Grape powder supplementation attenuates prostate neoplasia associated with Pten haploinsufficiency in mice fed high-fat diet

Abstract

Scope:

Previous studies have identified potent anticancer activities of polyphenols in preventing prostate cancer. The aim of the current study was to evaluate the chemopreventive potential of grape powder (GP) supplemented diets in genetically predisposed and obesity-provoked prostate cancer.

Methods and Results:

Prostate-specific Pten heterozygous (Pten^+/f) transgenic mice were fed low- and high-fat diet (LFD and HFD) supplemented with 10% GP for 33 weeks, ad libitum. Prostate tissues were characterized using immunohistochemistry and western blots, and sera were analyzed by ELISA and qRT-PCR. Pten^+/f mice fed LFD and HFD supplemented with 10% GP showed favorable histopathology, significant reduction of the proliferative rate of prostate epithelial cells (Ki67) and rescue of PTEN expression. The most potent protective effect of GP supplementation was detected against HFD-induced increase in inflammation (IL-1β; TGFβ1), activation of cell survival pathways (Akt, AR) and angiogenesis (CD31) in Pten^+/f mice. Moreover, GP supplementation reduced circulating levels of oncogenic microRNAs (miR-34a; miR-22) in Pten^+/f mice. There were no significant changes in body weight and food intake in GP supplemented diet groups.

Conclusions:

GP diet supplementation could be a beneficial chemopreventive strategy for obesity-related inflammation and prostate cancer progression. Monitoring serum miRNAs could facilitate the non-invasive evaluation of chemoprevention efficacy.

Graphical Abstract

1. Introduction

Prostate cancer is the most commonly diagnosed cancer and the second most common cause of cancer-associated death among men in the United States ^[1]. Epidemiological observations, including geographic and ethnic differences, suggest that lifestyle and specifically diet have a substantial impact on the development and progression of prostate cancer ^[2–5]. Moreover, both epidemiological data on obesity ^[6] and experimental studies using various diets in preclinical models ^{[3, 7, 8]} have implicated dietary fat as an imperative factor for prostate cancer risk and its aggressive progression. On the other hand, dietary bioactive polyphenols have been shown to possess anti-inflammatory, antioxidant and anticancer properties against prostate cancer development and progression ^[9–14].

Among polyphenols, stilbenes have been studied extensively for their potential as chemopreventive agents in prostate cancer ^[15–21]. Specifically, in our previous studies, we have reported that both resveratrol and pterostilbene restrained prostatic intraepithelial neoplasia (PIN) formation and reduced prostate tumor development and progression in transgenic and xenograft mouse models through inhibition of highly expressed metastasis-associated protein 1 (MTA1) or microRNAs (miRNAs), both epigenetic regulators that play an important role in prostate cancer progression and bone metastasis ^[22–29]. Since both resveratrol and pterostilbene are found in grapes, we sought to investigate the potential role of grape powder (GP), which is a composed of whole red, green and black California grapes, as a MTA1- and miRNA-targeted chemopreventive approach in prostate cancer. To this end, we aimed to test whether GP supplementation in a normal low-fat diet (LFD) or high-fat diet (HFD) protects the prostate against developing PIN, a precancerous stage of prostate cancer, which puts men at high risk of developing full-fledged disease that requires radical prostatectomy. Further, the increased evidence that miRNAs are involved in prostate cancer development and progression made them potential biomarkers for cancer diagnosis and prognosis ^[30–35]. We have evaluated specific miRNAs as potential chemoprevention predictive biomarkers in prostate cancer ^[36]. Detection of peripheral whole blood levels of specific miRNAs will enhance the accuracy of prostate cancer chemoprevention and provide a clinical benefit for patients, sparing them from invasive biopsies.

The best animal model for testing dietary chemopreventive agents against prostate cancer is an immune-competent prostate-specific Pten heterozygous mouse (Pb-Cre4⁺; Pten^+/f) (thereafter abbreviated as Pten^+/f ) that develop high grade PIN over a long period of time, namely 36 weeks, which mimics slow-growing prostate disease progression in men ^[37]. Upon accumulating 56 mice of the desired genotype, we randomized the mice into four groups and fed them either a LFD or HFD containing 0% or 10% GP supplemented diet ad libitum. The same groups were assigned for control mice (Pb-Cre4⁻; Pten^+/f), hereafter referred to as wild type (WT) mice. After 33 weeks on corresponding diets, mice were sacrificed and urogenital system (UGS) was collected for histological, immunohistochemical (IHC) and molecular analysis. Blood was also collected for cytokines’ evaluation and miRNA analysis. The main objective of this study was to evaluate the effects of GP supplementation to LF and HF diets for prostate cancer chemoprevention. Accordingly, we evaluated four major aspects: 1) whether altering fat content in the diet (LFD versus HFD) affects prostate cancer progression; 2) whether GP supplementation to the diets protects against prostate cancer progression; 3) whether MTA1 targeting is the main mechanism of GP action; and 4) whether specific MTA1-associated miRNAs linked to PIN may serve as predictive serum biomarkers.

2. Experimental Section

2.1. Materials

The California Table Grape Commission (Fresno, CA) provided the GP in a freeze-dried form. The GP is a composite of whole red, green and black California grapes in seeded and seedless varieties. The GP and grape placebo powder (GPP) were created using Good Manufacturing Practices and precautions to preserve the integrity of the biologically active compounds found in fresh grapes. As with fresh grapes, the GP is known to contain anthocyanins, resveratrol, catechins, flavonols, flavans and simple phenolics as well as sugars (www.grapesfromcalifornia.com/healthresearchgrants.php). The GP is considered to contain 3.71 kcal/g: 3% fat, 88.6% carbohydrates (as a 1:1 mixture of fructose and glucose), 3.58% protein and 9.73 g/kg K. The GPP (0% GP) contained 92.1% carbohydrates, 0.26% protein and 0.018% fat. Envigo Teklad Diets (Madison, WI) formulated custom- made LFD and HFD with 10% GPP (0% GP) and 10% GP. A standard chow, LFD, derives 10.5% calories from fat, 70% from carbohydrates and 19.5% from protein while HFD derives 42.4% calories from fat, 42.3% from carbohydrates and 15.4% from protein. Fat composition was the following for LFD: 52.7% saturated fatty acids (SFA); 28.4% monounsaturated fatty acids (MUFA), and 18.9% polyunsaturated fatty acids (PUFA), 3% milkfat and 1% soybean oil and for HFD: 63% SFA, 29% MUFA, 4% PUFA, and 21% milkfat. Of note, the 10% GP content of the LFD and HFD used in this study approximately translates to 3 cups of grapes (~453 g/day), or about six servings per day (1 serving of grapes is ½ cup), which is well tolerated and generally safe in humans.

