Cancer Cells with Irons in the Fire

Abstract

Iron is essential for the growth and proliferation of cells, as well as for many biological processes that are important for the maintenance and survival of the human body. However, excess iron is associated with the development of cancer and other pathological conditions, due in part to the pro-oxidative nature of iron and its damaging effects on DNA. Current studies suggest that iron depletion may be beneficial for patients that have diseases associated with iron overload or other iron metabolism disorders that may increase the risk for cancer. On the other hand, studies suggest that cancer cells are more vulnerable to the effects of iron depletion and oxidative stress in comparison to normal cells. Therefore, cancer patients might benefit from treatments that alter both iron metabolism and oxidative stress. This review highlights the pro-oxidant effects of iron, the relationship between iron and cancer development, the vulnerabilities of iron-dependent cancer phenotype, and how these characteristics may be exploited to prevent or treat cancer.

Introduction

Iron is crucial for many life processes, including cell growth and proliferation [1]. Moreover, iron facilitates oxygen delivery in the human body and is important for numerous other biological functions [2]. However, excess iron is associated with toxicity due to its pro-oxidant effects and has been associated with a number of diseases including cirrhosis of the liver, heart disease, diabetes, and cancer [3, 4]. Specifically, the oxidative effects of iron have been implicated in the development of cancer [4]. However, iron not only contributes to oncogenesis, it is also essential for maintaining the rapid growth rate of cancer cells that require the iron-dependent enzyme ribonucleotide reductase for DNA synthesis [5], Due to the high iron requirements of cancer cells, iron depletion has been investigated for its therapeutic potential. Moreover, mounting evidence suggests that altering iron metabolism may be an effective strategy for both cancer prevention and treatment. Several clinical studies presented in this review, assessing iron overload or cancer, exemplify the potential therapeutic benefits that can be obtained by directly or indirectly targeting iron metabolism and/or the redox effects of iron.

Iron and oxidative stress

Excess iron is generally associated with toxicity because it induces the hydroxyl radical (•OH), a type a reactive oxygen species (ROS) formed via the Fenton reaction (Fig. 1) [6]. Lipid peroxidation can be initiated from •OH, which is damaging to cell membranes [4]. Moreover, ROS such as superoxide anion (O₂•-) and hydrogen peroxide (H₂0₂) also play a role in the production of iron-induced free radicals [7]. Excess ROS increases oxidative stress, which overwhelms cellular defense systems and allows lethal oxidants to damage DNA and other biomolecules [4, 8]. High amounts of oxidative stress are associated with the development of many pathological conditions, including cancer [9, 10]. However, the pathways that link iron, oxidative stress, and pathological development remain to be fully elucidated.

Figure 1.

The Fenton reaction. The fenton reaction involves iron II (Fe ²⁺) reacting with hydrogen peroxide (H₂O₂) to yield iron II (Fe ³⁺) a hydroxyl radical (•OH) and a hydroxide ion (OH^-). The hydroxyl radical can induce lipid peroxidation; more reactive oxygen species (ROS) and oxidative stress; damage to DNA and other biomolecules; and if overexposed, carcinogenesis.

On the other hand, both iron and ROS also have positive effects in living systems. Iron is crucial for survival and development, and ROS has effects on cellular signaling that is important for proliferation, differentiation, and survival of the cell [11, 12]. Both iron depletion and ROS induction can also be used to selectively target cancer cells because these cells often have higher iron requirements and higher oxidative stress as a result of oncogenic transformation (Fig. 2) [5, 13, 14]. Ultimately, maintaining both iron and ROS homeostasis is crucial for preventing disease, whereas reducing iron and/or increasing ROS may be effective in cancer treatments (Fig. 3).

Figure 2.

Iron regulation in normal and cancer cells. The iron regulatory proteins or involved in cellular metabolism include: Tf = transferrin; TfR1 = transferrin receptor; Fe^III-Tf = iron III bound to transferrin; FPN = ferroportin; LIP = labile iron pool; TAM = tumor associated macrophages; and HAMP = hepcidin. For a cancer cell compared to a normal cell: iron influx is higher, iron efflux is lower, LIP is higher, oxidative stress is higher, HAMP is higher, FPN is lower, and there are more TfR1 and extracellular sources of iron, including ferritin from tumor associated macrophages (TAM).

