This secondary analysis of 50 CAFs was undertaken to explore potential mechanisms by which the low fat diet or flaxseed supplementation may influence prostate cancer development and progression. The results suggest that while flaxseed supplementation influenced a few CAFs, the low-fat diet appeared to exert a greater impact and decreased levels of a number of circulating proinflammatory CAFs. In the LF arm, significant changes were observed for 6 CAFs, the majority of which were pro-inflammatory factors, involved in the myeloid (e.g., G-CSF, M-CSF, and eotaxin) or pro-angiogenic factors (e.g., SDF-1α). In comparison, in the combination arm (FS+LF) we only detected significant decreases in eotaxin and MIP-1α and increases in SCF and b-NGF. It is not known whether these changes are primarily due to the low fat diet itself, weight loss, or some other factor (s). The fact that we observed such a strong correlation between changes in BMI and changes in VEGF suggests that weight loss or low fat diet may impact inflammatory and angiogenic responses.
Moreover, the decreases in the six CAFs in the LF arm appeared to occur in a coordinated manner, suggesting that the expression of some or all of these CAFs may be governed by a common regulatory mechanism. Pathway analysis identified the NF-κB pathway as a signaling node that interacted with a majority of the CAFs that decreased in the LF arm. Each of these factors has previously been reported to be regulated by NF-κB (10
). In addition, the immunohistochemical analysis of a subset of prostatectomy tumor specimens revealed a trend toward lower levels of NF-κB and COX-2 expressions. For VEGF, lower expression was observed for the LF arm as compared to controls. Meanwhile, a slight increase in VEGF expression was observed for the FS and FS+LF arms as compared to the control. These findings are consistent with the decreased levels of proangiogenic factors detected in the plasma of these patients. Caution however is necessary in interpreting these findings since this analysis was highly exploratory and only was performed on a limited number of specimens. Thus, further study is needed.
NF-κB, a central mediator of the inflammatory response, angiogenesis, and invasion is constitutively activated in many cancers (35
). Several recent studies have indicated that the NF-κB pathway plays a critical role in regulating energy balance and provides a link between diet and inflammation. A high-fat diet has been demonstrated to increased NF-κB activation and further, knockout of the NF-κB target, IKKε, prevented diet-induced obesity and chronic inflammation in mouse models (11
). Observations from a another study found that human subjects experience increases in NF-κB activation in peripheral blood mononuclear cells within hours of eating a high-fat meal (41
). In light of these previous studies findings and the current study’s findings that the majority of CAFs that decreased in patients in the LF arm were known to be NF-κB-regulated, our data suggest that a LF diet induced downregulation of the NF-κB pathway may have contributed to changes in circulating proinflammatory and angiogenic CAFs. However, not all known NF-κB-regulated factors (e.g., TNF-α and IL-6) decreased between baseline and preoperative follow-up (just prior to prostatectomy) in the LF arm, indicating that other pathways known to impact inflammatory and angiogenic pathways, such as the COX-2 and hypoxia-inducible factor-1α (HIF-1α) pathways, may have contributed to the changes we observed (42
The results of this study raise several questions when compared to the previously published analysis of prostate cancer specimens, in which tumors from men on the flaxseed arm had a lower rate of proliferation based on Ki-67 staining than those in the LF or control groups (30
). The first issue is whether Ki-67 or the CAFs in this study are appropriate surrogate markers for clinical outcome in prostate cancer. It is worth noting that in the prior study, only post-operative Ki-67, and not changes in Ki-67, was assessed; therefore the impact of the dietary intervention on a particular patient could not be evaluated. Tumor proliferation, inflammation, and angiogenesis have all been linked to cancer progression in a variety of different settings, but given the design and size of the current study, it is not possible to determine which, if any, of the markers is associated with prostate cancer progression or clinical outcome in the pre-operative setting for prostate cancer. It is plausible that separate mechanisms govern the putative effects of flaxseed on tumor cell proliferation and of a low-fat diet on inflammatory and angiogenic CAFs. Further studies will be needed to identify these mechanisms and determine their impact on prostate cancer.
Another issue that remains unresolved is why the many changes in cytokines observed in the LF arm are not mirrored in the FS+LF arm. It is worth noting that while both the LF and FS+LF groups experienced a significant decrease in BMI (30
), the LF group had a slightly higher loss which may have impacted the cytokine changes. Furthermore, it is conceivable that the addition of flaxseed may have altered the pattern of CAF changes, or that at least some of the changes in the LF arm were due to chance alone. The observations that 19/20 of the CAFs in the LF arm changed in the same direction (decreased), however, and that many of these changes occurred in a coordinated manner as seen in , argue against these changes being solely due to chance.
