Dietary pterostilbene for MTA1-targeted interception in high-risk premalignant prostate cancer

Abstract

Prostate cancer remains one of the most prevalent cancers in aging men. Active surveillance subpopulation of prostate cancer patients includes men with varying cancer risk categories of precancerous disease due to prostatic intraepithelial neoplasia (PIN) heterogeneity. Identifying molecular alterations associated with PIN can provide preventable measures through finding novel pharmacological targets for cancer interception. Targeted nutritional interception may prove to be the most appropriate chemoprevention for intermediate- and high-risk active surveillance patients. Here, we have generated two prostate-specific transgenic mouse models, one overexpressing MTA1 (R26^MTA1) and the other overexpressing MTA1 on the background of Pten heterozygosity (R26^MTA1; Pten^+/f), in which we examined the potential chemopreventive efficacy of dietary pterostilbene. We show that MTA1 promotes neoplastic transformation of prostate epithelial cells by activating cell proliferation and survival, leading to PIN development. Moreover, MTA1 cooperates with PTEN deficiency to accelerate PIN development by increasing cell proliferation and MTA1-associated signaling. Further, we show that mice fed with a pterostilbene-supplemented diet exhibited more favorable histopathology with decreased severity and number of PIN foci accompanied by reduced proliferation, angiogenesis, and inflammation concomitant to reduction in MTA1 and MTA1-associated CyclinD1, Notch2, and oncogenic miR-34a and miR-22 levels.

Introduction

According to the American Cancer Society, prostate cancer is the most common of all cancers in men and it accounts for approximately 13% of total newly diagnosed cancers in the United States (1). While the incidence of prostate cancer has been rising over the last two decades, it is acknowledged that it is not only because of changes in the age distribution of the population but also due to advances in screening, detection, and diagnosis (2). On the other hand, because the United States Preventive Services Task Force issued a grade “D” recommendation against the use of routine prostate-specific antigen (PSA)-based screening in all men, the incidence significantly decreased but the recommendation also lead to a shift toward a higher prostate cancer grade and stage upon diagnosis (3). High-risk population includes men with a family history of prostate cancer, those older than 65 years, certain race and ethnicity, and men with obesity and metabolic disorders. The active surveillance subpopulation that include men with high-grade prostatic intraepithelial neoplasia (PIN) and low-grade prostate cancer with a small tumor and low PSA are widely recognized as patients with precursors to prostate cancer. During the active surveillance time, various signaling pathways are activated, which may lead to tumor initiation and promotion. The clinical management of patients in active surveillance is currently not satisfactory since on one hand, no treatment is available to prevent cancer and, at the same time, monitoring patients who may show signs of progression to cancer is a necessity (4). As a result, unfortunately, 30% of these patients eventually develop more aggressive disease that requires surgery and intensive treatment. Identification of specific molecular mechanisms for accurate stratification of cancer risk in these patients is an urgent need for developing targeted interceptive measures to stop the progression at the precursor “favorable-risk” stages.

One of the most significant modifiable risk factors for developing prostate cancer is diet (5, 6). The major rationale for prostate cancer dietary interception relies on the specific characteristics of prostate cancer such as its stage-defined and slow-growing nature. In addition, due to the well-documented heterogeneity of prostate cancer in its development and progression, careful consideration must be given to the targeted subpopulation of patients that may benefit from particular dietary intervention. Reducing dairy and fat intake, for example, have been associated with decreased prostate cancer risk in both epidemiological and preclinical studies (7–9). Moreover, some epidemiological and case-control studies have found that high consumption of green tea, fruits, vegetables or soy foods was associated with a decreased risk of prostate cancer (10, 11). Further, distinct dietary phytochemicals have been shown to exert anti-inflammatory, antioxidant, and anticancer properties against prostate cancer progression through various cellular pathways and epigenetic mechanisms (12, 13). Since the time that a reduced prostate cancer risk was epidemiologically associated with red wine consumption and resveratrol (14), various signaling mechanisms of stilbene-mediated cytotoxic and anticancer effects have been reported in prostate cancer (15–17). Pterostilbene, a natural dimethoxy analog of resveratrol, which accumulates in grapes and blueberries, has shown enhanced bioavailability and superior biological activity to resveratrol in mechanistic and preclinical studies in cancer (18–20). Particularly, our group systematically reported on more potent metastasis-associated protein 1 (MTA1)-mediated effects of pterostilbene against prostate cancer (21–25).

The role of MTA1 signaling in prostate cancer is highly context dependent (23, 26, 27). MTA1, a master transcriptional co-regulator and chromatin modifier (26, 28) is strongly associated with clinically aggressive prostate cancer (27, 29, 30), and the loss of MTA1 reduces aggressiveness and metastatic potential of prostate cancer cells (31, 32). Preclinical studies with subcutaneous and orthotopic MTA1 knockdown xenograft models as well as with prostate-specific Pten deletion models, which exhibit increased MTA1 expression, have shown the tumor promoting role of MTA1 and associated survival pathways in prostate cancer (21, 23, 32). The role of MTA1 in the early neoplastic pathobiology of prostate cancer is less understood. In our published studies, we detected MTA1 expression in PIN lesions of human samples (29) and an increased epithelial and stromal MTA1 expression together with associated markers of inflammation in PIN lesions in conditional Pten-deficient mice (9, 23).

