Meat Heme and the Tumor microenvironment Reprogramming
The Multifaceted Role of Heme in Cancer
- Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, Turin, Italy
Heme, an iron-containing porphyrin, is of vital importance for cells due to its involvement in several biological processes, including oxygen transport, energy production and drug metabolism. Besides these vital functions, heme also bears toxic properties and, therefore, the amount of heme inside the cells must be tightly regulated. Similarly, heme intake from dietary sources is strictly controlled to meet body requirements. The multifaceted nature of heme renders it a best candidate molecule exploited/controlled by tumor cells in order to modulate their energetic metabolism, to interact with the microenvironment and to sustain proliferation and survival. The present review summarizes the literature on heme and cancer, emphasizing the importance to consider heme as a prominent player in different aspects of tumor onset and progression.
The onset and progression of cancer rely on the ability of tumor cells to channel different biological processes toward the promotion of cell proliferation, the escape from immunosurveillance and the resistance to drugs. Inorganic iron has been reported to play pivotal roles in several aspects related to cancer metabolic adaptation and tumor microenvironment reprogramming (1–3). Similarly, organic iron, in the form of the iron-containing porphyrin heme, is potentially a best candidate molecule exploited/controlled by tumor cells in order to modulate the energetic metabolism, to interact with the microenvironment and to sustain proliferation and survival. Heme bears a series of functions that are far beyond those mediated by its iron atom, including oxygen transport and storage, drug and steroid metabolism, transcriptional and translational regulation, signal transduction and microRNA processing (4, 5). In addition, heme synthesis is a cataplerotic pathway for the tricarboxylic acid cycle, as it consumes succynil-CoA. However, among the different metabolic processes that cancer cells can regulate to meet their specific demands, heme metabolism has been so far marginally studied, and frequently the importance of heme for cancer has been attributed to the iron atom contained in heme, rather than to specific functions mediated by the entire heme molecule itself. The present review will summarize the literature on heme and cancer highlighting both positive and negative effects of heme on cancer cells and on components of the tumor microenvironment.
Dietary Heme and Cancer
Historically, the role of heme in cancer has been studied focusing on the effects mediated by exogenous dietary heme on the organism. These studies contributed to the current notion that dietary heme is a risk factor for cancer. Heme is an iron coordinating porphyrin contained predominantly in red and processed meat in the form of hemoglobin and myoglobin. Red meat refers to unprocessed mammalian muscle meat, while processed meat refers to meat that has been transformed through salting, curing, fermentation, smoking, or other processes to enhance flavor or improve preservation. Recently, the International Agency for Research on Cancer (IARC), following an assessment of over 800 studies performed world-wide, classified processed meat as group 1 “carcinogenic to humans” and fresh red meat as group 2A “probably carcinogenic to humans” (6). Conversely, no link between white meat and fish and cancer has been found (7–9). For this reason, heme has been proposed as the key molecule contributing to tumorigenesis upon red and processed meat intake.
The role of dietary heme in cancer has been highlighted in different types of carcinomas. Indeed, high consumption of red and processed meat has been associated with increased incidence of esophageal, gastric, breast, endometrial, pancreas and lung tumor (10–15), while no clear link was found for bladder and prostate cancer (16–18). However, the majority of studies focused on the role of dietary heme in the pathogenesis of colorectal cancer (CRC), still a leading cause of cancer deaths in Western Countries (19–21). Dietary heme is absorbed mostly in the upper part of the small intestine. Once absorbed, heme is degraded by the action of the enzymes heme oxygenases (HMOXs) into biliverdin, carbon monoxide (CO), and iron (Fe2+), that is then scavenged by the protein ferritin (4). However, if red/processed meat is assumed in large amounts, all the ingested heme cannot be absorbed by the small intestine and it accumulates for a considerable time in the large intestine (22, 23).
