Broccoli sprouts delay prostate cancer formation and decrease prostate cancer severity with a concurrent decrease in HDAC3 protein expression in TRAMP mice.

Background: Cruciferous vegetables have been associated with the chemoprevention of cancer. Epigenetic regulators have been identified as important targets for prostate cancer chemoprevention. Treatment of human prostate cancer cells with sulforaphane (SFN), a chemical from broccoli and broccoli sprouts, inhibits epigenetic regulators such as histone deacetylase (HDAC) enzymes, but it is not known whether consumption of a diet high in broccoli sprouts impacts epigenetic mechanisms in an in vivo model of prostate cancer. Objective: In the transgenic adenocarcinoma of the mouse prostate (TRAMP) model, we tested the hypothesis that a broccoli sprout diet suppresses prostate cancer, inhibits HDAC expression, alters histone modifications, and changes the expression of genes regulated by HDACs. Methods: TRAMP mice were fed a 15% broccoli sprout, or control AIN93G diet; tissue samples were collected at 12 and 28 weeks of age. Results: Mice fed broccoli sprouts had detectable levels of SFN metabolites in liver, kidney, colon, and prostate tissues. Broccoli sprouts reduced prostate cancer incidence, and progression to invasive cancer by 11- and 2.4-fold at 12 and 28 weeks of age, respectively. There was a significant decline in HDAC3 protein expression in the epithelial cells of prostate ventral and anterior lobes at 12 weeks. Broccoli sprout consumption also decreased histone H3 lysine 9 tri-methylation in the ventral lobe (12 week), and decreased histone H3 lysine 18 acetylation in all prostate lobes (28 weeks). A decline in p16 mRNA levels, a gene regulated by HDAC3, was associated with broccoli sprouts consumption, but no significant changes were noted at the protein level. Conclusions: Broccoli sprout intake caused a decline in prostate cancer occurrence and HDAC3 protein expression in the prostate, extending prior work that implicated loss of HDAC3/corepressor interactions as a key preventive mechanism by SFN in vivo.


Introduction
Prostate cancer is the second most frequently diagnosed cancer among men globally, and is a leading cause of cancer-related deaths in the United States (1, 2). The disease is typically slow growing, and although abnormalities in the prostate epithelium can be observed in males in their twenties or thirties, prostate cancer generally does not become of clinical concern until later in life (3-5). The long latency period of prostate cancer suggests that therapeutic strategies that slow disease progression could be beneficial by delaying full disease onset and possibly decreasing invasive surgical Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.3945/cdn.117.002378/4804442 by guest on 28 July 2018 procedures like prostatectomy. Increasing the latency period of prostate cancer could also be beneficial by increasing the period of time during which a therapeutic intervention could occur. Characterization of the molecular mechanisms that delay prostate cancer formation will be beneficial to facilitate the development of effective chemopreventive strategies.
An association between increased cruciferous vegetable intake and a reduced risk of developing, or being diagnosed with prostate cancer has been reported (6).
Cruciferous vegetables, such as broccoli and broccoli sprouts, are a rich source of glucosinolates (7). When broccoli sprouts are chopped or chewed, the glucosinolate glucoraphanin interacts with the enzyme myrosinase, producing the phytochemical sulforaphane (SFN) (7). Broccoli sprouts and SFN have chemopreventive and cancer suppressive properties in carcinogen-induced and genetic models of prostate cancer (7)(8)(9); however, the mechanisms by which they act in vivo are not completely understood.
In the transgenic adenocarcinoma of the mouse prostate (TRAMP) model of prostate cancer, broccoli consumption and/or SFN treatment has been shown to slow prostate cancer growth and metastasis (8,9,14,15). Several potential mechanisms have been implicated, including the induction of Nrf2 related pathways, inhibition of the cancer promoting Akt signaling cascade, suppression of a chemokine receptor (CXCR4), and through augmenting the lytic activity of natural killer cells (8,9,14,15). In contrast to these results, Liu et al. did not find a significant decrease in prostate cancer in TRAMP Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.3945/cdn.117.002378/4804442 by guest on 28 July 2018 mice feed a diet high in broccoli sprouts, highlighting a degree of controversy regarding cruciferous vegetable intake and the prevention of prostate cancer (16).