It is widely acknowledged that specific genetic aberrations define variable phenotypes of premalignant condition, some of which, such as MTA1 overexpression, may define which patients are high-risk. However, the exact role of MTA1 in prostate cancer inflammation and carcinogenesis remains unknown and its value as a target for cancer interception has yet to be clearly defined. Accordingly, we sought to determine the role of MTA1 in the neoplastic transformation of prostate epithelial cells and the beneficial interceptive effects of diet supplemented with pterostilbene acting through the MTA1-targeted mechanisms against progression to cancer using an adequate mouse model of premalignant high-risk prostate cancer.

Materials and Methods

Materials

Pterostilbene (PTER) was synthesized by the late Dr. Agnes M Rimando according to a protocol described previously (23) and partially was purchased from Sigma-Aldrich. The purity of PTER was determined to be ≥ 99%. Pterostilbene powder was shipped to Envigo Teklad Diets for formulation of pterostilbene supplemented AIN 76A diet (PTER-Diet) at a concentration of 100 mg/kg Diet as previously described (23). Briefly, we used DD = (SD x BW) / FI formula (Research Diets), where DD is Diet Dose (mg PTER / kg Diet); SD is single Dose (mg PTER / kg bw/day); BW is Body Weight (g bw / animal) and FI is daily Food Take (g Diet / day) for our calculations. Of note, the 100 mg/kg Diet dose converts to 14.4 mg/kg BW for mice, which is equivalent to 1.17 mg/kg BW for humans according to formula: Human Equivalent Dose (HED) (mg/kg) = Animal dose (mg/kg) x K_m ratio, where K_m ratio for mice is 0.081 (33). Considering that average human male BW is 70 kg, the dose used in this study for mice approximately translates into 82 mg /day in humans, which is within the well tolerated and generally safe dose of up to 250 mg/day as shown in human clinical trial with PTER (34). AIN-76A diet was used as the control diet (Ctrl-Diet). Nutritional composition of diets is provided in Supplementary Table S1. Both Ctrl-Diet and PTER-Diet were stored at 4°C protected from light.

Animals

All animal protocols were approved in advance by the Institutional Animal Care and Use Committee (IACUC) at Long Island University. Mice had free access to drinking water and designated diets and were monitored daily for their general health.

Establishment of prostate-specific MTA1 transgenic mice:

We generated a conditional mouse with the human MTA1 transgene knocked into the mouse Rosa26 (R26) locus using CAG-LoxP-Stop-LoxP(LSL)-2HA-MTA1-T2A-GFP-pA construct (Supplementary Fig. S1). Briefly, after the generation of a genetically modified founder mouse line Rosa26-3attP (R26P3), which was used for MTA1 sequence integration, resulting MTA1 transgene embryos were injected into foster mice to produce MTA1 founders, designated as R26-LSL-MTA1 (Applied StemCell). We crossed the R26-LSL-MTA1 female mice with wild-type (WT), probasin promoter positive Pb-Cre4 (Cre⁺) male mice (Jackson Laboratories), to generate transgenic lines that express MTA1 upon Cre-mediated removal of the LSL cassette, specifically in the prostate epithelium, R26^+/MTA1; Cre⁺ (R26^+/MTA1). The male R26^+/MTA1 mice was then backcrossed with R26-LSL-MTA1 female mice to generate mice with biallelic overexpression of MTA1, R26^MTA1/MTA1; Cre⁺ (R26^MTA1). To monitor formation and development of tumors in live animals by using In Vivo Imaging System (IVIS) as before (35), we crossbred WT, Cre⁺ mice with R26-LSL-Luciferase (Luc) mice (a generous gift from Dr. A. Atfi, Virginia Commonwealth University, VA), allowing expression of Luc specifically in the prostate (R26^Luc; Cre⁺). By crossing R26-LSL-MTA1 mice with R26^Luc; Cre⁺ mice, we have generated transgenic lines that express MTA1 and Luc upon Cre-mediated removal of the LSL cassette, specifically in the prostate epithelium (R26^+/MTA1; R26^+/Luc; Cre⁺). Bioluminescent measurements were taken using IVIS Lumina LT instrument (Perkin Elmer). Briefly, mice were anesthetized with 2% isoflurane, injected with 150 mg/kg d-luciferin i.p. and placed inside the camera box as described previously (21, 35, 36). Image analysis and bioluminescent quantitation was performed using Living Image software (Perkin Elmer, RRID:SCR_014247). Background controls were used appropriately.

Generation of prostate-specific MTA1 overexpression on the background of Pten heterozygous mice: breeding and genotyping:

C57BL/6J female mouse homozygous for the “floxed” Pten allele was purchased from Jackson Laboratories and bred with our MTA1 transgenic mice (R26^MTA1) to create “high-risk” PIN-prone cohort of mice mimicking active surveillance patient subpopulation, R26^MTA1; Pten^+/f; Cre⁺. Mice with normal prostates (Cre-negative) or intermediate Cre⁺ genotypes such as R26^+/MTA1, R26^MTA1 or Pten^+/f served as control groups in different experiments.

Genotyping was performed using tail-genomic DNA by PCR-based screening using the primers listed in Supplementary Table S2.

Treatment groups:

Forty R26^MTA1; Pten^+/f; Cre⁺ mice at 3 weeks of age were randomized into two major groups: mice on PTER-Diet and mice on Ctrl-Diet, ad libitum. Animals were monitored daily for signs of toxicity and their body weights were measured twice weekly. After 17 weeks on diet, mice were sacrificed and urogenital system (UGS) including prostate, seminal vesicle, and bladder were removed en bloc and fixed with formalin for histology and IHC evaluation. Prostate tissues were dissected, snap frozen and kept at −80°C until use. Blood was collected by cardiac puncture; serum samples were prepared and stored at −80°C.