In presence of high free heme levels both ferritin and HMOXs are saturated and cells accumulate free heme and labile iron that exert a variety of cytotoxic effects on intestinal mucosa (24). For example, heme is able to induce cytotoxic damage to surface epithelial cells that changes surface to crypt signaling, resulting in hyperproliferation and finally hyperplasia of crypt cells in heme-fed mice (25). Furthermore, free heme and labile iron accumulation result in the production of reactive oxygen species (ROS) that pathologically oxidize DNA, lipids and proteins.
It has been well-demonstrated that ROS-induced DNA damage and gene mutations cause CRC and that proteins involved in CRC development are redox-sensitive (26). Additionally, ROS are able to induce lipid peroxidation of intestinal cells. Reactive lipid peroxides, formed by the action of ROS, covalently bind to the protoporphyrin ring of heme giving rise to an extremely lipophilic molecule, named cytotoxic heme factor (CHF) that induces cytotoxic damage on intestinal epithelial cells (27). To note, processed meat when in contact with gastric acid can also give rise to lipid hydroxiperoxide (LOOHs). LOOH is then broken down by the iron released by heme to produce free radicals and, subsequently, aldehyde molecules like malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) (28). MDA is toxic and it is able to bind DNA forming mutagenic adducts. 4-HNE induces apoptosis and kills normal cells, but not precancerous cells that are mutated on Adenomatous Polyposis Coli (APC) gene (29).
In addition to the processes described above, heme is also able to catalyze the formation of N-nitroso compounds (NOC) in the gastrointestinal tract (30). NOC are known carcinogens that can determine DNA mutation through alkylation, and high NOC concentrations have been associated to increased red meat consumption (31, 32). However, it is important to underline that NOC found after ingestion of red meat in humans consist mainly of nitrosyl iron and nitrosothiols, products that have profoundly different chemistries as compared to some tumorigenic N-nitroso species (33). Therefore, more studies are required to clarify the real involvement of heme in NOC pro-tumor effects.
Finally, it has been demonstrated that heme alters the normal intestinal bacterial flora especially by decreasing the number of gram-positive bacteria (34) leading to a state of dysbiosis (microbial imbalance or maladaptation) that exacerbates colitis and adenoma formation in mice (35) and is correlated to the insurgence of CRC (35–37).
Moreover, the gut microbiota can induce per sehyperproliferation via mechanisms occurring in the colon lumen, such as modulation of oxidative and cytotoxic stress or by influencing the mucus barrier, and these effects are intensified in presence of heme (38). Indeed, a recent study showed that mice receiving a diet with heme show an increased population of mucolytic bacteria in their colon. These bacteria, synergistically with heme-produced CHF, damage gut epithelium and lead to a compensatory aberrant hyperproliferation. Conversely, mice receiving heme together with antibiotics do not show this phenotype (38).
Overall, the studies on dietary heme and cancer support the idea that heme contained in food can sustain cancer by different mechanisms (Figure 1). However, it must be recognized that methodologies employed in some of these studies have been challenged. Indeed, some animal studies took advantage of diets low in calcium and high in fat, combined with the exposure of heme frequently at doses higher than that expected with a normal dietary consumption of red meat. Moreover, pork meat shows low heme levels, but it has been associated with CRC. Finally, it cannot be excluded that carcinogenesis could be ascribed to other molecules contained mostly in red meat, not related to heme (33). Therefore, further research is required to clarify the role of heme present in red and processed meat in cancer.
Heme Synthesis and Cancer
Heme can be acquired by dietary sources, but in addition, all the cells in the organism are able to synthesize heme. Heme synthesis includes eight different reactions occurring partly in mitochondria and partly in the cytosol. The first rate limiting step is based on the condensation of succynil-CoA and glycine to produce 5-aminolevulinic acid (ALA), a reaction catalyzed by the enzyme 5-Amilolevulinate Synthase 1 (ALAS1). Then, by subsequent reactions involving the enzymes ALA dehydratase (ALAD), porphobilinogen deaminase (PBGD), uroporphyrinogen III synthase (UROS), uroporphyrinogen decarboxylase (UROD), coproporphyrinogen oxidase (CPOX), protoporphyrinogen oxidase (PPOX) and ferrochelatase (FECH), heme is finally produced (4, 39).