2.2. Cell culture

Prostate cancer cells lines DU145 and PC3M were cultured in RPMI-1640 (ThermoFisher Scientific, Waltham, MA) containing 10% FBS and maintained in a 5% CO₂ incubator at 37°C. MTA1 knockdown DU145 and PC3M cells were generated using three different shRNA constructs as described previously, and the clone with the most efficient knockdown was chosen for current studies ^{[28, 38]}. Cells were authenticated using short tandem repeat profiling at the Research Technology Support Facility, Michigan State University.

2.3. Animals

Animal housing, care and experimental design were in accordance with approved protocol (# AL Grape Diet) by the Institutional Animal Care and Use Committee of Long Island University (LIU). All animals were housed three to four together in individually ventilated cages on wood chip bedding with a 12 h light/dark cycle, room temperature of 22°C and 40-60% relative humidity. During the study, animals were permitted free access to drinking water and specified diet. The body weight and food intake of the animals were monitored weekly. Animals were monitored daily for their general health.

2.4. Generation of prostate-specific Pten heterozygous mouse model

Female C57BL/6J mice homozygous for the “floxed” allele of Pten gene (Pten^f/f) were purchased and bred with Pb-Cre4 (Cre⁺) male mice from the B6.Cg genetic background (Jackson Laboratories, Bar Harbor, ME) that specifically express Cre recombinase in the prostate epithelium ^[39]. We performed tail-genotyping using the following primers: PTEN geno olMR9554F:5′-CAA GCA CTC TGC GAA CTG AG-3′ and PTEN geno olMR9555R:5′-AAG TTT TTG AAG GCA AGA TGC-3′ with 156 bp wild type band and 328 bp mutant band; Cre F: 5′-TCG CGA TTA TCT TCT ATA TCT TCA G-3′ and Cre R: 5′-GCT CGA CCA GTT TAG TTA CCC-3′ with a band of 393 bp. PCR was performed on an Eppendorf thermocycler. We used only male mice in experiments with GP dietary supplementation. Normal prostates from Cre-negative Pten^+/f (WT) male mice were processed as controls.

2.5. Mice feeding

After a series of breeding and genotyping, we collected 56 male mice in total with a body weight range of 23-25 g. Twenty eight Pten^+/f mice were randomized into two major groups, namely LFD and HFD, and into 2 subgroups each (n=7) with 0% (10% GPP) or 10% GP supplementation (see Materials/Diet). As an overall control, WT mice (n=28), which possess normal prostates, were also put on corresponding diets. At weaning (3-weeks old), mice were fed ad libitum with corresponding diets until sacrifice. Necropsy was performed for Pten^+/f mice and diet-matched WT mice at 36 weeks-of age, UGS were isolated and directly fixed in formalin. For protein isolation, prostate tissues were dissected and stored at −80°C prior to sample preparation for immunoblots. Blood was collected via cardiac puncture and serum was isolated and stored at −80°C until use.

2.6. Histopathology and immunohistochemistry

Formalin-fixed paraffin embedded UGS blocks were processed (4 μm thickness) and stained with hematoxylin & eosin (H&E) by Reveal Biosciences (San Diego, CA). A certified pathologist (VD) and other co-authors analyzed slides independently using the accepted classification scheme for mouse PIN (mPIN) ^[40]. Immunohistochemical staining of slides with Ki67, MTA1, CK8, SMA, PTEN, AR, and CD31 antibodies (Supporting Information, Table S1) was performed as per the protocol described previously ^{[25, 27, 41]}. The Vectastain ABC Elite kit and ImmPACT DAB kit (Vector Laboratories, Burlingame, CA) were used to visualize staining. Images were recorded on an EVOS XL Core microscope (Themo Fisher Scientific, Waltham, CA). MTA1 and AR were calculated by evaluating the staining intensity, which was scored using a four-tier system: -, no staining (0); +, weak (1); ++, moderate (2); +++, strong (3). MTA1, Ki67, and AR stained nuclei were quantitated using ImageJ software (NIH, Bethesda, MD) and analyzed by GraphPad Prism (San Diego, CA).

2.7. Western blot

Western blot was carried out as described recently ^{[28, 42]}. Briefly, lysates were prepared from cells and homogenized prostate tissues (Fisher Scientific, Hampton, NH) and total protein was isolated. Protein estimation was performed using Bio-Rad protein assay reagent on a SmartSpec 3000 spectrophotometer (Bio-Rad Laboratories, Hercules, CA). Protein was resolved on 10% or 15% polyacrylamide gels and transferred to a polyvinylidene fluoride membrane. Membranes were blocked for non-specificity and then probed with primary antibodies listed in Table S1 (Supporting Information, Table S1). Signals were visualized using enhanced chemiluminescence. ImageJ software (NIH, Bethesda, MD) was used to perform densitometry measurements. The quantifications were carried out using the ratio of the intensity of the protein of interest versus loading control (β-Actin or HSP70).

2.8. ELISA

At sacrifice, peripheral blood was collected by cardiac puncture and serum was isolated. Serum IL-1β level was analyzed using a commercial sandwich SimpleStep ELISA™ kit (Abcam, Cambridge, MA) according to the manufacturer’s protocol. The assay uses pre-coated 96 well strip microplates covered with anti-tag antibody. Samples (50 μl) or standards were added to the wells, followed by the antibody mix, and incubated for 1 hr at 37°C. After washings, 3.3’,5,5’-tetramethylbenzidine substrate was added for 10 min at 37°C followed by the stop solution to each well. Reaction was read at 450 nm using a BioTek Synergy-4 microplate reader (BioTek, Winooski, VT). Using the standard titration curve, a concentration of IL-1β in serum was calculated based on their optical density values.