Cell Lines

DU145 (purchased from ATCC) and PC3M (RRID:CVCL_9555), a generous gift from Dr. R. Bergman, University of Nebraska Medical Center, NE, prostate cancer cell lines were grown in RPMI 1640 media (Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS) and maintained in a 37°C with 5% CO₂ incubator. MTA1 knockdown DU145 and PC3M cells were generated as described previously (27). Cells were authenticated using short tandem repeat (STR) profiling at the Research Technology Support Facility (RTSF) of Michigan State University (MSU) and regularly tested for mycoplasma using the Universal Mycoplasma Detection Kit (ATCC) and found to be mycoplasma-free.

Western blot

Western blots of cancer cells and homogenized prostate tissues were performed as described previously (23, 35). Briefly, protein lysates from cells or prostate tissues were prepared using RIPA buffer (Thermo Fisher Scientific), protein was estimated, and samples were separated using 10-15% polyacrylamide gels and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% milk/TBS/0.1% Tween and then probed with corresponding primary antibodies listed in Supplementary Table S3. β-actin and/or Hsp70 were used as a loading control. Signals were detected using enhanced chemiluminescence (Thermo Fisher Scientific). Band intensity was measured using Image J (NIH, RRID:SCR_003070).

Histology and immunohistochemistry (IHC)

Urogenital system tissues were fixed in 10% neutral-buffered formalin, processed and embedded in paraffin and cut into 4-μm sections (Reveal Biosciences). H&E-stained sections were assessed for mouse PIN (VD). Sections were subjected to IHC analysis for Ki67, SMA, CD31, and MTA1 using antibodies listed in Supplementary Table S3 as described previously (23, 35). Images were taken using EVOS XL Core microscope (Thermo Fisher Scientific). Ki67, MTA1 and CD31 positively stained cells were counted in five randomly selected fields using ImageTool software (NIH, RRID:SCR_015574). For Figure 2B, tissues were stained with Ki67 antibody (1:50, Abcam, RRID: AB_2861195) following anti-rabbit Alexa Fluor 488 secondary antibody (1:500, Thermo Fisher Scientific). Cover slips were mounted in Vectashield with DAPI (Vector Laboratories) and imaged using a Nikon Eclipse 80i microscope (Nikon Instruments).

Figure 2.

Prostate-specific MTA1 overexpression leads to increased cell proliferation and changes in histopathology in R26^+/MTA1; Cre⁺ mice. A, representative H&E of the prostate from mice showing PIN with characteristic hypercellularity. B, representative immunofluorescent staining of prostate tissues for Ki67 is shown. Blue is nuclear DAPI counterstain; white arrows show Ki67-positive nuclei. Scale bar: 20 μm; C, immunoblots showing association of high MTA1 levels in the representative prostate tissues from 2 different mice in each group with increased proliferation (PCNA, p27, CyclinD1), increased inflammation (NF-κB, IL-1β), activation of survival pathways (Notch2, Akt) and EMT (E-Cad) markers. Hsp70 was used as a loading control. D, Left, representative bioluminescent images showing increased luciferase (Luc) signal in the prostate. 1: R26^+/MTA1; R26^+/Luc; Cre⁻, 2: R26^+/Luc; Cre⁺ (NP, normal prostate), 3: R26^+/MTA1; R26^+/Luc; Cre⁺. 1 and 2 were used as Luc background controls (Ctrl). Right, quantitative analysis of prostate light emission in total flux (p/s) is plotted for each representative group. ****p < 0.0001 (one-way ANOVA). E, comparison of MTA1 expression in the prostate of R26^+/MTA1 transgenic mice to MTA1 expression in human prostatectomy specimens (n = 4). Coomassie staining is a loading control. F, representative H&E (top) and IHC staining for SMA (second from top), Ki67 (third from top), and MTA1 (bottom) in the prostate of 17-week-old monoallelic R26^+/MTA1; Cre⁺ mice compared to age-matched biallelic R26^MTA1; Cre⁺ mice are shown. Scale bar, 50 µm. G, immunoblot (top) and quantitation (bottom) showing higher MTA1 levels in the prostate of a 17-week-old R26^MTA1; Cre⁺ mouse compared to age-matched R26^+/MTA1; Cre⁺ mouse.

ELISA

Serum IL-6 and IL-1β levels were analyzed using commercially available IL-1β SimpleStep ELISA™ kit and Il-6 Mouse ELISA kit (Abcam) according to the manufacturer’s protocol. The assay uses pre-coated 96 well strip microplates covered with an 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). Using the standard titration curve, a concentration of IL-6 and IL-1β in serum was calculated based on their optical density values.

qRT-PCR

Total RNA was isolated from the cell lines using miRNeasy kit (Qiagen) as described previously (23, 35, 37). The quality of RNA was determined on a NanoDrop spectrophotometer (Shimadzu Scientific Instruments). Real-time PCR was performed on a Lightcycler 480 II Real-Time PCR instrument (Roche Diagnostics) using primers specific for MTA1, Notch2, and CyclinD1 listed in Supplementary Table S4. For evaluation of PIN-derived circulating miRNAs in murine serum, RNA was isolated using miRNeasy serum/plasma kit as previously described (9, 20, 37). Real-time PCR was performed using custom primers for miR-22-3p, miR-34a-5p, and miR-103a-3p (internal reference control) and miRCURY LNA™ Universal RT microRNA PCR kit or 2x miRCURY LNA SYBR green PCR Kit (Qiagen). Sera from 6-7 mice in each group were combined to obtain 200 μl volume. Fold changes in mRNA and miRNA expression were estimated by the 2^−ΔΔCt method.

