Modulation of prostate cancer genetic risk by omega-3 and omega-6 fatty acids

Although a causal role of genetic alterations in human cancer is well established, it is still unclear whether dietary fat can modulate cancer risk in a predisposed population. Epidemiological studies suggest that diets rich in omega-3 polyunsaturated fatty acids reduce cancer incidence. To determine the influence of fatty acids on prostate cancer risk in animals with a defined genetic lesion, we used prostate-specific Pten-knockout mice, an immune-competent, orthotopic prostate cancer model, and diets with defined polyunsaturated fatty acid levels. We found that omega-3 fatty acids reduced prostate tumor growth, slowed histopathological progression, and increased survival, whereas omega-6 fatty acids had opposite effects. Introducing an omega-3 desaturase, which converts omega-6 to omega-3 fatty acids, into the Pten-knockout mice reduced tumor growth similarly to the omega-3 diet. Tumors from mice on the omega-3 diet had lower proportions of phosphorylated Bad and higher apoptotic indexes compared with those from mice on omega-6 diet. Knockdown of Bad eliminated omega-3–induced cell death, and introduction of exogenous Bad restored the sensitivity to omega-3 fatty acids. Our data suggest that modulation of prostate cancer development by polyunsaturated fatty acids is mediated in part through Bad-dependent apoptosis. This study highlights the importance of gene-diet interactions in prostate cancer.

A causal role of genetic alterations in human cancer is well established; however, environmental influence on cancer risk is not clearly understood. Over 2 decades, epidemiologic studies have been reported on the effect of dietary fat on prostate cancer risk (1). However, the mechanistic role of dietary fat in prostate cancer remains ill defined. Prostate cancer is the most frequently diagnosed cancer and a leading cause of cancer death in men in the US. There is a wide variation in international prostate cancer mortality rates; these are particularly high in Northern Europe and North America and much lower in Japan and other Asian countries (2). Yet small, latent carcinomas of the prostate diagnosed at autopsy are as common in Asian countries as in Western countries (3). Immigrants from Poland and Japan exhibit a significant increase in the risk of developing clinical prostate cancer when resident in the US (4–6). These findings implicate environmental variables, and possibly diet, as significant contributing factors.

Omega-3 and omega-6 polyunsaturated FAs (PUFAs) are essential FAs: mammals can neither synthesize them de novo nor interconvert them; therefore, they have to be taken in from diet. Many vegetable oils contain high levels of omega-6 PUFAs, whereas fish oil is a rich source of omega-3 PUFAs, mainly eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3). Diets with an omega-6/omega-3 PUFA ratio of 1 are recommended for human consumption by a panel of nutritionists (7), and diets with a similar ratio are consumed by some populations such as the Inuit (8). Current Western diets have omega-6/omega-3 ratios of approximately 30 (9), although they can be as high as 50 (10) (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI31494DS1). Some evidence suggests that cyclooxygenase inhibitors, which block the metabolism of omega-6 PUFAs, are beneficial in prevention of colon (11) and prostate (12) cancer. However, the cardiovascular toxicity (13) of cyclooxygenase-2 inhibitors has jeopardized the clinical utility of these drugs. Reducing the intake of omega-6 PUFAs and increasing the proportion of dietary omega-3 is an attractive approach.

Inaccuracy in reporting dietary intake and difficulties in conducting mechanistic studies on human populations have hampered investigations on the role of dietary fat in prostate cancer development. Most experiments using animals have been performed in xenograft models and are limited by the fact that tumors grow at an ectopic site in an immune-deficient host environment. Moreover, dietary composition in animal diets is often poorly defined, and dietary intake is inadequately monitored. In an effort to overcome these limitations, we used prostate-specific Pten-knockout mice, an immune-competent, orthotopic prostate cancer model, and designed diets to investigate the influence of dietary PUFAs on prostate cancer risk in animals with this defined genetic lesion.

Omega-3 and -6 PUFAs differentially modulate prostate tumor weight in Pten^P–/– mice. Mice with a defined genetic lesion, in this case loss of the tumor suppressor gene Pten, develop cancer spontaneously. To determine whether genetic cancer risk can be modified by dietary factors, wild-type mice and mice with Pten deletion were fed diets with different amounts of omega-3 and omega-6 fatty acids. Prostate-specific deletion of floxed Pten was achieved by expressing Cre recombinase under the control of a probasin promoter (PB). Mouse genotypes are Pten^loxP/loxPPB-cre4^–/– (wild-type), Pten^loxP/+PB-cre4^T/– (heterozygous), and Pten^loxP/loxPPB-cre4^T/– (homozygous). For simplicity, we hereafter refer to these mice as Pten^P+/+, Pten^P+/–, and Pten^P–/–, respectively. Experimental diets included high–omega-3, low–omega-3, and high–omega-6 diets, with ratios of omega-6/omega-3 PUFAs of 1, 20, and 40, respectively. Diets were designed to be nutritionally balanced, with the same caloric input and percentage of energy from fat (Supplemental Table 1). Omega-6/omega-3 FA ratios in mouse blood and prostate tissues were similar to those in the respective diet (Supplemental Figure 2). In addition, phospholipid profiling demonstrated that approximately 5-fold more omega-3 PUFAs were incorporated at the sn-2 position in mice fed the high–omega-3 diet compared with mice fed with the low–omega-3 or the–high omega-6 diet (Supplemental Figure 2C). The 3 diets were absorbed equally well in mice of different genotypes at various ages and did not affect body weight (Supplemental Figure 3).

