of the BRCA1 and/or other potential genes on chromosome 17q in prostate cancer. Twenty-three patients were screened by PCR for loss of heterozygosity at five microsatellite loci spanning the region of 17q12-21. One of the loci (i.e., D17S855) studied is intragenic to the BRCA1. Forty-four and 40% of the informative cases showed loss of heterozygosity at the BRCA1 (D17S855) and D17S856 loci, respectively, whereas 10%, 10%, and 11% of the informative cases were positive for loss of heterozygosity at the D17S250, D17S579, and D17S588 loci, respectively. Overall, 52% (11/21) of the informative cases have allelic loss of at least one locus on chromosome 17q. Our data suggest that the BRCA1 and/or other genes within the interval between BRCA1 and D17S856 on 17q21 may be important in the pathogenesis of prostate cancer.

A M(r) 55,000 tumor cell-secreted cytokine has been described which influenced the migration of the producing cells and was called autocrine motility factor (AMF). Activation of the cell surface receptor for AMF (gp78) was shown to stimulate production of a 12-lipoxygenase metabolite of arachidonic acid, 12-(S)-hydroxyeicosatetraenoic acid [12-(S)-HETE], in highly metastatic murine melanoma cells. AMF stimulated the motility of the high-metastatic (K1735-M1) but not the low-metastatic variant (K1735-Cl.11) of the K1735 murine melanoma and increased expression of the 12-lipoxygenase enzyme predominantly in the high-metastatic counterpart. The K1735-M1 cells responded to motile stimulation with increased endogenous 12-(S)-HETE production, and, reciprocally, exogenous 12-(S)-HETE up-regulated surface gp78 and caused gp78 translocation from an intracellular perinuclear pool to tubulovesicles which extended to the cell periphery in the K1735-M1 cells exclusively. These results suggest that differences in AMF responses may be due to alterations in the capacity of low-metastatic cells to transduce signals through 12-lipoxygenase or to involve downstream effector(s) of 12-(S)-HETE after gp78 activation.

12(S)-Hydroxyeicosatetraenoic acid [12(S)-HETE] is the 12-lipoxygenase metabolite of arachidonic acid. Previously, we have demonstrated that exogenous 12(S)-HETE can activate protein kinase C, increase cell surface expression of integrins, enhance adhesion, induce endothelial cell retraction, and increase experimental metastasis of tumor cells. Because of these prominent effects of exogenous 12(S)-HETE on tumor cell metastatic potential, it is important to determine whether there is endogenous 12(S)-HETE production by tumor cells. In the present study, mRNAs from human, rat, and mouse platelets as well as human colon carcinoma (Clone A), rat Walker carcinoma (W256), and mouse melanoma (B16a) and lung carcinoma (3LL) were reverse transcribed and amplified by polymerase chain reaction with platelet 12-lipoxygenase specific primers. Identity of the polymerase chain reaction fragments was confirmed by sequencing. 12-Lipoxygenase protein was detected by Western blotting. Tumor cell-derived 12-HETE was determined by reverse phase-high performance liquid chromatography analysis. In addition, the effect of endogenous 12(S)-HETE on tumor cells was studied by using a platelet-type 12-lipoxygenase selective inhibitor (N-benzyl-N-hydroxy-5-phenylpentanamide). Our results suggest that some tumor cells express platelet-type 12-lipoxygenase mRNA, protein and metabolize arachidonic acid to 12(S)-HETE and that endogenous 12(S)-HETE, like the exogenous 12(S)-HETE, may play an important role in tumor cell adhesion to matrix in vitro and lung colonization in vivo.