Clinical specimens

Previously archived prostatectomy specimens were obtained and processed according to approved University of Mississippi Medical Center (UMMC) Institutional Review Board (IRB) protocol as previously described (27, 35). Briefly, protein was extracted from the pool of core biopsies from each sample/subject and run on the same gel as mice samples.

Statistical analysis

The data from each treatment group was summarized as the mean ± SEM. To determine the significance of differences between groups, a two-sample two-tailed t-test or one-way ANOVA was applied. All the in vitro experiments were performed at least three times and data is shown as mean ± SEM. A p value of ≤ 0.05 was considered statistically significant. Graphs were generated using GraphPad Prizm v9 software (RRID:SCR_002798).

Data Availability Statement

Genetically modified cell lines and unique transgenic mice generated through this research will be shared with research community per request through properly executed Material Transfer Agreements (MTA), which are managed by the Office of Sponsored Research and Sponsored Programs (OSR-SP) of LIU.

Results

The prostate-specific MTA1 transgenic mice develop PIN

The known functional role of MTA1 as an epigenetic reader and master-coregulator indicated that overexpression of MTA1 in the prostate may produce specific cell signaling changes that can influence the neoplastic transformation of cells and/or progression of prostate cancer. In order to study the biological role of MTA1 in prostate tumorigenesis, we generated a conditional mouse model with the MTA1 transgene knocked into the Rosa26 locus (R26^+/MTA1) (Supplementary Fig. S1). We then crossed R26^+/MTA1 with prostate specific Pb-Cre4 (Cre⁺) mice to express MTA1 specifically in the prostate (R26^+/MTA1; Cre⁺ and R26^MTA1/MTA1; Cre⁺, hereafter referred as R26^+/MTA1 and R26^MTA1, respectively). Fluorescent ex-vivo imaging shows GFP expression specific to the prostate of R26^+/MTA1 mice (Fig. 1A). Fig. 1B also shows GFP expression concomitant with elevated levels of MTA1 specifically in the prostate tissue of R26^+/MTA1 mice compared to their control littermates (Cre⁻) with normal prostates. Consistent with the above observation, the prostate of R26^+/MTA1 mice show significantly higher levels of MTA1 transcript (Fig. 1C). Our detailed evaluation of the MTA1 protein overexpression in R26^+/MTA mouse prostate glands reveal high expression in ventral prostate (VP) and anterior prostate (AP) lobes (Fig. 1D). Histological evaluation revealed that the prostate glands from 13 week-old R26^+/Mta1 mice show signs of PIN characterized by glandular structures composed of atypical proliferating luminal cells that fill out the lumen (Fig. 2A). Immunohistochemical staining of prostate tissues demonstrated proliferative activity in the prostates of R26^+/Mta1 mice compared to controls (Fig. 2B). We also detected differences in cell proliferation as determined by increase in PCNA and decrease in p27 levels along with upregulation of inflammatory markers such as NF-κB, IL-1β, increase in CyclinD1 and Notch2, activation of Akt signaling, and changes in epithelial-to-mesenchymal (EMT) markers (reduced E-cadherin) in the prostates of R26^+/MTA1 mice compared to Cre⁻ controls (Fig. 2C). For purposes of noninvasive monitoring of prostate histopathology, we crossed R26^+/MTA1 mice with conditional mice with Luc transgene knocked into the Rosa26 locus (R26^+/Luc) to generate R26^+/MTA1; R26^+/Luc; Cre⁺ transgenic mice, which demonstrate higher luciferase signal specific to the prostate (Fig. 2D). However, it is important to remember that due to limitations of luciferase technology, the detected image gives the appearance of luciferase signal not being confined to the organ, i.e. prostate per se. The image has only “velocity” value showing dynamics of changed histology. We also found that levels of MTA1 in the mouse model were physiologically relevant as it was comparable with MTA1 expression in human prostatectomy samples (Fig. 2E). Further, to address whether monoallelic or biallelic overexpression of MTA1 makes a difference in prostate histopathology, we compared levels of MTA1 protein and prostate gland histology in 17-week-old R26^+/MTA1 and R26^MTA1 mice. As expected, R26^MTA1 mice had higher MTA1 protein expression leading to increased clusters of PIN lesions with characteristic disorganized glandular structures, hypercellularity, and cribriform and tufting (Fig. 2F and G). Although substantial histological and molecular differences were noted as early as 17 weeks of age, and severity of PIN lesions increased with the higher MTA1 expression and the age of the mice, histologic and IHC examinations of prostates from 6 to12-month-old animals failed to indicate development of invasive adenocarcinoma or metastasis. However, importantly, overexpression of MTA1 in mouse prostate activated cell proliferation and survival programming indicating a possible causal role in the neoplastic transformation of prostate epithelial cells, therefore MTA1 can be considered as a target for prostate cancer interception.

Figure 1.

Prostate-specific overexpression of MTA1 in a 13-week-old R26^+/MTA1; Cre⁺ mice. A, ex vivo images of the representative UGS from mice showing GFP expression localized in the prostate (fluorescent mode, rainbow palette format). B, immunoblots showing specific overexpression of MTA1 in the representative prostate compared to other organs as confirmed by higher MTA1 levels and specific GFP expression. C, overexpression of MTA1in the representative prostate detected by qRT-PCR showing higher MTA1 mRNA levels, ***p < 0.001 (two sample two-tailed t-test). D, immunoblots showing higher MTA1 levels in the representative anterior (AP), ventral (VP) and dorso-lateral (DLP) prostate lobes of 1: R26^+/MTA1; Cre⁻, 2: WT; Cre⁺, and 3: R26^+/MTA1; Cre⁺ mice.

