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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Int J Cancer. Author manuscript; available in PMC 2010 August 2.
Published in final edited form as:
PMCID: PMC2913418
NIHMSID: NIHMS98738

Downregulation of Vascular Endothelial Growth Factor and Induction of Tumor Dormancy by 15-lipoxygenase-2 in Prostate Cancer

Abstract

The enzyme 15-lipoxygenase-2 (15-LOX-2) utilizes arachidonic acid, a polyunsaturated fatty acid, to synthesize 15(S)-hydroxyeicosatetraenoic acid (HETE). Abundantly expressed in normal prostate epithelium but frequently suppressed in the cancerous tissues, 15-LOX-2 has been suggested as a functional suppressor of prostate cancer, but the mechanism(s) involved remains unknown. To study the functional role of 15-LOX-2 in prostate cancer, we expressed 15-LOX-2 as a fusion protein with GFP in DU145 and PC-3 cells and found that 15-LOX-2 increased cell cycle arrest at G0/G1 phase. When injected into athymic nu/nu mice, prostate cancer cells with 15-LOX-2 expression could still form palpable tumors without significant changes in tumorigenicity. But, the tumors with 15-LOX-2 expression grew significantly slower than those derived from vector controls and were kept dormant for a long period of time. Histological evaluation revealed an increase in cell death in tumors derived from prostate cancer cells with 15-LOX-2 expression, while in vitro cell culture conditions, no such increase in apoptosis was observed. Further studies found that the expression of vascular endothelial growth factor A (VEGF-A) was significantly reduced in prostate cancer cells with 15-LOX-2 expression restored. Our studies suggest that 15-LOX-2 suppresses VEGF gene expression and sustains tumor dormancy in prostate cancer. Loss of 15-LOX-2 functionalities, therefore, represents a key step for prostate cancer cells to exit from dormancy and embark on malignant progression in vivo.

Keywords: tumor dormancy, angiogenesis, lipoxygenase, prostate cancer, VEGF

Introduction

Prostate cancer is one of the most common cancers affecting men in the United States with approximately 234,000 new cases and 30,000 deaths in year 2006 alone 1. While some prostate tumors are aggressive and deadly, many others can be indolent and remain dormant for many years, without obvious symptoms of diseases 2, 3. Elucidation of mechanisms underlying the transition from latent tumors to full malignancy will yield effective approaches for management of prostate cancer. Lipoxygenases (LO or LOX) are a family of non-heme iron containing proteins that catalyze the dioxygenation of polyunsaturated fatty acids containing the 1-cis-4-cis-pentadiene moiety to form bioactive lipids. Metabolism of arachidonic acid by LOXs leads to the formation of regioisomeric cis/trans conjugated hydroperoxyeicosatetraenoid acids (HpETEs), which subsequently give rise to hydroxyeicosatetraenoic acids (HETEs), leukotrienes, lipoxins, and hepoxilins 4. Dependent on the predominant position of the incorporation of hydroperoxy group, LOXs are classfied as 5-, 8-, 12-, and 15-LOXs, whose main products are 5(S)-, 8(S)-, 12(S), and 15(S)-HETE, respectively.

Human 15-LOX-2 expression is frequently lost in prostate cancer 57. In normal or benign prostate tissues, 15-LOX-2 expression is mainly located in luminal cells of the gland 5. In prostate tumors, 15-LOX-2 immunostaining was completely absent in 23 of 70 cases and further the levels of 15-LOX-2 were inversely correlated with the grade and Gleason scores of the patients 57. In addition to prostate carcinogenesis, 15-LOX-2 expression is also silenced in lung neoplasm 8, skin-derived sebaceous neoplasms or carcinoma 6, and esophageal cancer 9. Tang and his colleagues investigated the mechanism of silencing of 15-LOX-2 in prostate cancer cells 10. Cloning and analysis of 15-LOX-2 promoter revealed that Sp1 positively, while Sp3 negatively, regulates the promoter activities of 15-LOX-2 in prostate epithelial cells 11. However, neither hypermethylation of the promoter region of 15-LOX-2 or histone deacytylation, was a significant cause of 15-LOX-2 silencing in prostate cancer cells 10. It should be pointed out that although absent in approximately 30% cases of prostate tumor specimens examined, 15-LOX-2 expression was still present in other 70%.