Mice were sacrificed at 5, 8, 12, and 24 weeks of age. After recording mouse body weight, we dissected, photographed, and weighed the anterior, dorsolateral, and ventral prostate (AP, DL, and VP) lobes. In the Pten^P+/+ and Pten^P+/– groups, the relative prostate weight (expressed as mg/25 g body weight) increased from approximately 29 to approximately 35 between 5 and 8 weeks and remained constant thereafter (Figure 1). Diets did not affect the weight or gross appearance of these wild-type prostates. By contrast, the average prostate weight of Pten^P–/– mice was significantly higher than that of Pten^P+/+ or Pten^P+/– mice starting at 8 weeks of age (Figure 1). Remarkably, the prostate weight gain from 5 to 24 weeks was significantly less in mice fed the high–omega-3 diet compared with mice fed the high–omega-6 diet, with intermediary gains for mice on the low–omega-3 diet (Figure 1). Age, treatment, and age/treatment interactions were all statistically significant (treatment, P < 0.001; age, P < 0.001; interaction, P = 0.018).

Figure 1

Suppression of prostate tumor proliferation by omega-3 PUFAs in vivo. Pten^P+/+, Pten^P+/–, and Pten^P–/– mice were fed the high–omega-3, low–omega-3, and high–omega-6 diet for a period of up to 24 weeks. Mouse AP, DL, and VP lobes were weighed, and the sums were expressed as milligrams per 25 gram body weight. Five mice were used per data point in a cohort of 180 mice. Open circles represent mice on the high–omega-3 diet; shaded squares, mice on the low–omega-3 diet; filled triangles, mice on the high–omega-6 diet. Horizontal bars represent averages. SDs are shown for Pten^P–/– mice fed with the high–omega-3 and high–omega-6 diet.

Omega-3 and -6 PUFAs differentially affect prostate tumor progression to carcinoma in Pten^P–/– mice. Prostate lobes from Pten^P+/+ and Pten^P+/– mice appeared morphologically normal at 5–24 weeks of age, regardless of diet. However, prostate lobes from Pten^P–/– mice were clearly enlarged and showed increased vascularity as well as cellularity (Figure 2A). Pathological evaluation was performed to determine the presence of hyperplasia, carcinoma in situ (CIS), and invasive carcinoma in AP, DL, and VP lobes of Pten^P–/– mice (Figure 2, B and C, and Supplemental Figure 4). DL lobes developed a greater proportion of advanced lesions as compared with AP and VP lobes. Prostates from 8-week-old mice on the high–omega-3 diet were more likely to have normal or benign histology. For instance, all VP lobes from 8-week-old mice on the high–omega-3 diet were either normal or hyperplastic, whereas VP lobes from mice on the high–omega-6 diet frequently contained CIS and invasive carcinoma. Even in the DL lobe, where Pten deletion occurs earliest, only half of the mice fed the high–omega-3 diet developed invasive carcinoma, whereas 80% of mice fed the high–omega-6 diet had invasive carcinoma. Mice fed the low–omega-3 diet generally developed prostate lesions with intermediate histopathology.

Figure 2

Pathological evaluation of prostate. (A) Gross appearance of 8-week-old prostates. (B) Histological evaluation of AP, DL, and VP lobes. Histology was evaluated for the cohort of 180 mice used in Figure 1, but qualitative differences were observed for 8-week-old Pten^P–/– mice when the high–omega-3, low–omega-3, and high–omega-6 diets were compared. Two sections of AP, DL, and VP from each mouse (5 mice per group) were sampled from separate areas of each tissue block and evaluated by 2 veterinary pathologists. When complex histology was found, the most advanced type was indicated. Hyper, hyperplasia; cis, carcinoma in situ; Inv ca, invasive carcinoma. (C) Representative H&E-stained sections from VPs of Pten^P–/– mice. Scale bar: 100 μm. Additional sections are shown in Supplemental Figure 3.

It is noteworthy that omega-3 PUFAs slowed the histological progression of prostate tumors in 5- to 8-week-old Pten^P–/– mice. Once carcinoma developed, omega-3 PUFAs induced apoptosis and decreased the growth of tumor mass. The probasin promoter, which drives the prostate-specific expression of cre, is activated by androgens. Levels of sex hormones increase in male mice around 6 weeks of age, resulting in efficient Pten deletion. Activity of the probasin promoter is highest in the lateral prostate followed by anterior and ventral lobes. Accordingly, invasive carcinoma developed earlier in the DL than the VP and AP lobes. To determine whether diets affected the Pten deletion rate, which may account for the difference in prostate cancer progression, we used 3 different approaches: (a) real-time quantification of the inactive Pten allele (Figure 3, A and B); (b) enzymatic assay of a lacZ gene whose expression depends upon Cre activity (Figure 3C); and (c) Western blotting of the active form of Akt, which is indicative of Pten inactivation (Figure 3C). Our data showed a similar Pten deletion rate in mice on the high–omega-3 diet and the high–omega-6 diet, indicating that this was not a factor in the rate of cancer development.