The study of heme synthesis in tumors has raised interest since many years. Indeed, in nineties, it was discovered that tumors, upon ALA administration, are able to accumulate remarkably higher amount of protoporphyrin IX (PpIX) as compared to normal tissues, and this property was demonstrated to be exploitable for tumor fluorescence-guided surgery (FGS) and to kill cancer cells by photodynamic therapy (PDT) (40–42). Since then, extensive research has been performed to determine the molecular mechanism involved in enhanced ALA-PpIX accumulation in tumor cells. Particularly, the rate of heme biosynthesis in different kinds of tumor was dissected in several works, leading to accumulation of conflicting results. However, ALAS1, PBGD and UROD expression and/or activity were frequently found up-regulated in cancer (43). Consistently, repression of heme biosynthesis by the ALAD inhibitor succinylacetone was shown to reduce tumor cell survival and proliferation (44–46). Conversely, FECH levels were found often down-modulated in tumor cells as compared to normal cells (43, 47).
Taking together these discoveries, it can be concluded that tumors are characterized by high porphyrins synthesis, not necessarily associated to final heme production. This conclusion, however, is controversial. Indeed, cancer cells have been shown to display high heme levels (45, 48), increased activity of heme containing proteins (45, 46, 49) and enhanced expression of heme exporters (45, 50), suggesting that heme, and not only its precursors, is produced and that the entire heme biosynthetic pathway is promoted in tumors. Therefore, the reason why tumors accumulate more ALA-mediated PpIX than surrounding normal tissues and why tumors enhance heme synthesis remains a fundamental question to be answered (Figure 2).
Heme and Tumor Microenvironment
Other than acting directly on tumor cells, heme can exerts its functions in cancer by modulating the tumor microenvironment (TME) (Figure 3). The TME is composed by tumor cells, endothelial cells of surrounding blood vessels, myofibroblasts, stellate cells, adipose cells, peritumor nerve cells, immune cells, endocrine cells, fibroblasts, and the extracellular matrix (104, 105). All the cell types in the TME contribute to tumor progression mainly by releasing factors, which establish a favorable environment for the cancer cell and promote tumor cell survival and migration, metastasis formation, chemo-resistance, and the ability to evade the immune system responses (104).
Heme has been reported to modulate the activity of tumor-associated macrophages (TAMs). Macrophages have heterogeneous phenotypes that range from the “classically-activated” pro-inflammatory M1-cells to the “alternatively-activated” anti-inflammatory M2-cells (106). If on one hand M1-like macrophages have anti-tumor properties, on the other hand macrophage polarization toward a M2-like phenotype correlates with pro-tumor activities, such as enhanced angiogenesis, matrix remodeling, and immune suppression (107, 108). Due to the specific cues that characterize the tumor microenvironment, TAMs are induced to preferentially acquire a M2-like specialized phenotype that protects cancer cells from targeted immune responses (109). Notably, M. Costa da Silva and colleagues demonstrated that TAMs exposed to haemolytic red blood cells (RBCs), a condition sometimes observed in cancer due to the extravasation of RBCs from the abnormal tumor-associated vessels (110), accumulate iron intracellularly and acquire a M1 pro-inflammatory phenotype, which in turn promotes tumor cell death (111). This is in line with studies on haemolytic diseases, such as sickle cell disease, where it has been observed that macrophage exposure to free heme, released by damaged erythrocytes, leads to M1-reprogramming (112). Moreover, these findings are in agreement with additional studies reporting the concept that iron can drive macrophages polarization toward a pro-inflammatory M1-phenotype (113, 114). The effects on TAMs described can be mainly ascribed to the iron atom contained in heme. However, another important aspect that should be taken into account is the role exerted by CO in the control of myeloid cell differentiation. Indeed, the CO produced upon heme degradation by HMOXs has been reported to cause tumor regression and increased drug sensitivity in prostate and lung cancer models (115), and this can be partly attributed to CO effects on macrophages within the TME. Specifically, it has been shown that CO treatment in mice leads to an increased number of M1-like macrophages and reduced tumor growth (116). Taken together, these works have led to the implementation of strategies able to modulate the exposure of macrophages to heme or iron, as the use of iron oxide nanoparticles (109, 111), for cancer therapy.
