"VSports在线直播" Iron and cancer: recent insights

Iron uptake and storage

Transferrin receptor 1 (TFRC1), a transmembrane glycoprotein homodimer, binds diferric transferrin and is essential for iron uptake and the regulation of cell growth. Many studies have shown that cancer cells have increased expression of transferrin receptor 1 when compared to their normal counterparts.^15,16 This observation has led to the exploration of TFRC1-targeted therapeutic approaches that target TRFC1 either directly, using antibodies, or indirectly, using transferrin conjugates that bind to TFRC1 to achieve tumor-specific drug delivery.^15,16 High levels of TFRC1 expression in cancers such as breast,¹⁷ renal cell,¹⁸ and nonsmall cell lung cancer¹⁹ are correlated with poorer patient prognosis. The expression of TFRC1 is regulated at the posttranscriptional level by IRPs in response to intracellular iron levels.²⁰ TFRC1 is also a direct transcriptional target of the oncoprotein c-Myc.²¹ Recently it was reported that cancer stem-like cells (CSCs) in glioblastoma express increased levels of TFRC1 and its ligand transferrin compared to non-CSCs.²² Iron-tracing experiments on these CSCs showed increased iron uptake in CSCs compared with non-CSCs, indicating that enhanced iron uptake may also be a property of cancer stem-like cells.

In addition to increasing iron uptake, cancer cells can increase intracellular iron by modulating levels of the iron-storage protein ferritin. Ferritin sequesters excess iron for storage, allowing iron to accumulate intracellularly while simultaneously preventing iron-mediated free radical–generating reactions. Ferritin is overexpressed in many cancer tissues, including breast,^23–25 pancreatic,²⁵ and hepatocellular cancers,²⁶ Hodgkin’s lymphoma,²⁷ and glioblastoma multiforme.²² However, the specific cell types within the tumor that overexpress ferritin have not always been examined. In tumors of the breast, there is strong ferritin expression in stromal cells within tumor tissue.²⁸ Further analysis revealed that the ferritin-rich stromal cells are CD68⁺ macrophages that infiltrate into tumors and can secrete ferritin.²⁹ These tumor-associated macrophages may release ferritin locally. Interestingly, locally secreted ferritin appeared to contribute to tumor cell proliferation through an iron-independent mechanism.²⁹ An iron-independent function of ferritin in the induction of inflammatory pathways has been previously suggested.³⁰ In addition to stromal cells, ferritin is increased in glioblastoma CSCs compared to non-CSCs and the non-neoplastic brain, similar to the results for TFRC1 described above.²² Targeting both the H and L subunits of ferritin with small hairpin RNA (shRNA) resulted in dramatic reduction in CSC growth both in vitro and in vivo, suggesting that storage of iron is crucial for tumor growth.²²

Iron efflux

Iron efflux mediated by FPN-1 and controlled by the peptide hormone hepcidin plays an important role in tumor growth and metastasis. Many cancer types exhibit an altered regulation of FPN-1. FPN-1 is significantly diminished in breast, prostate, and hepatocellular cancer cells as compared to the normal counterparts.^31–34. In breast cancer, diminished FPN-1 is associated with reduced metastasis-free and disease-specific survival and is predictive of patient outcome. Low levels of FPN-1 are significantly correlated with poor prognostic markers, such as absence of estrogen receptors, high histological grade, and lymph node metastasis.³² Studies from several groups have shown that forced expression of FPN-1 in breast cancer cells inhibits growth of cancer xenografts in mice.^32,35 Further, FPN-1 overexpression in breast cancer cells impaired metastasis to lung and liver in mice and significantly decreased the levels of epithelial mesenchymal transition (EMT) markers, such as Snail1, Twist1, ZEB2, and Vimentin.³⁵

