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
), 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
). 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 ) and progression (Figure ), and prolong survival (Figure ) 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
). 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
), 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 PtenP–/–
mice (Figure ). Knockdown of Bad in prostate cancer cells reduced the percentage of dead cells upon omega-3 PUFA treatment to baseline levels (Figure B), and introduction of exogenous Bad restored the phenotype (Figure C). 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
). Why is the Bad protein differentially phosphorylated in tumors from mice on omega-3 and omega-6 diet? Preliminary data indicated that in PtenP–/–
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
). 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
), 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
). 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.