Another fundamental component of the tumor microenvironment is represented by tumor-associated endothelial cells (TECs). TECs strongly differ from their normal counterparts (117) as they display a high pro-angiogenic potential that mostly relies on their enhanced ability to proliferate and migrate. The switch of TECs from quiescence to a highly active state is favored by a specific metabolic reprogramming (118–120). The cross-talk between cancer cells and TECs promotes aberrant neo-angiogenesis, which is required to sustain tumor growth, by providing oxygen and nutrients, and to favor metastatization (117, 121, 122). To our knowledge, there are no studies in literature analyzing the role of heme in TECs. However, we demonstrated that alterations in endothelial intracellular heme metabolism strongly affect the angiogenic process during development (98, 123). Consistently, another study highlighted the critical role of the heme biosynthetic pathway in supporting endothelial functions (124). In addition, tumor angiogenesis is also promoted by the increased stiffness of the extracellular matrix (ECM) found in TME (125, 126). High ECM stiffness is mainly due to increased collagen deposition and increased cross-linking within the tumor stroma. Matrix remodeling is primarily controlled by the activity of a group of zinc-dependent endopeptidases, named matrix metalloproteinases (MMPs), which are mainly released by cancer-associated fibroblasts (CAFs). However, recent studies highlighted the involvement in this process of peroxidases released by immune cells, such as myeloperoxidase (MPO) and eosinophil peroxidase (EPO). In particular, MPOs and EPOs have been reported to be able to directly induce the secretion of collagen I and collagen VI by CAFs (127), thus increasing matrix stiffness and promoting tumor angiogenesis and metastatization (128). Notably, MPOs and EPOs are heme-containing enzymes, thus suggesting that changes in the amount of available heme within the cells could affect the activity of these enzymes in matrix remodeling. Taking together all these considerations, it is tempting to speculate that heme could affect different processes involved in tumor angiogenesis and future studies will help to verify this hypothesis.
Finally, an additional promising aspect to dissect in the future is the possible implication of heme in the control of tumor innervation. The peripheral nervous system is nowadays gaining growing interest in cancer research due to its role in modulating both cancer cells and TME. This is achieved through the reciprocal interaction between nerves and cancer cells (nerve-cancer cell cross-talk), as well as between nerves and the TME (129–131). Indeed, recent data clearly indicate that tumor onset and progression is accompanied by increased innervation, through a mechanism largely dependent on the secretion of neurotrophic factors by cancer cells. Furthermore, nerves influence tumor onset, progression and metastasis formation, mainly through the secretion of neuropeptides and neurotransmitters in TME, where they interact with receptors expressed by cancer cells and by other cells of the TME (131–136).
Heme is required for the survival of different types of neuronal cells (137, 138); however, the specific role of heme in the peripheral nervous system and its potential implication in tumor innervation is completely unknown. Several evidences suggest that heme is crucial for the maintenance of the peripheral nervous system, particularly for sensory neurons, one of the types of nerves that actively innervates tumors (139–141). Notably, mutations in genes encoding proteins involved in heme synthesis and export have been reported in diseases characterized by the degeneration of sensory neurons (94, 142–145). Finally, heme may also regulates pathways important for nerve-cancer cell cross-talk. Indeed, heme is involved in the regulation of gene expression in neurons via nerve growth factor (NGF) signaling (146), thus suggesting that heme may modulate nerve outgrowth in the tumor microenvironment. Furthermore, heme also regulates the metabolism of some neurosteroids and neurotransmitters (137), with a potential implication in nerve-cancer cross-talk. In addition, other than sustaining cancer, tumor innervation is also one of the main cause of chronic pain in oncologic patients, particularly those in the advanced stage of the disease (147, 148). Interestingly, mutations in the heme exporter FLVCR1 have been reported in patients with peripheral sensory neuropathy (94, 149, 150), thus indicating an involvement of heme in pain perception. Furthermore, CO produced upon heme catabolism can act as an atypical neurotransmitter or neuromodulator in the nervous system and is involved in nociception regulation (151). Overall, these evidences support the idea that heme could be implicated in different aspects of tumor innervation. Future targeted in vitroand in vivo experiments will definitively verify whether this hypothesis is correct.