15-LOX-2 has been suggested as a functional tumor suppressor in PC-3 cells 12. PC-3 cells transfected with a 15-LOX-2 expression construct had diminished tumor development when injected into a mouse prostate 12. Among mechanisms proposed for the tumor suppression is the ability of 15-LOX-2 to inhibit DNA replication as indicated by a decrease in the incorporation of BrDU 12. It is unknown whether 15-LOX-2 expressing PC-3 cells failed to initiate tumor formations or whether the tumors derived failed to maintain or progress.

Here we present novel findings suggesting that, while 15-LOX-2 did not abrogate the tumorigenicity of prostate cancer cells when expressed in PC-3 or DU145 cells, 15-LOX-2 expression significantly reduced the growth of prostate tumors in vivo by keeping tumors in a state of dormancy. Histological evaluation revealed an increase in apoptotic index in tumors with 15-LOX-2 expression. Further studies suggest that VEGF expression was significantly downregulated by 15-LOX-2, evidencing 15-LOX-2 as a novel negative regulator of tumor growth via downregulating angiogenesis.

Materials and Methods

Materials

Prostate cancer PC-3 and DU145 cells were obtained from ATCC (Manassas, VA). All cell culture and immunochemistry reagents were purchased from Invitrogen unless otherwise indicated. The GenePORTER® liposome transfection reagent was from Genlantis (San Diego, CA). Declere® was supplied by Cell Marque™ Corporation (Hot Springs, AR). MTS solution, luciferase assay system, and cell culture lysis reagent were from Promega (Madison, WI). 15-LOX-2 antibodies were purchased from Oxford Biomedical Research Cooperation (Oxford, MI) or Santa Cruz Biotechnologies (Santa Cruz, CA).

Cell culture and transfection

PC-3 and DU145 cells were cultured in RPMI-1640 with 10 % FBS. PC-3 or DU145 cells were plated in 75 cm2 flasks, cultured for 24 h and then transfected with a 15-LOX-2 expression construct pEGFP-15LOX-2 (provided by Dr. Colin Funk, Queens’ College) which fuses 15-LOX-2 with GFP at its C-terminal end or empty vector pEGFP-C2 (Clontech), using Geneporter reagent according to the manufacturer’s instructions. After 48 h, cells were harvested by trypsin-EDTA digestion and the transfected cells were selected by florescence activated cell sorting (FACS). The selected cells were cultured in RPMI-1640-10% FBS in the presence of 0.4 mg/ml of G418.

Determination of 15-LOX-2 activities by HPLC-tandem mass spectrometry

Enzymatic activity of 15-LOX-2 was determined by measuring 15-HETE production by HPLC-tandem MS. PC-3 cell with stable 15-LOX-2 expression were grown to 90% confluence in 100 mm petri dishes and serum starved overnight prior to experimental use. Cells were trypsinized and washed with PBS once. Cell pellets were dissolved in 400 µl RPMI 1640 medium and sonicated for 10 sec at 4 °C to achieve homogenates. The homogenates were incubated at 37 at 4°C for 30 min and the incubation was terminated by adding 50 µl glacial acetic acid. The homogenates were extracted twice with 3 ml extraction buffer (hexanes: ethyl acetate: acetic acid=1:1:0.05). The organic extracts were dried under a stream of nitrogen and reconstituted in acetonitrile.