Prostate-specific MTA1 overexpression enhances and accelerates PIN development in Pten^+/f mice

PTEN loss is a relatively common abnormality in human prostate cancer with approximately 30-70% of cases showing genetic loss or mutation of one PTEN allele (38, 39). The pathogenic role of Pten loss in prostate tumorigenesis in genetically engineered mouse models is well-established (40). We have previously shown that prostate-specific heterozygous deletion (Pten^+/f) results in PIN, and that the expression of murine MTA1 is drastically increased in prostates of these mice (23). We have also shown the strong inverse correlation between MTA1 and PTEN expression in human prostate tumors and cell lines (20, 23, 41). To determine the biological significance of MTA1/PTEN cooperativity, we crossed our R26^MTA1 with Pten^+/f mice to generate prostate-specific mice overexpressing MTA1 on the background of Pten heterozygosity (R26^MTA1; Pten^+/f; Cre⁺, hereafter referred as R26^MTA1; Pten^+/f).

Since prostate-specific Pten^+/f mice develop PIN in 28-36 weeks (7-8 months) (9, 23), we hypothesized that R26^MTA1; Pten^+/f mice may develop PIN earlier (Fig. 3A). Analysis of prostate tissues from mice at 20 weeks showed PIN lesions characterized by more disorganized glandular structures with hypercellularity, the cribriform and micropapillary patterns in R26^MTA1;Pten^+/f compared to both R26^MTA1 and Pten^+/f mice. However, no signs of microinvasion were detected in R26^MTA1;Pten^+/f prostates as evident by the smooth muscle actin (SMA) staining (Fig. 3B). The number of glands involved in PIN was significantly increased by approximately 30-40% in R26^MTA1; Pten^+/f compared to both R26^MTA1 and Pten^+/f mice (Fig. 3C). Immunohistochemical analysis of prostate tissues revealed increased proliferation and MTA1 levels as indicated by more Ki67-positive and MTA1-positive cells, respectively, in prostates from R26^MTA1; Pten^+/f compared to both R26^MTA1 and Pten^+/f mice (Fig. 3B and C). MTA1 expression was the highest in the R26^MTA1; Pten^+/− mice prostates (Fig. 3B and C, bottom). Taken together, the results confirmed our initial hypothesis that prostate-specific overexpression of MTA1 promotes and accelerates development of PIN representing premalignant heterogeneity and corresponding changes that correlate with the risk of progressing to cancer.

Figure 3.

Prostate-specific overexpression of MTA1 cooperates with Pten deficiency to accelerate PIN development. A, schematic depicting time-line of PIN development in prostate-specific Pten^+/f and R26^MTA1; Pten^+/f mice. B, representative H&E (top) and IHC staining for SMA (second from the top), Ki67 (second from the bottom) and MTA1 (bottom) are shown for WT, R26^MTA1, Pten^+/f , and R26^MTA1; Pten^+/f mice. Scale bar, 50 µm. C, quantitative analysis of number of glands involved in PIN (top), Ki67-positive (middle), and MTA1-positive (bottom) cells. Values are mean ± SEM of cells counted in five randomly selected fields per sample for n=3 (WT, R26^MTA1, and Pten^+/f) and n = 6 (R26^MTA1; Pten^+/f) mice in each group. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA). D, representative immunoblots from three different mice (left, middle) and quantitative analysis (right) of MTA1, CyclinD1, and Notch2 expression in prostate tissues of WT, R26^MTA1 and R26^MTA1; Pten^+/f mice. ***p < 0.001 (two sample, two-tailed t-test). E, F and G, H, representative immunoblots (E, G) and quantitation of mRNA (F,H, top) and protein (F,H, bottom) levels in DU145 (E,F) and PC3M (G,H) cells expressing MTA1 (NS) and silenced for MTA1 (shMTA1) are shown. β-actin was used as a loading control. Changes in mRNA expression 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 (two sample, two-tailed t-test).

We have previously identified MTA1-associated genes by ChIP-Seq, among which were CyclinD1 and Notch2 (23). We have shown that both oncogenic molecules were directly regulated by MTA1 in prostate cancer MTA1 knockdown cells and prostate-specific Pten heterozygous mice (23). To determine whether prostate-specific MTA1 overexpression in Pten deficient condition further promoted oncogenic signals and survival events, we compared prostate protein extracts and found significantly increased expression of MTA1, CyclinD1, and Notch2 in R26^MTA1; Pten^+/f compared to R26^MTA1 mice (Fig. 3D). In order to further confirm direct transcriptional regulation of these two molecules by MTA1, independent of Pten status, we used two prostate cancer cell lines silenced for MTA1 expression as described previously (21, 27, 31). Figures 3E, F (DU145) and G,H (PC3M) show that consistent with reduced levels of MTA1 in both cell lines (shMTA1), CyclinD1 and Notch2 expression were significantly decreased at both mRNA and protein levels, indicating that CyclinD1 and Notch2 regulation by MTA1 is independent of PTEN status. Taken together, these data suggested a more aggressive precancerous phenotype of prostate-specific Pten deficiency in the presence of MTA1 overexpression.