2.9. Quantitative real-time RT-PCR

Total RNA was isolated from the cell lines using miRNeasy kit (Qiagen, Hilden, Germany) as described previously ^{[27, 28, 38]}. Real-time PCR was performed using primers specific for MTA1, p53, p21 and custom primers for miR-34a-5p and miR-22-3p, and Snord44 (Supporting Information, Table S2) and miRCURY LNA™ Universal RT microRNA PCR kit or 2x miRCURY LNA SYBR green PCR Kit (Qiagen, Hilden, Germany). For estimation of PIN-derived circulating miRNAs in murine serum, sera from 4-7 mice in each group were combined to obtain 200 μl volume. RNA was isolated using miRNeasy serum/plasma kit as previously described ^[36]. Real-time PCR was performed using custom primers mentioned above and miR-103a-3p was used as an internal reference control (Qiagen, Hilden, Germany). Fold changes in miRNA/mRNA expression were estimated by the 2^−ΔΔCt method ^[43].

2.10. Statistical analysis

The results for each treatment group were summarized as the mean value ± SEM. The differences between the groups were analyzed by one-way or two-way ANOVA or the two-tailed two-sample t-test based on the experiment design. All analyses were performed using GraphPad Prism v7 software. Statistical significance was defined as a p value < 0.05.

3. Results

3.1. Pten deficient mice fed HFD gained more body weight than WT mice

For the current studies, we chose the prostate-specific Pten heterozygous (Pten^+/f) mouse model that is best suitable for testing novel chemopreventive options. This model is the most appropriate due to its close resemblance to the stage-defined human disease ^[37], intact immune system and the fact that Pten is the most commonly lost tumor suppressor gene in men diagnosed with prostate cancer ^[44]. At the age of 3 weeks, prostate-specific Pten-deficient and WT mice, were allocated to either LFD or HFD with corresponding 0% or 10% GP supplementation (Figure 1A). The representative gross anatomy images of LFD- and HFD-fed Pten^+/f and WT mice showed large visceral fat deposits in all HFD-fed mice increased compared to LFD-fed mice (Figure 1B). Interestingly, Pten^+/f mice on HFD accumulated more visceral fat compared to WT mice fed HFD. Comparison of the prostate sizes showed that the prostates in HFD-fed Pten^+/f mice were slightly larger compared to the normal prostates in WT groups (Figure 1B). All individual mice, both Pten heterozygous and WT, gained body weight continuously on both LFD and HFD. However, as seen in Figure 2A, the mean body weight of Pten^+/f mice on HFD was higher compared to their WT littermates, reaching statistical significance at 15 weeks, which continued through the end of the experiment at 33 weeks of diet. The LFD and HFD supplemented with 10% GP had no effect on body weight of either Pten heterozygous or WT mice (Figure 2B, C). We then evaluated the food intake (kCal) and found no differences between the HFD- and LFD-fed mice of both genotypes (Figure 2D). However, GP supplementation had no detected effect on food intake (Figure 2E, F). Next, we calculated the percent of total fat intake in all groups and, as expected, fat intake was significantly higher in HFD group compared to LFD group (Figure 2G). Moreover, fat intake was the same in all HFD-fed mice irrespective of their genotype and GP supplementation had no impact on fat intake (Figure 2G–I). Taken together, these results indicate that despite the same HFD food intake in both Pten^+/f and WT groups, the body weight gain was more prominent in Pten heterozygous mice, indicating the possible impact of prostate genotype on body weight. However, the addition of GP to either LFD or HFD had no significant effect on body weight irrespective of the genotype.

Figure 1.

(A) Schema depicting the experimental design used for in vivo chemopreventive effect of GP in LFD and HFD fed Pten^+/f and WT mice. In total 56 mice (n=7/each group) were fed with LFD and HFD supplemented with 0% or 10% of GP for 33 weeks. At sacrifice, the prostate tissues were isolated and subjected to histopathological, IHC and molecular analysis. (B) Representative images of abdominal cavities of LFD and HFD fed Pten^+/f and WT mice showing increased large visceral fat deposits in all HFD fed mice compared to LFD fed mice. Lower panel shows images of UGS: the prostates (marked) in Pten^+/f mice were slightly larger compared to the WT group but there were no differences in size between LFD and HFD groups in each genotype.

Figure 2.

(A) Average body weight gain of LFD and HFD fed Pten^+/f and WT mice. Mouse weights were monitored weekly. The body weight gain of each mouse was determined by the body weight at each week minus the initial body weight at the beginning of the diet schedule. (B) Effects of GP supplementation on body weight in LFD and HFD fed Pten^+/f mice. (C) Effects of GP supplementation on body weight in LFD and HFD fed WT mice. (D) Food intake of LFD and HFD fed Pten^+/f and WT mice. (E) Effects of GP supplementation on food intake in LFD and HFD fed Pten^+/f mice. (F) Effects of GP supplementation on food intake in LFD and HFD fed WT mice. (G) Fat intake of LFD and HFD fed Pten^+/f and WT mice. (H) Effects of GP supplementation on fat intake in LFD and HFD fed Pten^+/f mice. (I) Effects of GP supplementation on fat intake in LFD and HFD fed WT mice. Values are means ± SEM, n=7/each group. *, p < 0.05; ****, p < 0.0001 (one-way ANOVA).

3.2. Grape powder supplementation improves the prostate neoplastic phenotype in Pten deficient mice

Prostate-specific monoallelic deletion of Pten in mice causes early stage neoplasia, represented by PIN ^{[27, 39]}. Mouse PIN is usually classified based on micro-architecture, differentiation pattern and degree of nuclear atypia but differs from invasive carcinoma by retaining an intact basement membrane surrounding the prostate gland. To investigate whether HFD causes progression of neoplasia and whether supplementation with GP in the LFD and HFD improves phenotype, 3-week- old male Pten^+/f mice and their WT littermate controls were fed corresponding diets for 33 weeks prior to histological and IHC analysis of the prostate. Figure 3A and B show the representative images of H&E sections of each cohort of mice. Examination of prostates from LFD- and HFD-fed WT mice mostly showed individual benign glands with clear lumen and normal continuous lining of the epithelial cells with occasional minor infolding in HFD-fed mice (Figure 3B). In contrast, LFD- and HFD-fed Pten^+/f mice exhibited predominantly PIN characterized by disorganized glandular structures with papillary infolding and tufting and proliferation of CK8-positive epithelial cells inside the lumen along with an intact basal layer comprise of SMA-positive cells (Figure 3A). Importantly, Pten^+/f mice on LFD and HFD supplemented with 10% GP showed more favorable histopathology with restored ductal structures and 50-60% reduction in the numbers of glands involved in mPIN (Figure 3A, C). To assess whether the restoration of a more favorable histopathology by 10% GP supplementation in Pten^+/f mice fed with LFD and HFD was associated with reduction in cell proliferation, we stained the prostate tissues with Ki67 antibody (Figure 3A, B). First, the number of Ki67-positive cells in Pten^+/f mice fed with LFD and HFD was significantly higher compared to their corresponding control WT littermates. Importantly, the effect of 10% GP supplementation was evident in Pten^+/f mice showing substantial and highly significant decrease in Ki67 staining (Figure 3D). Taken together, 10% GP supplementation irrespective of the diet fat content reduced the proliferation of glandular epithelial cells and restored favorable histopathology of mPIN in the genetically predisposed mice with conditional monoallelic deletion of Pten.