One microliter of each sample aliquot was injected into a ThermoFinnigan Surveyor HPLC equipped with a Gemini® narrow bore column (150×20 mm) packed with 3 µM C18 (110Å) at a flow rate of 0.150 mL/minute; a binary mobile phase gradient at 50:50 ([Delta with dot below] 2.5%) A:B in 20 minutes (Mobil Phase A=0.01% Formic Acid; Mobil Phase B=acetonitrile) was utilized providing proper peak shape, separation and reduction of interferences. A ThermoFinnigan (TSQ7000) triple stage quadrupole (TSQ) mass spectrometer equipped with electrospray ionization source (ESI) was tuned to provide mass collection of a [M-H] parent ion (mp •−) through a quadrupole filter (Q1) for each lipid product of interest, e.g. m/z 319.5 for 15(S)-HETE and m/z 327.5 for the internal standard 12(S)-HETE d8. The capillary temperature was 250°C and maintained at 4.5 kV. The mp •− from Q1 were passed through a collision chamber (Q2), operating in radio-frequency-only mode, and focused for subsequent ionization as a consequence of the collision of the rapidly moving mp •− with an inert gas (Ar) at ~1.8 mTorr to produce the unique product fragmentation spectrum that was subsequently scanned through a third mass filter (Q3) where selected daughter ions (md •−) were collected in multiple resonance monitoring. All quantitation of analytes were performed using relative response factors of the analyte:internal standard (i.e. 15(S)-HETE:12(S)-HETE d8).

Western blot

Stable transfected cells were characterized for the 15-LOX-2 expression by western blot. Semi-confluent (70% to 80%) PC-3 and DU145 cells stably transfected with expression construct pEGFP-15LOX-2 (denote as 15-LOX-2) or vector pEGFP-C2 (denote as vector control) were harvested in RIPA buffer. 30 µg protein samples were subjected to electrophoresis in SDS-PAGE and transferred to a PVDF membrane. The membranes were incubated with the primary rabbit antibody (Oxford Biomedical Research Corp, Oxford, MI) overnight, probed with HRP-conjugated secondary antibodies for 1 h, and visualized by ECL reagents (Amersham).

Flow cytometry analysis of cell cycle and apoptosis

Cell cycle and apoptosis in vector control and 15-LOX-2 cancer cells were analyzed using the APO-DIRECT kit from Pharmingen (San Diego, CA). Cells were harvested, fixed with 1%(w/v) paraformaldehyde in PBS at 4°C for 30 minutes, washed twice in 5 ml of 1x PBS, resuspended in ice cold 70% ethanol, and subjected to terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay, according to manufacturer’s instruction. Cells were further stained with 0.5 ml of PI/RNase staining solution (0.1% Triton X-100, 0.2 mg/ml DNase-RNase A, and 20 µg/ml propidium iodide) and incubated at room temp for 30 minutes in the dark before flow cytomtric analysis. At least 5 × 104 cells were analyzed for each sample.

Animal model and histology

For each mouse, a total of 3 × 106 15-LOX-2 transfectants or vector control cells in 200 µl of diluted Matrigel (5X) were injected s.c. into the right flank of 4 – 6 week old male athymic nu/nu mice (Harlan). The resulting palpable tumors were measured using a vernier caliper, and tumor volume was calculated using the formula: (width)2 X Length X 0.5 13. For DU145 cells, the mice were sacrificed six to seven weeks after injection. For PC-3 cells, mice were maintained for 10 weeks for evaluation of tumor dormancy for an extended period of time. Tumors were resected and fixed in 10% neutral buffered formalin and embedded in paraffin, and 5 µm sections were prepared for histology staining. Sections were stained with H&E to examine the presence of mitotic figures, necrosis and apoptosis. Mitotic and apoptotic figures in tumor sections stained with H&E were counted through the microscope at a magnification of 400 by a trained technician with a mechanical tabulator, in a double blind approach. Non-overlapping fields were selected with the aid of an ocular grid. Total background cells in each field were also counted and the percentage of positive cells [(X positive/Y total count)×100] was calculated for each tumor.

Immunohistochemistry

For tissue immunostaining, paraffin-embedded tissue sections were deparaffinized, rehyrated and antigen retrieved by placing in Declere® working solution in an electric pressure cooker for 15 min. The slides were processed for immunohistochemical staining using a Zymed® Histostain-SP kit, according to manufacturer’s instructions. The mouse monoclonal antibody against VEGF or Ki67 was purchased from Zymed. Cleaved caspase-3 (Asp175) antibody (Cell Signaling) was applied as primary antibody to detect active form of casepase-3 distribution within the tumors derived from Vector or 15-LOX-2 transfected PC-3 cells. The stained tissue slides were observed under BX41 system microscope (Olympus) at 200X magnification. Cleaved caspase-3 positively stained cells were counted against the total cells within the slide and the percentages were calculated.

