Metastasis-associated protein 1-mediated antitumor and anticancer activity of dietary stilbenes for prostate cancer chemoprevention and therapy

Abstract

Dietary bioactive polyphenols that demonstrate beneficial biological functions including antioxidant, anti-inflammatory, and anticancer activity hold immense promise as effective and safe chemopreventive and chemosensitizing natural anticancer agents. The underlying molecular mechanisms of polyphenols’ multiple effects are complex and these molecules are considered promising targets for chemoprevention and therapy. However, the development of novel personalized targeted chemopreventive and therapeutic strategies is essential for successful therapeutic outcomes. In this review, we highlight the potential of metastasis-associated protein 1 (MTA1)-targeted anticancer and antitumor effects of three dietary stilbenes, namely resveratrol, pterostilbene, and gnetin C, for prostate cancer management. MTA1, an epigenetic reader and master transcriptional regulator, plays a key role in all stages of prostate cancer progression and metastasis. Stilbenes inhibit MTA1 expression, disrupt the MTA1/histone deacetylase complex, modulate MTA1-associated Epi-miRNAs and reduce MTA1-dependent inflammation, cell survival, and metastasis in prostate cancer in vitro and in vivo. Overall, the MTA1-targeted strategies involving dietary stilbenes may be valuable for effective chemoprevention in selected subpopulations of early stage prostate cancer patients and for combinatorial strategies with conventional chemotherapeutic drugs against advanced metastatic prostate cancer.

1. Introduction

Natural products offer promising options to reduce the progress of cancer. In fact, most chemotherapeutics currently in use are derived from natural sources, which continue to provide key scaffolds for drug development [1]. Natural products mainly consist of bioactive polyphenols present in vegetables, fruits, or seeds. Polyphenols’s chemical composition varies from simple to highly polymerized structures containing multiple phenol rings [2, 3]. Different classes of polyphenols include flavonoids, phenolic acids, lignans, and stilbenes. Polyphenols are known to exert antioxidant, anti-inflammatory, and anticancer effects in in vitro and in vivo experimental settings through pleotropic mechanisms affecting various signaling pathways and cell functions [4, 5]. The potential mechanisms of action include phenol groups’ nonspecific scavenging abilities as well as specific interactions based on the particular structural and conformational properties of select polyphenols and the biological target. Importantly, there is epidemiological evidence for an association between polyphenol intake and low cancer risk [6].

Prostate cancer is the second most commonly diagnosed cancer and the second leading cause of cancer death among men in developed countries [7, 8]. The occurrence of prostate cancer is increasing because of population aging, the primary risk factor for slow- growing prostate cancer. Prostate cancer is unique in that if a man lives long enough he could develop some form of the disease that may never progress. On the other hand, due to other risk factors that include family history, ethnicity and Western diet [9, 10], men may develop aggressive prostate cancer that requires treatment. Treating patients who have developed high-grade localized prostate cancer can have a survival benefit but usually is associated with toxicity and significant adverse effects. Understandably, an interest in cost-effective and nontoxic nutritional chemopreventive measures is rising with the confidence that a substantial portion of prostate cancer cases could be prevented by applying effective “prostate cancer-specific diets” that contain bioactive dietary polyphenols and micronutrients [11, 12]. Indeed, epidemiological data and certain case-control studies have found that high consumption of vegetables, fruits, or soy foods was associated with reduced prostate cancer risk [13–18]. Dietary strategies targeting various mechanisms that are associated with prostate cancer progression also have been suggested [19–21].

A large number of cellular and molecular targets have been identified that could be involved in the cytotoxic and anticancer actions of dietary polyphenols. A combination of phytochemicals rather than any single polyphenol is likely responsible for any health benefits. Even so, it is vital to identify specific molecular targets of a particular class of polyphenols for a given type of cancer to develop targeted precision chemopreventive and therapeutics strategies. The objective of the current review is to present evidence about the role and potential mechanism of the action of dietary stilbenes in the inhibition of epigenetic modifier metastasis-associated protein 1 (MTA1) and MTA1-associated cell signaling, tumor progression, and metastasis in prostate cancer. Identifying the oncogenic MTA1 as a molecular target for stilbenes offers a novel advantage of stilbene-rich diets for chemoprevention as well as for effective combined strategy with approved drugs against advanced metastatic prostate cancer.

2. Stilbenes: Resveratrol, Pterostilbene, and Gnetin C

Stilbenes are known to possess a wide range of biological activities including antioxidative, anti-inflammatory, antifungal, antibacterial, antiviral, and antitumor activities. Readers who are interested in a detailed discussion of these effects are directed to several excellent articles and book chapters that cover those topics [3, 22–31]. Stilbenes have a common C6-C2-C6 structure, consisting of two aromatic rings linked through a two-carbon bridge with a double bond. Stilbenes are produced by certain plants in response to different environmental stressors such as UV light or infections [32–34]. The parent compound of this family is resveratrol, which occurs as a monomer and as oligomers [23, 34, 35]. Other stilbenes such as pterostilbene, piceatannol, or trimethoxy-resveratrol are structurally related to resveratrol with the same backbone but differing in the type, number, and position of substituents [36].