Pterostilbene supplemented diet diminishes PIN progression in R26^MTA1; Pten^+/f mice

Upon accumulating forty mice of R26^MTA1; Pten^+/f genotype, we randomized mice into two major groups: mice on a diet supplemented with pterostilbene (PTER-Diet) and mice on a control diet (Ctrl-Diet) ad libitum. Nutritional composition of both diets is provided in Supplementary Table S1. For comparison, Pten^+/f mice were fed with Ctrl-Diet. Mice were fed corresponding diets for 17 weeks, during which their body weights and food intake were measured twice weekly. We found that PTER-Diet had no effect on the mean body weight and food intake in R26^MTA1; Pten^+/f mice (Supplementary Fig. S2A and S2B).

Mice were sacrificed at 20 weeks of age and prostate tissues were used for histological and molecular evaluation. Comparison of the UGS from mice revealed that the prostates from R26^MTA1; Pten^+/f mice on Ctrl-Diet were larger than the prostates of Pten^+/f mice on the same diet. Remarkably, the prostates of R26^MTA1; Pten^+/f on PTER-Diet were smaller in size and reiterated anatomical features of healthy UGS with normal prostate (Fig. 4A).

Figure 4.

Diet supplemented with PTER shows high efficacy in blocking MTA1-associated progression of PIN. A, representative images of UGS (top), H&E (second from top) and IHC analyses of prostate tissues from 20-week-old R26^MTA1; Pten^+/f mice fed Ctrl-Diet versus PTER-Diet in comparison to age-matched Pten^+/f mice fed Ctrl-Diet: changes in histology (SMA; scale bar, 50 µm), proliferative activity (Ki67; scale bar, 20 µm ), angiogenesis (CD31, arrows indicate microvessels; scale bar, 50 µm), and MTA1 expression (scale bar, 50 µm). B, quantitative analyses of number of glands involved in PIN (top), Ki67-positive cells (second from the top), vessel area (third from the top), and MTA1-positive cells (bottom) showing beneficial effect of PTER-Diet on R26^MTA1; Pten^+/f mice. Values are mean ± SEM of cells counted in five randomly selected fields per sample for n = 6 (R26^MTA1; Pten^+/f) and n = 3 (Pten^+/f) mice in each group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (one-way ANOVA).

Fig. 4A shows that prostates of R26^MTA1; Pten^+/f mice on Ctrl-Diet exhibited predominantly PIN characterized by disorganized glandular structures with cribriform, papillary folding and tufting. Highly proliferative epithelial cells filled the lumen but showed intact membranes composed of basal SMA-positive cells. In contrast, mice fed PTER-Diet demonstrated favorable histology with mostly restored ductal structures, only occasional tufting and unoccupied lumen. The number of glands involved in PIN was reduced by approximately 60-65% in R26^MTA1; Pten^+/f mice fed PTER-Diet compared to Ctrl-Diet fed cohort and the difference in numbers of glands involved in PIN was also statistically significant between R26^MTA1; Pten^+/f and Pten^+/f mice on Ctrl-Diet (Fig. 4B, top). Further, prostates of mice on PTER-Diet showed significantly reduced number of Ki67-positive cells and CD31 staining, indicating a significantly decreased proliferation and angiogenesis, respectively. The differences between CD31 staining in prostates of R26^MTA1; Pten^+/f and Pten^+/f mice on the same Ctrl-Diet was also statistically significant, conforming the previously identified role of MTA1 overexpression in promoting angiogenesis (32, 35). Finally, consistent with our earlier reports (23), we also noted a significant decrease of MTA1 levels in the prostates of mice on PTER-Diet (Fig. 4B, bottom).

We have previously shown that MTA1 and MTA1-associated genes were responsive to pterostilbene treatment in cell lines and prostate-specific Pten heterozygous mice (23) as well as in PC3M xenografts (36). In order to extend our observations of MTA1-associated oncogenic network induction and pterostilbene efficacy in inhibiting MTA1 signaling, we analyzed expression of CyclinD1 and Notch2 in treatment groups by western blot. Although demonstrating noticeable heterogeneity, MTA1 and MTA1-associated CyclinD1 and Notch2 levels were significantly downregulated in the prostates of mice on PTER-Diet compared to mice on Ctrl-Diet (Fig. 5A and B). Taken together, these data demonstrated that dietary supplementation with pterostilbene restored favorable histopathology of PIN in genetically predisposed high-risk premalignant model of prostate cancer with reduced proliferation of glandular epithelial cells, reduced neovascularization, and statistically significant reduction in MTA1 expression, accompanied with downregulation of CyclinD1 and Notch2.

Figure 5.

Diet supplemented with PTER shows high ability in targeting MTA1 and associated CyclinD1 and Notch2. A, representative immunoblots and B, densitometric analysis of MTA1, CyclinD1 and Notch2 levels detected in prostates of 20-week-old R26^MTA1; Pten^+/f mice fed PTER-Diet and Ctrl-Diet in comparison to age-matched Pten^+/f mice fed Ctrl-Diet are shown. β-actin was a loading control. Values are mean ± SEM of protein levels from each group of mice, n = 8 (R26^MTA1; Pten^+/f) and n =3 (Pten^+/f). *, p < 0.05; **, p < 0.01; ***p < 0.001 (one-way ANOVA).