ELISA determination of VEGF levels

2 × 105 PCa cells were plated in 35 mm well in 6-well culture plates and cultured in RPMI-1640 containing 1% FBS. The culture media were then harvested for determination of the levels of VEGF or other cytokines. The levels of VEGF-A were measured in conditioned cell culture media using ELISA immunoassay kits purchased from R&D Systems as described previously 14.

Transient transfection to measure VEGF promoter activities

Cells, vector control or cells transfected with 15-LOX-2 or other isoforms of LOX such as 5-LOX or 15-LOX-1, were plated at 2 × 105 cells per 35-mm dish 2 days before transfection with the p − 1176/+54 VEGF promoter-luciferase construct and a LacZ expression plasmids using GenePorter transfection reagents as described previously 14. Cells were harvested for luciferase assay 36 ~ 40 h after transfection. Luciferase activity was measured using the luciferase assay kit (Promega) and normalized by assaying galactosidase activity as internal control for transfection efficiency. In other studies, PC-3 cells were co-transfected with 15-LOX-2 and VEGF promoter constructs to study the effects of transient expression of 15-LOX-2 on VEGF promoter activities.

Statistical analysis

Student’s t-test (two-tailed) was used for statistical analysis between two groups, unless otherwise indicated. A P value, less than 0.05, is considered significant.

Results

Restoration of functional 15-LOX-2 in human prostate cancer cells

To study the role of 15-LOX-2 in prostate tumor progression, wild-type 15-LOX-2 was expressed as a fusion protein with GFP at its C-terminal end 15 in PC-3 and DU145 cells. Transfected cells were selected by FACS and maintained by G418 selection. As shown in figure 1A, PC-3 cells transfected with the 15-LOX-2 expression construct presented an increased expression of 15-LOX-2 shown by western blot with GFP antibody (right panel) or antibody against 15-LOX-2 (left panel). DU145 cells transfected with the 15-LOX-2 expression construct showed a similar pattern of expression (not shown). Lipid analysis revealed that there was ~100 fold increase in the level of 15(S)-HETE in the prostate cancer cells expressing 15-LOX-2 (Figure 1B) as compared to the control vector. 12(S)-HETE d8 was used as the internal standard used for quantitation and relative response factor quantitation. It was calculated that there was 0.816 µg of 15(S)-HETE extracted from 50 million vector control cells, compared to 67.3 µg extracted from the same number of 15-LOX-2 transfected cells.

Figure 1
Expression and activity of 15-LOX-2 in prostate cancer cells

Inhibition of the growth, but not the formation, of tumors by 15-LOX-2

To elucidate the role of 15-LOX-2 in prostate carcinoma progression in vivo, we injected 15-LOX-2 transfected DU145 or PC-3 and their vector control cells s.c. into the right flank of athymic nu/nu male mice and monitored the formation and growth of tumors regularly. The incidence of formation of palpable tumors from DU145 cells with 15-LOX-2 expression restored was 100% (n = 6), compared to 80% in control DU145 cells (n = 5). The tumorigenesis incidence for 15-LOX-2 transfected PC-3 cells was 83% (n = 6), compared to 100% in control PC-3 cells. Therefore, there was no difference in the formation of palpable tumors by prostate cancer cells with or without 15-LOX-2 expression, suggesting that 15-LOX-2 did not abrogate the tumorigenic potential of PC-3 or DU145 cells. However, despite the lack of effects of 15-LOX-2 on the ability of PC-3 or DU145 cells to initiate tumor formation, tumors derived from 15-LOX-2 transfected DU145 cells progressed slowly when compared to those from their vector controls (GFP), as evidenced by the different growth kinetics of tumors (Figure 2A). In fact, the tumors with 15-LOX-2 transfection were kept dormant in vivo during entire six weeks of experiments. In contrast, tumors derived from vector controls started to grow rapidly within three weeks after injection (Figure 2A). We repeated the experiments with PC-3 cells. As shown in figure 2B, tumors derived from 15-LOX-2 transfected PC-3 cells were mostly dormant and progressed much more slowly than those from vector controls, during the entire 10 weeks of experiments. The results suggest that expression of 15-LOX-2 in prostate carcinoma cells keeps prostate tumors dormant.