Major dietary sources of stilbenes include grapes, wine, blueberries, peanuts, Itadori tea common in China and Japan, and edible melinjo plant that is used in Southeast Asian and western Pacific Islands’s cuisines [23, 33, 37].

We herein focus on the targeted antitumor and anticancer activities of three stilbenes: resveratrol, pterostilbene, and gnetin C. Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is one of the best characterized stilbenes, found mostly in grapes, wine, and peanuts. Pterostilbene (3,5-dimethoxy-4′-hydroxy-trans-stilbene) is a natural methoxylated analog of resveratrol, found mostly in blueberries and grapes. Gnetin C is a dimer-resveratrol (5-{(2S,3S)-4-hydroxy-2-(4-hydroxyphenyl)-6-[(E)-2-(4-hydroxyphenyl)vinyl]-2,3-dihydro-1-benzofuran-3-yl}−1,3-benzenediol) , found in the melinjo plant Gnetum gnemon (Fig. 1).

Figure 1.

Chemical structures of resveratrol, pterostilbene, and gnetin C.

3. MTA1, MTA1/nucleosome remodeling and deacetylase (NuRD) complex, and cancer

MTA1 belongs to the MTA family of cancer progression-related proteins that are part of the NuRD co-repressor complex, which is involved in chromatin remodeling via histone deacetylation and gene-specific transcriptional regulation [38–40]. The MTA family includes three major proteins (MTA1, MTA2, and MTA3) and several isoforms, which are engaged in numerous functions of NuRD complex. They interact with a variety of chromatin remodeling factors and enzymes leading to nucleosome remodeling and transcriptional repression or activation of target genes [41, 42]. MTA1 itself does not have intrinsic enzymatic activity and has not been shown to directly interact with DNA [40, 43, 44]. In cancer, MTA1 can interact with histone deacetylase (HDAC 1/2) complex but may also perform histone-independent functions including deacetylation of non-histone proteins [41, 45–48]. In addition, MTA1 regulates the expression of target genes through NuRD-independent mechanisms [40].

MTA1’s structural organization includes six functional modular domains by which the functions of MTA1 were extrapolated (Fig. 2). The functional significance of the BAH, ELM, and SANT domains were hypothesized from studies of proteins containing similar homologous domains [49–51]. Although experimental evidence in support of direct MTA1’s DNA-binding activity is lacking, a GATA-like zing-finger domain commonly found in the GATA transcription factors [52, 53] suggests that MTA1 may be a bona fide transcription factor. MTA1 also contains one monopartite and two bipartite nuclear localization signals (NLSs) in addition to a proline-rich Src-homology domain, which is essential for interacting with other proteins [43, 54].

Figure 2.

Schematic representation of the MTA1 protein structure and correlated putative functions. BAH, bromo-adjacent homology; ELM, egl-27 and MTA1 homology; SANT, SWI, ADA2, N-CoR, TFIIIB-B (shares homology with DNA-binding domain of Myb-related proteins); ZnF, zing finger motif (GATA-type); NLS, nuclear localization signals; BP1 and BP2, bipartite NLS; SH, src-homology binding motifs (proline-rich). aa: amino acids

MTA1 is highly expressed in various cancers and both nuclear and cytoplasmic accumulation of MTA1 has been reported [47, 55–57]. In human prostate cancer, immunohistochemical staining has documented predominantly nuclear staining associated with higher Gleason scores and metastasis [42, 58, 59], which explains MTA1’s epigenomic and genomic functions. On the other hand, MTA1 localization in the cytoplasm suggests possible nongenomic functions for MTA1 in prostate cancer (yet to be discovered), analogous to those reported in breast cancer that are a hyperstimulation of extranuclear activities of the estrogen receptor α (ERα) [60, 61]. MTA1 has been also detected in normal prostates, bladders, and brains isolated from wild-type (wt) mice with the highest MTA1 expression in testes [41, 42]. In prostate-specific Pten (phosphatase and tensin homolog) - loss mouse models, MTA1 was detected in both prostate epithelial glands and reactive stroma compartments, indicating its role in inflammation-driven processes [62]. The role of MTA1 in normal prostates remains unknown.

4. MTA1’s clinical significance and functional role in prostate cancer progression and metastasis

The clinical significance of MTA1 in human prostate cancer has been revealed [58, 59, 63, 64]. MTA1 is expressed in early stage prostate cancer but its overexpression is closely associated with the clinico-pathological characteristics of prostate cancer progression: high Gleason scores, development of castrate resistance, recurrence, and metastasis. Amplification of Mta1 gene was detected in neuroendocrine prostate cancer [65, 66]. MTA1 plays a special role in prostate cancer bone metastasis [58, 59, 67]. We found a strong association between high MTA1 levels and disease recurrence and metastasis, particularly in African American patients, which suggested to us MTA1’s possible race-specific driver role of aggressive prostate cancer progression and metastasis [59]. These observations warrant further studies for a better understanding of the specific role of MTA1 in each stage of prostate cancer and its potential value as a prognostic and predictive biomarker as well as chemopreventive and/or therapeutic target for prostate cancer progression and metastasis.