A hallmark of cancer development is the global modification of epigenetic marks (17). These marks regulate chromatin structure and thus participate in the regulation of gene expression and genome stability. Cancer cells often have dysregulated expression of genes that control epigenetics, such as upregulated histone deacetylase (HDAC) enzymes (18,19). This contributes to cancer development and progression by turning off tumor suppressor genes, or promoting the expression of oncogenes (20). We and others have shown that SFN can alter epigenetic endpoints in cancer cell lines and tissues, including suppression of HDAC expression, changes in DNA methylation, and increased expression of epigenetically repressed genes like p21 and p16 (21)(22)(23)(24)(25)(26)(27)(28)(29). In an in vitro study of TRAMP C1 cells, SFN was shown to restore Nrf2 expression through epigenetic modifications and attenuated the expression of several HDAC proteins (13).
While there is substantial evidence that SFN exposure can influence epigenetic endpoints in cancer cells, it has not yet been shown in an in vivo model of prostate cancer that consumption of a whole food rich in SFN, such as broccoli sprouts, can induce changes in epigenetic regulators and contribute to chemoprevention. We sought to test the hypothesis that consumption of a diet high in broccoli sprouts suppresses prostate cancer, inhibits HDAC expression, alters histone modifications, and changes expression of genes regulated by HDACs. We show that consumption of a diet high in broccoli sprouts decreased the incidence and severity of prostate cancer, reduced HDAC3 protein, and altered epigenetic related endpoints.

Husbandry and Study Design
Custom AIN93G diet with 15% broccoli sprout powder and matched control diet was prepared by Research Diets (Supplemental Table 1) (New Brunswick, NJ). This 15% broccoli sprout diet had 400 mg SFN / kg of diet, which was chosen because it is equivalent to 1 mg SFN / day which has been used in previous studies (14,15,30).
Broccoli sprout powder was purchased from Natural Sprouts Company, LLC (Springfield, MO). Diets were stored protected from the light at -20 °C. Male TRAMP mice in C57BL/6 background were obtained from Jackson Lab and bred in the Oregon Health & Science University (OHSU) animal facility (31)(32)(33). Animal protocol was approved by the OHSU Institutional Animal Care and Use Committee. Animals were housed with a 12-h light and 12-h dark cycle, in a temperature-and humidity-controlled environment and fed standard lab chow. At 4 weeks of age the animals were placed on either the broccoli sprout or AIN93G control diet. Food consumption was measured over the course of the study and no difference was found in the intake of food between the control and broccoli sprout fed groups.
Animals were sacrificed in the morning during a 3-4 hour window at 12 and 28 weeks of age. Lung, liver, spleen, kidney, colon and urogenital tract were removed.
Weights of the urogenital tract and prostate lobes were recorded. The prostate lobes were then formalin fixed, paraffin embedded, sectioned and stained with hematoxylin and eosin (H&E) and scored for cancer incidence and severity by multiple pathologists (CVL from Oregon Veterinary Diagnostic Laboratory at Oregon State University and GT Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.3945/cdn.117.002378/4804442 by guest on 28 July 2018 from Oregon Health & Science University). Some prostates were further dissected to separate the anterior, dorsolateral, and ventral lobes and analyzed separately because in this TRAMP model the cancer is driven by the T antigen oncoprotein primarily in the ventral and dorso-lateral lobes (reviewed in (34)). Individual lobes were snap frozen or put into RNA later for subsequent molecular assays.

Results
Broccoli sprout consumption slowed prostate cancer formation and decreased cancer In the TRAMP model, the SV40 transgene expression is turned on at sexual maturity between 8 and 10 weeks of age. Following this, mouse prostatic intraepithelial neoplasia (mPIN) lesions are seen at 12 weeks of age. By 28 weeks adenocarcinomas and metastasis can occur (31)(32)(33). As expected both the urogenital tract and prostate weights increased with age in control mice (Figure 2A and 2B). At the 12 week time point, urogenital tract weight and prostate weight in animals on the high broccoli sprout diet were 2.8-fold and 2.3-fold, respectively, lower than in mice on the control diet.