Figure 3.

Potential therapeutic strategies for cancer prevention and cancer progression. Iron overload increases oxidative stress by the Fenton reaction. Cancer may be prevented in patients with iron overload by reducing iron (1) or reducing iron and oxidative stress (2). On the other hand, high oxidative stress and high iron represents the cancer cell phenotype. Cancer patients might benefit from iron depletion (3), an increase in ROS (4), or both (5). These effects can selectively induce apoptosis in tumor cells, which is especially vulnerable to iron depletion and increased oxidative stress.

Some of the major players in iron metabolism that may have therapeutic potential for the prevention or treatment of cancer include the iron regulator hepcidin, the iron exporter ferroportin, the iron transporter transferrin (Tf), the transferrin receptor (TfR), and the iron storage protein ferritin (Fig. 2). Hepcidin is a protein secreted from the liver that binds to ferroportin and causes this iron exporter to degrade and prevent iron from being absorbed into the body or from being exported out of the cells [15, 16]. When iron is absorbed into the blood stream it is transported by the protein Tf, which binds to TfR on the membrane of cells to provide iron [2, 17]. By receptor-mediated endocytosis, iron enters the endosome, where acidification releases iron from Tf and the six-membrane epithelial antigen of prostate 3 (STEAP3), which reduces iron (III) to iron (II) before it is released to the cytosol by the divalent metal transporter 1 (DMT1) [17-19]. Once in the cytosol, iron may be stored in the form of ferritin, used for heme synthesis, or contribute to the labile iron pool (LIP) [17]. Moreover, the iron responsive element-binding protein 1 and 2 (IRP1 and IRP2) are involved in translational regulation of proteins that control iron import, storage, and efflux [20, 21]. Many of these iron regulatory proteins and pathways are altered in cancer cells (Fig. 2) and are described extensively in several review papers [21, 22].

Moreover, oxidative stress targets are also being explored for their therapeutic value in cancer prevention and treatment. The nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor of interest because it regulates the expression of several antioxidant enzymes, such as glutathione S transferase and NAD(P)H quinone oxidoreductase 1 [23, 24]. Regulation of Nrf2 and endogenous antioxidants are considered to be particularly important for the prevention of iron-induced oxidative stress that contributes to cancer development [25, 26]. Moreover, Rachmilewitz et al. have suggested that antioxidants may ameliorate the deleterious effects of ROS that are associated with iron overload in diseases such as β-thalassemia [27]. In particular, fermented papaya (Carica papaya) was shown to decrease oxidative stress in red blood cells of patients with β-thalassemia [28]. These effects may be due to polyphenolics in papaya, a class of compounds that have antioxidant and iron chelation effects [29, 30].

In addition, phosphatidylinositol kinases such as type 2 phosphatidylinositol-5 phosphate 4-kinases α and/or β (PI5P4Ka and β) have been shown to protect against ROS in several p53-null breast cancer cells [31]. Therefore, inhibition of PI5P4Kα and β may exert anti-cancer effects by reducing ROS protection in a specific subset of cancer types. Furthermore, a diverse array of natural compounds such as phenylethyl isothiocyanate (PEITC) found in many cruciferous vegetables, piperlongumine from the pepper plant (Piper longum), parthenolide from the feverfew plant (Tanacetum parthenium), and curcumin, a polyphenolic compound from turmeric (Curcuma longa), all have been shown to at least partially induce ROS and selectively target tumor cells [13, 32-35]. Moreover, the cancer preventative and therapeutic effects of curcumin are also attributed to its ability to alter iron metabolism by functioning as an iron chelator [36].

Cancer and Iron

The role of iron in cancer development

Excess iron is associated with the development of cancer, mainly due to its pro-oxidant effects, which can contribute to DNA damage and promotion of oncogenesis (Fig. 1) [4, 10]. Patients with hereditary hemochromatosis (iron overload) have been shown to be at greater risk for developing liver cancer compared to matched-control patients with non-iron related chronic liver disease [37]. Although it has been debated, there is some evidence that hereditary hemochromatosis also increases the risk for other cancers [37]. In particular, one study showed that mutations in the gene for hereditary hemochromatosis (HFE), specifically the homozygous C282Y mutation, increased the risk of colon cancer in men and breast cancer for women [38]. The majority of this group of men and women also had increased ferritin levels, an indicator of iron overload.