Almost a decade ago, my group identified MTA1 as a part of the “bone metastatic signature” in an experimental setting where we incubated various prostate cancer cells, namely LNCaP, C4–2, DU145, PC3, and PCa2b lines with bone chunks isolated from rats’ tibias [58]. Analysis of differential gene expression profiling revealed MTA1 as a novel putative component of the bone metastatic “vicious cycle”, which suggested MTA1’s explicit role in prostate cancer bone-specific metastasis. Although we proceeded to detect striking nuclear overexpression of MTA1 in bone lesions of human metastatic prostate cancer samples [58, 59], only recently were we able to demonstrate the functional role of MTA1 in the formation of bone metastasis using an experimental bone metastatic model in prostate cancer xenografts [67]. We found that MTA1 silencing (shMTA1) in PC3M prostate cancer cells impaired tumor growth and reduced colonization and development of bone metastasis in subcutaneous and intracardiac prostate cancer xenografts, respectively. Moreover, we demonstrated the critical role of MTA1 as an upstream regulator of Cathepsin B, a cysteine protease actively involved in prostate cancer bone metastasis [67–69]. MTA1’s role is not exclusive to bone metastasis since we detected overexpression of MTA1 in other metastatic lesions obtained from prostate cancer patients [59]. This was expected, since the active involvement of MTA1 in two major biological processes such as epithelial-to-mesenchymal transition (EMT) and angiogenesis, which are characteristics of prostate cancer progression and metastasis, is well documented. The role of MTA1 as an inducer and promoter of EMT pathways and metastatic progression has been documented in many cancers [42, 70–73]. In prostate cancer, mechanistic studies with loss-of-function and overexpression of MTA1 have demonstrated MTA1’s involvement in the malignant phenotype of cells via pAkt/E-cadherin pathway [74]. E-cadherin, in turn, regulates MTA1-dependent tumor cell adhesion, mobility, invasion and cellular polarity [75]. We detected an inverse correlation between MTA1 and E-cadherin in prostate tissues of prostate-specific Pten knockout mice (Pb-Cre4; Pten^f/f), in which increased levels of MTA1 were concomitant with decreased levels of E-cadherin and increased levels of vimentin [62]. We also have reported modulation of EMT markers upon MTA1 knockdown in LNCaP and DU145 prostate cancer cells [62]. Further, MTA1 silencing led to a significant increase in the E-cadherin mRNA and protein levels in PC3M cells and PC3M-xenograft tumor tissues [67]. One additional mechanism, by which MTA1 regulates E-cadherin, involves activation of MTA1-associated microRNAs [76] (see section 6.3).

The pro-angiogenic character of MTA1 has been well documented in various cancers [47, 48, 77–81]. One early observation about the link between MTA1 and angiogenesis was that the hypoxia inducible factor 1α (HIF-1α) is regulated by a MTA1/HDAC1 complex, which stabilizes and transcriptionally activates HIF-1α [48]. Reports demonstrated that MTA1 is overexpressed under hypoxic and in certain treatment conditions, physically interacts with HIF-1α causing its deacetylation and stabilization [47, 48, 81]. Moreover, there is a positive correlation between high MTA1 levels and transcriptional activation of HIF-1α leading to enhanced expression of vascular endothelial growth factor (VEGF), a target molecule of HIF-1α [47, 80]. In addition to HIF-1α and VEGF, there are other angiogenic factors such as angiopoietin1/2, cyclooxygenase 2 (COX-2), matrix metalloproteinases (MMPs), transforming growth factor β (TGFβ), and interleukins (IL6, IL8, and IL-1β) that are involved in prostate cancer angiogenesis [82]. We have shown the contribution of MTA1 in tumor neovascularization and in HIF-1α-, VEGF-, IL-1β-induced angiogenesis in prostate cancer in vitro and in vivo. In cultured MTA1 knockdown PC3/PC3M prostate cancer cells, VEGF levels in conditioned media and HIF1-α mRNA and protein levels in cell extracts were significantly decreased [58, 83]. Moreover, MTA1 silencing considerably reduced the ability of PC3 cells to induce endothelial cell chemotaxis in migration assay [58]. Furthermore, LNCaP and DU145 MTA1 knockdown cells showed decreased levels of IL-1β, a known pro-angiogenic factor that potentiates VEGF expression [62, 82]. All three of these angiogenic factors, i.e. HIF-1α, VEGF-C, and IL-1β, are directly regulated by MTA1, according to our differential MTA1 chromatin IP based deep sequencing (ChIP-Seq) data, in which tracks for each gene loci showed decreased MTA1 occupancy of respective promoters in MTA1 reduced prostate tissues [62, 83]. Most importantly, we demonstrated the link between MTA1 and tumor angiogenesis in vivo [58, 62, 83, 84]. Subcutaneous tumors from LNCaP and DU145 MTA1 knockdown xenografts showed decreased VEGF staining and a reduction in microvessel density (MVD) assessed by CD31 staining of blood vessel’s endothelial cells, compared to control xenografts expressing high levels of MTA1 [58]. In DU145 MTA1 knockdown orthotopic xenografts, tumors showed significant reduction in CD31-positive areas accompanied by lesser metastasis and in fewer organs [84]. In contrast, mice prostate tissues from autochthonous prostate cancer models, namely prostate-specific Pten heterozygous (Pb-Cre⁺; Pten^+/f;) and Pten knockout (Pb-Cre⁺; Pten ^f/f;) mice, which express high levels of MTA1, exhibit higher levels of pro-angiogenic factors TGFβ, HSP90, and IL-1β [62] and high levels of circulating VEGF-C and IL-1β detected in mouse sera [83]. Likewise, prostate tissues from prostate-specific MTA1 transgenic mice, MTA1-tg, (Pb-Cre⁺; Rosa26^+/MTA1 and Pb-Cre⁺; Rosa26^MTA/MTA1), which express high levels of MTA1 showed significantly higher levels of HIF-1α at both mRNA and protein levels compared to normal prostates [83]. Collectively, this data demonstrate MTA1’s active involvement in prostate cancer angiogenesis through the regulation of pro-angiogenic factors.