Tumor cell interaction with endothelial cells is a crucial step leading to organ-selective metastasis. Adhesion of murine B16 amelanotic melanoma cells (B16a) to murine microvascular endothelial cells (CD3) was enhanced, in a dose- and time-dependent manner, by pretreating CD3 cells with 12(S)-hydroperoxyeicosatetraenoic acid [i.e., 12(S)-HETE], a 12-lipoxygenase metabolite of arachidonic acid. The metabolic precursor of 12(S)-HETE, 12-HPETE (12-hydroperoxyeicosatetraenoic acid) also enhanced B16a cell adhesion to CD3 monolayers, whereas other lipoxygenase products, i.e., 5(S), 11(S), and 15(S)-HETEs were ineffective. 12(S)-HETE-enhanced tumor cell adhesion was blocked by treating endothelial cells with antibodies against the alpha v beta 3 complex or against individual subunits but not with antibodies against alpha 5 beta 1. In contrast, neither of these two integrins appeared to be involved in tumor cell adhesion to unstimulated endothelium. Flow cytometric analysis, immunofluorescent labeling, and image analysis indicated that 12(S)-HETE induced a time- and dose-dependent increase in the surface expression of alpha v beta 3 but not alpha 5 beta 1 on CD3 cells. The increased surface expression of alpha v beta 3 on endothelial cells did not result from an increased transcription or translation of alpha v beta 3 message as confirmed by quantitative reverse transcription-polymerase chain reaction, Northern blotting, and quantitative Western blotting. Instead, subcellular fractionation studies revealed an increased translocation of alpha v beta 3 integrins from the cytosolic pool to the membrane fractions. Pretreatment of endothelial cells with several cytoskeleton-disrupting agents (i.e., cycloheximide or acrylamide to disrupt intermediate filament vimentin, cytochalasin D to disrupt microfilaments, colchicine or Nocodazole to disrupt microtubules) abolished the 12(S)-HETE-enhanced alpha v beta 3 surface expression as well as tumor cell adhesion to endothelial cells. Also, pretreatment of CD3 cells with protein kinase C inhibitor calphostin C, but not with protein kinase A inhibitor H8, blocked 12(S)-HETE-enhanced alpha v beta 3 surface expression and tumor cell adhesion. Collectively, these results suggest that eicosanoid 12(S)-HETE modulates tumor cell interaction with endothelium via protein kinase C- and cytoskeleton-dependent up-regulation of the surface expression of alpha v beta 3 integrin.

Our previous work demonstrated that the 12-lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] induced a nondestructive and reversible retraction of cultured endothelial cells. In the current study we tested the hypothesis that tumor cells produce 12(S)-HETE during their interactions with endothelial cells which in turn induces endothelial cell retraction. Coincubation of Lewis lung carcinoma cells or elutriated B16 amelanotic melanoma (B16a) cells but not 3T3 fibroblasts with microvascular endothelial cells (CD3) resulted in a time- and concentration-dependent retraction of the CD3 monolayers as revealed by quantitative binding assays and phase contrast microscopy. Lewis lung carcinoma cell-induced endothelial cell retraction was blocked by specific lipoxygenase inhibitors but not by cyclooxygenase inhibitors, suggesting the involvement of a lipoxygenase metabolite(s). Radioimmunoassay and high-performance liquid chromatography analysis of tumor cell extracts identified 12(S)-HETE as the major lipoxygenase metabolite of arachidonic acid and tumor cell generation of 12(S)-HETE was specifically blocked by a select 12-lipoxygenase inhibitor N-benzyl-N-hydroxy-5-phenyl-pentamide. The identity and stereochemistry of tumor cell-derived 12-HETE was substantiated by gas chromatography-mass spectrometry analysis and chiral phase high-performance liquid chromatography, respectively. Lewis lung carcinoma cell adhesion to CD3 monolayers was accompanied by an enhanced 12(S)-HETE biosynthesis by tumor cells, which paralleled the tumor cell-induced endothelial cell retraction in a cell number-dependent manner. Pretreatment of tumor cells with N-benzyl-N-hydroxy-5-phenylpentamide inhibited both increased 12(S)-HETE biosynthesis and tumor cell-induced endothelial cell retraction. Highly metastatic variants of elutriated B16a cells which had been shown to produce large quantities of 12(S)-HETE induced significant CD3 cell retraction, while low metastatic subpopulations of B16a cells which synthesized no or little 12(S)-HETE did not induce endothelial cell retraction. These results suggest that 12(S)-HETE synthesis during tumor cell-endothelial cell interactions may represent a key contributory factor in cancer metastasis.