Additional Heme Functions Potentially Relevant for Cancer
In the previous paragraphs, we discussed the works that contributed to explore the role of heme in tumor growth and metastatization. However, we believe that heme could be involved in additional aspects of tumor biology, not investigated so far. Indeed, emerging evidences indicate that heme is required for the processing of microRNA (miRNA) that, by regulating more than 50% of the mammalian genome are implicated in several pathways crucial for cancer onset, growth and metastatization (152). Specifically, the RNA-binding protein DiGeorge critical region-8 (DGCR8), which is essential for the first processing step of pri-miRNAs, is a heme-binding protein (153–156). Heme binding to DGCR8 is required for its dimerization and activation (153) and the modulation of heme availability was reported to affect pri-miRNA processing in vitro (157). A correlation between alterations of heme and miRNAs expression was also reported. For instance, ALA-mediated sonodynamic therapy or PDT is associated with the altered expression of selected miRNAs (158–161). Although, further studies are required to fully understand the physio-pathological implications of these findings, these data suggest that mysregulation of miRNAs may represent an additional mechanism through which alterations of heme metabolism sustain and promote cancer progression.
In addition, several data support a potential involvement of heme in epigenetic modifications, that control multiple processes essential for cancer cells (162, 163). This is suggested by the observation that heme regulates the transcriptional and demethylase activity of the yeast histone demethylase GIS1 (GIS1), that belongs to the lysine demethylase 4 (jmjd-2/KDM4) subfamily of demethylases implicated in histone methylation, cellular signaling and tumorigenesis (164). The yeast GIS1 protein is conserved from yeast to mammals, suggesting a possible role for heme in the regulation of this protein also in mammals. Furthermore, several heme-regulated proteins of the circadian rhythms machinery are epigenetic modifying enzymes themself. For instance, clock circadian regulator (CLOCK) is a histone acetyltransferase and Rev-erbα functions as a transcriptional repressor by forming a complex with the nuclear receptor corepressor (NCOR) and the histone deacetylase 3 (HDAC3) (165). Finally, the activity of epigenetic modifying enzymes relies on the availability of specific metabolites (like α-ketoglutarate) and cofactors (acetyl-CoA, S-adenosylmethionine, and nicotinamide adenine dinucleotide). Most of them are produced by the TCA cycle. Since heme biosynthesis is a TCA cycle cataplerotic pathway (166), it is reasonable that alterations of heme homeostasis may affect the availability of metabolites to epigenetic modifying enzymes. Based on the role of heme in the control of cellular metabolism (166) and circadian rhythms (81), we propose that the alterations of heme metabolism observed in cancer may also contribute to the mysregulation of cancer epigenetics.
Finally, heme metabolism has been reported as an apoptosis modifying pathway in acute myeloid leukemia (AML) (167). This is relevant, because it suggests the possibility to target heme metabolism in order to increase drug sensitivity in cancer cells. In addition, we observed that the modulation of heme metabolism induces paraptosis (123), at least in endothelial cells. This suggests the possibility to potentially exploit the regulation of heme metabolism in cancer therapy, as the availability of compounds inducing alternative forms of programmed cell death could be very useful to counteract the resistance to apoptosis in tumors (168). Similarly, the identification of heme as an inhibitor of the proteasome (169) appears as a promising property to be exploited for therapeutic purposes, particularly considering that proteosome inhibitors are successfully currently used in cancer therapy (170).
