Long associated with decreased risk of cancer, broccoli and other cruciferous vegetables – the family of plants that also includes cauliflower, cabbage, collard greens, Brussels sprouts and kale – contain a molecule that inactivates a gene known to play a role in a variety of common human cancers.
In a new paper published today in Science, researchers, led by Pier Paolo Pandolfi, MD, PhD, Director of the Cancer Center and Cancer Research Institute at Beth Israel Deaconess Medical Center, demonstrate that targeting the gene, known as WWP1, with the ingredient found in broccoli suppressed tumor growth in cancer-prone lab animals.
“We found a new important player that drives a pathway critical to the development of cancer, an enzyme that can be inhibited with a natural compound found in broccoli and other cruciferous vegetables,” said Pandolfi.
“This pathway emerges not only as a regulator for tumor growth control but also as an Achilles’ heel we can target with therapeutic options.”
A well-known and potent tumor suppressive gene, PTEN is one of the most frequently mutated, deleted, down-regulated or silenced tumor suppressor genes in human cancers.
Certain inherited PTEN mutations can cause syndromes characterized by cancer susceptibility and developmental defects.
But because complete loss of the gene triggers an irreversible and potent failsafe mechanism that halts the proliferation of cancer cells, both copies of the gene (humans have two copies of each gene; one from each parent) are rarely affected.
Instead, tumor cells exhibit lower levels of PTEN, raising the question of whether restoring PTEN activity to normal levels in the cancer setting can unleash the gene’s tumor suppressive activity.
To find out, Pandolfi and colleagues identified the molecules and compounds regulating PTEN function and activation.
Carrying out a series of experiments in cancer-prone mice and human cells, the team revealed that a gene called WWP1 – which is also known to play a role in the development of cancer – produces an enzyme that inhibits PTEN’s tumor suppressive activity.
Cruciferous vegetables have been associated with the chemoprevention of cancer.
Epigenetic regulators have been identified as important targets for prostate cancer chemoprevention.
Treatment of human prostate cancer cells with sulforaphane (SFN), a chemical from broccoli and broccoli sprouts, inhibits epigenetic regulators such as histone deacetylase (HDAC) enzymes, but it is not known whether consumption of a diet high in broccoli sprouts impacts epigenetic mechanisms in an in vivo model of prostate cancer.
An association between increased cruciferous vegetable intake and a reduced risk of developing, or being diagnosed with, prostate cancer has been reported (6).
Cruciferous vegetables, such as broccoli and broccoli sprouts, are a rich source of glucosinolates (7).
When broccoli sprouts are chopped or chewed, the glucosinolate glucoraphanin interacts with the enzyme myrosinase, producing the phytochemical sulforaphane (SFN) (7).
Broccoli sprouts and SFN have chemopreventive and cancer-suppressive properties in carcinogen-induced and genetic models of prostate cancer (7–9); however, the mechanisms by which they act in vivo are not completely understood.
SFN has been shown to inhibit the initiation of cancer by blocking damage caused by carcinogens through the induction of phase 2 enzymes via kelch-like ECH associated protein 1 (Keap1) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling (10–13).
In the transgenic adenocarcinoma of the mouse prostate (TRAMP) model of prostate cancer, broccoli consumption and/or SFN treatment has been shown to slow prostate cancer growth and metastasis (8, 9, 14, 15).
Several potential mechanisms have been implicated, including the induction of Nrf2-related pathways, inhibition of the cancer-promoting Akt signaling cascade, suppression of a chemokine receptor (CXCR4), and through augmenting the lytic activity of natural killer cells (8, 9, 14, 15). In contrast to these results, Liu et al. (16) did not find a significant decrease in prostate cancer in TRAMP mice feed a diet high in broccoli sprouts, highlighting a degree of controversy regarding cruciferous vegetable intake and the prevention of prostate cancer.
A hallmark of cancer development is the global modification of epigenetic marks (17).
These marks regulate chromatin structure and thus participate in the regulation of gene expression and genome stability.