This study has the potential to establish a new paradigm in adhesion-mediated control of eicosanoid production and could be relevant to tumor migration, invasion, and metastasis. 12-Lipoxygenase metabolizes arachidonic acid to form 12(S)-HETE, a metabolite known to play a significant role in the process of tumor-induced angiogenesis and metastasis. The role of 12-LOX in proliferation, survival, and motility of tumor cells as well as in angiogenesis has been described for a variety of cancers. Similarly Alpha6Beta4 integrin also plays a role in both angiogenesis and tumorigenesis in addition to having a role in the formation of hemidesmosomes (HD). Evidence suggests a correlation between 12-LOX and integrin Beta4 since they play a similar role in cancer progression. The physical interaction between 12-LOX and Beta4 was first identified by a yeast two-hybrid screen using 12-LOX as bait against a library made from A431 cells that are known to have endogenous expression of both proteins. We verified this interaction by co-immunoprecipitation from A431 cells, and recapitulated it in CHO and PC-3 cells ectopically expressing 12-LOX and the cytoplasmic tail of Beta4 integrin. Additionally our data shows that ligation or clustering of Alpha6Beta4 integrin by the natural ligand laminin or an antibody mimic of laminin, 3E1, caused an increase in translocation of 12-LOX from the cytosol to membrane and an increase in 12-LOX enzymatic activities as assayed by immunofluorescence and 12(S)-HETE measurements respectively. 3E1-stimulated A431 cells showed increased cell migration but there was 40% decrease in presence of 12-LOX inhibitor BMD122 which confirms the role of 12-LOX in cell invasion. Here we describe for the first time the interaction domains that unite Beta4 integrin and 12-LOX. We mapped the domains in the cytoplasmic tail of Beta4 critical for interacting with 12-LOX and found that the 1126-1157 domain in the cytoplasmic tail of Beta4 is important for binding of 12-LOX. Also we have shown the mutant fragment (1126-1315) interacts with 12-lipoxygenase in the cytosol and prevent its binding to native, full-length membrane-associated Beta4 and thereby prevents 12-LOX activation. This physical interaction has functional implications with regard to decreased cell survival and migration. Determination of the structural basis for protein-protein interaction may provide potential means to manipulate or block such interaction, which may yield therapeutic benefits. We propose that the results obtained from these studies will provide a novel, and important dimension to both integrin and eicosanoid biology. Acknowledgements: This project is supported by NIH: NCI, 2 R01 CA029997-17A1 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2890. doi:10.1158/1538-7445.AM2011-2890

12-Lipoxygenase (12-LOX) converts arachidonic acid to 12(S)-hydroxyeicosatetraenoic acid 12(S)-HETE, an eicosanoid implicated in tumor angiogenesis, growth, and metastasis. We have demonstrated previously that 12-LOX interacts with integrin beta4 cytoplasmic domain by using yeast two hybrid system. Integrins are involved in metastasis by mediating attachment and migration of cells, as well as through transducing signals. Increased expression of 12-LOX and beta4 integrin have been documented in a number of carcinomas. The purpose of this study is to find out the mechanisms underlying the signaling pathways in 12-LOX mediated cancer progression. The phosphorylation of beta4 integrin, Src, and 12-LOX were analyzed using specific antibodies against phospho-tyrosine, phospho-beta4 integrin, Phospho-Src (pSrc). c-Src activity and association was determined in beta4 integrin immunoprecipitate. The HEK293 cells transiently transfected with wild type or dominant negative (DN) Src were used as in vitro models to study the regulation of 12-LOX activity. The 12-LOX product 12(S)-HETE was analyzed by using an analytical liquid chromatography-tandem mass spectrometry.12-LOX tyrosine phosphorylation sites mutants viz., 19, 295, and 614 were used to study the effect on its activity upon beta4 ligation. We show here that the cytoplasmic domain of beta4 integrin, can interact specifically with pSrc after beta4 integrin ligation in A431 cells. Herein we report that beta4 integrin ligation resulted in significant increase in 12(S)-HETE level in A431 cells, compared to that of unligated beta4 integrin. An increase in Src kinase activity was found in beta4 integrin ligation of A431 cells and inhibition of Src kinase by PP2 or DN Src significantly reduced 12(S)-HETE production. The ligation of beta4 integrin induces src activation, which resulted in tyrosine phosphorylation of 12-LOX. The residues that are important for 12-LOX activity and tyrosine phosphorylation were mapped to Y19 and Y614. We examined that 12-LOX mutants (Y19F and Y614F), showed 70% less enzymatic activity, compared to wild type 12-LOX. Furthermore, we showed that the 12-LOX activity modulated by these residues impacts migration in HUVEC cells. To our knowledge, this is the first report to show that Src kinase activity is required for beta4 integrin mediated regulation of 12-LOX, which may in turn regulate metastatic potential of cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2902. doi:10.1158/1538-7445.AM2011-2902

