Overexpression of 12/15-lipoxygenase, an ortholog of human 15-lipoxygenase-1, in the prostate tumors of TRAMP mice

Abstract

Changes in the expression and activity of lipid-metabolizing enzymes, including the linoleic acid (LA)-metabolizing enzyme 15-lipoxygenase-1 (15-LO-1), may play a role in the development and progression of human prostate carcinoma (PCa). We reported that human 15-LO-1 (designated as leukocyte type 12-LO or 12/15-LO in mouse) is expressed in human prostate and increased in PCa, particularly high-grade PCa. Genetically engineered mouse (GEM) models of PCa could facilitate the study of this gene and its regulation and function in PCa progression. In this study, we examine the protein expression and enzyme activity levels of 12/15-LO associated with PCa progression in the TRansgenic Adenocarcinoma of Mouse Prostate (TRAMP) model of PCa. This GEM model develops prostatic intraepithelial neoplasia (PIN), followed by invasive gland-forming PCa and invasive and metastatic less differentiated PCa, with neuroendocrine (NE) differentiation (NE Ca). In the wild-type and TRAMP prostates, the most prominent LA metabolite was 13-hydroxyoctadecadienoic acid (13-HODE). Lesser amounts of 12-hydroxyeicosatetraenoic acid and 15-hydroxyeicosatetraenoic acid (HETE) were made from arachidonic acid (AA). In TRAMP prostates, 12/15-LO activity was increased compared to wild type at 20, 29, 39, and 49 weeks, as assessed by LA conversion to 13-HODE, and by AA conversion to 12/15-HETE, respectively. Immunostaining demonstrated that the increased capacity to generate 13-HODE was paralleled by an increase in neoplastic epithelial expression of 12/15-LO in PIN and invasive carcinomas. In conclusion, although there is a basal 12/15-LO activity in the wild-type mouse prostate, there is a marked increase in the expression of 12/15-LO with TRAMP PCa progression, paralleling our previously reported increased expression of the ortholog 15-LO-1 in high-grade human PCa. Thus, 12/15-LO and LA metabolism in the TRAMP model shares similarities to human PCa, and may allow to confirm a role for LA metabolism and other biologic functions of 15-LO-1 in human PCa. In addition, the TRAMP model will serve as a tool for testing the suitability of 12/15-LO-and ultimately human 15-LO--as a therapeutic target during PCa progression.

Figure 1

Representative LA and AA metabolism in wild-type nontransgenic 20-week mouse prostate. [¹⁴C]HODE and [¹⁴C]12/15-HETE formation in wild-type mouse prostate (control) from 15-minute incubation with 25 µM [¹⁴C]LA or [¹⁴C]AA. The solvent system consisted of a methanol/water gradient at a flow rate of 1.1 ml/min for reverse-phase HPLC analysis, with in-line radiodetection (Y-axis as disintegrations per minute, or DPM). A prominent 13-HODE peak (panel A) elutes at ≈ 20 minutes, and a mixture of two metabolites coelutes at ∼18 to 19 minutes with standard 12-HETE and 12-HETE, respectively (12/15-HETE peak, panel B) (X-axis as minutes). The unmetabolized LA and AA elutes are the prominent peaks at far right (in both panels A and B) eluting at ≈ 54 and 55 minutes, respectively. Also, a prominent peak of a mixture of prostaglandins (PGs) is seen eluting at ≈ 6 to 7 minutes.

Figure 2

Representative LA and AA metabolism in 20-week TRAMP mouse prostate. [¹⁴C]HODE and [¹⁴C]12/15-HETE formation in wild-type mouse prostate (control) from 15-minute incubation with 25 µM [¹⁴C]LA or [¹⁴C]AA. The solvent system consisted of a methanol/water gradient at a flow rate of 1.1 ml/min for reverse-phase HPLC analysis, with in-line radiodetection ( Y-axis as DPM). A prominent 13-HODE peak (panel A) elutes at ≈20 minutes in a mixture of two metabolites: one major metabolite coelutes at ∼18 minutes with standard 12-HETE, as well as a minor metabolite that coelutes at ∼19 minutes with standard 15-HETE (panel B) (X-axis as minutes). The unmetabolized LA and AA elutes are the prominent peaks at far right (in both panels A and B) eluting at ≈54 and 55 minutes, respectively. Also, a prominent peak of a mixture of prostaglandins (PGs) is seen eluting at ≈ 6 to 7 minutes.