( Figure 2A and 2B). At 28 weeks of age the effect of broccoli sprout diet was not as apparent on urogenital tract and prostate weights, and there was no significant differences between the animals on control and broccoli sprout diet (Figure 2A and 2B).
Prostate cancer incidence and severity was significantly reduced in the broccoli sprout groups at both the 12 and 28 week time points (Figure 2C-E). At 12 weeks of age all control mice developed at least early neoplastic lesions (mPIN), while seven out of 20 of the broccoli sprout fed animals had normal prostates ( Figure 2C and 2D).
Furthermore, only one broccoli sprout mouse developed an adenocarcinoma while 10 out of 18 control animals had adenocarcinomas at 12 weeks of age ( Figure 2C and 2D).
By 28 weeks of age, 16 out of 18 control animals had an adenocarcinoma, while only 7 out of 19 broccoli sprout mice had cancer that had advanced to this state ( Figure 2C and 2E). It is also worth noting that at the point of sacrifice, two of the broccoli sprout fed animals never developed any prostate lesions ( Figure 2C). Importantly, consumption of a diet high in broccoli sprouts significantly reduced the incidence of invasive prostate cancer by 11-and 2.4-fold, at the 12 and 28 week time points, respectively ( Figure 2F).

Broccoli sprout consumption decreased HDAC3 expression in prostate epithelium
To determine if HDAC protein expression was altered in prostate epithelium we performed immunohistochemistry using antibodies against HDAC3 and HDAC6. We focused on these HDACs because HDAC3 is highly expressed in prostate cancer and HDAC6 regulates androgen receptor signaling, and both HDACs are decreased by SFN treatment in in vitro models of cancer (22,23,28,(38)(39)(40). A significant decline in HDAC3 protein expression was detected in the ventral and anterior lobes of the prostate of mice fed broccoli sprout diet ( Figure 3A and 3B). The broccoli sprout-induced decline in HDAC3 protein was more apparent at the 12-week time point ( Figure 3B). We did not detect a significant change in HDAC6 protein abundance in the prostates of broccoli sprout fed mice at 12 or 28-weeks of age ( Figure 4A and 4B). It is worth noting that HDAC6 protein appeared lower with broccoli sprouts at the 28-week time point in the ventral lobe of the prostate, and a t-test confirmed a trend for decreased HDAC6 with broccoli sprouts in this lobe ( Figure 4B, t-test, p=0.056).

Broccoli sprout-induced changes in histone modifications in TRAMP mouse prostates
HDACs regulate gene expression by removing acetylation marks from histones (41). Since histone H3 lysine 18 (H3K18) and histone H3 lysine 9 (H3K9) acetylation have been shown to be regulated by HDAC3, we tested if broccoli-sprout mediated decrease in HDAC3 expression resulted in alterations in H3K18 and H3K9 acetylation levels in the prostate (42). Surprisingly, broccoli sprout diet induced a significant 2 fold decline in H3K18 acetylation levels in all prostate lobes at the 28 week time point ( Figure 5A and B).There was no significant change in H3K18ac at 12 weeks when HDAC3 protein was significantly decreased, and thus no correlation between HDAC3 and H3K18 acetylation was detected (Figure 3 and 5B). We did not detect a significant change in acetylation of H3K9 residues in the prostate of mice fed broccoli sprouts, but we did find a significant age effect in the anterior lobe of the prostate (Supplemental Figure 1). Since we did not find the expected changes in acetylation of histones, we next examined if consumption of broccoli sprouts altered the tri-methylation status of histone H3 lysine 9 (H3K9me3), which has been previously reported to decrease in vitro following SFN treatment in PC-3 prostate cancer cells and is altered with HDAC3 deletion (26,43). More specifically, because the anterior lobe of the prostate exhibited a marked loss of HDAC3, we examined the area, intensity, and number of H3K9me3 foci, and noted H3K9me3 punctate staining in the nucleus. A significant decline in H3K9me3 also occurred with age, but there was no apparent effect of diet (Supplemental Figure   2A). We next focused our examination of H3K9me3 levels in the ventral lobe at the 12 week time point, because this was when HDAC3 was significantly downregulated. A significant 13% decrease in the mean area / foci for H3K9me3 staining was found with broccoli sprout consumption in the ventral lobe (Supplemental Figure 2B).