Figure 3.

(A) Representative images of H&E (top), SMA (second from top), CK8 (second from bottom), and Ki67 (bottom)-stained sections of the prostate from Pten^+/f mice. (B) Representative images of H&E (top), SMA (second from top), CK8 (second from bottom), and Ki67 (bottom)-stained sections of the prostate from WT mice. H&E, SMA and CK8 are 20X images (scale bar, 50 μm); Ki67 is 40X image (scale bar 20 μm). (C) Quantitation of prostate glands involved in mPIN formation in Pten^+/f and WT mice fed LFD and HFD supplemented with 0% or 10% GP. The glands were counted in five randomly selected fields per sample, and the average count was expressed as a percent. Values are means ± SEM, n=7/each group. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 (one-way ANOVA). (D) Quantitative analysis of Ki67 immunostaining showing drastic effect of GP supplementation on cell proliferation in Pten^+/f mice. Values are means ± SEM of cells counted in five separate fields per sample, and the average count is expressed as a percent. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 (one-way ANOVA).

3.3. Grape powder supplementation protects against hyperactive Akt survival signaling in prostate-specific Pten deficient mice and HFD-induced Akt activation in WT mice

Based on our earlier studies signifying an inverse relationship between PTEN and MTA1, the MTA1-signaling involvement in the Pten loss progression of high-grade PIN, and benefits of effective MTA1-targeted inhibition by pterostilbene ^{[26, 27]}, we examined PTEN and MTA1 levels in the prostate tissues in the present study (Figure 4A, B). GP supplementation resulted in increased PTEN staining in Pten^+/f mice in both LFD and HFD groups (Figure 4A). Although demonstrating noticeable heterogeneous expression of MTA1 in experimental and control mice analyzed by IHC, consistent with our previous observation ^[27], Pten deficient prostates showed higher expression of MTA1 compared to WT counterparts irrespective of diet (Figure 4A,C). Although there was a trend towards reducing MTA1 levels in Pten deficient group fed HFD supplemented with GP, the IHC results did not reach statistical significance (Figure 4C). On the other hand, while immunoblot analysis of MTA1 expression in these tissues was somewhat inconsistent (data not shown), normal prostates in WT mice steadily showed remarkable HFD-associated MTA1 increase, which was significantly diminished by GP supplementation (Figure 4D).

Figure 4.

(A) Representative images of PTEN (top), MTA1 (second from top), AR (second from bottom), and CD31 (bottom)-stained sections of the prostate from Pten^+/f mice. (B) Representative images of PTEN (top), MTA1 (second from top), AR (second from bottom), and CD31 (bottom)-stained sections of the prostate from WT mice. Images are 20X (scale bar, 50 μm). (C) Quantitative analysis of MTA1 immunostaining and (D) representative immunoblot images of MTA1 in prostate tissues from WT mice. (E) Representative immunoblot images (top) and densitometric analysis (bottom) of Akt kinase activity in the prostate lysates of GP supplemented LFD and HFD fed experimental mice. (F) Representative immunoblot images (top) and densitometric analysis (bottom) of AR levels and (G) quantitative analysis of AR immunostaining in the prostate tissues from experimental mice. (H) Quantitative analysis of CD31 immunostaining showing effects of GP supplementation in LFD and HFD fed experimental mice. Values are means ± SEM of cells counted in five randomly selected fields per sample. For immunoblots, β-Actin and HSP70 were used as loading controls. A.U., arbitrary units. *, p < 0.05; **, p < 0.01 (one-way ANOVA).

Pten heterozygosity causes an increased Akt signaling activity in the prostate ^{[27, 45]}. We have previously reported on an inverse relationship between PTEN and MTA1 ^[27] and on the ability of MTA1 to regulate the activity of the PTEN/Akt signaling pathway via deacetylation of PTEN ^[26]. In addition to reported MTA1 transcriptional repression of PTEN ^[46], we have demonstrated a direct link between MTA1 and Akt activation, in part through prospective MTA1 occupancy of Akt1 promoter ^[27]. Importantly, we have previously demonstrated the rescue of PTEN expression and inhibition of Akt signaling upon resveratrol- and pterostilbene-mediated downregulation of MTA1 in vitro and in vivo ^{[26, 27]}. In the current study, while PTEN expression was not affected by the diet in Pten^+/f mice, supplementation of diets with 10% GP was associated with increased PTEN staining, coincident with substantial reduction of Akt activity in these cohorts (Figure 4E). As expected, activated Akt levels in Pten^+/f mice fed with either LFD or HFD were higher than in WT mice. In addition, pAkt levels were also reduced in WT mice fed HFD with GP supplementation. This data demonstrate that Pten^+/f mice have hyperactive Akt signaling in the prostate compared to HFD-induced Akt activation in WT mice and indicate that GP supplementation may protect against “Pten deficient” genotype- associated as well as HFD-induced activation of Akt pathway.

3.4. Grape powder protects against HFD-induced AR increase in prostate-specific Pten deficient mice

Activation of androgen receptor (AR) signaling in response to androgens is a source for prostate cancer cells to grow. Therefore, keeping AR levels low can help to limit the progression of prostate cancer. We sought to investigate AR levels in mice fed LFD and HFD with and without GP supplementation. We have previously demonstrated that stilbenes inhibit AR expression in various prostate cancer cell lines and that AR levels were markedly reduced in prostate tissues from pterostilbene-treated Pten loss mice compared to control WT mice ^{[27, 47, 48]}. Here, we found that Pten ^+/f mice fed HFD had significantly higher levels of AR compared to the LFD-fed mice and HFD-fed WT mice (Figure 4F, G). Notably, supplementing the HFD with GP was associated with reduced expression of AR in Pten^+/f mice, even though the effects did not reach statistical significance (Figure 4F, G).