Figure 3

Pten deletion rate in mice on high–omega-3 and high–omega-6 diets. (A) Schematic representation of Pten and lacZ alleles before and after Cre-loxP–mediated recombination. In the ROSA26^LoxZ allele, recombination removes a sequence that interrupts the coding frame of β-galactosidase, resulting in activation of β-galactosidase enzyme. (B) Real-time quantification of Pten Δ5 allele. The inactive (exon 5 deletion) Pten allele and a control gene (wild-type Il-2) were quantified in 6-, 7-, and 8-week-old AP, DL, and VP lobes from mice on the high–omega-3 and high–omega-6 diet. Levels of the Pten Δ5 allele normalized to Il-2 are shown. Three mice were used for each data point, with bars representing SEM. Pten was deleted to similar extents in mice on either diet. (C) Prostates from 6-week-old Pten^loxP/loxPROSA26^Z/ZPB-cre4^T/– mice on the high–omega-3 and high–omega-6 diet were dissected, snap-frozen, and used for β-galactosidase staining as well as activity measurement. Three mice were used for each data point. Bars represent SEM. Pictures were taken at ×20 magnification. In 4 of the 6 mice that had sufficient protein remaining, the ratio of phosphorylated to total Akt, which is indicative of Pten inactivation, was quantified by Western blotting.

Genetic validation of the effects of PUFAs on prostate tumor growth. Although dietary ingredients were carefully formulated, the diets necessarily included other fats besides the omega-3 and -6 PUFAs to keep the energy from fat constant (Supplemental Table 1). To eliminate these possible confounding effects, the fat1 transgene (14), encoding an omega-3 desaturase from Caenorhabditis elegans that converts omega-6 into omega-3 FAs, was bred into the prostate-specific Pten-knockout genetic background. Pten^P–/– mice with or without the fat1 transgene (fat1^T/– and fat1^–/–, respectively) were both fed the high–omega-6 diet. Mice were sacrificed at 12 weeks of age, and prostates were dissected and weighed. The average omega-6/omega-3 ratio in the blood and prostate tissues of fat1^T/– mice was 5, compared with 40 in fat1^–/– mice (Table 1). Thus, fat-1 was able to convert most of the omega-6 PUFAs to omega-3. Relative prostate weights were significantly lower in Pten^P–/–fat1^T/– than that in Pten^P–/–fat1^–/– mice (Figure 4; P < 0.006, Student’s t test). This genetic approach confirms the suppressive effect of omega-3 PUFAs on tumor growth observed with diet manipulation. Thus, the fat1 mouse results are consistent with our conclusion that omega-3 and omega-6 FAs have opposite effects on tumor growth.

Figure 4

Effect of the fat-1 omega-3 desaturase on prostate tumor growth. Pten^P–/–fat1^T/– and Pten^P–/–fat1^–/– mice were fed the high–omega-6 diet. Prostate tumor weight was compared in 12-week-old mice, with 5 mice per group. Bars represent SD. P < 0.006, Student’s t test.

Table 1

PUFA ratios in fat1 transgenic mice fed with high–omega-6 diet

Omega-3 and -6 PUFAs differentially affect survival of Pten^P–/– mice. Overall survival was also monitored for mice on the 3 different diets. Pten^P+/+ mice remained free of tumors and had a survival rate of 100% within the time period monitored, regardless of diet. Pten^P+/– mice also had a survival rate of 100%, although some prostates from mice on the high–omega-6 diet were hyperplastic or neoplastic. By contrast, the 12-month survival rate for Pten^P–/– mice was 60% on the high–omega-3 diet, 10% on the low–omega-3 diet, and 0% on the high–omega-6 diet (Figure 5). In fact, no Pten^P–/– mice on the high omega-6 diet survived beyond 10 months of age in our experimental group. The main cause of death was bladder obstruction due to the prostate tumor compressing the urethra. Bladders were visibly dilated in some mice, and a few were even ruptured. These severe cases led to early death. For Pten^P–/– mice that survived 6 months or longer, retention of urine in the AP lobes was consistently seen and often accompanied by pyelonephritis. Cox proportional hazards model analysis revealed that diets exerted statistically significant effects on survival (P = 0.005). Compared with the high–omega-6 diet, the high–omega-3 diet significantly increased the survival of Pten^P–/– mice (hazard ratio 0.137; P = 0.0016), and, although to a lesser degree, the low–omega-3 diet significantly increased the survival as well (hazard ratio 0.337; P = 0.0338).

Figure 5

Survival rate of mice on different diets. A cohort of 90 mice (10 mice per group) on the high–omega-3, low–omega-3, or high–omega-6 diet was monitored for a period of 1 year. The Kaplan-Meier cumulative survival plot shows the cumulative probability of survival of each group of mice. Pten^P+/+ and Pten^P+/– mice had 100% survival regardless of diet. Twelve-month survival of Pten^P–/– mice was 60%, 10%, and 0% on the high–omega-3, low–omega-3, and high–omega-6 diets, respectively.

