Targeting cancer by binding iron: Dissecting cellular signaling pathways

Abstract

Newer and more potent therapies are urgently needed to effectively treat advanced cancers that have developed resistance and metastasized. One such strategy is to target cancer cell iron metabolism, which is altered compared to normal cells and may facilitate their rapid proliferation. This is supported by studies reporting the anti-neoplastic activities of the clinically available iron chelators, desferrioxamine and deferasirox. More recently, ligands of the di-2-pyridylketone thiosemicarbazone (DpT) class have demonstrated potent and selective anti-proliferative activity across multiple cancer-types in vivo, fueling studies aimed at dissecting their molecular mechanisms of action. In the past five years alone, significant advances have been made in understanding how chelators not only modulate cellular iron metabolism, but also multiple signaling pathways implicated in tumor progression and metastasis. Herein, we discuss recent research on the targeting of iron in cancer cells, with a focus on the novel and potent DpT ligands. Several key studies have revealed that iron chelation can target the AKT, ERK, JNK, p38, STAT3, TGF-β, Wnt and autophagic pathways to subsequently inhibit cellular proliferation, the epithelial-mesenchymal transition (EMT) and metastasis. These developments emphasize that these novel therapies could be utilized clinically to effectively target cancer.

Keywords: cancer; chelators; iron; signaling; thiosemicarbazones.

Figure 1

A. Cellular iron metabolism. Two atoms of ferric iron bind to transferrin (Tf) proteins present in the circulation to form the iron-saturated form of Tf (holo-Tf). Two molecules of holo-Tf can then bind to transferrin receptor 1 (TfR1) expressed at the cell membrane. The Tf-TfR1 complex undergoes endocytosis and is internalized into the endosome, inducing the release of Tf-bound ferric iron and reduction to its ferrous form by six-transmembrane epithelial antigen of the prostate 3 (STEAP3). Iron is then released into the cytosol via the divalent metal transporter 1 (DMT1) into the labile iron pool, where it can be: (i) incorporated into the active sites of proteins, such as ribonucleotide reductase; (ii) stored as cytoplasmic ferritin; (iii) utilized by the mitochondria; and (iv) exported out of the cell via the iron efflux pump, ferroportin 1 (Fpn1). The iron-deficient form of Tf (apo-Tf) and TfR1 molecules are subsequently recycled and returned to the cell surface. B. Chemical structures of key chelators discussed in this review. The structure of clinically available iron chelators, DFO and deferasirox, and anti-cancer thiosemicarbazone or PIH chelators, 3-AP and 311, respectively. The lead compounds of the first and second generation DpT thiosemicarbazones derived from PKIH analogues are shown, namely Dp44mT and DpC, respectively. Dp2mT is a structural analogue of Dp44mT that cannot bind iron and is used in studies as a negative control compound.

Figure 2. Iron chelation modulates multiple regulators of the cell cycle

Regulation of cell cycle progression is mediated by the integration of multiple cues from upstream signal transduction pathways. Iron chelation has been demonstrated to modulate the activities of ribonucleotide reductase (RR), cyclin/cyclin-dependent kinase (CDK) complexes, CDK inhibitors, and other cell cycle regulators including p53 and c-myc (see text for more detail). Molecules known to be negatively regulated by iron chelators are indicated by red boxes, while molecules known to be positively regulated by iron chelators are indicated by green boxes.

Figure 3. Iron chelators modulate PI3K/AKT signaling

Activated PI3K catalyzes the phosphorylation of PIP2 to PIP3, leading to phosphorylation and activation of AKT (p-AKT). PIP3 levels are controlled by the phosphatase PTEN to inhibit p-AKT, which affects multiple cascades that control cell growth, proliferation and apoptosis, including GSK3β, mTOR, cyclin D1, c-myc, BAD and Bcl-2 (see text for more detail). Molecules negatively regulated by iron chelators are indicated by red boxes, and those positively regulated are indicated by green boxes. Solid boxes indicate direct evidence for modulation by iron chelators, while dashed boxes indicate potential indirect modulation, e.g., through chelator-mediated up-regulation of NDRG1.