Effect of broccoli sprouts diet on the expression on HDAC3 target genes
To gain further insights into how decreases in HDAC3 by broccoli sprouts could slow prostate cancer progression we evaluated the expression of several genes which are known to be regulated by HDAC3. We examined the mRNA expression of p16 (also known as cyclin dependent kinase inhibitor 2A (CDKN2A)), signal transducer and activator of transcription 3 (STAT3), retinoblastoma binding protein 8, endonuclease (RBBP8 also known as CTIP), and serpin family B member 5 (SERPINB5) (38,44,45).
This work was done in TRAMP prostates of 12 week old animals when HDAC3 was decreased with broccoli sprouts. As a positive control we examined the expression of a known target of SFN, the NAD(P)H quinone dehydrogenase 1 gene (NQO1), and show that its mRNA level was significantly upregulated with broccoli sprout consumption in the dorsolateral lobe of the prostate ( Figure 6A, and (46)). We found significant detected although there was a trend for increased expression of STAT3 with broccoli sprout consumption (Supplemental Figure 3A-C). Unexpectedly, the tumor suppressor gene p16 was significantly decreased at the mRNA level with broccoli sprout consumption in all prostate lobes ( Figure 6B). This coincided with the time point when HDAC3 was decreased. Western blotting revealed no change in the amount of p16 at the protein level ( Figure 6C). We also examined p21 protein expression but it was not detectable in the prostate lobes (data not shown).

Discussion
Given the high incidence and mortality associated with prostate cancer worldwide, reducing prostate cancer incidence and slowing progression is of great importance. The World Health Organization has identified that between 30-50% of the current global cancer burden could be prevented, and indicate an unhealthy diet and low fruit and vegetable intake is a key modifying risk factor for cancer development (47).
Here we show in a preclinical model that consumption of a diet high in broccoli sprouts results in detectable levels of SFN metabolites in the prostate and reduced prostate cancer incidence and severity. We showed for the first time that a diet high in cruciferous vegetables can decrease HDAC expression, primarily HDAC3, in the prostate epithelial cells at a time when prostate cancer is developing. We also showed that broccoli sprout-diet caused significant changes in some epigenetic marks, with broccoli-induced declines in the acetylation of histone H3 lysine 18 being the most notable. The TRAMP model of prostate cancer was utilized because the tumors occur in the prostate epithelium and the tumor tissue histopathology closely mimics human disease. Additional advantages include that the tumors arise spontaneously and appear in ~100% of mice (31)(32)(33). The cancer is driven by the oncoprotein SV40 T antigen which binds to p53 and retinoblastoma proteins, disrupting their tumor suppressor function and the normal signaling circuitry that controls cell cycle (48). Our data are in agreement with several studies in TRAMP mice where a diet high in broccoli sprouts, or treatment with SFN, suppressed prostate cancer development and/or metastasis (8,14,15,49). Overall this literature suggests that broccoli sprouts (and/or SFN) are acting through multiple mechanisms to decrease prostate cancer development, including inhibition of cell cycle, inhibition of the chemokine receptor CXCR4, and increased apoptosis via mechanisms like inhibition of the Akt signaling pathway (8,14,49). In contrast to these studies, Liu et al. did not find a significant effect of broccoli sprout diet on prostate cancer, although they used a lower amount of broccoli sprouts (10% broccoli sprout powder), and started the animals on the diet at a later age than our study (16).