Pterostilbene supplemented diet reduces inflammation and PIN-derived circulating oncomiRs in R26^MTA1; Pten^+/f mice

The association between chronic inflammation and prostate cancer development is well-established (42). In our previous studies, we have shown the link between MTA1 and several pro-inflammatory factors and cytokines in vitro and their involvement in the induction of reactive stroma in the prostate tissues of Pten deficient mice (9, 22, 23). We have reported reduction of serum interleukin-6 (IL-6) levels in prostate cancer xenografts orally treated with resveratrol and analogs (22). Further, we detected a reduction of IL-1β serum levels in mice fed low-fat or high-fat diets supplemented with grape powder (9) and in mice treated with PTER or SAHA either alone or in combination (35). To investigate whether MTA1 overexpression in the prostate promotes inflammation and whether PTER-Diet could reduce inflammation, we examined levels of IL-6 and IL-1β in murine serum by ELISA (Fig. 6A). Although the levels of both the cytokines were significantly increased in the sera of R26^MTA1; Pten^+/f compared to WT mice on Ctrl-Diet, pterostilbene supplementation resulted in downregulation of the levels of both cytokines in sera of R26^MTA1; Pten^+/f mice, an indication that systemic inflammation was decreased in mice fed PTER-Diet compared to mice on Ctrl-Diet.

Figure 6.

Diet supplemented with PTER downregulates the levels of inflammatory cytokines and oncogenic miRNAs. A, analysis of IL-6 (top) and IL-1β (bottom) levels in the sera of 20-week-old R26^MTA1; Pten^+/f mice fed with PTER-Diet (n = 6) and Ctrl-Diet (n = 6) in comparison to age-matched WT mice fed Ctrl-Diet. Data represents the mean ± SEM of three independent experiments performed in triplicates. *, p < 0.05; **, p < 0.01; ***p < 0.001 (two sample, two-tailed t-test). B, qRT-PCR analysis of circulating miR-22 and miR-34a levels in the sera of R26^MTA1; Pten^+/f mice fed with Ctrl- Diet and PTER-Diet Serum was pooled from 6 mice on Ctrl-Diet and 7 mice on PTER-Diet for RNA isolation. Changes in miRNA were calculated by the 2-^ΔΔCt method. Data represent the mean ± SEM of three independent experiments performed in triplicates. *, p < 0.05; **, p < 0.01; ***p < 0.001 (two sample, two-tailed t-test). C, Schematic representation of MTA1-targeted interception by dietary pterostilbene for prostate cancer chemoprevention.

We have previously investigated the potential of miRNAs in responding to natural product treatment and serving as predictive and prognostic biomarkers for cancer progression (20, 43). Specifically, we have previously reported on pterostilbene regulation of miR-17-5p and miR-106a-5p oncomiRs in prostate cancer (20) and on MTA1-associated oncomiRs miR-34a and miR-22 on their regulation by low-fat and high-fat diets supplemented with grape powder (9, 37). Here, we examined the changes of miR-34a and miR-22 in sera of R26^MTA1; Pten^+/f mice fed with PTER-Diet in comparison with mice on Ctrl-Diet. Fig. 6B shows that there was a statistically significant reduction of both miRs in the sera of mice on PTER-Diet compared to their littermates on Ctrl-Diet.

Discussion

During the last decade, there have been extensive discussions in the high-level United Nations and World Health Organization meetings on the Plan for the Prevention and Control of noncommunicable diseases including cancer. Although the majority of cancer research focuses on treatment aspects of already developed cancer, there is continuously accumulating strong scientific evidence for developing effective cancer prevention strategies (44). “High-risk” based prevention may be especially successful for certain cancers such as prostate cancer due to its specific features (i.e. age-related, slow growing) and risk factors (i.e. diet). In addition, the relatively recent concept of interception is particularly relevant for prostate cancer because of its clinically defined active surveillance subpopulation of patients that can be prevented from developing cancer.

Although there are strong genetic similarities between PIN and cancer, which suggests clonal expansion of PIN leading to adenocarcinoma, there is also evidence of multiple foci of PIN, which can arise independently within the same prostate (45). However, current understanding of the molecular basis of PIN heterogeneity is limited, in part due to the fact that a limited amount of tissue needle biopsy is usually available.

Beside the well-known genetic alterations, e.g. the common region of allelic loss on chromosome 10 in prostate cancer that contains the PTEN tumor suppressor gene, epigenetic changes are among the most common molecular alterations in human neoplasia (46). Notably, the majority of cancer research focuses on epigenetic enzymes that regulate DNA methylation and histone modifications since epigenetic changes are reversible upon treatment with pharmacological agents. While the epigenetic enzymes are an obvious choice for targeted therapy, epigenetic readers such as MTA1, which “direct” HDAC1 and HDAC2 toward binding acetylated histones, play an essential role as master co-regulators of genes involved in cell proliferation, invasion and migration. Although such molecules are difficult to target pharmacologically, their inhibitors may have potential chemopreventive and therapeutic uses, as they may be more specific than broadly acting HDAC inhibitors (47).

In the current study, we have investigated the consequences of the combination of dual genetic alterations in the prostate, which leads to cooperative action of high MTA1 expression and PTEN deficiency resulting in establishment of high-risk premalignant phenotype representing one of the set-ups of clinically heterogeneous precancerous cases in prostate cancer. We then sought to target MTA1 pharmacologically for preventing the progression of PIN to a stage that is more aggressive.

