15-Lipoxygenase 2 (15-LOX2

15-Lipoxygenase 2 (15-LOX2 15-Lipoxygenase 2 (15-LOX2)1 is a recently cloned lipoxygenase that shows the highest homology (ϳ80% amino acid identity) to murine 8-LOX, with ϳ40% identity to human 5-LOX, 12-LOX, or 15-LOX1 (1). It has at least three splice variants (termed 15-LOX2sv-a/b/c) (2, 3) and metabolizes preferentially arachidonic acid (AA) to 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) (1). 15-LOX2 shows an interesting tissue expression pattern, i.e. mainly in prostate, lung, skin, and cornea (1-3). This tissue-restricted expression pattern suggests that 15-LOX2 may play a role in the normal development and its abnormal expression/function may contribute to tumorigenesis in these organs. Indeed, work by indicates that 15-LOX2 mRNA, protein expression, and enzymatic activity are decreased in high grade prostate intraepithelial neoplasia (PIN) and prostate cancer (PCa), and the expression levels of 15-LOX2 are inversely correlated with the pathological grade (Gleason scores) of the patients. We recently reported that 15-LOX2 is a negative cell-cycle regulator in normal human prostate (NHP) epithelial cells (3). These observations (3-6) together raise the possibility that 15-LOX2 may represent an endogenous prostate tumor suppressor, and its down-regulation may contribute to PCa development. Here we provide experimental data in support of this possibility as restoration of 15-LOX2 expression inhibits PCa cell proliferation in vitro and tumor development in vivo. We further show that the tumor-suppressive functions of 15-LOX2 do not necessarily depend on the AA-metabolizing activity and nuclear localization as 15-LOX2sv-b, a splice variant that does not metabolize AA and is mostly excluded from nucleus, demonstrates similar inhibitory effect on PCa development. MATERIALS AND METHODSCells and Reagents-Six primary NHP cell strains, NHP1-NHP6, were prepared from six different donors. NHP1, NHP3, NHP4, and NHP6 cells were obtained from Clonetics (Walkersville, MD), and NHP2 and NHP5 cells were generated as previously described (7-9). These cells were cultured in serum-free, PrEBM medium (Clonetics)

15-Lipoxygenase 2 (15-LOX2), a lipid-peroxidizing enzyme, is mainly expressed in the luminal compartment of the normal human prostate and often decreased or lost in prostate cancer. Previous studies from our lab implicate 15-LOX2 as a functional tumor suppressor. To better understand the biological role of 15-LOX2 in vivo, we established prostate-specific 15-LOX2 transgenic mice using the ARR2PB promoter. Unexpectedly, transgenic expression of 15-LOX2 or 15-LOX2sv-b, a splice variant that lacks the arachidonic acid metabolizing activity, resulted in age-dependent prostatic hyperplasia and enlargement of the prostate. Prostatic hyperplasia induced by both 15-LOX2 and 15-LOX2sv-b was associated with an increase in luminal and Ki-67+ cells; however, 15-LOX2-transgenic prostates also showed a prominent increase in basal cells. Microarray analysis revealed distinct gene expression profiles that could help explain the prostate phenotypes. Strikingly, 15-LOX2, but not 15-LOX2sv-b, transgenic prostate showed upregulation of several well-known stem/progenitor cell molecules including Sca-1, Trop2, p63, Nkx3.1 and Psca. Prostatic hyperplasia caused by both 15-LOX2 and 15-LOX2sv-b did not progress to prostatic intraprostate neoplasia (PIN) or carcinoma and, mechanistically, prostate lobes (especially those of the 15-LOX2 mice) showed a dramatic increase in senescent cells as revealed by increased SA-βgal, p27Kip1 and HP1γ staining. Collectively, our results suggest that 15-LOX2 expression in mouse prostate leads to hyperplasia and also induces cell senescence, which may, in turn, function as a barrier to tumor development.

In this project, we studied the gene regulation of 15-lipoxygenase 2 (15-LOX2), the most abundant arachidonate-metabolizing LOX in adult human prostate and a negative cell-cycle regulator in normal human prostate (NHP) epithelial cells. Through detailed in silico promoter examination and promoter deletion and activity analysis, we found that several Sp1 sites (i.e., three GC boxes and one CACCC box) in the proximal promoter region play a critical role in regulating 15-LOX2 expression in NHP cells. Several pieces of evidence further suggest that the Sp1 and Sp3 proteins play a physiologically important role in positively and negatively regulating the 15-LOX2 gene expression, respectively. First, mutations in the GC boxes affected the 15-LOX2 promoter activity. Second, both Sp1 and Sp3 proteins were detected in the protein complexes that bound the GC boxes revealed by electrophoretic mobility shift assay. Third, importantly, inhibition of Sp1 activity or overexpression of Sp3 both inhibited the endogenous 15-LOX2 mRNA expression. Since 15-LOX2 is normally expressed in the prostate luminal epithelial cells, we subsequently explored whether androgen/androgen receptor may directly regulate its gene expression. The results indicate that androgen does not directly regulate 15-LOX2 gene expression. Together, these observations provide insight on how 15-LOX2 gene expression may be regulated in NHP cells.