The expression of FPN-1 is regulated at transcriptional, posttranscriptional, and posttranslational levels.¹¹ In macrophages, FPN-1 is transcriptionally regulated by heme, which increases FPN-1 transcription by modulating Bach1.³⁶ Bach1 binds to the antioxidant response element (ARE) present in the promoter sequence of FPN-1 and represses its transcription.³⁶ Heme causes the degradation of Bach1, leading to the derepression of transcription. Another factor that affects FPN-1 transcription is hypoxia; activation of hypoxia-inducible factor (HIF) in enterocytes upregulates FPN-1 expression.³⁷ In breast cancer cells, transcription of FPN-1 is activated by the transcription factors Nrf2 (Nuclear factor erythroid 2–like 2)³⁸ and MZF1 (myeloid zinc finger-1).³¹ Posttranscriptionally, FPN-1 is regulated by IRPs. Most cell types predominantly express FPN-1 isoform 1a mRNA, which contains a 5′ IRE. Under conditions of iron deficiency, IRPs bind to a 5′ IRE, repressing FPN-1 translation and decreasing expression of FPN-1 on the cell membrane.³⁹ However, IRP regulation of FPN-1 is finely tuned to meet cellular and organismal needs. In enterocytes, depletion of FPN-1 by iron deficiency would reduce uptake from the diet and be detrimental to an organism that was iron deficient. Hence, enterocytes express FPN-1 isoform 1b mRNA, which lacks the 5′ IRE. FPN-1 is also regulated posttranslationally by the peptide hormone hepcidin, which binds to FPN-1 resulting in FPN-1 internalization and proteolysis in lysosomes.⁴⁰ Mechanisms underlying the reduction in FPN-1 expression in cancer cells are incompletely understood. However, aberrant DNA methylation of the FPN-1 gene has been reported to contribute to the downregulation of FPN-1 in breast tumors.³¹

Hepcidin–ferroportin axis in cancer

Hepcidin, the negative regulator of FPN-1, is also deregulated in neoplastic disease.^32,33,40,41 Serum hepcidin is elevated in patients with many cancers, including prostate cancer,⁴² breast cancer,⁴³ multiple myeloma,⁴⁴ non-Hodgkin’s lymphoma,⁴⁵ hepatocellular carcinoma (HCC),⁴⁶ ovarian cancer, and upper gastrointestinal tract tumor.⁴⁷ In addition to systemic hepcidin, cancer cells also synthesize hepcidin. For example, breast, colorectal, and prostate cancer cells synthesize higher amounts of hepcidin than their non-malignant counterparts.^32,33,41

Hepcidin is regulated by a number of signaling pathways, with major roles played by the bone morphogenetic protein (BMP) and interleukin-6 (IL-6) inflammatory pathways (see Refs. 12 and 48–50 for recent reviews). Dysregulation of these pathways in malignant cells^33,51–53 likely contributes to the dysregulation of hepcidin in cancer.^33,34,54 In particular, BMP, IL-6, and Wnt signaling all contribute to enhanced hepcidin production in prostate cancer.³³ BMP signaling through SMADs (vertebrate homologs of Sma and mothers against decapentaplegic) and IL-6 signaling through STAT3 (signal transducers and activators of transcription 3) have been further implicated in breast cancer.^34,54

Dysregulation of hepcidin in cancer results in altered iron homeostasis. High plasma hepcidin blocks the mobilization of iron into the circulation from enterocytes and macrophages (contributing to the anemia seen in cancer) and can also cause the accumulation of iron in tumor cells by degrading FPN-1, resulting in the activation of signaling pathways such as Wnt⁵⁵ and NF-κB,⁵⁶ which contribute to tumor progression.⁵⁷ The tumor-promoting role of circulating hepcidin was demonstrated using hepcidin (Hamp^−/−) knockout mice. Hamp^−/− mice implanted with Lewis lung cancer cells (LL/2 cells) displayed improved survival and fewer metastases than wild-type mice.³⁵ In addition to systemic hepcidin, cancer cells also synthesize hepcidin. For example, breast, colorectal, and prostate cancer cells synthesize higher amounts of hepcidin than their nonmalignant counterparts.^32,33,41. Hepcidin generated by cancer cells initiates a local autocrine signaling to degrade membrane FPN-1.^32,33 High hepcidin expression in tumors has been associated with metastatic progression and poor prognosis.^32,58 Accumulating evidence suggests that the pro-oncogenic nature of hepcidin may be due to its ability to increase intracellular iron content in tumor cells by initiating internalization and degradation of FPN-1.

"V体育ios版" Ferroptosis

Ferroptosis, named for its dependence on iron,^59,60 is a recently characterized form of non-apoptotic cell death (Fig. 2). Ferroptosis was first identified in a high-throughput screen of small molecules that selectively kill cells overexpressing oncogenic HRAS,⁶¹ although subsequent work has shown that ferroptosis is not limited to cells with activated HRAS.^60,62 In this study, the small molecule erastin induced cell death with distinct morphological and mechanistic characteristics,⁶¹ differentiating ferroptosis from other forms of cell death.²⁵ Ferroptosis is distinguished from apoptosis by the lack of chromatin margination, nuclear fragmentation, plasma membrane blebbing, and caspase activation.^60,61,63 Ferroptosis is differentiated from autophagy owing to a lack of autophagosome formation and changes in microtubule-associated protein light chain 3 (LC3).^61,64,65 Ferroptosis is also distinct from classical necrosis as there is no rapid depletion of ATP.⁶⁰ Furthermore, ferroptosis was not repressed by known inhibitors of apoptosis, autophagy, or necrosis.⁶⁰ Instead, ferroptosis is morphologically characterized by reduced mitochondrial size with increased membrane density.^60,63 Mechanistically, ferroptosis causes cell death through iron-mediated accumulation of lipid ROS.^59,60,66 Accumulation of lipid ROS interferes with cellular integrity and membrane fluidity and permeability, effects which can be lethal.⁶⁷

