The MENS (Molecular Effects of Nutritional Supplements) study was a double-blinded placebo-controlled randomized clinical trial that was developed based on the epidemiological and clinical evidence that linked lycopene and fish oil with reduced prostate cancer incidence and progression 
. This trial demonstrated the feasibility and safety of studying the effects of nutritional supplements on prostate gene expression in men with low-risk prostate cancer opting for active surveillance. In addition, this study provided further evidence that active surveillance can offer a unique window of opportunity for investigating potential chemopreventive agents 
. Other trials including the Men's Eating and Living Study (MEAL) also provide important data on the feasibility of implementing clinical trials of dietary intervention in men with low risk prostate cancer 
We examined the relationship between baseline gene expression patterns and self-reported dietary intake levels of fish and tomato, and studied the effects of short-term lycopene or fish oil supplementation on the change in prostate gene expression. Taking into account the adjustment for multiple comparisons required in microarray analysis, both analyses yielded no individual gene that was differentially expressed. These results suggest that there were no differences between the groups compared, or that changes in expression were too subtle to be detected given a threshold. Modest alterations in gene expression are difficult to distinguish from noise especially when there is a large number of genes tested, limited samples and high variability between individuals 
. In addition, potential changes in gene expression may have been dampened by a combination of biological and technical factors including: 1) the dosage and formulation of lycopene and fish oil administered in our study did not have the potency to cause changes in gene expression; 2) the intervention period of three months may have been too short; 3) variability due to diverse participants' diets; 4) detectable changes in gene expression may have occurred in tumors but were not available for analysis; 5) the sample size was relatively small and 6) the noise inherent to the cDNA microarray technology (e.g. print batch effect) may have masked the relatively subtle effects on gene expression.
Although no individual gene was significantly identified with either baseline intake levels or change with lycopene or fish oil supplement, we further explored our prostate gene expression data for potential biological clues by performing pathway analyses of genes (not adjusted for multiple comparisons) that showed the largest differences between groups compared 
. The unadjusted p-value calculated for each gene was used to order and select top ranking genes for pathway analyses. Canonical pathway analyses (in IPA) revealed candidate pathways that were associated with high dietary intake of tomato or fish at baseline as well as subtle gene expression changes after lycopene and fish oil supplementation. These analyses were done to identify potential genes for future study e.g., RT-PCR of selected genes in candidate pathways modulated by dietary or supplementation of these micronutrients.
We observed that high dietary intake of fish modulated genes involved in metabolic pathways including C21-steroid hormone metabolism and insulin receptor signaling in the normal prostate tissue. To the best of our knowledge, associations between fish intake and steroid hormone metabolism or insulin receptor signaling in the prostate have not been reported. However, the modulation of docosahexaenoic acid (DHA) signaling makes sense as DHA is a major component of fish oil. Studies have shown that DHA inhibits growth of prostate cancer cells 
and may enhance the efficacy of taxanes, and possibly other drugs, such as COX-2 inhibitors 
. Moreover, we observed that fish oil supplementation modulated genes involved in arachidonic acid metabolism. In vivo
and in vitro
experimental studies have suggested that arachidonic acid, an omega-6 fatty acid, plays a role in the stimulation of proliferation genes that may lead to prostate cancer 
. Lowering omega-6
omega-3 fatty acid ratio by increased intake of foods rich in omega-3 fatty acids (such as fish) and perhaps by fish oil supplementation may have inhibitory effects on prostate cancer as demonstrated in cell lines and xenografts 
Selenoamino acid metabolism pathway was modulated in men who had high tomato consumption. Selenoamino acids are hypothesized to be responsible for the anti-cancer properties of selenium compounds 
. For example, seleno-methionine selectively induced growth inhibition and apoptosis in prostate cancer cells but not in normal cells 
, consistent with some of the observational epidemiological data suggesting that selenium may prevent prostate cancer. Of note, selenium supplementation was not associated with reduction in incidence of prostate cancer in the Selenium and Vitamin E Cancer Prevention Trial (SELECT) 
. We also observed that lycopene supplementation modulated signaling pathways including apoptosis and ceramide signaling. Ceramide is a family of lipid molecules that promote apoptosis and cell cycle arrest 
. Direct associations between lycopene and ceramide signaling have not been reported, however, lycopene's apoptotic effects have been documented in both xenografts and cancer cell lines 
. Interestingly, modulation of androgen and estrogen metabolism was observed in men with high consumption of fish and tomatoes. Many dietary factors, including a high omega-3 fatty acid diet, have been documented to impact androgen or estrogen levels 
-mediated oxidative stress response pathway was observed to be modulated by both lycopene and fish oil supplement vs. placebo. Studies in mice have shown that the loss of Nrf2
function correlated with increased reactive oxygen species and DNA damage leading to the transformation of normal prostate tissue 
. In addition, a study in knock out mice revealed a link between the chemopreventive effects of soy isoflavones and the role of Nrf2
in modulating signaling pathways involved in the prevention of prostate cancer 
. Recent studies have shown that other food-based or pharmacological compounds also exert their chemopreventive effects via the Nfr2
signaling pathway by mediating increased activity of cytoprotective enzymes, e.g. phase II detoxifying and antioxidant enzymes 
. Taken together, the modulation of the Nrf2
signaling pathway may be an important molecular mechanism involved in chemoprevention by several agents including lycopene and fish oil. Interventions that target the Nrf2
pathway may offer a promising strategy for chemoprevention.