This contributes to cancer development and progression by turning off tumor suppressor genes, or promoting the expression of oncogenes (20).
We and others have shown that SFN can alter epigenetic endpoints in cancer cell lines and tissues, including suppression of HDAC expression, changes in DNA methylation, and increased expression of epigenetically repressed genes such as p21 and p16(21–29).
In an in vitro study of TRAMP C1 cells, SFN was shown to restore Nrf2 expression through epigenetic modifications and attenuated the expression of several HDAC proteins (13).
Although there is substantial evidence that SFN exposure can influence epigenetic endpoints in cancer cells, it has not yet been shown in an in vivo model of prostate cancer that consumption of a whole food rich in SFN, such as broccoli sprouts, can induce changes in epigenetic regulators and contribute to chemoprevention.
We sought to test the hypothesis that consumption of a diet high in broccoli sprouts suppresses prostate cancer, inhibits HDAC expression, alters histone modifications, and changes expression of genes regulated by HDACs.
We show that consumption of a diet high in broccoli sprouts decreased the incidence and severity of prostate cancer, reduced HDAC3 protein, and altered epigenetic related endpoints.
Anticancer Action Mechanisms of Sulforaphane
Components of the human diet usually have pleiotropic action mechanisms that interfere with multiple targets within the intracellular and extracellular microenvironment.
Such a divergent action is advantageous against multi-factorial diseases like cancer in which multiple pathways enter into an erroneous alternative. The succeeding sections briefly outline various mechanistic approaches responsible for the cancer chemopreventive properties of sulforaphane.
NRF2-Mediated Elevation of Antioxidant Defense by Sulforaphane Reduces the Incidence of ROS-Induced Genomic Insult
One of the hallmarks of cancer cells is their dependence on increased aerobic glycolysis resulting in oxidative stress due to the accumulation of reactive oxygen species (ROS) that may directly challenge the genomic stability or participate in alterations of signaling pathways .
The ROS at low to moderate levels are active participants in cellular functions acting as signaling molecules that sustain cellular proliferation and differentiation, along with activating responses to oxidative stress .
However, excessive production of ROS damages cellular components such as DNA, proteins and lipids and serves as one of the major culprits in the induction of cancer (pre-initiation stage). ROS are constantly produced by both enzymatic and non-enzymatic reactions. Thus, a constitutive balance in the intracellular ROS status is required to maintain normal cellular homeostasis.
The transcription faction NRF2 (nuclear factor erythroid 2-related factor 2) is the principal regulator of the expression of molecules with antioxidant functions within the cell . NRF2 stimulates anti-stress signaling with protective response to suppress oxidative or electrophilic stress and inhibits carcinogenesis .
In the resting state NRF2 is inactive due to proteasomal degradation induced by a negative regulator KEAP1 (Kelch-like ECH associated protein 1). Under condition of stress, the KEAP1 is oxidized leading to the stabilization and translocation of NRF2 into the nucleus and expression of genes critical to antioxidant defense (Figure 3) .