Effect of omega-3 and -6 PUFAs on the proapoptotic protein Bad. Pten encodes a phosphatidylinositol phosphatase that negatively regulates the PI3K/Akt signaling pathway. Akt promotes cell survival, protein synthesis, and proliferation when it is activated after being recruited to the plasma membrane by PI(3,4,5)P₃ (phosphatidylinositol [3,4,5] triphosphate). Pten converts PI(3,4,5)P₃ to PI(4,5)P₂, resulting in lower activity of Akt. Loss of Pten is expected to increase Akt activity; therefore, we examined the status of Akt and its downstream signaling effectors in prostate samples of wild-type and Pten-knockout mice on the high–omega-3 and high–omega-6 diets.

Pten status or diet had little effect on total Akt protein levels, as measured by Western blotting (Figure 6A). The ratio of active (pSer473) Akt to total Akt protein was approximately 3- to 7-fold higher in prostates from the Pten^P–/– mice than those from the Pten^P+/+ mice. However, there was no significant difference in the active/total Akt ratio between Pten^P–/– prostates from mice on the high–omega-3 and high–omega-6 diet (Figure 6A). Among the Akt effectors, the proapoptotic protein Bad was detected at higher levels in Pten^P–/– than in Pten^P+/+ prostates from mice on the high–omega-3 diet but not on the high–omega-6 diet. Additionally, the ratio of phosphorylated (pSer112) to total Bad was significantly lower in Pten^P–/– prostates from mice on the omega-3 diet compared to the omega-6 diet (Figure 6A). Phosphorylation of Bad inhibits its proapoptotic function. Therefore, in mice on the omega-3 diet, the elevated Bad levels combined with a lower fraction of phosphorylated Bad would favor apoptosis. Indeed, a higher number of apoptotic cells was seen in Pten^P–/– prostates from mice on the high–omega-3 than on the high–omega-6 diet as determined by immunohistochemistry of cleaved caspase-3 (Figure 6B and Table 2). Other Akt substrates, namely Foxo1 and Foxo3a and p70S6K (product of the gene Rps6kb1), were phosphorylated to similar degrees in Pten^P–/– prostates regardless of diet (Supplemental Figure 5).

Table 2

Apoptotic index in tumor glands

Omega-3, but not omega-6, PUFA induces prostate cancer cell apoptosis in a Bad-dependent manner. Omega-3 PUFA treatment inhibits tumor cell proliferation and induces apoptosis in culture (15). Since in vivo data suggest that omega-3 PUFAs may induce apoptosis through regulation of Bad, we further investigated the role of Bad in omega-3 PUFA–induced cell death. PC3 and LNCaP cells were mock infected or infected with lentiviruses expressing scrambled or Bad-specific short hairpin RNA (shRNA) (Figure 7A). Successful knockdown of Bad was confirmed by Western blotting (Figure 7A). These cells were then incubated with omega-3 or -6 PUFAs for 6 days, and the total cell number as well as the number of dead cells were determined by trypan blue exclusion. As shown in Figure 7B, in mock- or scrambled shRNA–infected cells, omega-3 PUFA treatment increased the percentage of dead cells, whereas omega-6 PUFA treatment had no significant effect. Strikingly, knockdown of Bad completely eliminated omega-3 PUFA–induced cell death, and the percentage of dead cells was similar to that in untreated cells. By contrast, knockdown of Bad had little effect on omega-6 PUFA–treated cells. To confirm the specificity of the Bad knockdown construct, we introduced mouse Bad cDNA, which cannot be silenced by our shRNA designed to target human Bad. Introduction of Bad cDNA restored prostate cancer cell sensitivity to omega-3–induced cell death but did not significantly increase cell death upon omega-6 treatment (Figure 7C). These results suggest that cell death induced by omega-3 PUFAs is dependent on Bad expression.

Figure 7

Dependence on Bad for omega-3 PUFA–induced cell death. (A) PC3 cells were infected with lentivirus expressing scrambled shRNA (C shRNA) or Bad-specific shRNA (Bad shRNA) or mock infected (no shRNA) for 2 days. Cells were seeded and incubated with FAs for 6 days, then photographed using a fluorescence microscope. The presence of lentivirus is evidenced by GFP expression. Western blotting was performed to confirm the successful knockdown of Bad in both PC3 and LNCaP cells. Day 0, prior to FA treatment; day 6, after incubation with media containing FA for 6 days. C, control. (B) Live and dead cells were enumerated by the trypan blue exclusion method using a hemocytometer. P values were determined by Student’s t test. LNCaP cells have higher background apoptosis compared with PC3 cells, but omega-3 FA treatment increased apoptosis approximately 4-fold in both cell lines.(C) LNCaP cells were transfected with control vector or mouse Bad expression vector. G418-resistant colonies were isolated, verified for successful expression of the HA-tagged mouse Bad by Western blotting, and pooled. Vector control– (V) and mouse Bad–expressing (mBad) LNCaP cells were infected with shRNA lentivirus as described above. Cells were incubated with FAs for 6 days, and live and dead cells were enumerated as described above. Cell lysates were used for confirmation of successful knockdown of the endogenous Bad but not the exogenous mouse Bad (whose sequence varies from human Bad in the region targeted by the shRNA).