3.5. Grape powder reduces angiogenesis in prostate-specific Pten deficient mice

In our previous studies, we have repeatedly found a close association between MTA1 and angiogenic network (VEGF, IL-1β, HIF-1α) and, more importantly, we have demonstrated that MTA1 inhibition by resveratrol/pterostilbene decreased angiogenesis, resulting in PIN or tumor volume reduction and tumor growth inhibition ^{[22, 24, 27, 41]}. To determine if GP supplementation to diet affects angiogenesis, we performed CD31 staining on the prostate tissues of experimental mice (Figure 4A, B, H). We did observe a decrease in CD31 staining reduction in the prostates from Pten^+/f mice fed GP supplemented diets, although the differences were not statistically significant (Figure 4A, H).

3.6. Grape powder attenuates increased inflammation in prostate-specific Pten deficient mice

Obesity-associated chronic inflammation and metabolic syndrome are often precursors of cancer development and the association between inflammation, reactive stroma and prostate cancer is well- established ^{[49, 50]}. We have previously shown that several pro-inflammatory factors and cytokines, including IL-1β and TGFβ1, were involved in the induction of reactive stroma in the prostate tissues of Pten^+/f mice ^[27]. In response to inflammatory stimuli, IL-1β accumulates in the cytosol but it is most often secreted in its active form and can be detected in serum. To investigate whether HFD promotes inflammation and whether GP supplemented diets could reduce inflammation in Pten deficient mice, we examined levels of IL-1β in prostate tissues by immunoblot (Figure 5A) and in murine serum by ELISA (Figure 5B). Figure 5A shows that prostate tissue levels of IL-1β were almost two times greater in Pten^+/f mice fed the HFD versus LFD and that the supplementation of the HFD with GP was associated with decrease in IL-1β levels in prostate tissue microenvironment. No noticeable changes in IL-1β was detected in WT mice. ELISA results demonstrated that the production of secreted IL-1β is considerably higher in the sera of Pten^+/f and WT mice fed HFD compared to their corresponding LFD- fed groups (p <0.0001 and p< 0.05, respectively). Further, HFD-fed Pten^+/f mice produced significantly higher IL-1β levels compared to HFD-fed WT mice (p < 0.001). Notably, GP supplementation resulted in statistically significant reduction of IL-1β levels in in Pten^+/f mice fed with HFD (p< 0.001) (Figure 4B).

Figure 5.

(A) Representative immunoblot images (top) and densitometric analysis (bottom) of IL-1β levels in prostate tissues of GP supplemented LFD and HFD fed experimental mice (B) Pro-inflammatory and pro-angiogenic IL-1β measured by ELISA (pg/ml) in murine sera. All values are indicated as the mean ± SEM (n = 7/each group).*, p <0.05; ***, p < 0.001; ****, p < 0.0001 (one-way ANOVA). (C) Representative immunoblot (top) and densitometric analysis (bottom) of TGFβ1 levels in the prostate tissues of GP supplemented LFD and HFD fed experimental mice. β-Actin was used as a loading control.

Transforming growth factor β1 (TGFβ1) is a multifaceted and multifunctional cytokine, which plays a role in inflammation, immune responses, regulation of cell proliferation and differentiation, wound healing, EMT, and angiogenesis ^[51]. To determine if GP supplementation to the diet affects prostate tissue levels of TGFβ1in Pten^+/f mice, TGFβ1 levels were detected by immunoblot (Figure 5C). Similar to our previous observation, TGFβ1 levels in prostates of Pten^+/f mice were elevated compared to WT counterparts ^[27]. Although Pten^+/f mice in LFD group had much higher TGFβ1 levels relative to HFD group at 33 weeks on respective diets, GP supplementation to diet decreased the levels of TGFβ1 in both Pten^+/f cohorts (Figure 5C). TGFβ1 levels were similar in the WT mice cohort regardless of the diet’s fat content but GP supplementation showed reduced TGFβ1 levels in HFD fed group. Altogether, the results show the inhibitory effects of GP on the levels of inflammatory cytokines in Pten^+/f mice, which suggests decreased local and systemic inflammation and reduced prostate cancer progression.

3.7. Grape Powder does not induce apoptosis in prostate-specific Pten deficient mice

Given that molecular mechanisms responsible for GP efficacy in Pten^+/f mice may involve apoptosis, we examined the impact of a HFD and GP supplementation on apoptotic markers. Apoptosis can be triggered through multiple signaling pathways including p53-activated apoptotic pathway derived from MTA1 inhibition in prostate cancer ^{[22, 24, 27]}. The anti- and pro-apoptotic members of Bcl-2 protein family regulate intrinsic mitochondrial apoptotic pathways, where permeabilization of the mitochondrial outer membrane by Bax/Bak, the subsequent release of cytochrome c and activation of caspase cascades ultimately leads to apoptosis. We performed immunoblot analysis of p53, p21, Bax, Bak, cleaved caspase 9, and cleaved caspase 3. As expected, p53 levels increased dramatically in prostates of Pten^+/f mice fed with LFD and HFD supplemented with GP (Figure 6A, B). However, interestingly, levels of p53-dependent Bax and p21 went down in prostate tissues of mice on diets supplemented with GP. Moreover, prostate lesions in the GP supplemented LFD and HFD groups had lower level of apoptotic markers (Bak, cleaved caspase 9, and cleaved caspase 3) compared to the diets not supplemented with GP in Pten^+/f mice. GP supplementation decreased levels of Bax and cleaved caspase 9 in WT mice, as well (Figure 6A, B). This finding was unexpected to us as anticancer effects are generally associated with induction of apoptosis. Nevertheless, in the present study, there was a trend towards decreased apoptotic markers in PIN lesions of Pten^+/f mice fed LFD and HFD supplemented with GP. Although data in the literature is conflicting with regards to the biological significance of apoptosis in prostate cancer ^{[52, 53]}, several studies demonstrated decreased apoptosis in prostate cancer cells in response to dietary fat reduction ^{[54, 55]}. In the current study, similar to our previous observations, we found a reduction in p21 and induction of cleaved caspase 3 levels in Pten^+/f compared to normal prostate. However, in contrast to the pterostilbene-diet-induced increase in both apoptotic markers in Pten^+/f mice observed by us earlier ^[27], GP supplementation showed a decrease in p21 and cleaved caspase 3 (Figure 6A, B). The data indicate that GP supplemented in LFD and HFD did not cause mitochondrial-pathway apoptosis in PIN lesions of mice.

Figure 6.

