Studies on the effects of dietary components, such as isothiocyanates and fish omega-3 fatty acids, on cancer progression have focused on reporting their effects on tumour growth and development [20–22]. Likewise studies on gene expression associated with carcinogenesis in animal models have frequently only centred on changes associated with the later stages of tumour growth and progression to metastasis . In this study we are concerned with changes in gene and protein expression that occur in the very early stages of prostate carcinogenesis, prior to tumour development and how they may be modulated by diet. Up to now preliminary results indicate that green tea catechins are promising in suppressing cancer progression in individuals with HG-PIN but a mechanistic explanation for this effect is not yet clear . In the present study, we sought to explore the possibility that the dietary isothiocyanate sulforaphane has contrasting effects on cells and tissues with differential PTEN expression. This may provide a partial explanation of the inverse association between diets rich in broccoli and the risk of prostate cancer [12, 24], and also suggest potential therapeutic applications of sulforaphane and related compounds.
Initially, we found that in men that are at risk of prostate cancer following a previous diagnosis of HGPIN, there is considerable variation in PTEN and pAKT expression that is not associated with obvious histological abnormalities. Heterogeneity of expression would be consistent with the characteristic emergence of several cancer foci in the human prostate. PTEN also varied in its localisation of expression. While intense pAKT expression was associated with lack of both nuclear and cytoplasmic PTEN expression, we also observed loss of nuclear expression of PTEN without activation of pAKT. While the function of cytoplasmic PTEN is to counteract increased levels of PIP3 by dephosphorylating to PIP2, thereby preventing activation of AKT and downstream signaling, the function of nuclear PTEN is not as well defined but may be independent of its phosphatase activity . Nuclear PTEN function leads to p53-mediated G(1) growth arrest, cell death, and reduction of reactive oxygen species production . Recently, nuclear PTEN has also been linked in controlling chromosomal integrity by acting on chromatin to regulate expression of Rad51, which reduces the incidence of spontaneous DNA double-strand breaks .
To determine whether PTEN and subsequent perturbation of the PI3K/AKT signaling pathway would play a significant role in sulforaphane-mediated effects on prostate carcinogenesis, we initially studied the human prostate cancer cell line PC3, which has a PTEN deletion, and found that it is more sensitive to growth inhibition by SF than the PNT1A cell line that has wild type PTEN expression. At low concentrations, SF enhanced the growth of PNT1A, similar to that previously reported for non-cancerous prostate primary epithelial cells . However, as PC3 and PNT1A cell lines would have other differences in their genetic background, we sought to investigate the biological activity of SF in the mouse prostate-specific PTEN deletion model, where progression to carcinogenesis parallels that of human disease. By comparing PTEN-deficient and WT littermates, which differ only in the expression of Cre recombinase and PTEN, we unambiguously showed that SF has much greater activity in a PTEN-deficient background, indicating selectivity towards cells that are at risk of carcinogenesis or tumour tissue itself. In addition, these effects are occurring at the early initiating stages of prostate carcinogenesis before malignant transformation has occurred.
The effects of SF on gene expression in PTEN-deficient prostate tissue were complex. After five weeks, the main effect is to ameliorate PTEN null-mediated gene expression, so that SF itself acted as a surrogate PTEN tumour suppressor, although its effects were not sufficient in this model to reverse the histopathological changes induced by the knock out of PTEN in all prostate epithelial cells. At a later stage, while SF continued to inhibit a subset of PTEN null-mediated gene expression, it also changed the expression of other genes that are associated with cell cycle arrest and apoptosis. For example, the HSF diet up-regulated cyclins A2, B1, B2, E1 and E2, down-regulated cyclin D2 and induced their associated cyclin-dependent kinases, Cdk2 and 6 (Additional file 5 - Table S5). These results are consistent with previous studies. Cell cycle regulation by SF has been reported in prostate and colon cell lines in which G1 phase cell cycle arrest was associated with protein down-regulation of cyclin D1 [28, 29], and in non-transformed T lymphocytes where SF reduced cyclin D2 . Gene expression of cyclin D2 was also down-regulated in small intestinal polyps of ApcMin/+ mice after 3 days of SF treatment. Increased protein expression of cyclin B1 has been reported to be induced by SF in human colon and breast cells [32, 33]. SF also resulted in a greater than 2-fold increase in expression of caspases 3 and 7 (Additional file 6 - Table S6), consistent with induction of apoptosis in a caspase-dependent manner previously observed in a variety of cell and animal models by SF . For example, SF increased caspase-3 activity in cultured PC3 human prostate cancer cells [5, 34] and broccoli sprouts - a rich source of SF - retarded tumour growth associated with caspase-3 cleavage in TRAMP mice, an alternative model of human prostate cancer .