Figure 4

A. Iron chelation modulates mitogen-activated protein kinase (MAPK) signaling. Activated by growth factors and cytokines, the Ras/Raf/MEK/ERK cascade regulates cell growth and proliferation through multiple downstream effectors. ERK can also inhibit canonical TGF-β signaling by phosphorylating SMAD2 at the linker region (p-SMAD2L), preventing nuclear translocation of the SMAD2/3/4 complex and subsequent tumor-suppressive effects. The JNK and p38 pathways are two parallel MAPK cascades that are also classically activated by stress stimuli and may primarily mediate pro-apoptotic processes. Iron chelation is known to modulate the activities of ASK by dissociating its complex with thioredoxin (Trx), as well as JNK, p38, ERK and p-SMAD2L (see text for more detail). B. Modulation of STAT3 signaling by iron chelators and integrated pathway crosstalk between the MAPK and AKT pathways. STAT3 signaling is classically activated by cytokine receptor interactions, e.g., IL6/JAK, but can also be induced by growth factor receptor tyrosine kinases, e.g., EGFR, and non-receptor kinases, e.g., Src and c-Abl. Evidence also exists for potential STAT3 pathway crosstalk between the ERK and AKT pathways. Iron chelation has been demonstrated to inhibit STAT3 activity, which could be mediated by chelator-mediated effects on upstream regulators of STAT3 (see text for details). Molecules that are negatively regulated by iron chelators are indicated by red boxes, and those positively regulated are indicated by green boxes. Solid boxes indicate direct evidence for modulation by iron chelators, while dashed boxes indicate potential indirect modulation, e.g., through chelator-mediated up-regulation of NDRG1. Dashed arrows indicate interactions that remain to be fully characterized (see text for details).

Figure 5. Iron chelators regulate the epithelial-to-mesenchymal transition (EMT) and molecular motors involved in cancer cell migration and metastasis

Non-canonical activation of TGF-β signaling promotes tumor progression, EMT and metastasis via SMAD, MAPK and PI3K/AKT pathways that up-regulate mesenchymal-associated transcription factors (e.g., Snail, Slug, Twist and ZEB) and repress E-cadherin expression. The Wnt signaling pathway also regulates EMT through its inhibition of GSK3β-mediated degradation of β-catenin, Snail and Slug. TGF-β can also activate Rho/ROCK/MLC signaling to drive dynamic actin reorganization required for metastasis. Iron chelators have been demonstrated to inhibit the EMT and metastasis by maintaining expression of E-cadherin and β-catenin at cell membranes, and may also mediate these effects through regulation of other upstream cascades (see text for more details). Molecules negatively regulated by iron chelators are indicated by red boxes, and those positively regulated are indicated by green boxes. Solid boxes indicate direct evidence for modulation by iron chelators, while dashed boxes indicate potential indirect modulation, e.g., through chelator-mediated up-regulation of NDRG1.

Figure 6. Iron chelation modulates ER stress and autophagy pathways

Autophagy involves the formation of double-membrane vesicles from phagophores, i.e., autophagosomes, which engulf cytoplasmic contents and organelles. Fusion of the autophagosome with a lysosome forms an autolysosome that results in the degradation of its contents, nutrient recycling and autophagic cell survival. However, prolonged autophagy in certain settings can also lead to non-apoptotic autophagic cell death. Two core regulators of autophagy include the PERK/eIF2α pathway that is activated by endoplasmic reticulum (ER) stress, and the PI3K/AKT/mTORC1 pathway. Iron chelators have been demonstrated to modulate both these pathways and subsequent autophagic processes, and also enhance expression of LC3-II, which is recruited to autophagosomal membranes during induction of autophagy (see text for more details). Molecules negatively regulated by iron chelators are indicated by red boxes, and those positively regulated are indicated by green boxes.