(A) Representative immunoblot images and (B) densitometric analysis of proteins involved in apoptosis (p53, p21, Bak, Bax, cleaved caspase 9, and cleaved caspase 3) from the prostate tissues of experimental mice fed with GP supplemented LFD and HFD.

3.8. Grape powder supplementation to the diet regulates circulating PIN-derived oncomiRs in prostate-specific Pten deficient mice

Aberrant expression of miRNAs in cancer is well- established ^{[35, 56, 57]}. A number of studies have assessed the potential of circulating miRNAs in diagnosis, prognosis and prediction for clinical applications in prostate cancer ^[58–60]. We have previously reported on the ability of miRNAs as predictors to treatment response. Particularly, we found that pterostilbene’s beneficial anticancer effects were reflected through changes in levels of miR-17-5p and miR-106a-5p oncomiRs detected in the serum of prostate cancer xenografts ^[36]. In our previous miRNA profiling and ChIP-Seq studies, we identified candidate MTA1-associated miRNAs that are also regulated by pterostilbene ^[61] (Supporting Information, Figure S1). In the current study, we narrowed down two miRNAs of interest, namely miR-34a and miR-22 that, according to publicly available prediction algorithms [TargetScan (www.targetscan.org); miRanda (https://omictools.com/miranda-tool], target tumor suppressors p53 and p21, respectively. To confirm a direct relationship between MTA1 and oncogenic miR-34a and miR-22 and an inverse relationship between MTA1 and p53 and p21, we used previously established prostate cancer cell lines silenced for MTA1 ^{[28, 38]}. MiR-34a and miR-22 expression are significantly reduced in DU145 and PC3M cells silenced for MTA1 (Figure 7A, D) with concomitant increase in p53 and p21 mRNA (Figure 7B, E) and protein levels (Figure 7C, F). To examine clinical value of targeting these miRNAs in prostate cancer, we sought to measure changes in circulating levels of these miRs upon exposure to LFD/HFD supplemented with GP. Figure 7G, H show the effect of GP supplementation on the levels of miR-22 and miR-34a in murine serum: Pten^+/f mice had significantly higher serum levels of miR-34a and miR-22 relative to WT mice and there was a statistically significant reduction of miR-34a and miR-22 in the both LFD and HFD groups supplemented with GP. Levels of miRs were not altered in WT mice. Altogether, GP supplemented diets reduced the levels of PIN-derived circulating oncomiRs, i.e. miR-34a and miR-22, detected in the murine serum.

Figure 7.

(A) qRT-PCR analysis of miR-34a and miR-22 and (B) MTA1, p53, and p21 in DU145-NS and DU145-shMTA1 prostate cancer cells. (C) Representative immunoblot (left) and densitometric analysis (right) of MTA1, p53, and p21 levels in DU145-NS and DU145-shMTA1 cells. (D) qRT-PCR analysis of miR-34a and miR-22 and (E) MTA1, p53, and p21 in PC3M-NS and PC3M-shMTA1 prostate cancer cells. (F) Representative immunoblot (left) and densitometric analysis (right) of MTA1, p53, and p21 levels in PC3M-NS and PC3M-shMTA1 cells. β-Actin was used as a loading control for immunoblots. (G, H) qRT-PCR analysis of circulating miR-34a and miR-22 levels in the sera of Pten^+/f and WT mice fed LFD and HFD supplemented with GP. Serum from 4-7 mice in each group was combined for RNA isolation. Changes in mRNA and miRNA were calculated by the 2-ΔΔCt method. Data represent the mean ± SEM of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 (one-way ANOVA).

4. Discussion

From both chemopreventive and therapeutic perspectives, dietary compounds are being intensively studied for their anti-inflammatory, antioxidant, and anticancer properties. The Vitis vinifera L. fruit (grape) contains various phenolic compounds including flavonoids and stilbenes and has been used traditionally against diarrhea, hypertension, neurodegeneration, and arthritis ^{[62, 63]}. In recent years, active constituents found in the stem, whole fruit, skin and seed of grapes have been experimentally shown to have various pharmacological effects including cardio-, neuro-, and hepato-protective as well as antibacterial, antioxidant, anti-inflammatory, and anticancer effects ^[64]. However, in contrast to drugs or bioactive polyphenols, which are mostly single chemical compounds, the evaluation of the biological effects of dietary products is challenging due to a wide variety of individual phytochemicals that may act differently when mixed together. Most of the literature on grape effects in cancer or other diseases have dealt with “grape extract” as the biological active entity ^[65]. However, even if extract is defined as “grape-derived bioactives”, due to the differences in the sources of raw material (whole fruit, seed or skin) from numerous grape varieties and diverse extraction methods, the composition and quantity of bioactive chemicals vary significantly ^{[65, 66]}. In prostate cancer, grape seed/skin extracts have been used to determine chemopreventive and possible therapeutic applications ^{[67, 68]}. Muscadine (Vitis rotundifolia) grape skin extract, whose main bioactive component is anthocyanin, has been shown to decrease prostate cells’ invasion and migration, revert epithelial-to-mesenchymal transition (EMT) and induce autophagy and apoptosis ^[69–71]. In a recent study, Ignacio et al ^[72] found yet another mechanism, by which muscadine grape skin extract significantly inhibited prostate tumor growth and migration. Unfortunately, a randomized, multicenter, placebo-controlled clinical trial with muscadine grape skin extract enriched with ellagic acid, quercetin, and resveratrol, in men with biochemically recurrent prostate cancer showed no significant benefit ^{[73, 74]}. Grape seed extract’s effects in prostate cancer have been studied extensively in vitro and in TRAMP mouse models ^[75–80]. Recent studies have also demonstrated that grape seed extract, whose most active constituent is procyanidin B2 3,3’-di-gallate (B2G2), induced apoptosis in prostate cancer cells via different signaling pathways ^{[81, 82]}, inhibited endothelial cell growth and motility ^{[83, 84]}, and targeted prostate cancer stem cells ^[84]. Lipophilic grape seed proanthocyanidin extract induced caspase-dependent apoptosis in PC3-derived mouse xenografts ^[85].