The primary objective of this study is to investigate the role of TXA2R signaling in prostate cancer progression and metastasis. Thromboxane A2 (TXA2) is a major arachidonic acid metabolite that signals through TXA2 receptors to induce platelet aggregation and smooth muscle contraction. TXA2 receptors (TP) are expressed as two different isoforms in humans- TP-alpha (TPα) and TP-beta (TPβ), which share both common and distinct signaling pathways. Previous studies have established the important role played by TXA2R isoforms in several cancers such as bladder, lung and breast cancer. But the role played by TXA2R signaling in prostate cancer (PCa) progression is not well understood. Our data indicates that human prostate cancer cell lines such as PC3, DU145, PC3M and LNCaP only express the TPα isoform but not the TPβ isoform. Also expression of TPα was higher in PCa cells compared to normal prostate epithelial cells. Results from the angiogenesis array indicated that activation of TXA2 receptors using TXA2 mimetic-IBOP resulted in an increase in angiogenesis related proteins such as VEGF, MMP9 and uPA in DU145 cells. To further confirm the role of TPα activation on VEGF, which is one of the most potent and predominant angiogenic factor, Real-time PCR and ELISA for human VEGF was performed that showed an increased expression of VEGF in response to IBOP in DU145 cells. To further understand the mechanisms behind TPα mediated VEGF expression, immunoblotting was performed on DU145 cells lysates treated with IBOP in order to analyze the phosphorylation status of downstream targets with phospho-specific antibodies. Activation of TXA2 signaling by IBOP did result in phosphorylation of downstream targets such as Src kinase, EGFR kinase and ERK. To verify whether EGFR kinase is involved in TPα induced VEGF expression, we pre-treated these cells with an EGFR kinase inhibitor AG1478 before treatment with IBOP. Results from Real-time PCR and ELISA suggest that inhibition of EGFR kinase did result in reduced VEGF mRNA and protein expression that confirmed its role in TPα mediated angiogenesis. Over expression of TPα in another PCa cell line, PC3 (PC3-TPα) that has lower endogenous expression of TPα also resulted in an increase in VEGF expression. Increased angiogenesis was further verified by a preliminary in vivo study that showed mice injected with PC3-TPα cells exhibited greater tumor growth and increased neovascularization compared to mice with PC3-Neo cells. Taken together, these data suggests that, activation of TPα receptor in PCa cells induced the expression of pro angiogenic factors such as VEGF and hence might play an important role in prostate cancer progression and metastasis. Acknowledgements: This research was supported by NIH Grant 1R01 CA114051-01A1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3483. doi:10.1158/1538-7445.AM2011-3483

The putative tumor suppressor gene DCC has been shown to be frequently lost or expressed at low levels in colorectal, gastric, pancreatic, and esophageal carcinomas. In the present study, the DCC gene and its mRNA expression in human and rat prostatic carcinoma cells as well as in prostatic carcinoma tissues were examined by reverse transcriptase-polymerase chain reaction and polymerase chain reaction-loss of heterozygosity. The DCC gene was present and expressed in normal prostatic cells. However, its expression was decreased or undetectable in all prostatic carcinoma cells from either humans (4 cell lines) or rats (5 cell lines). In patients, 12 of 14 cases (86%) showed reduced DCC expression and 5 of 11 informative cases (45%) showed loss of heterozygosity at the DCC locus. These results demonstrate that loss of DCC expression and loss of heterozygosity at the DCC locus are a frequent feature of prostatic carcinoma cells.

We recently reported that disruption of tumor cell microfilaments or intermediate filaments resulted in an inhibition of the ability of tumor cells to induce the aggregation of homologous platelets in vitro (H. Chopra et al., Cancer Res., 48: 3787-3800, 1988). Previous investigators demonstrated that disruption of the tumor cell cytoskeleton decreases the ability of these cells to form lung colonies. We proposed that this latter effect is due, in part, to decreased interaction of tumor cells with platelets, following their arrest in the microvasculature. To test this hypothesis, B16 amelanotic melanoma cell microtubules, microfilaments, or vimentin intermediate filaments were disrupted with colchicine (50 microns), cytochalasin D (50 microns), or cycloheximide (50 microns), respectively, and then cells were tail vein injected into syngeneic mice. Both cytochalasin D- and cycloheximide-treated cells formed fewer lung colonies than did control cells. Colchicine, however, failed to inhibit lung colony formation. Neither colchicine nor cycloheximide treatment altered initial pulmonary arrest; however, fewer cycloheximide-treated cells remained in the lungs 8 h postinjection. Greater than 90% of control or colchicine-treated cells were found to be associated with activated platelets, and they also demonstrated typical cell membrane process formation 10 min and 8 h post-tumor cell injection. In contrast, less than 10% of cycloheximide-treated cells were in contact with activated platelets 10 min postinjection. However, by 8 h approximately 90% of cycloheximide-treated cells were in contact with activated platelets. This recovery coincided with the reformation of the B16 amelanotic melanoma vimentin intermediate filament network and the reacquisition of the ability to induce platelet aggregation in vitro. Neither colchicine nor cycloheximide treatment altered initial B16 amelanotic melanoma cell adhesion to murine microvessel-derived endothelial cells. This study provides in vivo evidence in support of our previous findings that disruption of certain cytoskeletal elements (i.e., vimentin intermediate filaments) inhibits the tumor cell ability to activate platelets. This study also suggests that platelet activation may stabilize the initial tumor cell arrest in the microvasculature.