Figure 2
Induction of prostate tumor dormancy in vivo by 15-LOX-2

Increased apoptosis in tumors with 15-LOX-2 expression

To study how tumor dormancy was sustained as result of 15-LOX-2 expression, we first examined the effect of 15-LOX-2 on the steady state distribution of cell cycle in prostate carcinoma cells. As shown in figure 3A, expression of 15-LOX-2 caused an approximate 22% increase in G1/G0 arrest, with concomitant reduction in S and G2/M phases in DU145 cells cultured in serum free media. A similar result also was obtained from DU145 cells cultured in serum containing media, except that there was no significant difference in G2/M phase (Figure 3B). Similar results were also obtained with PC-3 cells (not shown). The reduced steady state level of cell population in the S phase, as result of 15-LOX-2 expression, is consistently with the observation that 15-LOX-2 inhibits DNA synthesis 10.

Figure 3
Mechanism of tumor dormancy by 15-LOX-2

However, histological analysis of tumor sections did not confirm a reduction in cell proliferation in tumors with 15-LOX-2 expression. Immunostaining with Ki67 also did not reveal significant difference in cellular proliferation between tumors with 15-LOX-2 expression and control tumors (Figure 3C). Also no statistically significant differences in the mitotic index were found in tumors derived from 15-LOX-2 transfected cells when compared to that of control tumors (Figure 3D). Therefore, although 15-LOX-2 can increase cell cycle arrest at G0/G1 and inhibit DNA synthesis in vitro, the reduced cellular proliferation did not become a rate limiting factor causing tumor dormancy as result of 15-LOX-2 expression.

Increased apoptosis were observed in sections of tumors derived from 15-LOX-2 transfected prostate cancer cells. When stained for active caspase-3, increased staining was observed in the tumor sections derived from 15-LOX-2 transfected cells when compared to those from vector controls (Figure 3E, F). The increased apoptosis was also confirmed by a significant increase in apoptotic index observed in H&E stained tumor sections from 15-LOX-2 transfected PC-3 cells (Figure 3G). To study whether 15-LOX-2 expression can directly cause apoptosis in prostate cancer cells, we determined the rate of apoptosis in prostate cancer cells transfected with 15-LOX-2 using TUNEL flow cytometric assay. We did not find any significant increase in apoptosis in prostate cancer cells (PC-3 or DU145), whether they were cultured in serum containing media or serum free media, as result of 15-LOX-2 expression (data not shown). The results suggest that the increased apoptosis in tumors with 15-LOX-2 expression was likely caused in an indirect manner. Taken together, while there was no reduction in mitotic index in tumors derived from 15-LOX-2 transfected cells, the increase in cell death in tumors with 15-LOX-2 expression may likely slow down the growth of tumors and cause dormancy for a sustained period.

Downregulation of VEGF expression by 15-LOX-2

The dormancy and increased apoptosis in tumors with 15-LOX-2 expression led us to examine whether 15-LOX-2 alters tumor angiogenesis. It has been reported that 12-LOX, a member of LOX family, stimulates tumor angiogenesis and growth in prostate or breast cancer 13, 16, 17, via regulating the expression of VEGF, an angiogenic factor involved in prostate tumor growth and progression 14. To study whether 15-LOX-2 causes prostate tumor dormancy though downregulation of tumor angiogenicity, we examined the effect of 15-LOX-2 on the expression of VEGF. First we measured the levels of VEGF in culture supernatants from prostate cancer cells using ELISA. As shown in figure 4A, the level of VEGF in culture supernatants was significantly reduced in prostate cancer cells with 15-LOX-2 expression. Next we evaluated the levels of VEGF in sections of tumors using immunohistochemical staining for VEGF. As shown in figure 4B, the immunoreactivities of VEGF were much stronger in vector control tumors (middle panel) than those in 15-LOX-2 tumors (Low panel). The results suggest that VEGF expression at protein levels were downregulated in vitro and in vivo in prostate cancer cells with 15-LOX-2 expression.