In terms of single compound effects in prostate cancer, the vast majority of in vitro and in vivo studies investigated anticancer effects of stilbenes, particularly resveratrol through multiple signaling pathways ^{[62, 86–93]}. Over the last decade, our group has largely contributed to understanding the chemopreventive and therapeutic potential of stilbenes, namely resveratrol and pterostilbene, in prostate cancer. We have demonstrated that both resveratrol and pterostilbene have potent anticancer and anti-inflammatory effects in prostate cancer through inhibition of MTA1-mediated signaling leading to posttranslational modification and reactivation of PTEN and p53, modulations of miRNAs, inhibition of Akt survival pathways and EMT as well as angiogenesis and metastasis ^{[15, 16, 22, 24, 26–28, 36, 41, 61, 94]}. Pterostilbene, which is found in grapes and blueberries ^{[95, 96]} showed more potent MTA1-mediated anticancer efficacy in prostate cancer relative to resveratrol ^{[24, 38]}. Moreover, in our recent studies with whole grape powder extract (GPE) using prostate cancer cell lines, we found that that GPE also inhibited MTA1 signaling and cell viability, survival, and migration ^[42].

In the current study, we have investigated the potential of whole GP dietary supplementation for MTA1-targeted chemoprevention of prostate cancer. The GP used in this study preserves the integrity of the biologically active compounds found in fresh grapes.

Here, we demonstrate that the most beneficial effects of GP was observed in Pten^+/f mice on HFD. High fat diet feeding alone had very little effect on PIN histopathology in Pten^+/f mice. However, Pten^+/f mice fed diets supplemented with 10% GP for 33 weeks were substantially protected from developing PIN compared with those fed diets without supplementation. Consistent with these observations, mice fed the GP supplemented diets showed a statistically significant decrease in epithelial cell proliferation of PIN lesions relative to those fed diets without supplementation. Consistent with our previous observations ^[27], the MTA1 levels were significantly higher in Pten^+/f mice compared to WT mice. Despite an overall increase in MTA1 levels in Pten^+/f mice compared to the WT group, HFD alone or with GP supplementation had no effects on MTA1 expression. Of note, we did find a reduction in MTA1 levels in prostate cancer cell lines treated with an extract from the same GP (California Table Grape Commission, Fresno, CA), albeit with less potency than resveratrol and pterostilbene alone ^[42]. The most consistent results on MTA1 expression in this study were observed in WT mice fed HFD, in which highly increased expression of MTA1 was reduced by GP. These results suggest, for the first time, that the observed increase in MTA1 in response to HFD in WT mice may act as a triggering factor for early carcinogenic events resulting from MTA1-mediated inflammation and MTA1/c-myc/Akt signaling activation ^[27]. This novel finding underlines one of the potential mechanisms by which obesity may provoke prostate cancer development and/or progression. In this case, GP supplementation might be beneficial due to its ability to inhibit MTA1 and Akt, although further research would be necessary to confirm this.

As already mentioned, we have previously demonstrated high levels of MTA1-dependent Akt activation in Pten^+/f mice ^[27]. Others have demonstrated that overexpression of Akt in the prostate promoted PIN in transgenic mice ^[97] and that Akt deficiency markedly inhibited development of PIN in Pten^+/f mice ^[98]. It was also shown that reduction of dietary fat may inhibit the transition from PIN to invasive cancer by affecting cell proliferation via the Akt-mTOR pathway in Hi-Myc transgenic mouse prostate cancer model ^[55]. In the current study, we found decrease in Akt signaling activity in Pten^+/f mice fed with diets supplemented with GP.

Several studies have suggested that fat-containing diets might modulate intraprostatic hormonal status and AR signaling in prostate cancer. For example, an association between low serum cholesterol levels and reduced tumor growth accompanied by decreased intratumoral androgens was reported in a Pten-null transgenic mouse model of prostate cancer ^[6]. On the other hand, the mechanisms by which polyphenols exert their antitumor effect have been shown to involve an inhibition of AR and its signaling ^{[47, 48, 87, 99–102]}. Herein, we found that HFD feeding had significant effects on increasing AR levels in Pten^+/f mice and to a lesser mark in WT mice. Importantly, GP supplementation reduced AR levels in HFD fed Pten^+/f and WT mice.

A number of studies have demonstrated the role of inflammation in HFD-induced prostate cancer accompanied by the prostate gland stromal infiltration with inflammatory cells and increased levels of serum proinflammatory cytokines ^{[45, 103–105]}. It has been shown that mice with Pten haploinsufficiency in the prostate exhibit an increase in macrophages and T-lymphocytes infiltrating the microenvironment, which is further increased by a high calorie diet ^[45]. Interleukin-1beta is a pro-inflammatory cytokine that plays an important role in immune response by activating T lymphocytes and natural killer cells and triggering the production of other pro-inflammatory cytokines in cancer. Microenvironmental IL-1β is also required for in vivo angiogenesis and tumor cell growth and invasion ^[106]. We found that HFD notably led to increased secreted IL-1β in Pten^+/f and WT mice as well as tissue IL-1β levels in Pten^+/f mice. Importantly, GP supplementation significantly reduced the levels of this pro-inflammatory and pro-angiogenic cytokine in HFD fed Pten^+/f mice.

TGFβ1 can play a contradictory role in normal physiology and pathology ^[107]. For example, TGFβ can induce apoptosis or inhibit the proliferation of nontransformed cells but accumulates and loses its growth-inhibitory functions in cancer. More than that, high levels of TGFβ in tumor cells and reactive stroma contribute to cell growth, invasion, and metastasis ^[51]. It is well established that prostatic inflammation and prostate cancer are associated with high levels of TGFβ1 and the activation of TGFβ1 cascade genes in tumor cells and inflamed stroma ^{[27, 51, 108, 109]}. However, the multifunctional role of TGFβ1 in a normal prostate as well as in the development of PIN remains controversial ^[110]. We found high levels of TGFβ1 in LFD fed Pten^+/f mice compared to normal prostates, which suggests the proinflammatory role for this cytokine during the very early stage of prostatic neoplasia. Similar to the beneficial effects of a pterostilbene-diet in Pten^+/f mice ^[27], GP supplementation inhibited TGFβ1 levels in Pten deficient mice irrespective of the diet’s fat content. However, the low level of TGFβ1 expression in HFD-fed Pten^+/f mice is difficult to interpret.