Our laboratories have previously demonstrated that the malignancy of human and animal tumors is associated with increases in cathepsin B activity, due in part to increases in cathepsin B-specific RNA transcripts and in part to decreased regulation by the endogenous low molecular weight cysteine proteinase inhibitors (CPIs). In this study we have extended these observations to tumor cell subpopulations of B16 amelanotic melanoma (B16a) and Lewis lung carcinoma (3LL) isolated by centrifugal elutriation. B16a subpopulations exhibited a 10-fold differential in lung colonization potential, whereas 3LL subpopulations exhibited no differential. In the B16a subpopulations, cathepsin B activities, total cellular and plasma membrane-associated, corresponded positively (4- and 10-fold increase, respectively) with their lung colonization potentials. CPI activities, total cellular and plasma membrane-associated, corresponded inversely (2- and 5-fold decrease, respectively) with the lung colonization potential of the B16a subpopulations. In the 3LL subpopulations, neither cathepsin B nor CPI activities changed. In the plasma membrane fractions of all 3LL subpopulations the ratio of cathepsin B activity to CPI activity was less than 1, whereas in the plasma membrane fractions of all B16a subpopulations the ratio was 1 or greater. In the plasma membrane fractions of the B16a subpopulations of higher lung colonization potential the ratios were 2.5 and 7, indicating that the levels of endogenous CPIs in these fractions may not be sufficient to regulate cathepsin B activity. Cathepsin B mRNA levels were not increased in the B16a subpopulations expressing increased cathepsin B activity. Thus increased cathepsin B activity in these subpopulations was apparently due not to increased synthesis but to decreased regulation by the endogenous CPIs. These results suggest that membrane-associated cathepsin B and CPIs may both play a role in the expression of the experimental metastatic phenotype.

We have developed a new in vitro model system to examine tumor cell-platelet-endothelial cell interactions under dynamic conditions. Using the same model, we can determine endogenous eicosanoid metabolism and alterations in the prostacyclin-thromboxane A2 balance associated with interactions among tumor cells, platelets, and endothelial cells. The model consisted of cloned rat aortic endothelial cells grown on gelatin microcarrier beads under dynamic conditions (i.e., spinner culture). Interactions of these endothelial cells with platelets (heparinized rat platelet rich plasma) and/or tumor cells (rat Walker 256 carcinosarcoma) were assessed in an aggregometer. Gelatin beads alone or microcarrier grown endothelial cells did not elicit spontaneous aggregation of platelet rich plasma over a time period of 30 min. Microcarrier grown endothelial cells inhibited tumor cell induced platelet aggregation in a dose dependent fashion (i.e., depending on endothelial cell number). The ability of microcarrier grown endothelial cells to inhibit tumor cell induced platelet aggregation depended on endogenous production of prostacyclin. This conclusion is based on the following results: an increased number of microcarrier grown endothelial cells caused a prolongation of the aggregation lag time; an increased number of microcarrier grown endothelial cells caused a proportionate increase in 6-keto-prostaglandin F1 alpha concentration; an increased number of microcarrier grown endothelial cells was inversely correlated with thromboxane A2 production by platelets; indomethacin pretreatment of microcarrier grown endothelial cells caused a decrease in prostacyclin production and therefore overcame the associated inhibition of tumor cell induced platelet aggregation; and the inhibition of tumor cell induced platelet aggregation in the presence of endogenous prostacyclin produced by microcarrier grown endothelial cells was the same as that observed in the presence of exogenous prostacyclin. Scanning electron microscopy of aggregometry samples revealed: little or no platelet or tumor cell adhesion to gelatin beads alone, a low basal adhesion of tumor cells to microcarrier grown endothelial cells, and large aggregates of platelets and tumor cells located primarily at gaps in the monolayer of indomethacin treated microcarrier grown endothelial cells. This new in vitro model provides a method for examining the effects of eicosanoid metabolism by endothelial cells on tumor cell-platelet-endothelial cell interactions under dynamic conditions.