Although a causal role of genetic alterations in human cancer is well established, epigenetic effects on cancer were not appreciated until recently (16). On the one hand, methylation, acetylation, and other molecular mechanisms implicated in epigenetics are being studied intensively (17, 18), but environmental factors causing these changes are largely unknown. On the other hand, factors such as diet are believed to affect cancer incidence, but molecular mechanisms have not been delineated. Here, we demonstrated the influence of defined dietary factors (omega-3 and -6 essential FAs) on a mouse model of prostate cancer with a known genetic risk (Pten deletion) and investigated underlying molecular mechanisms.

Epidemiological studies suggest that consumption of fish or fish oil reduces prostate cancer incidence (19, 20). One of the largest prospective studies, involving 6,272 men with 30 years of follow-up, indicated that fatty fish consumption was associated with decreased risk of prostate cancer (20). Serum levels of omega-3 PUFAs were reported to be significantly lower in patients with benign prostate hyperplasia and prostate cancer, and omega-6 PUFA levels were higher in patients with prostate cancer compared with age-matched controls (21). In prostate tissues, the percentage of total PUFAs was shown to be significantly lower in the presence of perineural invasion, seminal vesicle involvement, and stage T3 tumor. Levels of α linolenic acid (αLNA) omega-3 PUFAs were significantly lower when tumor extended to an anatomical or surgical margin. Total omega-3 levels and omega-3/omega-6 PUFA ratios were 1.5- to 3.3-fold lower in cases than in controls (22). In a 12-year prospective study of 47,882 men participating in the Health Professionals Follow-up Study, higher consumption of fish was strongly associated with a reduced risk of metastatic prostate cancer (23).

Nevertheless, molecular mechanisms of omega-3 PUFA effects on prostate cancer remain elusive. Our data show that the high–omega-3 diet, with an omega-6/omega-3 ratio recommended by nutritionists, could effectively deliver omega-3 PUFA to the prostate (Supplemental Figure 2), delay tumor formation (Figure 1) and progression (Figure 2), and prolong survival (Figure 5) as compared with the high–omega-6 diet. Mice on the low–omega-3 diet, with an omega-6/omega-3 ratio of 20 as compared with a ratio of 1 in the high–omega-3 diet, showed intermediary tumor growth, progression, and survival. Therefore, the omega-6/omega-3 ratio appears to be a critical factor in the effectiveness of prostate cancer suppression, with a higher proportion of omega-3 being more effective. However, the absolute amount of PUFAs may also be of consequence, since high total fat intake has been associated with cancer incidence (24). In the present study, all diets contained 13% fat with 30% energy from fat, mimicking the average Western diet, which is relatively high in fat. The development of normal prostate was not affected by the ratio of omega-6 to omega-3 in the Pten wild-type background. This demonstrates the importance of gene-diet interactions and that genetic cancer risk can be modified favorably by omega-3 PUFAs. It remains to be determined whether there is a critical omega-6/omega-3 ratio threshold for achieving maximal tumor suppression. Clinically, prostate cancer is usually diagnosed in men age 60 or older, and cancer cells proliferate slowly. Therefore, dietary and/or chemoprevention are of particular importance for the management of prostate cancer. Our data imply a beneficial effect of omega-3 PUFAs on delaying the onset of human prostate cancer. It will be interesting to determine whether any beneficial effects can also be achieved by supplementing the diet with omega-3 PUFAs after tumor initiation has occurred.

Lipid signaling plays a critical role in cancer of the prostate and many other human cancers. The Pten tumor suppressor gene is the most frequently mutated gene in metastases of prostate cancer (25, 26). A significant loss of Pten expression is also seen in prostate tumor tissues (27). Homozygous deletion of Pten in mouse prostate results in prostate cancer development and metastasis (28). In addition, the PI3K pathway appears to be a dominant growth factor–activated cell survival pathway in LNCaP prostate carcinoma cells (29). It was shown that PI3K/Akt stimulates the androgen pathway (30, 31), which further highlights the importance of lipid signaling in prostate cancer.