To our knowledge, the current study is the first to report changes in plasma levels of CAFs resulting from dietary changes in prostate cancer patients. Prior studies have suggested that dietary factors influence prostate cancer development and progression. In addition, given that previous studies have shown higher rates of prostate cancer with high-fat diets, as well as associations between obesity and more aggressive disease, the mechanisms identified in this exploratory analysis involving the low-fat diet may merit further investigation (43
). Indeed, mounting evidence suggests that obesity is associated with chronic inflammation, which is characterized by an influx of circulating inflammation-related markers (45
). Adipose tissue itself is highly vascularized and is capable of promoting angiogenesis through the production and release of angiogenic factors, such as VEGF (46
). Preclinical studies have demonstrated that mice on a low-fat diet experience a reduction in prostate cancer growth and have lower serum insulin and IGF-1 levels, compared with mice fed a Western diet (49
). Adding serum from men on a low-fat, high-fiber diet and an exercise intervention to androgen-independent LNCaP cells also has been found to decrease growth, increase apoptosis, and reduce NF-κB activation (47
). Taken together, these studies suggest that diet-induced changes in inflammatory and/or angiogenic factors may influence the growth of prostate cancer.
In the current study we also observed decreases in the proinflammatory cytokine TNF-β lymphotoxin) in the low-fat arm. TNF-β can bind to the TGF-βR1, the same receptor as TNF-α and TGF-R2 and is thus an important activator of NF-κB (51
). In a murine obesity model, elevated levels of TNF-α contributed to the development of hepatocellular carcinoma, suggesting obesity mediated increased TNF-1α contributes to the tumorigenic process (19
). TNF-α was also found to be elevated in parallel with activated NF-kB in the mammary gland in a diet-induced murine obesity model (14
). This is in line with our study, in which patients following a low-fat diet in the LF arm exhibited decreases in TNF-β that could potentially mediate decreased NF-κB activity and contribute to the changes in CAFs.
Additionally, significant decreases in eotaxin emerged in all three treatment arms. Eotaxin has pro-angiogenic effects through chemotaxis of microvascular endothelial cells and plays a role as a chemoattractant for eosinophils, basophils and Th2 lymphocytes (53
). Eotaxin expression is induced by proinflammatory cytokines such as TNF-α, IFN-α and glucocorticoids (22
). A previous study demonstrated that a link between obesity and eotaxin serum levels in obese (high-fat diet-induced) mouse models as well as in obese human serum and omental fat (54
). Similar to the results in the current study, serum eotaxin levels were reduced in patients who experienced diet-induced weight loss. An NF-κB binding site is present within the eotaxin promoter (along with STAT-6, IFN-α response and glucocorticoid response element) (53
). Suggesting that decreased NF-κB activity following a low-fat diet is a plausible mechanism for eotaxin changes observed in this study.
The current study had several potential limitations. Given the modest sample size, our findings might not be transferable to other studies. There was no adjustment for multiple comparisons given the exploratory nature of this analysis. A number of the CAFs we assessed can be affected by factors other than diet, such as stress, and may be greatly changed in a subset of individuals but would not reflect the effect of diet modulation on the cohort as a whole. The presence of a control diet arm, however, should reduce some of these confounding factors. Further, the biologic changes mediated by these changes in CAFs may not have been accurately demonstrated because of the short mean time period (30
days) of adhering to the diet on this study. Finally, it is not possible to determine whether changes in CAF were the result of changes in the tumor, the host, or a combination of the two and whether greater changes may have occurred in the tumor microenvironment than was observed in mixed venous blood. The findings from this study would therefore need to be validated in a larger study and a longer on-study duration time, and would ideally include paired tumor biopsies that would enable one to distinguish tumor vs host effects.
In summary, the current study was the first to investigate of the effects of LF and FS diets on circulating CAFs in prostate-cancer patients. While relatively few changes in CAFs were observed with flaxseed supplementation it does not negate the possibility that other mechanisms may be at play. In contrast, the low-fat diet appeared to spur a coordinated reduction among a set of inflammatory cytokines, particularly among those involved in myeloid-cell recruitment and proliferation, and several angiogenic factors. The changes with a low-fat diet occurred in factors regulated by NF-κB. Combined with emerging evidence that the NF-κB pathway plays a critical role in coupling energy balance with inflammatory and angiogenesis, our findings suggest that NF-κB is a potential mediator of the changes in circulating CAFs associated with a low-fat diet. Although these changes might be expected to reduce inflammation and tumor angiogenesis, the magnitude of these changes (detectable in mixed venous blood) were often modest and their impact on prostate cancer or other cancer types remains unknown and warrants further investigation. Therefore, the present study is hypothesis-generating, and additional studies are needed to investigate the potential links between diet, the NF-κB pathway, inflammation, angiogenesis and cancer biology.