While current evidence in the literature links MTA1 to processes associated with cancer metastasis, our studies suggest that MTA1 is involved in almost all stages of prostate cancer progression [42]. Prostate cancer is associated with inflammation, which is characteristic of an accepted precursor to prostate cancer, prostatic intraepithelial neoplasia (PIN) [85]. Levels of MTA1 were significantly higher in the prostate tissues from prostate-specific Pten heterozygous mice with high grade PIN characterized by the presence of reactive stroma compared to normal prostate, suggesting that MTA1 may be involved in the early inflammation stage of prostate cancer. In cancer-prone prostate-specific Pten knockout model, MTA1 expression was dramatically higher and was associated with the activation and promotion of oncogenic and survival pathways [62]. Mechanistically, it has been shown that MTA1 either regulates or is regulated by several key molecules that have been implicated in the induction and activation of reactive stroma leading to inflammation and tumor promotion. The link between MTA1 and NF-κB, TGFβ, and c-Myc has been demonstrated in various cancers [86–90]. In the prostate-specific Pten loss models of prostate cancer, high levels of MTA1 were accompanied with increased levels of AR, TGFβ, NF-κB (p65), IL-1β, HSP90, Akt/pAkt, Cyclin D1, Notch2, and ETS2 [62]. We also detected a reduction in the levels of Cyclin D1, c-Myc, pAkt/Akt, ETS2, Notch2, IL-1β, and HSP90 in MTA1 knockdown prostate cancer cell lines at both mRNA and protein levels confirming transcriptional regulation of these genes by MTA1 (MTA1 ChIP-Seq analysis) [62]. Therefore, MTA1, which is expressed in both prostate tumor and reactive stroma, has pro-inflammatory, pro-survival, pro-angiogenic and metastatic roles in Pten loss-driven prostate tumor growth and progression. Of a note, our MTA1-tg mice developed high grade PIN detected at 14 weeks of age but failed to develop invasive adenocarcinoma at up to 1 year of age, suggesting that MTA1 alone is insufficient to cause prostate cancer (unpublished data, Levenson). Nevertheless, the MTA1-targeted strategies may prove successful as chemopreventive and therapeutic options for early stage and metastatic prostate cancer, respectively.

5. MTA1-targeted chemoprevention and therapeutic potential of dietary stilbenes in prostate cancer

5.1. Targeted therapy in prostate cancer

Although the link between diet and cancer is complex, epidemiological data confirm diet as a risk factor for prostate cancer [91] and indicate a reduced prostate cancer risk associated with red wine consumption, attributable to high resveratrol content [92]. The fact that prostate cancer is an age-related slow-growing disease linked with diet as a risk factor makes prostate cancer the most appropriate for chemoprevention by dietary bioactive polyphenols with anti-inflammatory, antioxidant and anticancer properties. Primary prostate cancer chemoprevention would be particularly valuable for the subclass of patients for whom we usually recommend active surveillance or watchful waiting. These patients are not eligible for surgery or any other treatments due to their early stage, which might or might not progress to prostate cancer. Unfortunately, almost half of these patients develop prostate cancer, posing an urgent demand for developing effective chemoprevention tactics such as a “chemopreventive” diet. Now it became clear that it is imperative to customize chemopreventive strategies by defining the particular subset of patients (active surveillance) likely to respond to certain dietary compounds (dietary stilbenes) via specific signaling pathways (MTA1-signaling). In addition, various combination strategies with dietary stilbenes and chemotherapeutic drugs may show effective therapeutic effects by targeting MTA1 through the same or different signaling pathways. Thus, combinatorial approaches with other MTA1 pharmacological inhibitors should be considered for possible synergistic and minimizing adverse side effects in targeted patient populations with advanced prostate cancer. Table 1 summarizes MTA1-mediated anticancer effects of stilbenes and combinations with synthetic agents in prostate cancer.

Table 1.