Multiple steps or events have been described as essential in the metastatic cascade. Tail vein injection of single cell suspensions was used to study the ultrastructural details of the events involved in the initial arrest and attachment of circulating tumor cells. Lewis Lung Carcinoma (3LL) and a mammary adenocarcinoma (16c) were compared to a previous ultrastructural study of B16 amelanotic melanoma (B16a) detailing morphological events in the initial arrest and attachment of tumor cells in lung. The three murine tumors followed similar steps and varied only slightly in the time sequence of the steps. We observed the following steps: (a) initial arrest of tumor cells was characterized by an intimate tumor endothelial cell contact; (b) platelet activation and aggregation was noted by two minutes. Platelet aggregation continued for 1-4 h until a thrombus formed; (c) after approximately 4 h endothelial cell separation with extension of the tumor cell to the subendothelial matrix was noted; (d) at approximately 24 h the tumor cell associated thrombus dissipated and the attached tumor cells were exposed to a reestablished circulation. (e) mitoses were observed after 24 h with cell division and the development of intravascular tumor nodules; (f) the final step in the extravasation sequence was dissolution of the basement membrane by the attached tumor cells.

Tumor cell adhesion to subendothelial matrix in the presence of platelets and plasma has been examined in vitro using an entirely homologous system of rat Walker 256 carcinosarcoma cells, matrix laid down by rat aortic endothelial cells and rat platelets and plasma. In the presence of platelets or platelets plus plasma, tumor cell adhesion was significantly enhanced when compared to adhesion in the absence of platelets. In the presence of plasma alone (0.1%), we observed no significant increase in tumor cell adhesion. In order to determine which platelet factors contribute to the enhancement of tumor cell adhesion by platelets, we subjected washed rat platelets to mechanical lysis or thrombin stimulation followed by centrifugation. The membrane fractions and supernatant fractions containing platelet attachment proteins were compared for their abilities to support tumor cell adhesion to subendothelial matrix. Platelet membranes were also recombined with platelet supernatant fractions to determine if platelet attachment proteins or platelet membranes required the presence of the other to enhance tumor cell adhesion. Platelet supernatant fractions which contained release reaction proteins (confirmed by polyacrylamide gel electrophoresis) did not enhance tumor cell adhesion. Purified thrombospondin, fibronectin, beta-thromboglobulin, platelet derived growth factor, and serotonin had no effect on tumor cell adhesion. Platelet membrane containing fractions affected tumor cell adhesion to subendothelial matrix as follows: (a) platelets formed an adhesive bridge between tumor cells and the subendothelial matrix as demonstrated by scanning electron microscopy; (b) intact platelets and thrombin stimulated platelets were the most effective at facilitating tumor cell adhesion; (c) preparations containing partially lysed platelet ghosts were more effective in supporting tumor cell adhesion to subendothelial matrix than were preparations containing completely lysed platelet membrane fragments; (d) recombination of platelet supernatant fractions with mechanically lysed platelets did not enhance their ability to support adhesion; (e) fixed platelets, either alone or in combination with platelet supernatant fractions, failed to enhance adhesion. These data indicate that platelet enhanced tumor cell adhesion appears to be dependent on platelet membrane factors including receptor mobility, rather than intraplatelet components.

In solid s.c. tumors of a variant of the murine B16 melanoma with high metastatic potential (B16F10), there was a 2- to 7-fold elevation of lysosomal cathepsin B activity when compared to the B16F1 variant with low metastatic potential. The highest activities (based on either protein or DNA) of cathepsin B were found in tumors of less than 1 g. When B16F1 and B16F10 melanoma variants were grown in tissue culture, the metastatic differential in cathepsin B activity was lost as the cells were subcultured. However, this differential in cathepsin B activity could be restored by reestablishing the cultured cells as s.c. tumors. The activities of four other lysosomal enzymes (cathepsin D, beta-N-acetylglucosaminidase, beta-glucuronidase, and acid phosphatase) showed little evidence of a positive correlation with the metastatic potential of the B16 melanoma variants. Eighty to 90% of cathepsin B activity has been localized to a fraction containing viable tumor cells which was isolated by centrifugal elutriation. In contrast, only 50% of cathepsin D activity was in the viable tumor cell fraction, and from 30 to 70% of beta-N-acetylglucosaminidase, beta-glucuronidase, and acid phosphatase. Elevated levels of cathepsin B in the high metastatic B16F10 variant are consistent with the idea that cathepsin B may play a direct or a regulatory role in tumor metastasis.