Figure 3

12/15-LO enzyme activity (% conversion to metabolic product) at different time points [20 weeks (n = 5), 29 weeks (n = 5), 39 weeks (n = 5), and 49 weeks (n = 3)] with AA and LA in wild-type nontransgenic and TRAMP mouse prostates. The values for 12/15-LO are the sum of 12-HETE and 15-HETE, respectively.

Figure 4

Representative 12/15-LO immunostaining in TRAMP mice. (A) Positive control, showing normal cytoplasmic immunostaining in the bronchial epithelium in the lung, with clean background in surrounding alveolated parenchyma. (B) Negative immunostaining in prostate ducts with PIN lesions (arrowheads) in 24-week TRAMP mouse. Included are occasional foci (arrowhead) in which distinct smaller gland profiles do not as obviously connect up to the central duct lumen and conform less obviously to the normal duct lining, raising considerations of well-differentiated adenocarcinoma. (C) Strong cytoplasmic 12/15-LO immunostaining in stratified atypical cells in focus of PIN (*) in the prostate of 18-week TRAMP mouse. Residual (but immunonegative) normal-appearing prostate-secretory epithelium is noted (arrowhead). (D) 12/15-LO immunostaining in focal PIN (arrowheads) in the prostate of 29-week TRAMP mouse. Residual normal prostatic epithelium is noted (arrowhead). (E) Strong uniform 12/15-LO immunostaining in focus of PIN of 18-week TRAMP mouse. Although there are small and apparently increased gland spaces, the connection of these to the central normal duct lining, the uniformity of this lesion, and the preservation of the normal surrounding contractile periprostatic stroma support the designation of PIN. Other similarly involved duct/gland profiles with less prominent or absent immunostaining (e.g., bottom left). (F) Strong extensive 12/15-LO immunostaining in atypical epithelium (arrows) of polypoid focus of combined epithelial and stromal hyperplasia (“phyllodes like tumor”) of 29-week TRAMP mouse. Duct lumen is toward top left. Surrounding rim of pre-existing prostatic contractile stroma is at bottom right (arrowhead). (G) Strong, uniform 12/15-LO immunostaining in microscopic focus of invasive, poorly differentiated (NE) carcinoma (white*) in a background of focally but less prominently immunostaining PIN in 29-week TRAMP mouse. (H) Strong, uniform, diffuse 12/15-LO immunostaining in invasive, poorly differentiated (NE) carcinoma in the prostate of 29-week TRAMP mouse. A residual entrapped PIN containing the prostate duct is noted (*). (I) Strong, uniform, diffuse 12/15-LO immunostaining in extensively invading, poorly differentiated (NE) carcinoma in 39-week TRAMP mouse. Tumor focus comprises areas previously referred to as moderately differentiated and compatible with NE carcinoma in the current designation (top right), as well as areas previously referred to as poorly differentiated carcinoma that are morphologically compatible with small cell carcinoma (bottom left), showing focal “crush” artifact typical of such tumors. Tumor massively infiltrates surrounding pelvic soft tissues (adipose tissue noted*). (J) Strong, uniform 12/15-LO immunostaining in metastatic poorly differentiated (NE) carcinoma (arrowheads) in retroperitoneal lymph node from 39-week TRAMP mouse. Immunonegative residual normal lymphoid germinal centers are noted (white*). (K) 12/15-LO immunonegative liver metastasis in 39-week TRAMP mouse. Benign liver parenchyma is seen at bottom right. (L) 12/15-LO negative lung metastasis of poorly differentiated (NE) carcinoma (arrowheads) in 20-week TRAMP mouse. Note moderately strong positive control immunostaining in adjacent normal bronchial epithelium (arrow). (M) Strong 12/15-LO immunostaining in lung micrometastasis (arrowhead) in 29-week TRAMP mouse.