Iron, which plays a role in several types of cell death,⁶⁶ has long been known to participate in the generation of ROS through the Fenton reaction.⁴ It is likely that iron-catalyzed ROS production is primarily responsible for ferroptotic death, although iron may also function as a cofactor for enzymes involved in ROS production.⁶⁶ Indeed, the sensitivity of cells to ferroptosis can be modulated by cellular iron status (Fig. 2). Increasing intracellular iron by iron treatment or through transferrin and transferrin receptor promotes ferroptosis.^60,68 Heme oxygenase-1, which promotes iron availability⁶⁹ but also functions as an antioxidant,⁷⁰ has an unclear effect on ferroptosis. Conversely, reducing intracellular iron through iron chelation reduces ferroptosis.⁵⁹ Heat shock protein β-1 (HSPB1) protects cancer cells from ferroptosis through reduction of intracellular iron and lipid ROS.⁷¹. In vivo, HSPB1 knockdown enhanced the antitumor activity of the ferroptosis inducer erastin.⁷¹ Iron loading of cancer cells, such as through aminoferrocence-based therapies,^72,73 may prove useful therapeutically by enhancing ferroptosis in cancer cells.

In addition to iron status, other sources of ROS seem to play roles in ferroptosis. In some cells, ROS are derived from nicotinamide adenine dinucleotide phosphate (NADPH) generated by NADPH oxidase 1 (NOX1).⁶⁰ NAPDH is produced by several cellular reactions, but the pentose phosphate pathway is particularly important for ferroptosis in RAS-mutated cancer cells.^60,74 However, ROS generated from the mitochondrial electron transport chain, which was originally found to not contribute to ferroptosis,^60,65 may contribute under certain circumstances.⁷⁵

The importance of ROS in ferroptosis led to the study of antioxidant pathways as potential regulators of ferroptosis. Glutathione (GSH) is a potent antioxidant⁷⁶ whose production is limited by the availability of cystine/cysteine.⁷⁷ Erastin reduced GSH production through inhibiting cystine uptake by system X_c^−.60 Recently, p53, a well-characterized tumor suppressor,⁷⁸ was also shown to sensitize cells to ferroptosis. Similar to erastin, p53 reduces cystine uptake and GSH by repression of the SLC7A11 component of system X_c^−.79,80 In contrast, increases in cysteine and GSH following knockdown of cysteinyl-tRNA synthetase (CARS) protected cancer cells from erastin-induced ferroptosis.⁸¹ Furthermore, GSH is a cofactor essential for glutathione-dependent peroxidase (GPX) function.⁸² GPX4, which normally reduces lipid peroxides formed by ROS,⁸² is particularly important in ferroptosis.⁸³ GPX4 was identified in a screening for target proteins of a known ferroptosis inducer, RSL3.⁸³ GPX4 overexpression rescued ferroptotic cell death induced by a panel of 12 structurally diverse ferroptosis inducers, indicating a central role for GPX4 in regulation of ferroptosis⁸³ (Fig. 2). In addition to its role in cancer, modulation of ferroptosis by GPX4 seems to play an important role in ischemia/reperfusion-induced tissue damage^{62, 84} as well as motor neuron survival.³¹

Several modulators of ferroptosis have been identified through the study of ferroptotic mechanisms (Fig. 2). Most ferroptosis inducers can be classified based on their mechanism of action⁸³ (Table 1). Class I ferroptosis inducers indirectly reduce GPX4 function by depleting GSH. Class I ferroptosis inducers include erastin, erastin derivatives (aldehyde erastin, morpholine erastin II, and piperazine erastin), buthionine sulfoximine, DPI2, glutamate, lanperisone, sulfasalazine, SRS13-45, SRS13-60, and sorafenib.^{60,70,83,85,86} Class 2 ferroptosis inducers directly inhibit GPX4 function and include DPI7, DPI10, DPI12, DPI13, DPI17, DPI18, DPI19, ML162, and RSL3.^59,83,87 Ferroptosis inducers that function by mechanisms not involving direct depletion of GSH or inhibition of GPX have also been identified. Sorafenib, a small molecule kinase inhibitor that is approved by the U.S. Food and Drug Administration for treatment of renal cell, hepatocellular, and differentiated thyroid carcinomas was also found to induce ferroptotic cell death by inducing ROS.^75,88 Similarly, artemisinin derivatives, including artesunate, induce ferroptosis through iron-mediated ROS generation.^89,90