Exploring the top genes (most differentially expressed) selected after applying specified cut-offs revealed intriguing results. For example, KLK3
(aka prostate specific antigen or PSA) was up regulated in the lycopene arm (6.2 fold change) compared to the placebo group (Table S3
) while KLK3
was down regulated (−2.5 fold change) in men who had high fish consumption at baseline compared to men who consumed less fish (Table S2
). Of note, IGF1
was down regulated in the fish oil arm (−1.5 fold change) compared to the placebo group (Table S4
). Down regulation of IGF1
was not observed in the lycopene arm. While provocative, care should be exercised when interpreting these results since these genes were not significant after adjustment for multiple comparisons.
Except for Nrf2
-mediated oxidative stress response, none of the pathways identified after supplementation overlapped with the pathways identified at baseline. Interestingly, there are on-going debates 
whether obtaining nutrients from whole food has the same health impacts as supplementation. Besides lycopene and fish oil (DHA/EPA), other naturally occurring nutrients in whole tomatoes (e.g. other carotenoids) 
and fish (e.g. alpha-linolenic acid) may also be important to the positive health effects of these foods. However, interpretations made from comparing the results between the analysis of baseline and supplementation groups in this study should be done with caution because the baseline and intervention analyses were originally designed to measure different outcomes.
Recent research in personalized nutrition has demonstrated that nutrients may interact with an individual's genotypic and phenotypic background 
. For example, supplements may affect gene expression in different directions (i.e. up- or down regulation) depending on the genotype/phenotype of the individual. Furthermore, nutrients may produce different phenotypes in patients with different genotypes. A P450 cytochrome allele, for instance, may metabolize a dietary substrate to a bioactive form, in contrast to another allele that produces an inactive metabolite 
. Hence, variability among patients may cancel out the effects of the dietary factors if analyzed at the global level resulting in an undetectable net change. As more examples of diet-gene interactions are discovered, increased power and sophistication of clinical trials will become possible.
A more recent report from the Selenium and Vitamin E Cancer Prevention Trial (SELECT) demonstrated the cell and tissue specific effects of selenium and vitamin E on gene and protein expression in the prostate 
. They detected differentially expressed genes (relative to the placebo group) in men who were taking selenium or vitamin E or in combination, only when cell type (normal epithelium, stroma or tumor) was taken into consideration. In the MENS study, morphologically normal tissue used for gene expression contained both stroma and normal epithelium; hence, cell type specific analysis was not possible.
Although differential expression analysis did not detect significant individual genes, our exploratory analysis revealed candidate in vivo pathways that may be modulated by dietary fish and tomato intakes or by lycopene and fish oil supplementation. Our study provides a platform to investigate the bioactivity and relevance of nutrients in prostate cancer. Understanding molecular mechanisms by which micronutrients affect gene expression would have a great impact on the development of prevention and treatment strategies in prostate cancer, especially for men electing active surveillance. Finally, improvements in commercial RNA amplification, oligonucleotide microarray platforms, potency and longer intervention periods are important factors to consider when performing gene expression studies for agents that have subtle effects on the human transcriptome.