Studies on HFE indicate that other diseases that contribute to iron overload may also increase the risk for cancer. For example, as the life expectancy of β-thalassemia has improved over the years with better treatment options, there has been a greater incidence of hepatocellular carcinoma in the aging population with this condition [39, 40]. β-thalassemia patients often have iron overload because of decreased hepcidin expression and increased iron absorption, which is further exacerbated by transfusion dependency [41]. Iron overload at least partially contributes to the development of hepatocellular carcinoma and therefore may be prevented by depleting excess iron. Currently, the treatments used to manage iron overload in β-thalassemia require iron chelators [41]. However, potential new treatments that target iron absorption might utilize minihepcidins and/or drugs that increase hepcidin expression. [42-44]. These treatments may not only treat β-thalassemia patients, but they may also be effective at preventing cancer in patients with this disease and other iron overload conditions.

Additionally, one study demonstrated that patients with peripheral arterial disease that underwent a phlebotomy had a lower risk for subsequent cancer and those that did develop cancer had lower cancer-specific deaths than the control group [45]. Other research has indicated that iron may also contribute to the development of leukemia in patients with myelodysplastic syndrome (MDS). It has been found that transfusion dependence in MDS patients is associated with an increased risk for developing leukemia, and that those transfusion-dependent patients that developed iron overload had an even lower chance for survival [46]. However, these associations need to be evaluated carefully, since it could be argued that these effects are attributable to more advanced bone marrow failure. Nevertheless, iron chelation therapy significantly improved survival time in low-risk MDS patients and was associated with a trend toward longer leukemia-free survival [47].

Furthermore, iron chelation therapy improved hemoglobin levels, transfusion independence or reduced transfusion requirements in several MDS patients [48]. These studies suggest that not only does iron chelation prevent iron overload and potentially cancer, but it may also improve the disease itself from progressing. Interestingly, it has also been found that the iron chelator deferasirox (Exjade™;DFX) selectively reduced the proliferation of MDS hematopoietic cells and not normal hematopoietic cells in vitro [49]. In this case, ROS induction was suggested as the potential mechanism [49, 50]. However, more studies are needed to confirm these effects.

Overall, several clinical studies evaluating iron overload suggest that excess iron contributes to cancer development. In terms of tissue specificity, the most compelling studies suggest iron overload mostly contributes to cancer in the liver, colon, and breast tissues (Table 1) [38, 40, 51]. The liver is a major organ for iron storage and therefore may be more susceptible to the pro-oxidant effects of iron overload and carcinogenesis [3]. The intestinal epithelium is potentially exposed to dietary iron more than other tissues, which may explain why colon cancer is especially associated with high dietary iron intake [21, 51]. Moreover, iron and estrogen may work synergistically and contribute to the development of breast cancer. In particular, it has been speculated that post-menopausal women with increased iron concentrations and lower estrogen levels may be at higher risk for breast cancer, due to the increase in oxidative stress pathways [52]. Interestingly, premenopausal women with iron deficiency and high estrogen may also be at increased risk for breast cancer as a result of pro-angiogenic pathways that are beyond the scope of this review and described more extensively by others [52, 53].

Table 1.

Conditions associated with iron and cancer development

Condition	Cancer	References
Hereditary hemochromatosis (homozygous C282Y)	Liver, colon, breast	[37,38]
β-thalassemia	Liver	[40]
Myelodysplastic syndrome (MDS)	Leukemia (AML)	[47]

Furthermore, cancers from prostate, kidney, gastric, and brain tissues may also be induced by iron overload but there is less evidence supporting these relationships and further studies are needed. Mounting evidence also suggests that altered iron metabolism plays a role in the transformation of MDS to AML, although the pathways associated with these effects remain to be identified. In addition, the microenvironment may also play a role in promoting tumor growth by providing iron to the tumor cells by tumor-associated macrophages (TAM) that secrete ferritin (Fig.2) [54] and potentially by senescent erythrocytes and other extracellular iron sources [55]. Although more studies are warranted, it appears that treatments that involve iron depletion (e.g., iron chelators), complemented with ROS reduction (e.g., activation of Nrf2 pathways, antioxidants), may prevent the development of cancer in patients with iron overload diseases (Fig. 3).