Ferroptosis inhibitors have also been identified (Table 1). While ferroptosis inducers are envisioned as cancer therapeutics, inhibitors of ferroptosis may prove useful in minimizing ischemia/reperfusion-induced tissue damage,⁸⁴ or perhaps in protecting normal cells from being targeted by ferroptosis inducers. Ferroptosis inhibitors exhibit varied mechanisms of action, all primarily involving a reduction of ROS. Antioxidants (vitamin E, Trolox, U0126)^{60, 83} and inhibitors of ROS production, such as ferrostatin-1 and its more potent derivatives (SRS8-72, SRS11-92, SRS12-45, SRS13-35, SRS13-37, and SRS16-86),^60,62,91 also show antiferroptotic function. β-Mercaptoethanol inhibits ferroptosis by increasing cystine uptake.^60,86 The iron chelators desferoxamine, 2,2-bipyridyl, and ciclopiroxolamine are able to inhibit ferroptosis by reducing iron and ROS.⁶⁰ Cycloheximide, aminooxyacetic acid, and ebselen––a protein synthesis inhibitor, a transaminase inhibitor, and a GPX mimetic, respectively––also inhibit ferroptosis.^60,92 These agents may provide leads to clinically relevant therapeutics.

Ferritinophagy (VSports)

Autophagy is a lysosomal-mediated cellular self-digestion process that is upregulated in response to stress stimuli. Autophagy involves formation of double-membrane autophagosomes, which engulf cellular components and deliver them to lysosomes for degradation.⁹³ Autophagy helps in maintaining normal cellular homeostasis by acting as a recycling mechanism: cells degrade damaged organelles and protein aggregates by autophagy and use the products to refuel supplies of amino acids, lipids, nutrients, and metals.⁹⁴ Recent evidence suggests that selective autophagy is critical for iron homeostasis.^95,96 The iron-storage protein ferritin plays a key role in this process. Ferritin is a 24-subunit protein comprised of ferritin light and heavy chains. Ferritin is able to store up to 4500 iron atoms.⁹⁷ Under conditions of low intracellular iron, ferritin is degraded to release iron for use by the cell. This degradation of ferritin occurs in lysosomes through a targeted autophagic process referred as ferritinophagy.^95,96 Nuclear receptor coactivator 4 (NCOA4) was recently identified as an important protein in ferritinophagy. NCOA4 binds to ferritin and delivers it to lysosomes for degradation. Ncoa4^−/− mice exhibited significant accumulation of iron in spleen, indicating the disruption of iron storage and recycling.⁹⁵ The role of this process in cancer has not been studied; however, we speculate that activation of this pathway may represent an additional source of iron in malignant cells.

MicroRNAs

MicroRNAs (miRNAs) are a class of small, noncoding RNAs that function to regulate gene expression posttranscriptionally by sequence-specific binding to target mRNAs, resulting in their degradation.⁹⁹ Since deregulation of miRNAs is implicated in cancer, alterations of miRNAs that control iron metabolism may represent a novel mechanism through which cancer cells alter their iron phenotype.¹⁰⁰

One study designed specifically to identify miRNAs that posttranscriptionally regulate FPN-1 identified 44 miRNAs that were differentially expressed after exogenous addition of iron by ferric ammonium citrate (FAC) or chelation of iron by deferoxamine (DFO).¹⁰¹ After analyzing the predicted targets of these miRNAs, two miRNAs, miR-485-3p and miR-194, were predicted to target the 3′ UTR of FPN-1. Of these two, miR-485-3p was the most induced during cellular iron deficiency across cell types. The functional significance of FPN-1 targeting by miR-485-3p was demonstrated by an increase in ferritin, a protein that is translationally induced by intracellular iron and is frequently used as an indirect indicator of high intracellular iron. Presumably, the miR-485-3p–mediated decrease in FPN-1–dependent iron efflux was the cause of increased ferritin. Thus, miR-485-3p may act as a mechanism to restore iron homeostasis in cells challenged by iron deficiency. Reduced FPN-1 is also observed in cancer cells, as tumor cells require increased iron to support their proliferative demands.^32,33 It is plausible that this may in part be due to increased levels of miR-485-3p. It will be interesting to determine if miR-485-3p is upregulated in cancer cells, and if that contributes to the basal reduction of FPN-1 present in tumors.