We showed that the high–omega-3 PUFA diet reduced phosphorylation of the downstream Akt target Bad and increased tumor cell death compared with the high–omega-6 PUFA diet in Pten^P–/– mice (Figure 6). Knockdown of Bad in prostate cancer cells reduced the percentage of dead cells upon omega-3 PUFA treatment to baseline levels (Figure 7B), and introduction of exogenous Bad restored the phenotype (Figure 7C). It is well documented that phospho-Bad protein is sequestered by 14-3-3 and that dephosphorylation allows Bad to interact with antiapoptotic proteins Bcl-2 and/or Bcl-xL, thereby increasing the Bax/Bcl-2 ratio and favoring apoptosis (32). Expression of a nonphosphorylatable form of Bad induces apoptosis in various cell lines, including prostate cancer cells (33–35). Why is the Bad protein differentially phosphorylated in tumors from mice on omega-3 and omega-6 diet? Preliminary data indicated that in Pten^P–/– prostates from mice on the omega-6 diet, a large proportion of active Akt was localized to the plasma membrane, whereas on the omega-3 diet, active Akt tended to be distributed through the cytoplasm. The pattern of PI(3,4,5)P₃ localization coincided with that of the active Akt protein. The Akt kinase is activated by phosphoinositide binding (36, 37). In mammals, the sn-1 position on the glycerol backbone of phospholipids, including phosphoinositides, is usually linked to a saturated FA such as stearic acid and the sn-2 position often to an omega-6 PUFA such as arachidonic acid. Feeding cells or animals with omega-3 PUFAs results in replacement of omega-6 with omega-3 FAs at the sn-2 position (38), which was verified in the blood and prostate tissue of our experimental mice (Supplemental Figure 2C). Therefore, it is possible that incorporation of omega-3 PUFAs into phospholipids could alter the localization of both PI(3,4,5)P₃ and the active form of Akt protein, resulting in differential Bad phosphorylation. On the other hand, it is also possible that this differential Bad phosphorylation is caused by a phosphatase. However, the nature of the enzymes involved in Bad dephosphorylation is somewhat controversial. Calcineurin and protein phosphatase 2A have been shown to dephosphorylate Bad in different cell types (39, 40). Experiments exploring the mechanism of Bad dephosphorylation in our system are ongoing.

We believe Bad-dependent modulation of apoptosis by omega-3 PUFAs to be a novel finding that could account, at least in part, for the proposed tumor-preventive properties of these FAs. In addition, omega-3 and -6 PUFAs can be metabolized by cyclooxygenases and lipoxygenases (41), and the resulting eicosanoids have different biological functions in processes such as proliferation, inflammation, and angiogenesis. These processes may contribute to carcinogenesis directly or indirectly. Indeed, we have noticed a reduction in microvessel density as well as CD3⁺ lymphocyte levels in tumors from mice fed the omega-3 diet compared with those fed the high–omega-6 diet. The significance of these changes in relation to the suppression of prostate cancer by omega-3 PUFAs is currently under investigation.

View Supplemental data

We are grateful to Bioriginal (Saskatoon, Saskatchewan, Canada), which generously provided omega-3 FAs used in this study. We thank Lingyi Lu (Department of Biostatistical Science) for statistical analysis and Karen Klein (Grant and Manuscript Development, Office of Research, Wake Forest University School of Medicine) for manuscript editing. This work was supported in part by NIH grants R01CA107668 and P01CA106742, the Chinese National Natural Science Foundation (no. 20576047), and the Chang Jiang Scholars Program.

Address correspondence to: Yong Q. Chen, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157, USA. Phone: (336) 713-7655; Fax: (336) 713-7660; E-mail: yqchen@wfubmc.edu.

Nonstandard abbreviations used: AP, anterior prostate; DL, dorsolateral prostate; PB, probasin promoter; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PUFA, polyunsaturated FA; shRNA, short hairpin RNA; VP, ventral prostate.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J. Clin. Invest.117:1866–1875 (2007). doi:10.1172/JCI31494