Stilbenes’ MTA1-mediated anticancer effects in prostate cancer

Stilbene	Cell Type	Mouse Model / Route	Effects	Signaling / Markers	Reference #
Resveratrol	LNCaP DU145		Apoptosis⭡	MTA1⭣; HDAC1⭣ p53 activation via acetylation⭡ p21, Bax promoter activation Bax⭡; BCL-2⭣ p53 acetylation⭡⭡	[46]
Resveratrol + SAHA	LNCaP DU145		Apoptosis⭡⭡
No treatment	PC3 PC3-siMTA1		Invasion⭣ Angiogenesis⭣	VEGF⭣	[58]
		LNCaP-siMTA1 DU145-siMTA1 s.c xenografts / i.p.	Tumor volume⭣ Tumor weight⭣	Ki67⭣; CD31⭣
Resveratrol Trimethoxy-resveratrol Piceatannol Pterostilbene	LNCaP DU145 PC3M		Cell viability⭣ Colony formation⭣		[36]
		LNCaP-Luc s.c. xenografts / oral gavage	Tumor volume⭣	Serum IL-6⭣
Resveratrol Trimethoxy-resveratrol Piceatannol Pterostilbene	LNCaP DU145 PC3M			MTA1⭣ MTA1⭣⭣	[84]
Pterostilbene	DU145-EV DU145-shMTA1	DU145-Luc orthotopic xenografts / i.p.	Tumor volume⭣ Apoptosis⭡ Angiogenesis⭣ Metastasis⭣	MTA1-mediated p53 acetylation⭡ Ki67⭣; M30⭡; CD31⭣
Resveratrol Pterostilbene	DU145 22Rv1 DU145-EV DU145-shMTA1			MTA1/HDAC ½⭣ PTEN acetylation⭡ pAkt/Akt⭣	[45]
Resveratrol	DU145-EV DU145-shMTA1	DU145- Luc Orthotopic xenografts / i.p.	Tumor volume⭣ Apoptosis⭡	MTA1⭣; PTEN⭡; pAkt⭣ Ki67⭣
No treatment	LNCaP-EV LNCaP-shMTA1 DU145-EV DU145-shMTA1			MTA1-mediated NF-κB⭣; IL-1β⭣ Hsp90⭣; E-cad⭡; Vimentin⭣ c-Myc⭣; CyclinD1⭣; Notch2⭣ ETS2⭣; pAkt/Akt⭣; p21⭡; p27⭡	[62]
Pterostilbene		Pb-Cre+; Pten+/f / diet	PIN formation⭣ Proliferation⭣ Apoptosis⭡	MTA1⭣; PTEN⭡; SMA⭡; CK8⭡ pAkt/Akt⭣; c-Myc⭣; Cyclin D1⭣ Notch2⭣; TGFβ⭣; ETS2⭣; Hsp90⭣; AR⭣; p21⭡; p27⭡ Ki67 ⭣; cl caspase 3⭡
		Pb-Cre+; Ptenf/f / i.p.	Adenocarcinoma⭣ Proliferation⭣ Apoptosis⭡ Angiogenesis⭣	MTA1⭣; pAkt/Akt⭣; AR⭣ E-cad⭡; IL-1β⭣; Hsp90⭣ Ki67⭣; CD31⭣; cl caspase 3⭡ Ac p53⭡; Bak⭡; VEGFc⭣
Pterostilbene Pterostilbene + SAHA	LNCaP PC3M PC3M-NS PC3M-shMTA1		Cell viability⭡	MTA1-mediated HIF1α⭣ VEGFc⭣; IL-1β⭣	[83]
		Pb-Cre+; Ptenf/f; Rosa26Luc/+ / i.p.	Hg PIN formation⭣ Apoptosis⭡ Angiogenesis⭣	MTA1/HIF1α⭣; p27⭡ SMA⭡; CK8⭡; Ki67⭣ Cl caspase 3⭡; CD31⭣ Serum VEGFc⭣ Serum IL-1β⭣
		Pb-Cre+; Rosa26MTA1/+		MTA1/HIF1α⭡
Resveratrol Pterostilbene Gnetin C	DU145 PC3M DU145-NS DU145-shMTA1 PC3M-NS PC3M-shMTA1		Cell viability⭣ Colony formation⭣ Migration⭣	MTA1/ETS2⭣	[105]
No treatment	PC3M-NS PC3M-shMTA1		Proliferation⭣ Colony formation⭣ Migration⭣ Invasion⭣	MTA1/CTSB⭣; E-cad⭡	[67]
		PC3M-shMTA1-Luc s.c. xenografts / i.p. PC3M-shMTA1-Luc intracardiac xenografts	Tumor growth⭣ Bone metastasis⭣	Ki67⭣ MTA1/CTSB⭣; E-cad⭡
		Pb-Cre+; Rosa26MTA1/+		MTA1/CTSB⭡
No treatment	LNCaP-EV LNCaP-shMTA1		MTA1-regulated Epi-miRNAs	MTA1⭣; Epi-miR-22⭣ E-cad⭡	[76]
		Pb-Cre+; Rosa26MTA1/+ Pb-Cre+; Rosa26MTA1/MTA1		MTA1⭡; Epi-miR-22⭡ E-cad⭣
Resveratrol	LNCaP		OncomiRs⭣ TS miRs⭡	miR-17~92⭣; miR-106a⭣ miR-106b⭣	[126]
Resveratrol Pterostilbene	LNCaP DU145			PTEN⭡ miR-17–5p⭣; miR-20a⭣ miR-106a⭣; miR-106b⭣	[127]
Pterostilbene	DU145-EV DU145-miR-17/106a	DU145-miR- Luc s.c.xenografts / i.p.	Tumor growth⭣ Apoptosis⭡	PTEN⭡ Ki67⭣; M30⭡; cl caspase 3⭡ Tumor & serum miR-17–5p⭣ Tumor & serum miR-106a-5p⭣