Sulforaphane induces the phase II carcinogen detoxification enzymes, mediated via ARE-NRF2 pathway such as glutathione transferases, UDP-glucuronyltransferase, NAD(P)H:quinone oxidoreductase I and heme oxygenase-1 (HO-1), thereby allowing a diverse array of electrophilic and oxidative toxicants to be eliminated or inactivated before they cause damage to critical cellular macromolecules . Sulforaphane has been shown to interact with KEAP1 by covalent binding to thiol groups of this inhibitory protein . It was observed that sulforaphane modified multiple Keap1 domains , whereas the model electrophiles, but less potent pathway activators dexamethasone mesylate and biotinylated iodoacetic acid, modified Keap1 preferentially in the central linker domain . Further, gene-expression profiles by an oligonucleotide microarray revealed that sulforaphane upregulated the expression of NQO1, GST and GCL in the small intestine of wildtype mice, whereas the Nrf2-null mice displayed diminished levels of these enzymes . In another study, knockdown of Nrf2 with siRNA attenuated SFN-induced heme oxygenase-1 (HO-1) up-regulation . In vitro studies have reported the time- and dose-dependent responses with sulforaphane treatment on the induction of phase II enzyme demonstrating the positive effect of 25 μM dose on the enzymatic activities of GST, NQO1, aldo-keto reductase (AKR) and glutathione reductase (GR) in several mammalian cancer cell lines: HepG2, MCF7, MDA-MB-231, LNCaP, HeLa and HT-29 . Similar effects were observed in in vivo studies showing SFN to be effective at inducing the phase II enzyme response in rats and mice which were given SFN for four to five days at higher doses (up to 1000 mmol/kg per day), resulting in increased phase II enzyme activities in the liver, lung, mammary gland, pancreas, stomach, small intestine and colon of the animals . An important aspect that need to be considered is that ROS depletion via NRF2 by any agent including sulforaphane would be eligible to block the incidence of genomic insult in order to prevent initiation of cancer, whereas the activation of the NRF2 pathway at a later stage might interfere with the efficacy of certain chemo- and radio-therapies that rely on ROS production .
Sulforaphane as Inhibitor of HDACs Challenges the Pro-Oncogenic Epigenetic Pattern in Cancer Cells
Studies have implicated the anticancer effect of sulforaphane in its inhibitory activity against histone deacetylases (HDACs)  thus extending its chemopreventive activities to post-initiation stages.
The mechanism of histone acetylation depends on the balance between the enzymes with histone acetyltransferase (HAT) activity and enzymes that deacetylate histones (HDACs) .
The reversible acetylation of nuclear histones is an important mechanism of gene regulation. Histone acetylation is associated with an open chromatin conformation, allowing for gene transcription, whereas HDACs maintain the chromatin in the closed, non-transcribed state (Figure 4) .
A tightly regulated balance exists in normal cells between HAT and HDAC activities, and factors influencing this balance may contribute to cancer development.
These enzymes have many critical roles in regulation of gene expression, cell proliferation, cell migration, cell death and angiogenesis. HDACs are over-expressed in cancer cells making them one of the promising targets for the development of anticancer drugs .
It has been reported that HDAC inhibitors (HDACi) can induce growth arrest, apoptosis, reactive oxygen species facilitated cell death and mitotic cell death in cancer models . Many of the studies relating sulforaphane with HDAC inhibition come from the laboratory of Dashwood and Ho .
In many cases, it has been established that decline in the histone acetylation state corresponds with increased grade of cancer and risk of prostate cancer recurrence .
Moreover, inhibitors of HDAC, including suberoylanilidehydroxamic acid (SAHA), valproic acid, depsipeptide, and sodium butyrate have been demonstrated to be effective against prostate cancer cell lines and xenograft models [40,41].
Sulforaphane treatment in prostate cellular models have shown a reduction in HDAC activity and down-regulation of HDAC proteins followed by an increase in acetylation of histone H3 at the p21 promoter and increased acetylation of alpha-tubulin (specifically in hyperplastic and cancer cells) leading to cell death .
Interestingly, the enhancement in the acetylation status of alpha-tubulin demonstrates the potential of sulforaphane in the regulation of non-histone proteins also, which might have critical role in cell survival and cell death.
It is known that colon cancer cells over-express HDAC3, a protein required for enhanced genomic stability.
Studies have reported the ability of sulforaphane to retard HDAC3 protein expression in human colon cancer cells .
Another study reported that in mice treated with a single oral dose of 10 μmol SFN, there was significant inhibition of HDAC activity in the colonic mucosa and suppression of tumor development in APCmin mice (mouse model of multiple intestinal neoplasia with APC gene mutation) .
Furthermore, considering the challenge of breast cancer treatment of patients with estrogen receptor (ER)-negative tumors, studies have addressed the strategy of reactivating ERα expression and subsequent treatment with conventional anti-estrogen therapy [44,45].