Therapeutic Implications of Targeting Iron Metabolism in Cancer

Therapeutics that target iron metabolism may not only prevent the onset of cancer, but they may also improve the outcome of cancer patients. Cancer cells have higher iron demands in comparison to their healthy counterpart cells [5, 21]. Several studies have reported that cancer cells have increased expression of iron regulatory proteins that facilitate iron uptake (e.g., transferrin receptor 1) and/or have decreased expression of the iron exporter ferroportin, relative to normal cells (Fig. 2). In fact, patients that had breast cancer cells with low ferroportin gene expression had a significant reduction in disease-specific survival compared to those that had higher ferroportin expression [56]. Similarly, high levels of TfR1 expression in breast cancer tissues correlated with a poorer prognosis [57]. These and other studies suggest that increased iron influx and reduced iron efflux in cancer cells provides a net increase in LIP and a favorable environment for tumor growth (Fig. 2).

Targeting iron regulatory proteins or the LIP by iron depletion may slow or prevent the growth of cancer cells. Cancer cells are especially vulnerable to iron depletion and ROS induction, both of which can lead to apoptotic events [7]. Several studies indicate that the cancer phenotype (high ROS and iron requirements) can be selectively targeted by reducing iron or increasing ROS (Fig. 3), while normal cells remain unharmed or less affected [13, 14, 22]. In particular, an increasing number of studies are evaluating the iron depletion effects of iron chelators for cancer therapy. Iron chelators not only have effects on iron metabolism, but they also have effects on oxidative status. Iron chelators can function as antioxidants by preventing the Fenton reaction (ROS production) and can also form redox-active iron complexes that increase ROS, which is cytotoxic to cancer cells, depending on the structure of the compound and the environment [4, 58, 59].

In one study that demonstrated the role of iron in cancer progression, patients with head and neck squamous carcinoma cells (HNSCC) with high HFE expression (increases hepcidin and intracellular iron) had reduced survival compared to patients with cells that had low HFE expression [60]. Furthermore, HNSCC cells that were treated with the iron chelator, ciclopirox olamine (CPX), had significantly reduced viability and clonogenic survival. This study suggests that HFE expression could be a potential prognostic marker for tumor progression of HNSCC and that iron depletion may be a treatment option for this type of cancer. Similarly, studies on breast cancer cells indicated that the iron chelator deferoxamine (DFO) increased the sensitivity of these cells to chemotherapeutic agents [61].

Human trials that have assessed the anticancer effects of iron chelators have mostly been evaluated in hematologic malignancies and neuroblastoma (Table 2) [62]. Iron chelators showed promising effects in preclinical studies and in a few case studies of patients with acute myeloid leukemia (AML) [63, 64]. The iron chelator DFX induced complete cytogenetic remission in a 73-year old male patient with relapsed, refractory AML after 12 months of iron chelation with no additional chemotherapy [65], Moreover, a 69-year-old male patient, with relapsed AML that was treated with the iron chelator DFO and vitamin D, had decreased peripheral blast counts, decreased requirement for transfusion support, and increased monocytic differentiation [63], In addition to AML, a six-week-old infant with pre-B acute lymphoblastic leukemia (ALL), that failed induction chemotherapy, had peripheral blast counts reduced to zero and monocytic differentiation after 15 and 20 days, respectively, after treatment with DFO and cytarabine [66].

Table 2.

Cancers that may improve with iron chelation based on patient studies

Cancer type	Patient Study Data	Chelatorsa	Reference
Head and neck squamous carcinoma (HNSCC)	High HFE expression = reduced survival. Iron chelator reduced viability and clonogenic survival of cells.	Ciclopirox olamine	[60]b
Breast cancer	Lower ferroportin expression = poorer prognosis. Iron chelator increased breast cancer cell line sensitivity to chemotherapeutics.	DFO	[56,61]b
Leukemia (AML, ALL)	Several patients had improved survival time	DFX DFO + vitamin D DFO + AraC Triapine® + AraC Ciclopirox olamine	[63,64,65,67,68]
Neuroblastoma	Decreased bone marrow infiltration in patients.	DFO	[68]

^aThe abbreviations for the iron chelators/treatments: deferoxamine (DFO), deferasirox or Exjade^TM(DFX), and cytarabine AraC.