By the present review, we attempted to provide a comprehensive overview of the literature on heme in cancer, highlighting heme participation in multiple processes that sustain tumor growth and metastatization, encompassing the control of mitochondrial metabolism, the function of hemoproteins and P53 signaling.
Summarizing, the studies on dietary heme and cancer, although affected by some limitations, support the idea that heme contained in food can sustain cancer by different mechanisms. Conversely, the impact of endogenous heme in cancer is much more complex to envisage. On the one hand, heme biosynthesis is frequently enhanced in tumors, but this phenomenon could serve different and sometimes conflicting purposes. Moreover, both heme import and export are increased in tumor cells, but the reason is unclear. In addition, heme degradation by HMOX1 in tumor cells seems to support cancer by counteracting oxidative stress during tumorigenesis and upon anti-tumor therapies, but concomitantly to promote TAMs acquisition of a M1-like phenotype, favoring tumor regression and increased drug sensitivity. Finally, heme-containing enzymes like MPOs and EPOs can promote tumor angiogenesis and metastatization, and heme could also potentially affect cancer epigenetics, miRNAs and tumor innervation. Therefore, targeting heme metabolism is promising because it could have a broad impact on different aspects of cancer. For this reason, we envision that future work should be directed to the development of novel therapeutic strategies based on heme (Figure 4). However, the choice of the appropriate strategy is challenging, due to possible conflicting effects obtained by the block or the promotion of heme-related processes. To overcome these problems, further work is required in order to classify the precise tumor subtypes that can benefit of each single strategy.
Anyhow, according to present literature, the targeting of heme metabolism has already been exploited for cancer therapy. In particular, the stimulation of heme synthesis with ALA has been widely used for PDT (171). In addition, the indirect inhibition of heme synthesis through iron chelation therapy has been recently proposed for selected cancer types. Iron chelation therapy has emerged as an important chemotherapeutic strategy, because of the strong link between iron excess and tumorigenesis. However, iron-deprivation therapy was successful only on selected tumor types. The discovery that heme directly regulates P53 stability (87) explained the selective therapeutic efficacy of iron deprivation-based chemotherapy. Indeed, Shen et al. demonstrated that this selectivity was due to the P53 status of the tumor types (87). Specifically, iron chelation therapy, by decreasing heme levels, leads to the stabilization of P53 proteins only in tumors with wild-type P53, and not in case of P53 mutations. As already suggested by Shen et al., these findings will allow the discrimination of the types of tumor that should benefit of iron chelation therapy (87). The targeting of heme synthesis through the deprivation of iron required for heme synthesis (iron chelation therapy) remains the best therapeutic strategy to date. However, because iron is essential for multiple processes beyond heme synthesis, a key challenge of chelation therapy is to balance iron levels in order to avoid excessive iron chelation. Moreover, it has to be underlined that emerging studies demonstrate that some kinds of tumor could be counteracted by iron supplementation, rather than by iron chelation therapy (172). Therefore, more specific strategies to blunt heme synthesis are required, not based on iron. The discovery that the beneficial effects of iron-chelation therapy on the growth of certain tumor types depends on the regulation of P53 by heme raises the possibility to directly target heme synthesis to counteract tumor growth. Several compounds, like succinylacetone or N-methyl protoporphyrin, are used to inhibit heme synthesis in vitro. Although these compounds are not yet used in the clinic, we cannot exclude that new drugs based on them will be developed. Similarly, it could be possible that, in the future, drugs aimed at blocking/stimulating heme importer/exporter/degrading proteins will be identified, in order to perturb heme-related mechanisms in cancer.
In conclusion, we hope that, in the next future, the growing awareness on heme role in processes relevant for cancer will stimulate research aimed at implementing innovative therapeutic approaches and at identifying the tumor subtypes sensitive to these treatments.