1. Kolonel, L.N. 2001. Fat, meat, and prostate cancer. Epidemiol. Rev. 23:72-81.
   View this article via: PubMed Google Scholar
2. Wynder, E.L., Mabuchi, K., Whitmore (Jr.), W.F. 1971. Epidemiology of cancer of the prostate. Cancer. 28:344-360.
   View this article via: CrossRef PubMed Google Scholar
3. Breslow, N., et al. 1977. Latent carcinoma of prostate at autopsy in seven areas. The International Agency for Research on Cancer. Lyon, France. Int. J. Cancer. 20:680-688.
   View this article via: CrossRef PubMed Google Scholar
4. Dunn, J.E. 1975. Cancer epidemiology in populations of the United States — with emphasis on Hawaii and California — and Japan. Cancer Res. 35:3240-3245.
   View this article via: PubMed Google Scholar
5. Haenszel, W., Kurihara, M. 1968. Studies of Japanese migrants. I. Mortality from cancer and other diseases among Japanese in the United States. J. Natl. Cancer Inst. 40:43-68.
   View this article via: PubMed Google Scholar
6. Staszewski, J., Haenszel, W. 1965. Cancer mortality among the Polish-born in the United States. J. Natl. Cancer Inst. 35:291-297.
   View this article via: PubMed Google Scholar
7. Simopoulos, A.P., Leaf, A., Salem (Jr.), N. 1999. Essentiality of and recommended dietary intakes for omega-6 and omega-3 fatty acids. Ann. Nutr. Metab. 43:127-130.
   View this article via: CrossRef PubMed Google Scholar
8. Dewailly, E., et al. 2001. n-3 fatty acids and cardiovascular disease risk factors among the Inuit of Nunavik. Am. J. Clin. Nutr. 74:464-473.
   View this article via: PubMed Google Scholar
9. Simopoulos, A.P. 1999. Essential fatty acids in health and chronic disease. Am. J. Clin. Nutr. 70(3 Suppl.):560S-569S.
10. Weber, P.C. 1989. Are we what we eat? Fatty acids in nutrition and in cell membranes: cell functions and disorders induced by dietary conditions. InFish, fats and your health. Proceedings of the International Conference on Fish Lipids and Their Influence on Human Health. August 9–11. Svanoy, Norway. Svanoy Foundation. 9–18.
11. Giardiello, F.M., et al. 1993. Treatment of colonic and rectal adenomas with sulindac in familial adenomatous polyposis. N. Engl. J. Med. 328:1313-1316.
    View this article via: CrossRef PubMed Google Scholar
12. Jacobs, E.J., et al. 2005. A large cohort study of aspirin and other nonsteroidal anti-inflammatory drugs and prostate cancer incidence. J. Natl. Cancer Inst. 97:975-980.
    View this article via: PubMed Google Scholar
13. Solomon, S.D., et al. 2005. Cardiovascular risk associated with celecoxib in a clinical trial for colorectal adenoma prevention. N. Engl. J. Med. 352:1071-1080.
    View this article via: CrossRef PubMed Google Scholar
14. Kang, J.X., Wang, J., Wu, L., Kang, Z.B. 2004. Transgenic mice: fat-1 mice convert n–6 to n–3 fatty acids. Nature. 427:504.
    View this article via: PubMed Google Scholar
15. Edwards, I.J., et al. 2004. Differential effects of delivery of omega-3 fatty acids to human cancer cells by low-density lipoproteins versus albumin. Clin. Cancer Res. 10:8275-8283.
    View this article via: CrossRef PubMed Google Scholar
16. Feinberg, A.P., Tycko, B. 2004. The history of cancer epigenetics. Nat. Rev. Cancer. 4:143-153.
    View this article via: PubMed Google Scholar
17. Feinberg, A.P. 2004. The epigenetics of cancer etiology. Semin. Cancer Biol. 14:427-432.
    View this article via: CrossRef PubMed Google Scholar
18. Lund, A.H., van Lohuizen, M. 2004. Epigenetics and cancer. Genes Dev. 18:2315-2335.
    View this article via: CrossRef PubMed Google Scholar
19. Norrish, A.E., Skeaff, C.M., Arribas, G.L., Sharpe, S.J., Jackson, R.T. 1999. Prostate cancer risk and consumption of fish oils: a dietary biomarker-based case-control study. Br. J. Cancer. 81:1238-1242.
    View this article via: CrossRef PubMed Google Scholar
20. Terry, P., Lichtenstein, P., Feychting, M., Ahlbom, A., Wolk, A. 2001. Fatty fish consumption and risk of prostate cancer. Lancet. 357:1764-1766.
    View this article via: CrossRef PubMed Google Scholar
21. Yang, Y.J., Lee, S.H., Hong, S.J., Chung, B.C. 1999. Comparison of fatty acid profiles in the serum of patients with prostate cancer and benign prostatic hyperplasia. Clin. Biochem. 32:405-409.
    View this article via: CrossRef PubMed Google Scholar
22. Freeman, V.L., et al. 2000. Prostatic levels of fatty acids and the histopathology of localized prostate cancer. J. Urol. 164:2168-2172.
    View this article via: CrossRef PubMed Google Scholar
23. Augustsson, K., et al. 2003. A prospective study of intake of fish and marine fatty acids and prostate cancer. Cancer Epidemiol. Biomarkers Prev. 12:64-67.
    View this article via: PubMed Google Scholar
24. Hursting, S.D., Thornquist, M., Henderson, M.M. 1990. Types of dietary fat and the incidence of cancer at five sites. Prev. Med. 19:242-253.
    View this article via: CrossRef PubMed Google Scholar
25. Suzuki, H., et al. 1998. Interfocal heterogeneity of PTEN/MMAC1 gene alterations in multiple metastatic prostate cancer tissues. Cancer Res. 58:204-209.
    View this article via: PubMed Google Scholar
26. Vlietstra, R.J., van Alewijk, D.C., Hermans, K.G., van Steenbrugge, G.J., Trapman, J. 1998. Frequent inactivation of PTEN in prostate cancer cell lines and xenografts. Cancer Res. 58:2720-2723.
    View this article via: PubMed Google Scholar
27. McMenamin, M.E., et al. 1999. Loss of PTEN expression in paraffin-embedded primary prostate cancer correlates with high Gleason score and advanced stage. Cancer Res. 59:4291-4296.