⭡, upregulation;

⭣, downregulation

5.2. Resveratrol and prostate cancer

The anticancer effects of resveratrol acting through various signaling pathways in prostate cancer have been reported in numerous publications [93–100]. Our first report on resveratrol’s inhibition of MTA1/NuRD complex, which restored p53 acetylation/activation causing apoptosis in prostate cancer cells, opened the door for comprehensive studies of MTA1-mediated epigenetic and genetic molecular mechanisms of stilbene compounds’ action in vitro and in vivo [46]. We found that by disrupting the MTA1/HDAC1 unit and deregulating its deacetylation function, resveratrol reverses p53 acetylation, leading to the activation of pro-apoptotic Bax and p21, and ultimately causing apoptosis in DU145 (mut p53) and LNCaP (wt p53) prostate cancer cells. Interestingly, the clinically approved HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), alone did not have any effect on p53 acetylation but amplified resveratrol-induced increase of the acetylated p53 in both cell lines. Combined treatment with resveratrol and SAHA significantly increased apoptosis [46]. These results suggested the epigenetic reader MTA1 as a novel molecular target of resveratrol and the combination of resveratrol and HDAC inhibitor(s) as a possible effective therapeutic approach against prostate cancer. Due to resveratrol’s known low bioavailability, we began a search for analog(s) with better pharmacokinetic properties and potent biological effects. We compared the ability of six different analogs of resveratrol to inhibit prostate cancer cell proliferation, colony formation, and tumor growth in subcutaneous (s.c.) xenografts and found that some analogs, including pterostilbene, are more potent in inhibiting prostate cancer cells compared to resveratrol [36].

5.3. Pterostilbene and prostate cancer

In contrast to resveratrol, publications on the effects of pterostilbene in prostate cancer are limited [101–104]. We found that pterostilbene was the most potent inhibitor of MTA1 in cultured prostate cancer cells and showed MTA1-dependent tumor regression and inhibition of metastasis in orthotopic DU145 xenografts through increased p53 acetylation, higher apoptotic index, and decreased angiogenesis [84]. Resveratrol and pterostilbene also rescued PTEN acetylation through inhibition of MTA1/HDAC 1/2, which leads to lower pAkt levels and inhibition of the Akt survival-signaling pathway [45]. The relevant in vivo efficacy of pterostilbene as a MTA1-targeted chemopreventive and intervention strategy has been demonstrated using prostate-specific Pten loss immunocompetent pre-clinical mouse models [62]. Pten loss driven prostate cancer progression in prostate-specific Pten heterozygous and Pten-null mice bear a close resemblance to the human disease with an intact immune system. Primary chemopreventive design included diet supplementation with 100 mg/kg diet pterostilbene in Pten heterozygous mice that develop PIN at 7–8 months of age. The intervention strategy involved Pten-null mice that mimic stage-defined progression of human prostate cancer, developing characteristics of invasive adenocarcinoma by 15–20 weeks of age. Because Pten loss resulted in a marked increase in MTA1 expression leading to activation of MTA1-dependent survival and metastasis-associated signaling pathways, we found that pterostilbene, both as a dietary supplement and daily injection, caused MTA1-dependent inhibition of inflammation (NF-κB, HSP90, and IL-1β), tumor growth (pAkt/Akt, AR, Cyclin D1, ETS2, TGFβ, c-Myc, and NOTCH2), EMT (E-cadherin, and vimentin), and angiogenesis (VEGF, CD31, and IL-1β). Pterostilbene induced marked apoptosis (Acp53, p21, p27, cleaved caspase 3), resulting in reduction of PIN lesions in chemopreventive experiments and adenocarcinomas in cancer-prone Pten-null mice [62]. We also provided preclinical evidence for the benefits of MTA1-targeted combinatorial pterostilbene with SAHA strategy. Data showed that pterostilbene sensitized tumor cells to SAHA treatment causing additional decline of tumor progression in Pten-null mice. The effects were dependent on the reduction of MTA1/HIF-1α signaling [83].