In our previous in vivo studies, we identified the critical but indirect role of MTA1 in early stages of prostate cancer associated with MTA1-mediated inflammation and activation of survival pathways in prostate-specific Pten heterozygous and Pten knockout mouse models of prostate cancer (23). Although suggestive, these studies in PTEN-deficient mice that showed elevated levels of MTA1 in prostate tissues compared to normal prostates (23) were not sufficient to conclude whether MTA1 is playing a causal role in prostate neoplasia and progression. To answer this question, we generated transgenic mice expressing MTA1 in prostate epithelial cells (R26^MTA1). Even though the PIN histological features and MTA1-associated molecular changes were detected in the prostate tissues of R26^MTA1 mice as early as 13 weeks of age, the lesions did not progress to invasive adenocarcinoma even in mice monitored for over 1 year. Nevertheless, when combined with Pten-deficiency, R26^MTA1; Pten^+/f mice showed accelerated PIN development with a significant increase in glands that are involved in neoplastic lesions, a substantial histological and proliferative change (Ki67) and more aggressive molecular characteristics such as increase in MTA1, CyclinD1, and Notch2 levels. Curiously, although the histological features of PIN lesions in these mice were characterized by disorganized glandular structures with epithelial cells almost fully occupying lumen and similar to the cribriform patterns, the lesions retained intact basement membranes. Together, this data demonstrate that MTA1 alone may play an important causal role in neoplastic transformation of epithelial cells comparable with events induced by deletion of one Pten allele (Pten^+/f). Combination of these two neoplastic alterations in the mouse prostate exemplifies a unique preclinical model of a specific high-risk premalignant PIN condition representing a precursor to prostate cancer.

Our interceptive strategy using R26^MTA1; Pten^+/f mice fed a diet supplemented with pterostilbene exhibited more favorable histopathology with decreased severity of PIN accompanied by decreased proliferative activity of epithelial cells. Further, a diet supplemented with pterostilbene reduced MTA1 levels and associated angiogenesis in R26^MTA1; Pten^+/f mice. Moreover, a pterostilbene supplemented diet reduced oncogenic CyclinD1 and Notch2 expression, molecules previously identified by us as part of a pterostilbene responsive signature associated with MTA1 (23).

The development of minimally invasive biomarkers for monitoring nutritional interception during human clinical trials are urgently needed. As part of inflammation response, serum cytokines secreted by tissue cells into bloodstream can be measured in serum/plasma as clinical predictive and prognostic biomarkers in prostate cancer patients. Interleukin-6 is increased in prostate cancer tissue at early stages of the disease and is highly elevated in the serum of patients with refractory and metastatic prostate cancer (48). Direct or indirect association between MTA1 and IL-6 have been reported (49, 50). While the classic IL-6 signaling is known to activate the JAK/STAT3 pathway, it can also activate the PI3K/AKT and other pathways (51). The association between MTA1 and STAT3 as well as MTA1 and PI3K/AKT pathways is well established (23, 52). The reduced levels of IL-6 under pterostilbene treatment either direct or partially linked to the inhibitory effects on MTA1 may lead to inactivation of oncogenic STAT3 and PI3K/AKT pathways. Another pro-inflammatory cytokine, IL-1β, drives an inflammatory microenvironment in prostate cancer through various mechanisms (53), one of which is its direct transcriptional regulation by MTA1 (23). Consistent with our previous reports about the effects of stilbenes on cytokine levels in prostate cancer cells and in vivo (9, 22, 23, 35), a diet supplemented with pterostilbene significantly reduced the levels of both IL-6 and IL-1β in serum of R26^MTA1; Pten^+/f mice.

In the last decades, among the potential biomarkers detectable in liquid biopsies, miRNAs are gaining increasing attention, since they are stable, easily detectable, and highly sensitive (54). Specifically, it has been demonstrated that dietary polyphenols, particularly stilbenes, modulate inflammation-related and oncogenic miRNAs (20, 43, 55). For the current study, we chose two miRNAs, namely miR-34a and miR-22, previously identified by our group as MTA1-associated and responsive to resveratrol and pterostilbene (20, 37, 43). We have also shown MTA1 regulated miR-34a/p53, miR-22/p21 and miR-22/E-cad axes in prostate cancer (9, 37). Consistent with our recently published paper on the effects of a diet supplemented with grape powder on the levels of circulating miR-34a and miR-22 in murine serum (9), we found that both miR-34a and miR-22 were responsive to pterostilbene treatment and indicated beneficial effects in reducing levels of these oncogenic miRs in the high-risk premalignant model.

In summary, our results highlight the importance of dietary pterostilbene in preventing further progression of precancerous prostate lesions in a preclinical model that may represent a significant and currently overlooked subpopulation of prostate cancer patients who may benefit from interceptive nutritional approaches for chemoprevention (Fig. 6C). Our data suggest the future clinical trials investigating protective effects of pterostilbene on prostate cancer risk should be conducted in active surveillance subpopulations of patients at increased risk of prostate cancer based on high MTA1 expression detected in prostate biopsy during their annual screening. Certain MTA1-associated cytokines and miRNAs could serve as noninvasive predictive biomarkers and perhaps a blood test with MTA1 detection would be useful as a noninvasive test when available (56).

In conclusion, although the exact molecular mechanisms connecting MTA1 to epigenetic alterations, such as histone hypoacetylation leading to neoplasia, remain to be elucidated (57), the role of MTA1 per se as a prospective interceptive target for pterostilbene is undeniable. Pterostilbene, which is recognized as a potent anticancer agent (58) and exhibits good tolerability and no toxicity in humans when used in a dose up to 250 mg/day (34), should be urgently validated for clinical relevance in blocking prostate cancer progression in active surveillance subpopulation of patients.

Prevention relevance:

Developing novel interceptive strategies for prostate cancer chemoprevention is a paramount goal in clinical oncology. We offer preclinical evidence for the potential of pterostilbene as a promising natural agent for MTA1-targeted interceptive strategy in future cancer prevention trials towards protecting select prostate cancer patients under active surveillance from developing cancer.