Our results of broccoli-induced decrease in HDAC3 protein is significant because HDAC3 is highly expressed in carcinomas of prostate cancer patients, and upregulation of class I HDACs are thought to be an early event in prostate carcinogenesis (40). Our results are consistent with previous work showing inhibition of HDAC3 with broccoli related supplements in preclinical models of colon and skin cancer, and in clinical studies looking at human breast tissue and blood cells (27)(28)(29). Together, these studies show that HDAC3 is suppressed following broccoli sprout consumption across multiple tissue types and species. The mechanism by which SFN induces HDAC3 degradation has been previously described in colon cancer cells and is likely similar in prostate tissue, involving disruption of corepressor interactions and increased nuclearcytoplasmic trafficking (23). We did not see a significant decrease of HDAC3 when the prostate cancer was more advanced. It is not clear why this effect was lost, and future work will have to explore this phenomenon. One issue that may contribute to the loss of some of the expected effects is the adaption of the organism and/or cancer to a high broccoli sprout diet that was consumed over the majority of the animals' life. HDAC6 has been observed to be inhibited and/or decreased by SFN in cultured prostate cancer cells (22,39). We saw only a trend of decreasing HDAC6 protein with broccoli sprout consumption. It will be interesting for future work to determine if HDAC3 or HDAC6 expression is suppressed in prostate biopsies of men who have consumed broccoli sprout or related supplements.
We encountered limitations with the TRAMP model when we found a diet high in broccoli sprouts significantly decreased H3K18ac, and p16 mRNA levels. Inhibition of HDAC3 is generally thought to increase histone acetylation, and increased expression of p16 has previously been observed in human PBMCs following consumption of broccoli sprout extracts, and in colon tumors of wild-type mice treated with SFN (28).
We cannot rule out effects of the dietary treatments on histone acetyltransferases (HATs), which coordinate with HDACs to regulate overall histone acetylation status. note though that in the TRAMP model the large T antigen is known to upregulate p16 expression and promote global H3K18 hypoacetylation through interactions with the histone acetyltransferases p300 and CBP, and this is likely effecting the epigenetic targets studied here (50)(51)(52)(53). Our study cannot directly confirm large T antigen effects on P16 or H3K18ac because we did not follow these endpoints over a continuum of prostate cancer development. Nevertheless, it is encouraging that the broccoli-induced alterations in p16 mRNA and H3K18ac levels we observed were correcting for changes that are thought to contribute to cancer promotion in this model (52,53). The changes in cell signaling induced by the large T-antigen are also the likely mechanism for why no changes in p16 protein abundance was found (50,51). Interestingly, p16 overexpression has been found in human benign tumors, high-grade malignancies, and in a specific mouse model of colon cancer, where SFN treatment decreased p16 protein levels when the animals were heterozygous for the gene Nrf2 (28, 54, 55).
Broccoli sprout consumption also decreased the area of H3K9 tri-methylation in the ventral prostate lobe at the same time when HDAC3 was decreased. This decline in H3K9 tri-methylation is consistent with a previous report in our lab showing SFN decreased global H3K9me3 by modifying the histone methyltransferase SUV39H1 (26).
Taken together, the data from this study support that broccoli-induced alteration of the epigenetic landscape is likely one important mechanism by which a diet high in broccoli sprouts contributes to prostate cancer chemoprevention. The study also highlights that the cellular context in which a chemopreventive treatment is given is critical in determining the expected molecular endpoints, and point to the need to conduct studies using human clinical samples when possible.   determine differences between control and broccoli sprout groups where ** indicates a significant differences between the groups where p < 0.01.  acetylation was quantified on a scale from 0-3. Significant differences between samples  mRNA levels of p16, or C) p16 protein levels in prostate lobes of mice feed a control diet (white bars), or 15% broccoli sprout diet (green bars). Tissue was collected at 12 weeks of age. A-B) Significant differences between groups was calculated using twoway ANOVAs with results detailed in individual tables for each gene. Bonferroni posttests were used to determine differences between control or broccoli sprout groups where * and *** indicate significant differences between the groups where p < 0.05 and p < 0.001 respectively and n=4-7. C) Images are representative Western blots of mouse prostate tissue analyzed for p16 protein abundance, with corresponding densitometry results were no significant differences between control and broccoli sprout groups was detected (t-test p > 0.05).