^bThese studies evaluated patient cancer samples in relation to survival and prognosis, as well as patient samples or cell lines with iron chelators.

Furthermore, 2 phase-1 studies with iron chelators indicated efficacy for some patients with hematologic malignancies. The combination treatment of the iron chelator 3-aminopyridine-2-carboxaldehyde (Triapine®) and cytarabine showed that 4 (3 AML and 1 ALL) out of31 evaluable patients (the majority had primary refractory AML) demonstrated a complete response with a median survival of 30.9 weeks compared to 12.6 weeks for all patients [67], Moreover, treatment with an oral formulation of the intracellular iron chelator ciclopirox olamine once a day for 5 days (40 mg/m²), was well tolerated, demonstrated sustained pharmacodynamic activity, and induced disease stabilization and/or hematologic improvements in 2/3 patients with relapsed or refractoryhematological malignancies (mostlyAML) [68], Moreover, at least one clinical studies of neuroblastoma, with the iron chelator DFO, has indicated promising anti-cancer effects [69].

In terms of mechanistic effects, the intracellular iron chelators Triapine® and DFX have been reported to have anticancer effects that are associated with ROS induction [63, 70] However, other reports suggest that the extracellular chelator DFO is unlikely to induce ROS because of its structure [59], In addition, ciclopirox olamine is an intracellular iron chelator that does not increase ROS levels in vitro, suggesting both DFO and ciclopirox olamine may exhibit different anticancer effects that may be less harmful for patients with iron overload diseases [68, 71], Additionally, the iron chelator deferiprone (DFP) has been shown to reduce iron overload and redistribute iron availability for hemoglobinization [72]. This kind of chelator may help prevent cancer and anemia from developing in patients with disorders involving iron maldistribution. These and other studies suggest more research is needed on the specific effects of iron chelation therapy, whether it be redox and/or iron depletion effects.

Conclusion

More and more studies suggest that altering iron metabolism is a valid approach for prevention and treatment of cancer. The cancer phenotype is generally associated with high iron requirements and high oxidative stress, which suggest that cancer cells may be more vulnerable to reaching iron deficiency and ROS levels that induce apoptosis in comparison with normal cells. Iron chelators have been shown to target either one of these vulnerabilities or may deplete iron and induce ROS concomitantly. However, depending on the redox potential of iron chelators and the environment, some chelators may be more appropriate for iron overload treatments while others may be more effective for cancer treatments. Such differences need to be clarified in future studies.

Furthermore, the effects of iron chelators need to be carefully evaluated in regard to the benefits and risks for patients with cancer, iron overload diseases, or within specific age groups or genders (e.g., post menopausal women and premenopausal women have different iron levels and different types of risks for breast cancer). Such therapeutics should also be further evaluated for iron sequestration, iron distribution, and other potential effects that may stave off cancer or contribute to tumor cell death. It also remains to be seen if there are clear differences between iron metabolism effects in specific types of cancers, such as carcinomas vs. sarcinomas, and whether specific cancers are more susceptible to ROS or iron depletion effects. In addition to iron chelators, manipulation of iron regulatory proteins or moderators of oxidative stress also have promising therapeutic implications for both the prevention and treatment of cancer. Overall, there are many irons in the fire for both cancer prevention and treatment strategies that target iron metabolism; however more studies are warranted that fully assess the safety, efficacy, and the mechanisms of these therapeutics.

Highlights.

• Iron is crucial for cellular growth and proliferation.

• Iron overload leads to high oxidative stress and carcinogenesis.

• Iron depletion and antioxidants may prevent cancer in iron overload diseases.

• Both iron depletion and pro-oxidants may selectively target cancer cells.

• Targeting iron metabolism may be beneficial to patients with iron overload and cancer.