Figure 4
Down-regulation of VEGF expression in prostate cancer cells by 15-LOX-2

As a key regulator of angiogenesis, VEGF (VEGF-A) is closely associated with angiogenesis in prostate cancer 18. The expression of VEGF is tightly regulated by both transcriptional and posttranscriptional mechanisms. At the transcriptional level, HIF, AP1, AP2, and Sp1 are known to regulate the transcription of VEGF gene by binding to their respective recognition sites located in the human VEGF promoter region 19. To study how 15-LOX-2 downregulates VEGF expression, the promoter activities in prostate cancer cells, with or without 15-LOX-2 expression, were evaluated by reporter gene assay. DU145 cells (GFP control, 5-LOX, 15-LOX-1 or 15-LOX-2 transfected) were transfected with a VEGF promoter luciferase construct (p− 1176/+45), along with a lacZ construct for normalization of transfection efficiency. As shown in Figure 4C, there was a significant decrease (p = 0.017, Student’s T test) in DU145 cells with 15-LOX-2 expression, when compared to the vector control (GFP). No statistically significant reduction in VEGF promoter activities was found in DU145 cells expressing 5-LOX or 15-LOX-1, suggesting a specific reduction of VEGF gene expression by 15-LOX-2. A similar reduction in VEGF promoter activities by 15-LOX-2, but not by 5-LOX or 15-LOX-1, also was found in PC-3 cells (Figure 4D). Transient transfection of PC-3 cells with 15-LOX-2 expression construct also reduced the VEGF promoter activity (p− 1176/+45) (Data not shown). The results suggest suppression of VEGF promoter activities by 15-LOX-2.

Discussion

The clinical progression of human prostate tumors is quite heterogenous. While some cancers are rapidly progressing, many others can remain indolent for many years without overt symptoms of diseases. A dilemma for physicians and prostate cancer patients, once diagnosis is made, is to determine whether aggressive treatment, which often has debilitating side effects, is needed, or watchful waiting can be afforded. In the present study, we demonstrated that 15-LOX-2 inhibited the growth, but not the formation, of prostate tumors. It was found that tumors with 15-LOX-2 expression were kept dormant for a long period of times. A possible mechanism for tumor dormancy is the downregulation of VEGF expression by 15-LOX-2. Our studies suggest that a gain in 15-LOX-2 expression keeps prostate tumors dormant at least partly through downregulating VEGF expression. Loss of 15-LOX-2 functionalities, therefore, may have significant bearings on the exit of prostate tumors from dormancy. Our studies lend a strong rationale to utilize 15-LOX-2 as a potential biomarker to categorize prostate tumors regarding their possible clinical progressions.

Emerging evidence suggests 15-LOX-2 as a tumor suppressor. It has been shown that 15-LOX-2 is a negative regulator of cell cycle as evidenced by the reduced incorporation of BrDU label in prostate cancer cells 10 or in mouse keratinocytes 20. Increased expression of 15-LOX-2 was found related to replicative senescence in normal prostate epithelial cells 21. We found that restoration of 15-LOX-2 expression in both PC-3 and DU145 cells increased cell cycle arrest at G0/G1 phase, suggesting that 15-LOX-2 may regulate cell cycle progression. However, further studies are needed to determine how 15-LOX-2 regulates cell cycle progression and causes G0/G1 arrest.