    View this article via: PubMed Google Scholar
28. Wang, S., et al. 2003. Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer. Cancer Cell. 4:209-221.
    View this article via: CrossRef PubMed Google Scholar
29. Lin, J., Adam, R.M., Santiestevan, E., Freeman, M.R. 1999. The phosphatidylinositol 3ι-kinase pathway is a dominant growth factor-activated cell survival pathway in LNCaP human prostate carcinoma cells. Cancer Res. 59:2891-2897.
    View this article via: PubMed Google Scholar
30. Sharma, M., Chuang, W.W., Sun, Z. 2002. Phosphatidylinositol 3-kinase/Akt stimulates androgen pathway through GSK3beta inhibition and nuclear beta-catenin accumulation. J. Biol. Chem. 277:30935-30941.
    View this article via: CrossRef PubMed Google Scholar
31. Manin, M., et al. 2002. Androgen receptor expression is regulated by the phosphoinositide 3-kinase/Akt pathway in normal and tumoral epithelial cells. Biochem. J. 366:729-736.
    View this article via: PubMed Google Scholar
32. Danial, N.N., Korsmeyer, S.J. 2004. Cell death: critical control points. Cell. 116:205-219.
    View this article via: CrossRef PubMed Google Scholar
33. Datta, S.R., et al. 2000. 14-3-3 proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation. Mol. Cell. 6:41-51.
    View this article via: CrossRef PubMed Google Scholar
34. Sastry, K.S., Smith, A.J., Karpova, Y., Datta, S.R., Kulik, G. 2006. Diverse antiapoptotic signaling pathways activated by vasoactive intestinal polypeptide, epidermal growth factor, and phosphatidylinositol 3-kinase in prostate cancer cells converge on BAD. J. Biol. Chem. 281:20891-20901.
    View this article via: CrossRef PubMed Google Scholar
35. She, Q.B., et al. 2005. The BAD protein integrates survival signaling by EGFR/MAPK and PI3K/Akt kinase pathways in PTEN-deficient tumor cells. Cancer Cell. 8:287-297.
    View this article via: CrossRef PubMed Google Scholar
36. Klippel, A., Kavanaugh, W.M., Pot, D., Williams, L.T. 1997. A specific product of phosphatidylinositol 3-kinase directly activates the protein kinase Akt through its pleckstrin homology domain. Mol. Cell. Biol. 17:338-344.
    View this article via: PubMed Google Scholar
37. Stocker, H., et al. 2002. Living with lethal PIP3 levels: viability of flies lacking PTEN restored by a PH domain mutation in Akt/PKB. Science. 295:2088-2091.
    View this article via: CrossRef PubMed Google Scholar
38. Chabot, M.C., Schmitt, J.D., Bullock, B.C., Wykle, R.L. 1987. Reacylation of platelet activating factor with eicosapentaenoic acid in fish-oil-enriched monkey neutrophils. Biochim. Biophys. Acta. 922:214-220.
    View this article via: PubMed Google Scholar
39. Wang, H.G., et al. 1999. Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD. Science. 284:339-343.
    View this article via: CrossRef PubMed Google Scholar
40. Chiang, C.W., et al. 2003. Protein phosphatase 2A dephosphorylation of phosphoserine 112 plays the gatekeeper role for BAD-mediated apoptosis. Mol. Cell. Biol. 23:6350-6362.
    View this article via: CrossRef PubMed Google Scholar
41. Larsson, S.C., Kumlin, M., Ingelman-Sundberg, M., Wolk, A. 2004. Dietary long-chain n-3 fatty acids for the prevention of cancer: a review of potential mechanisms. Am. J. Clin. Nutr. 79:935-945.
    View this article via: PubMed Google Scholar
42. Lesche, R., et al. 2002. Cre/loxP-mediated inactivation of the murine Pten tumor suppressor gene. Genesis. 32:148-149.
    View this article via: CrossRef PubMed Google Scholar
43. Wu, X., et al. 2001. Generation of a prostate epithelial cell-specific Cre transgenic mouse model for tissue-specific gene ablation. Mech. Dev. 101:61-69.
    View this article via: CrossRef PubMed Google Scholar
44. Berquin, I.M., Min, Y., Wu, R., Wu, H., Chen, Y.Q. 2005. Expression signature of the mouse prostate. J. Biol. Chem. 280:36442-36451.
    View this article via: CrossRef PubMed Google Scholar
45. Shappell, S.B., et al. 2004. Prostate pathology of genetically engineered mice: definitions and classification. The consensus report from the Bar Harbor meeting of the Mouse Models of Human Cancer Consortium Prostate Pathology Committee. Cancer Res. 64:2270-2305.
    View this article via: CrossRef PubMed Google Scholar
46. Bligh, E.G., Dyer, W.J. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917.
    View this article via: PubMed Google Scholar
47. Metcalfe, L.D., Schmitz, A.A., Pelka, J.R. 1966. Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Anal. Chem. 38:514-515.
    View this article via: CrossRef Google Scholar
48. Schwenke, D.C., Rudel, L.L., Sorci-Thomas, M.G., Thomas, M.J. 2002. Alpha-tocopherol protects against diet induced atherosclerosis in New Zealand white rabbits. J. Lipid Res. 43:1927-1938.
    View this article via: CrossRef PubMed Google Scholar
49. Rouser, G., Siakotas, A.N., Fleisher, S. 1966. Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots. Lipids. 1:85-86.
    View this article via: CrossRef Google Scholar
50. DeLong, C.J., Baker, P.R., Samuel, M., Cui, Z., Thomas, M.J. 2001. Molecular species composition of rat liver phospholipids by ESI-MS/MS: the effect of chromatography. J. Lipid Res. 42:1959-1968.
    View this article via: PubMed Google Scholar
51. Liu, J., et al. 2002. Mediation of the DCC apoptotic signal by DIP13 alpha. J. Biol. Chem. 277:26281-26285.
    View this article via: PubMed CrossRef Google Scholar