Components of the tumor cell cytoskeleton (i.e., microtubules, microfilaments, and intermediate filaments) have been reported to affect metastatic ability, since disruption of these components leads to a decrease in metastasis. One mechanism of metastasis which has not been previously considered is the decreased interaction of tumor cells with platelets. We present evidence that disruption of the tumor cell cytoskeleton decreases the ability of tumor cells to aggregate homologous platelets. This effect is dependent upon the disruption of microfilaments/intermediate filaments but not disruption of microtubules. In addition, tumor cell platelet interactions require the lateral mobility of specific receptors (i.e., clustering) on the tumor cell plasma membrane. A membrane glycoprotein immunologically related to the platelet glycoprotein IIb/IIIa complex was identified on Walker 256 carcinosarcoma cells using specific polyclonal and monoclonal antibodies and Northern blot analysis using complementary DNA probes for IIb and IIIa. Mobility of this receptor is dependent upon tumor cell microfilaments/intermediate filaments, but not microtubules. Furthermore, treatment of tumor cells with specific antibodies to the platelet glycoprotein IIb/IIIa complex inhibits tumor cell-platelet interaction at the macroscopic level (i.e., aggregation) and at the ultrastructural level (i.e., platelet adhesion to the tumor cell surface). These results suggest that this immunologically related glycoprotein IIb/IIIa is a receptor for platelet binding to the tumor cell surface, an event which precedes overt platelet aggregation and is dependent upon an intact tumor cell microfilament and intermediate filament network. Therefore, the decreased metastasis observed by others following disruption of the tumor cell cytoskeleton may be due, in part, to a decreased tumor cell-platelet interaction.

We have examined nifedipine, a dihydropyridine class calcium channel blocker, for ability to overcome cis-diamminedichloroplatinum(II) (cisplatin) resistance in a murine tumor line variant, B16a-Pt, which we developed for resistance to cisplatin. Nifedipine significantly enhanced the antitumor actions of cisplatin against primary subcutaneous B16a-Pt tumors and their spontaneous pulmonary metastases. We have characterized, in vivo, the pharmacokinetics and dose-response interactions between nifedipine and cisplatin. We now report our studies designed to compare, in vivo, the efficacy of nifedipine and other calcium active compounds including: (a) structurally similar calcium channel blockers (nimodipine, nicardipine) from the dihydropyridine class, (b) structurally different calcium channel blockers from the benzothiazepine (diltiazem) and the phenylalkylamine (verapamil) classes, and (c) calmodulin antagonists (trifluoperazine and calmidazolium) for ability to enhance the antitumor action of cisplatin. Nifedipine was included as the standard or reference compound. In these studies verapamil and diltiazem failed to enhance the antitumor actions of cisplatin as did both calmodulin antagonists. Our findings suggest that nifedipine has a greater degree of specificity for B16a-Pt cells than structurally different calcium channel blockers from other chemical classes (i.e., diltiazem and verapamil), or the two calmodulin antagonists (i.e., trifluoperazine and calmidazolium). We concluded that nifedipine interacts with specific target site(s) which are not accessible by verapamil, by diltiazem, or by the calmodulin antagonists. Surprisingly, the two dihydropyridine class calcium channel blockers, nimodipine and nicardipine, also failed to enhance cisplatin's antitumor actions despite the fact that their specificity and kinetics for binding to the dihydropyridine receptor component of the calcium channel favors them (nimodipine and nicardipine) over nifedipine. Therefore, we postulate that the synergism between cisplatin and nifedipine is independent of the latter's effect on the voltage sensitive, slow inward calcium channel. We suggest that cisplatin cytotoxicity is enhanced by nifedipine's interaction with an as yet unidentified specific "target site," as opposed to nonspecific interactions with the tumor cell plasma membrane or specific interactions with calmodulin or the P-glycoprotein (which is responsible for pleiotropic resistance).