The absence of ERα gene expression in ER-negative breast cancer is largely due to epigenetic silencing instead of DNA mutation or deletion of the ERα gene . T
reatment of ER-negative breast cancer cells with histone deacetylase inhibitors such as trichostatin A (TSA) leads to the reactivation of ER expression.
HDAC inhibitors and sulforaphane epigenetically reactivates ERα expression in ERa-negative MDA-MB-231 cells.
Additionally, combined treatment of green tea polyphenols and SFN along with tamoxifen therapy in hormonal refractory breast cancer significantly reduced cellular proliferation, likely due to the pronounced effect of histone modifications as well as DNA demethylation-mediated ERα activation in MDA-MB-231 cells .
How to disable this PTEN kryptonite?
By analyzing the enzyme’s physical shape, the research team’s chemists recognized that a small molecule – formally named indole-3-carbinol (I3C), an ingredient in broccoli and its relatives – could be the key to quelling the cancer causing effects of WWP1.
When Pandolfi and colleagues tested this idea by administering I3C to cancer-prone lab animals, the scientists found that the naturally occurring ingredient in broccoli inactivated WWP1, releasing the brakes on the PTEN’s tumor suppressive power.
When Pandolfi and colleagues tested this idea by administering I3C to cancer prone lab animals, the scientists found that the naturally occurring ingredient in broccoli inactivated WWP1, releasing the brakes on the PTEN’s tumor suppressive power.
The image is in the public domain.
But don’t head to the farmer’s market just yet; first author Yu-Ru Lee, PhD, a member of the Pandolfi lab, notes you’d have to eat nearly 6 pounds of Brussels sprouts a day – and uncooked ones at that – to reap their potential anti-cancer benefit.
That’s why the Pandolfi team is seeking other ways to leverage this new knowledge.
The team plans to further study the function of WWP1 with the ultimate goal of developing more potent WWP1 inhibitors.
“Either genetic or pharmacological inactivation of WWP1 with either CRISPR technology or I3C could restore PTEN function and further unleash its tumor suppressive activity,” said Pandolfi.
“These findings pave the way toward a long-sought tumor suppressor reactivation approach to cancer treatment.”
In addition to Pandolfi and Lee, authors include, Ming Chen, Jonathan D. Lee, Jinfang Zhang, Tomoki Ishikawa, Jesse M. Katon, Yang Zhang, Yulia V. Shulga, Assaf C. Bester, Jacqueline Fung, Emmanuele Monteleone, Lixin Wan, John G Clohessy, and Wenyi Wei, all of BIDMC; Shu-Yu Lin, Shang-Yin Chiang and Ruey-Hwa Chen of Institute of Biological Chemistry; Tian-Min Fu and Chen Shen of Harvard Medical School; Chih-Hung Hsu, Hao Chen and Hao Wu of Boston Children’s Hospital; Antonella Papa of Monash University; Julie Teruya-Feldstein of Icahn School of Medicine at Mount Sinai; Suresh Jain of Intonation Research Laboratories; and Lydia Matesic of University of South Carolina.
Funding: This work was supported by the National Institutes of Health (R01CA82328 and R35 CA197529), granted to Pandolfi. Lee was supported in part by the Postdoctoral Research Abroad Program Fellowship, Taiwan National Science Council (NSC), and DOD Prostate Cancer Research Program (PCRP) Postdoctoral Training Award (W81XWH-16-1-0249).
Disclosures: Pandolfi, Wei and Suresh Jain are cofounders of Rekindle Pharmaceuticals. The company is developing novel therapies for cancer. All other authors declare no competing interests.
Beth Israel Deaconess Medicial Center
Jacqueline Mitchell – Beth Israel Deaconess Medicial Center
The image is in the public domain.
Original Research: Closed access
“Reactivation of PTEN tumor suppressor for cancer treatment through inhibition of a MYC-WWP1 inhibitory pathway”. Yu-Ru Lee, et al.