In addition to controlling iron export, miRNAs also regulate proteins that control iron import. TFR1 was shown to be a direct target of miRNA-210: overexpression of miRNA-210 inhibited TFR1 expression and decreased uptake of transferrin-bound iron and proliferation.¹⁰² In addition, TFR1 was identified as a target of miRNA-152 in a computational analysis of HCC.¹⁰³ Using the TCGA database, an inverse correlation was identified between TFR1 and miRNA-152, where miRNA-152 levels were reduced and TFR1 was overexpressed in human HCC tissue compared to non-tumor liver tissue.¹⁰³ This inverse relationship was confirmed experimentally, as transfection of HCC with miRNA-152 resulted in suppression of TFR1, identifying a novel means to target TFR1 and restore normal iron levels in cancer cells.¹⁰³ MicroRNAs also control expression of iron-storage proteins, as miR-200b was shown to target the iron-storage protein ferritin heavy chain (FTH).¹⁰⁴ This study revealed that FTH is upregulated in the aggressive, triple-negative breast cancer cell line MDA-MB-231.¹⁰⁴ The increase of FTH corresponds to decreased levels of miR-200b, and reintroduction of miR-200b experimentally resulted in decreased FTH expression, suggesting that FTH is a direct target of miR-200b.¹⁰⁴ Interestingly, the reduction in FTH by miR-200b was associated with increased sensitivity to doxorubicin chemotherapy treatment, suggesting that reducing iron storage may sensitize cancer cells to apoptosis.¹⁰⁴ Other miRNAs that may prove useful in targeting the disrupted iron metabolism observed in cancer cells are displayed in Table 2.

VSports在线直播 - Epigenetic control

Epigenetic mechanisms alter the accessibility of chromatin to transcriptional regulation through modifications of DNA or modification or rearrangement of nucleosomes.¹¹⁶ Alterations in epigenetic profiles are an important mechanism through which cancer cells modify gene expression.¹¹⁶ Specific epigenetic alterations to iron-related genes have been reported in cancer cells.

DNA hypermethylation observed at CpG islands in proximal promoters is an epigenetic mechanism of gene silencing.¹⁰⁵ One study observed CpG island methylation at the FPN-1 promoter, which resulted in reduced basal FPN expression in breast cancer cells.³¹ Hepcidin, the negative regulator of FPN-1, is also controlled by promoter hypermethylation, although in this case the regulation is exerted indirectly, through methylation of HAMP regulators.³³ One such regulator, SOSTDC1, a negative regulator of hepcidin, was found to be epigenetically silenced through promoter hypermethylation in prostate cancer.³³ SOSTDC1 silencing increased expression of hepcidin, reduced FPN-1, and increased intracellular iron in prostate tumor cells. Methylation of certain histone sites, such as histone H3 at lysine 4 (H3K4me), are epigenetic markings for gene activation and have also been observed in the HAMP promoter.¹¹⁷ Specifically, exogenous addition of TGF-β or BMP-4, positive regulators of hepcidin, resulted in increased H3K4 methylation at the hepcidin promoter, consistent with increased hepcidin expression associated with this activating mark.¹¹⁸

Acetylation of histones is another epigenetic mechanism for controlling gene expression.¹¹⁶ Generally, histone acetylation corresponds to transcriptional activation by providing an open chromatin structure for active transcription of genes.¹¹⁹ Malignant cells often exhibit upregulation of histone deacetylases (HDAC), enzymes responsible for removing acetyl groups from histones, promoting closed chromatin state and thus repressing transcription of tumor suppressor genes.¹¹⁹ For this reason, HDAC inhibitors have emerged as promising anticancer therapeutics. HDAC inhibitors function by inhibiting HDAC enzyme activity, thus maintaining histone acetylation and subsequent gene expression of critical tumor suppressor genes.¹¹⁹ Interestingly, the H subunit of the iron-storage protein ferritin was found to be induced by HDAC inhibitors, through recruitment of NF-Y transcription factor to the ferritin H promoter.¹²⁰ In this case, restoring histone acetylation results in increased ferritin H. Increased ferritin H was hypothesized to promote apoptosis through its ability to sequester and bind iron, limiting metabolically active iron cancer cells require to proliferate. It is expected that other genes encoding proteins involved in iron metabolism may be modified through epigenetic mechanisms in cancer to allow for increased iron availability.