5.4. Gnetin C and prostate cancer

So far, there are only two publications available for the effects of gnetin C in prostate cancer cells including our own study on MTA1/ETS2-mediated effects of gnetin C [105, 106]. We have recently revealed that gnetin C exhibited potent MTA1 inhibition in prostate cancer cells compared to resveratrol and pterostilbene. In addition, we reported for the first time that gnetin C demonstrated significant MTA1-mediated induction of apoptosis in prostate cancer cells while inhibiting cell viability, colony formation, and migration with more efficacy than resveratrol or pterostilbene. Notably, gnetin C also inhibited oncogenic ETS2 in prostate cancer cells via MTA1-dependent and independent mechanisms [105]. As expected, gnetin C showed potent inhibition of subcutaneous prostate cancer xenograft tumors (unpublished data, Levenson).

6. Main MTA1-mediated mechanisms of stilbenes’ action in prostate cancer

6.1. Stilbenes and MTA1/HDAC complex-associated epigenetic mechanisms

The MTA1-mediated molecular mechanisms that contribute to the chemopreventive and therapeutic capacities of natural stilbenes are many-sided including epigenetic effects associated with the MTA1/HDAC1/2 inhibition leading to posttranslational modifications of tumor suppressors and microRNAs (miRNAs, miRs) modulation [107] (Fig. 3). As an epigenetic reader, MTA1 in complexes with HDAC1 and HDAC2 participates in DNA sliding of NuRD and promotes the deacetylation of histones and certain non-histone proteins [38, 39, 46, 108]. As a “master co-regulator”, MTA1 plays a role in recruiting the protein complexes to the promoters of specific genes for transcriptional activation or silencing [42, 43, 109–111]. As we already mentioned, stilbenes exhibit epigenetic actions through disruption of the NuRD co-repressor complex (Fig. 3A). We have shown that both resveratrol and pterostilbene promote acetylation and reactivation of tumor suppressors p53 and PTEN through downregulation of the MTA1/HDACs units of the NuRD complex, which lead to inhibition of survival pathways and induction of apoptosis in prostate cancer cells [45, 46]. Another notable target of the MTA1 and HDAC1 is HIF-1α, a key transcriptional regulator of pro-angiogenic VEGF. It has been shown that due to deacetylation by MTA1/HDAC complex, HIF-1α becomes stabilized and transcriptionally active leading to tumor angiogenesis and MTA1-associated metastasis [47, 48]. We too accumulated data on the tight link between high levels of MTA1 and angiogenesis and demonstrated the MTA1-mediated inhibitory effects of stilbenes on HIF-1α and pro-angiogenic factors VEGF and IL-1β in prostate cancer [58, 62, 83, 84]. Further, studies of combinatorial resveratrol /SAHA or pterostilbene/SAHA approach demonstrated benefits of chemosensitization of cancer cells, associated with more efficacy of treatment [46, 83].

Figure 3.

Main MTA1-mediated mechanisms of stilbenes’ action in prostate cancer. (A) Through the epigenomic pathway, stilbenes downregulate MTA1, HDAC1, and HDAC2 disrupting NuRD corepressor complex deacetylation function, which leads to changes in chromatin conformation. Histone acetylation defines the “open” chromatin landscape, which is characterized by transcriptionally active DNA sequences that serve as docking sites for transcription factors and other effector proteins. Non-histone tumor suppressor proteins p53 and PTEN become acetylated and activated, which leads to inhibition of survival pathways and induction of apoptosis. In contrast, acetylation of HIF-1α causes its destabilization and inactivation resulting in decreased angiogenesis. (B) Through the genomic pathway, stilbenes reverse MTA1’s transcriptional co-repressor or/and co-activator functions. p21, E-cadherin are upregulated by stilbenes in prostate cancer whereas the inhibition of MTA1/ETS2, CyclinD1, and Notch2 is also recorded. (C) Left, Stilbenes inhibit oncogenic miRNAs in prostate cancer: targeting oncomiRs of the miR-17 family restored PTEN expression. Right, MTA1-activated Epi-MiR-22 regulates E-cadherin leading to promotion of EMT and prostate cancer progression.

6.2. Stilbenes and MTA1-associated genomic pathways

A transcriptional link between MTA1 and specific genes such as NF-κB, TGFβ, SMAD7, c-Myc, and PTEN has been shown [89, 90, 112–114]. To identify new transcriptional targets of MTA1 and changes in genetic landscape associated with response to pterostilbene, we performed genome-wide analysis by ChIP-Seq using prostate tissue chromatin from prostate-specific Pten loss mice treated or not with pterostilbene (Fig. 3B) [62, 67]. Genes of potential significance were identified by mapping MTA1-binding peaks to RefSeq-annotated transcripts. Along with MTA1 associated genes already reported by others [115–117], we prioritized novel candidate MTA1 transcriptional targets in prostate cancer, namely Cathepsin B, E-cadherin, HIF-1α, Cyclin D1, and ETS2 and validated their strong direct or inverse correlation with MTA1 and responsiveness to stilbene treatment in vitro and in vivo [62, 67, 83, 105]. Mechanistic studies such as ChIP and ChIP-re-ChIP experiments will further validate MTA1’s direct involvement in transcriptional regulation of these genes.