The cathepsin B-like cysteine proteinase activity which has been implicated in tumor malignancy has been attributed to several cellular sources, including viable tumor cells, necrotic tumor cells, and host-inflammatory cells. We have isolated subpopulations of cells from eight rodent tumors of five histological types, using centrifugal elutriation, and verified the cellular composition of the subpopulations cytologically. Ninety-two % or greater of the cathepsin B-like activity was associated with the isolated fractions containing greater than or equal to 95% tumor cells of 86 +/- 2% (SE) viability (beta fractions). The isolated fractions consisting of necrotic tumor cells and inflammatory cells (alpha fraction) apparently contain a cysteine proteinase inhibitor, since both cathepsin B-like and cathepsin H activities in the beta fraction of B16 amelanotic melanomas could be inhibited by addition of the alpha fraction.

Prostacyclin has been evaluated for its ability to inhibit tumor cell-induced platelet aggregation (TCIPA) induced by several rodent tumor lines: B16a (melanoma); 3LL (carcinoma); 15091A (adenocarcinoma); and W256 (carcinosarcoma). Aggregation of human platelets by all four lines was inhibited by prostaglandin I2 (PGI2) in a dose-dependent manner, with complete inhibition observed at 10 ng/ml. However, higher PGI2 concentrations were required to inhibit aggregation of homologous rat platelets induced by W256 cells. Prostacyclin was compared to other icosanoids known to inhibit platelet aggregation and was found to be 100-fold more potent than either prostaglandin E1 or prostaglandin D2 and 1000-fold more potent than its stable nonenzymatic metabolite (6-ketoprostaglandin F1 alpha). Prostaglandin E2 in contrast to prostaglandin E1 and prostaglandin D2, did not inhibit TCIPA; however, both prostaglandin E2 and its enzymatic metabolite (13,14-dihydro-15-ketoprostaglandin E2) prevented PGI2 inhibition of TCIPA. The addition of prostaglandin I2 (100 ng/ml) after initiation of TCIPA (50% of maximum response) resulted in immediate arrest of TCIPA followed by reversal of platelet aggregation. Prostacyclin partially reversed platelet aggregation when added at 100% of maximum response. Platelets enhanced the adhesion of [125I]uridine-labeled W256 cells to plastic culture dishes under both aggregatory and nonaggregatory conditions. Prostacyclin in vitro inhibited platelet-facilitated tumor cell adhesion. These in vitro results demonstrated that PGI2 is a potent inhibitor of TCIPA and of tumor cell adhesion; we suggest that these are possible mechanisms to explain the antimetastatic effects of PGI2 in vivo [Honn, K. V., Cicone, B., and Skoff, A. Science (Wash. D.C.), 212: 1270-1272, 1981].

Lewis lung carcinoma cells express a plasma membrane receptor (i.e., IRGpIIb/IIIa) which is immunologically and functionally related to the platelet aggregation receptor complex (i.e., GpIIb/IIIa). Both fluorescence microscopy and flow cytometric analysis reveal that surface expression and/or activation of this tumor cell receptor is enhanced by a phorbol ester [i.e., 12-O-tetradecanoylphorbol-13-acetate (TPA)] and a lipoxygenase metabolite of arachidonic acid; 12-hydroxyeicosatetraenoic acid (i.e., 12-HETE). TPA-enhanced expression appears to be mediated by a lipoxygenase metabolite, as this effect can be reversed by lipoxygenase inhibitors but not by cyclooxygenase inhibitors. In parallel with these results both TPA and 12(S)-HETE [but not 12(R)-HETE] enhance tumor cell adhesion to endothelial cells, subendothelial matrix and fibronectin, but not to type IV collagen. TPA-enhanced adhesion can be reduced by lipoxygenase inhibitors but not by cyclooxygenase inhibitors and in addition, stimulated adhesion can be blocked by pretreatment of tumor cells with specific polyclonal or monoclonal antibodies which react against IRGpIIb/IIIa. 12(S)-HETE-enhanced adhesion can also be inhibited by these same antibodies. In contrast, a lipoxygenase product of linoleic acid, 13(S)-hydroxyoctadecadienoic acid, inhibited TPA and 12(S)-HETE-enhanced tumor cell adhesion to endothelial cells, subendothelial matrix, and fibronectin. These results suggest that (a) IRGpIIb/IIIa is a multifunctional receptor which mediates tumor cell adhesion to a variety of biological substrata, (b) TPA enhances surface expression and/or activation of this receptor possibly via a lipoxygenase metabolite of arachidonic acid, and (c) these effects are opposed by a lipoxygenase metabolite of linoleic acid.