Isothiocyanates such as SF are known activators of the NF-E2-related factor 2 (NRF2) transcription factor-signaling pathway , and previous studies have reported enhanced expression of NRF2-regulated genes in small intestine and liver of wild type mice [35, 36]. Surprisingly, we did not find any enhanced expression of NRF2-regulated genes in either wild type or PTEN null prostate tissue. This may be due to the exposure of prostate tissue in our mouse model to SF being significantly lower than that used in cell studies and other mouse studies, in order to better reflect routine dietary exposure, combined with the probable greater sensitivities of liver and small intestine compared to prostate to any given dose of SF due to topological exposure and first pass effects, respectively. However, it is noteworthy that a previous study also reported lack of enhanced expression of NRF2-regulated genes through dietary intervention with SF in intestinal polyps of APCMin/+ mice , suggesting tissue specific effects. Moreover, an acute intervention with standard broccoli and a 12-month dietary intervention with a broccoli-rich diet did not induce NRF2-regulated genes in gastric mucosa  and prostate tissue , respectively, in human volunteers.
In this study, using the prostate-specific PTEN deletion mouse model we have provided evidence that in a PTEN null background, addition of SF to the diet can ameliorate the effects of the PTEN deletion. It is, therefore, of considerable interest to know whether dietary intervention in men who are at risk of prostate cancer, partly through PTEN deletion or activation of PI3K/AKT signaling, may have a similar effect. To test this we compared changes in gene expression induced by SF in PTEN-null mice with previously reported changes in human prostate tissue following diets enriched in either broccoli (a dietary source of SF) or peas. We unexpectedly found evidence that both diets may induce similar changes, although the overlap with the broccoli diet was of greater statistical significance. It is conceivable, then, that in addition to SF derived from broccoli, some dietary component common to both diets may be able to suppress the downstream effects of PTEN deletion. Possible factors may be lignans, such as secoisolariciresinol, and flavonoids which are present in both peas and broccoli, and which have been associated with reduction in prostate cancer risk [38, 39]. Lignans are converted by intestinal microflora to enterodiol and enterolactone, which have been shown to suppress pAKT expression in cell models  and certain flavonoids have been shown to modulate PI3K/AKT signalling .
The development of exon arrays has facilitated global analysis of alternative gene splicing, and enables more precise interpretation of microarray data. Alternative splicing of primary RNA transcripts may have a variety of functions, including the expression of different protein isoforms with different functional properties and the regulation of gene expression through nonsense-mediated decay . Serine-rich (SR) proteins function as important regulators of alternative mRNA splicing; kinases and protein phosphatase 1 control the reversible phosphorylation status of these proteins that determines splice site selection and cellular location [43, 44]. pAKT has been shown to phosphorylate the SR proteins SRp40, SF2/ASF and 9G8, which determine alternative spicing of PKC and fibronectin mRNA [45, 46]. Furthermore, insulin has been shown to induce the alternative splicing of PKC via PI3K/AKT activation . Thus, it may be expected that hyperactivation of PI3K/AKT signaling through PTEN deletion may lead to alternative splicing of genes, which may have important biological consequences. While exon arrays have been used to compare tissue- and tumour-specific splicing in human tissues [48, 49], we are unaware of any reported analyses that have specifically focussed on PTEN deletions or assessed the role of diet in inducing alternative splicing.
We provide evidence that the interaction between PTEN deletion and supplementation of diet with SF can result in the alternative splicing of many genes. This is likely to be a complex interaction of both PTEN deletion and SF affecting phosphatase activity, as has been shown for other isothiocyanates  and overall changes in gene transcription induced by SF. Indeed, the HSF diet altered expression of two regulatory subunits of protein phosphatase 1 that may partly explain the observed effects. An example of perturbed alternative splicing is the DMBT1 gene where PTEN deletion promoted the expression of the longer isoform and SF affected this process. The human DMBT1 gene, involved in terminal differentiation of epithelial cells, is located on chromosome 10 q, a region often deleted in prostate cancer and also containing the PTEN tumour suppressor . It is also important to note that in a previous study bioinformatic analyses of the changes in gene expression in men with a broccoli-rich diet reported changes in mRNA processing, indicative of alternative splicing . However, as this previous study did not use exon arrays, it is not yet possible to quantify the effects of diet on splicing in human prostate tissue.