6.3. Stilbene- regulated and MTA1- regulated miRNAs

MicroRNAs (miRNAs, miRs) are small non-coding RNAs that play a role in gene regulation [118, 119] and act as oncogenes or tumor suppressors [120]. Aberrant miRNA expression is implicated in prostate cancer and correlates with high Gleason score and clinical recurrence [121–123]. Importantly, miRNAs have remarkable stability in serum/plasma, which makes them outstanding “liquid biopsy” biomarkers for cancer diagnosis, prognosis, and therapy response [124, 125]. The first report on resveratrol-regulated miRNAs in prostate cancer using miRNA microarray analysis of LNCaP cells showed significant inhibition of oncogenic miRs and upregulation of tumor suppressor miRs [126]. We further demonstrated that resveratrol and pterostilbene restore PTEN expression in prostate cancer cells by inhibiting miR-17~92, miR-106a~363, and miR-106b~25 clusters, which are members of the miR-17 oncogenic family (Fig. 3C) [127]. Moreover, pterostilbene through downregulation of these miRs in murine tumors and systemic circulation rescued PTEN mRNA and protein levels, leading to reduced tumor growth in xenografts [127]. Accumulating evidence suggests dietary stilbenes as attractive miRNA-mediated chemopreventive and therapeutic agents, and circulating miRNAs as potential chemopreventive and predictive biomarkers for clinical development in cancer [25, 128]

Of particular interest in understanding the MTA1-associated epigenetic network is the area of regulatory miRs that are associated with MTA1. MiRNAs that are regulated by or are regulating epigenetic factors are called “Epi-miRs” [120, 129]. Specifically, HDAC and HAT inhibitors regulate mature miRNAs [130, 131]. We identified Epi-miRNAs regulated by MTA1 using miRNA microarrays differential profiling of LNCaP MTA1 knockdown cells [76]. Among Epi-miRNAs induced by MTA1, we identified miR-22 and demonstrated its direct targeting of the 3′-untranslated region (3ˊUTR) of E-cadherin, mechanistically presenting the MTA1/Epi-miR-22/E-cadherin axis as a novel epigenetic signaling pathway that promotes tumor invasion in prostate cancer (Fig. 3C, right). MTA1-induced overexpression of miR-22 diminished E-cadherin expression resulting in increased cell invasiveness and migration in prostate cancer. Further, we also found positive correlation between MTA1 and miR-22 expression and their inverse correlation with E-cadherin in human prostate cancer samples [76]. The regulation of MTA1/miR-22/E-cadherin axis by stilbenes warrants further investigation to validate Epi-miR-22 as a predictive biomarker and potential therapeutic target in advanced prostate cancer. In addition, miRNAs that target MTA1 have been identified as well [132–134] and it would be interesting to test the responsiveness of those miRNAs to stilbenes.

In summary, we suggest MTA1 and MTA1-associated signaling pathways targeting stilbenes as a novel epigenetic personalized chemopreventive and therapeutic strategy to be used either alone or in combination with Food and Drug Administration-approved anticancer drugs for prostate cancer management.

7. Conclusions and perspectives

This review highlighted the potential of dietary stilbenes, namely resveratrol, pterostilbene and dimer-resveratrol gnetin C, in the context of MTA1-targeted chemoprevention and therapy in prostate cancer. Although all three stilbenes target MTA1, they do it with different potencies with gnetin C being the most potent. Studies have reported pterostilbene’s and gnetin C’s superior pharmacokinetics compared to resveratrol [135–140], which may explain the observed more potent biological activity of pterostilbene and gnetin C over resveratrol [25, 105]. Notably, we detected accrual of pterostilbene in the murine prostate tissues in Pten loss mice treated with pterostilbene indicating that pterostilbene reaches the target organ [62].

The precise mechanism by which stilbenes regulate MTA1 expression is not completely elucidated and more research should focus on understanding the complex mechanisms of stilbene interaction with MTA1. Nevertheless, current findings on potent inhibition of MTA1 justifies its exploitation as a chemopreventive and therapeutic target in prostate cancer.

Clinical studies in humans have been conducted with all three stilbenes showing their safety [138, 139, 141, 142], however, no human clinical trial has been conducted to evaluate the effects of resveratrol or pterostilbene or gnetin C specifically in prostate cancer. At the time this review was submitted, there was only one somewhat relevant reported trial, in which researchers evaluated the effects of high dose resveratrol on the serum levels of androgens, circulating prostate specific antigen levels, and prostate size in middle-aged men with metabolic syndrome and found only lowered serum levels of the androgen precursors [143]. More attempts were made with combination phytotherapy in prostate cancer, concluding that further exploration of combination with other phytochemicals and/or grape skin extracts for convincing clinical proof is necessary [144–146]. Evidently, there are number of challenges in the translational success of natural chemopreventive agents including stilbenes [21].

There is a clear need for personalized targeted approaches for dietary chemoprevention and combinatorial strategies of chemosensitization with approved drugs for better outcomes in prostate cancer. Well-designed human chemoprevention and intervention trials in adequate subpopulation of prostate cancer patients will prove the promise of MTA1-targeted efficient management of prostate cancer.