Available data in the literature are conflicting concerning proliferation versus apoptosis in different stages of prostate cancer, specifically concerning PIN or premalignant tissue associated with prostate cancer. The role of apoptotic markers, such as cleaved caspase 3, was found to be complex as it showed a significant decrease in high-grade PIN and cancer lesions in human radical prostatectomy samples ^[111]. Authors concluded that active apoptosis in high-grade PIN might accelerate cell turnover in the development of premalignant lesions of the prostate. Finally, a retrospective clinical study found that higher proliferative activity was positively correlated with higher apoptotic rates in aggressive phenotype with an increased mortality from prostate cancer ^[52]. In the present study, we too observed the expected increased proliferative activity in PIN lesions of Pten^+/f mice together with increased apoptosis. Similar to our current observations, simultaneous increase in cell proliferation and high Bax-associated apoptotic rate was observed in PIN lesions of the rat prostate ^[53]. In agreement with other studies, we found no difference in apoptotic markers in PIN lesions in the LFD and HFD groups of Pten^+/f mice suggesting that dietary fat had no impact on apoptosis during the PIN stage of prostate carcinogenesis ^[55].

Although polyphenols have been shown to exert anticancer activity by stimulating apoptosis through mitochondrial pathways ^{[22, 27, 36, 96, 112–114]}, surprisingly, GP supplementation increased only p53 levels in Pten^+/f mice, while it reduced other apoptotic markers including p53-dependent p21 and Bax. High levels of p21 in WT mice reflect the complex role of p21 as an anticancer target since p21 acts as both a tumor suppressor and an inhibitor of apoptosis in different microenvironments ^[115]. The results were unexpected as anticancer agents are generally linked with increased apoptosis. One can argue, though, that because levels of caspase 9 and 3 are much lower in WT mice compared to Pten^+/f mice, significant reduction of caspase 9 and 3 by GP supplementation may indicate the somewhat beneficial effect of GP in Pten^+/f mice.

Dietary recommendations for the purpose of prostate cancer chemoprevention would be dramatically improved if serum markers that reflect patient responsiveness to diet intervention could be identified. MiRNAs could serve as such biomarkers, since they are easily detectable in liquid biopsies ^[116–118]. Importantly, available experimental and clinical data have suggested that dietary polyphenols may be effective miRNA-modulating chemopreventive and therapeutic agents ^{[16, 119–121]}. In particular, resveratrol and its analogs’ modulation of specific miRNAs caused apoptosis, cell cycle arrest, and cell growth, invasion and migration inhibition in various cancer types ^{[36, 122–128]}. However, only limited studies with bioactive dietary compounds have used changes in miRNA levels as predictive biomarkers in prostate cancer ^[129–131]. Relevant to our study, the effects of a grape extract containing resveratrol on the modulation of inflammation-related miRNAs in the peripheral blood was demonstrated ^[132] . Given our previous reports on the identification of MTA1-associated and resveratrol- and pterostilbene- regulated miRNAs in prostate cancer ^{[36, 61, 133]}, we found that GP supplementation caused significant reduction in the circulating levels of miR-34a and miR-22 in Pten^+/f mice. As mentioned above, we observed GP- induced upregulation of p53, which is a miR-34a target, suggesting at least in part the miR-mediated mechanism of p53 rescue by GP. Incidentally, miR-34a was among the altered miRNAs regulating inflammatory response in a clinical trial using a grape extract ^[132]. Others also reported miR-34a-5p downregulation potential as a therapeutic target to improve outcomes for cancer patients through restoring p53 signaling ^[134], although the role of miR-34a and its complex functional relationship with p53 in cancer remains somewhat controversial ^{[135, 136]}. Oncogenic miR-22 was shown to target E-cadherin in prostate cancer ^[61]. Surprisingly, in this study, significant inhibition of circulating miR-22 by GP was not reflected in increases in p21 expression in prostate tissues in Pten^+/f mice, which is in accordance with a report suggesting a contradictory and dual role for miR-22/p21 on causing cell cycle arrest or inducing apoptosis in cancer ^[137].

To summarize, HFD promoted inflammation, changes in hormonal status, and activation of cell survival pathways in prostate-specific Pten deficient mice. We demonstrated, for the first time to our knowledge, that GP supplementation to diet has protective effects against prostate epithelial cell proliferation, inflammation, activation of cell survival signaling pathways, and angiogenesis leading to improved histopathology of PIN in these genetically predisposed mice. The whole grape polyphenols in GP also reduced serum cytokines and oncomiRs’ levels, defining them as potential non-invasive predictive biomarkers. Mechanistically, we did not observe expected MTA1 inhibition by the GP in Pten deficient mice, which can be simply explained by the fact that GP contains negligible quantities of resveratrol and no pterostilbene. Irrespective of the upstream mechanisms of action, we suggest that the ability of GP to modulate genetic cancer risk favorably by restoring PTEN and inhibiting activated Akt signaling plays a critical role in preventing obesity-associated progression of PIN in mice. In conclusion, given that we tested physiological doses of GP for dietary grape intake in humans (2.1 Materials), the results of this study may have potential applications to human health providing support for future clinical trials on prostate cancer chemoprevention. Successful studies with recruitment of an “active surveillance” subpopulation of prostate cancer patients diagnosed with high-grade PIN or atypical small acinar proliferation (ASAP) and/or men at high risk for developing obesity-induced prostate cancer will offer a long-anticipated chemopreventive measure for management of early neoplastic lesions in the prostate.

Abbreviations

Akt

serine/threonine kinase

AR

androgen receptor

ASAP

atypical small acinar proliferation

B2G2

procyanidin B2 3,3’-di-gallate

Bak

Bcl2 antagonist/killer

Bax

Bcl2 associated X protein

Bcl2

B-cell leukemia/lymphoma 2

CD31

cluster of differentiation 31

CK8

cytokeratin 8

Cre

recombinase enzyme

DU145

prostate cancer cell line

EMT

epithelial-to-mesenchymal transition

GP

grape powder

GPE

grape powder extract

H&E

hematoxylin and eosin

HFD

high -fat diet

IHC

immunohistochemistry

IL-1β

interleukin 1β

kCal

kilocalorie

Ki67

cellular protein marker of proliferation

LFD

low-fat diet

mRNA

messenger RNA

miRNA/miR

micro RNA

MTA1

metastasis-associated protein 1

mTOR

mammalian target of rapamycin

Myc

myelocytomatosis oncogene

Pb

probasin

PC3M

prostate cancer cell line

PIN

prostatic intraepithelial neoplasia

PTEN

phophatase and tensin homolog

Pten^+/f

Pten heterozygous mice

RPMI-1640

Roswell Park Memorial Institute 1640 cell culture media

SFA

saturated fatty acid

shRNA

short hairpin RNA

SMA

smooth muscle actin

TGFβ1

transforming growth factor β1

TRAMP

transgenic adenocarcinoma of the mouse prostate

UGS

urogenital system

VEGF

vascular endothelial growth factor

WT

wild type