The tumor suppressing activities of 15-LOX-2 was firstly proposed on the observation that PC-3 cells stably transfected with a 15-LOX-2 expression construct blocked tumor development when injected into a mouse prostate 12. However, without histological data of the prostate glands injected with tumor cells or tumor imaging data demonstrating the temporal progression of tumors, it is unknown whether the tumor cells with 15-LOX-2 expression just failed to form tumors, or 15-LOX-2 expressing tumors failed to progress significantly. The subcutaneous model chosen in this study enabled us to evaluate the temporal growth and progression of tumors, especially those derived from 15-LOX-2 transfected prostate cancer cells. In our study, the tumor suppressing activities of 15-LOX-2 were extended to an additional prostate cancer cell line, DU145. Interestingly, 15-LOX-2 did not abrogate the tumorigenicity of prostate cancer cells when restored in PC-3 or DU145 cells. Instead, 15-LOX-2 expression significantly reduced the growth of prostate tumors in vivo and kept the tumors in a state of dormancy. Further studies suggest that VEGF expression was significantly downregulated by 15-LOX-2. As a key regulator of angiogenesis, VEGF is closely associated with angiogenesis in prostate cancer 18 and is required for prostate cancer cells to acquire angiogenic phenotype 22. Our study suggests a novel link between 15-LOX-2, VEGF expression, and tumor dormancy.

Abundantly expressed in normal or benign prostate tissues, 15-LOX-2 expression is found frequently silenced during prostate carcinogenesis. In prostate tumors, 15-LOX-2 immunostaining was completely absent in 23 of 70 cases, with negative staining in more than 50% of the tumor in 45 of 70 cases 5. It should be pointed out that although absent in approximately 30% cases of prostate tumor specimens examined, 15-LOX-2 expression was not completely lost; the majority of cases (70%) still retained 15-LOX-2 expression to certain extents 5. Data from a number of clinical studies establishes that the expression levels of 15-LOX-2 are inversely correlated with the pathological grade and Gleason scores of cancer patients57. With our data suggesting that loss of 15-LOX-2 may lead to the exit of tumors from dormancy, it will be interesting to determine whether the expression or functionality of 15-LOX-2 can be used as a marker to predict the clinical progression of human prostate cancer.

In addition to prostate cancer, 15-LOX-2 expression is also frequently lost or suppressed in lung, esophageal, and sebaceous cancers 5, 6, 8, 9. Therefore, the novel observation regarding 15-LOX-2 in suppressing VEGF gene expression may have significant implications in other cancers. However, further studies are required to determine whether 15-LOX-2 can keep those cancers dormant, and whether a loss of 15-LOX-2 expression or functionality has significant bearings on the exit of tumors from dormancy.

In summary, the present study describes a novel link between 15-LOX-2, VEGF expression, and tumor dormancy. A gain in 15-LOX-2 expression led to prostate tumor dormancy in vivo, which was accompanied with increased necrosis and apoptosis, and with a reduction in VEGF expression. Further studies suggest that 15-LOX-2 downregulates the gene expression of VEGF in prostate cancer. The findings add a novel dimension for the functions of 15-LOX-2, a tumor suppressor whose expression or functionality is frequently suppressed in a variety of cancers.

Acknowledgement

We thank Drs. Colin Funk and Dean Tang for providing 15-LOX-2 expression constructs and Dr. Giles Page for the VEGF promoter construct. This work was partially supported by National Institute of Health Grant R01CA029997 (K.V.H) and R01CA131445 (D.N), start-up funds from Southern Illinois University School of Medicine and SimmonsCooper Cancer Institute (D.N), United States Department of Defense Prostate Cancer Research Program New Investigator Award No.W81XWH-04-1-0143 (D.N), and a grant from Illinois Department of Public Health Prostate Cancer Research Program (D.N).

Abbreviations used

AA
arachidonic acid
HETE
hydroxyeicosatetraenoic acid
LOX
lipoxygenase
15-LOX-2
15-lipoxygenase-2
VEGF
vascular endothelial growth factor

Footnotes

Novelty and Impact: The present study describes a novel function of 15-LOX-2 in suppressing VEGF expression and maintaining prostate tumor dormancy. Loss of 15-LOX-2 expression or functionality, as observed in prostate and other cancers, may represent a key step in the acquisition of malignancy or accelerated clinical progression.

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