Transferrin receptor 2 is frequently expressed in human cancer cell lines

Abstract

Different proteins ensure the fine control of iron metabolism at the level of various tissues. Among these proteins, it was discovered a second transferrin receptor (TfR2), that seems to play a key role in the regulation of iron homeostasis. Its mutations are responsible for type 3 hemochromatosis (Type 3 HH).

Although TfR2 expression in normal tissues was restricted at the level of liver and intestine, we observed that TfR2 was frequently expressed in tumor cell lines. Particularly frequent was its expression in ovarian cancer, colon cancer and glioblastoma cell lines; less frequent was its expression in leukemic and melanoma cell lines.

Interestingly, in these tumor cell lines, TfR2 expression was inversely related to that of receptor 1 for transferrin (TfR1).

Experiments of in vitro iron loading or iron deprivation provided evidence that TfR2 is modulated in cancer cell lines according to cellular iron levels following two different mechanisms: (i) in some cells, iron loading caused a downmodulation of total TfR2 levels; (ii) in other cell types, iron loading caused a downmodulation of membrane-bound TfR2, without affecting the levels of total cellular TfR2 content. Iron deprivation caused in both conditions an opposite effect compared to iron loading.

These observations suggest that TfR2 expression may be altered in human cancers and warrant further studies in primary tumors. Furthermore, our studies indicate that, at least in tumor cells, TfR2 expression is modulated by iron through different biochemical mechanisms, whose molecular basis remains to be determined.

Introduction

Iron is essential for almost all living organisms as iron-containing proteins catalyze a wide variety of key metabolic processes, including hemoglobin synthesis in erythroid cells, oxygen transport, DNA synthesis, and electron transport. The best characterised mechanism for iron uptake is the binding of the serum iron carrier protein transferrin to the transferrin receptor 1 (TfR1). In 1999, Kawabata and colleagues cloned a second TfR-like molecule known as the transferrin receptor 2 (TfR2) [1]; unlike TfR1, TfR2 is not regulated by intracellular iron levels and appears to be regulated in accordance with the cell cycle [2], [3]. Major differences between the two TfRs concern their expression patterns. TfR1 is expressed on all cells, except for mature erythrocytes and terminally differentiated cells, while human TfR2 mRNA is highly expressed in the liver and to a lesser extent in spleen, lung, muscle, prostate and peripheral mononuclear cells [1], [2]. Moreover, TfR2 transcript is found to be highly expressed in erythroid precursor cells [2], whereas the protein is not expressed at any stage of normal erythroid differentiation [4]. As TfR2 is able to bind transferrin and internalize iron [5], even if with a lower affinity compared to TfR1, it was initially considered as a second mediator for iron uptake. This first interpretation is however not convincing, since TfR2 is not able to compensate for TfR1 function in TfR1-knockout mice, which died before embryonic day 12.5 because of severe anemia and neurological abnormalities [6]. Furthermore, mutations of the TfR2 gene produce hemochromatosis type 3, with significant hepatic iron loading [7], [8], and TfR2-deficient mice show a phenotype of parenchymal iron overload and features of human TfR2-related hemochromatosis [9]. These observations clearly indicate that TfR2 has a unique but yet unknown role in the regulation of iron homeostasis rather than a simple contribution to cellular iron uptake.

Iron is a basic requirement for cellular proliferation and viability of both normal and neoplastic cells and its depletion leads to G₁/S arrest and apoptosis [10]. Given its essential role in cell metabolism and its pro-oxidant capacity, it is not surprising that altered iron homeostasis may be involved in the development of cancer. Indeed, tumor cells generally have higher levels of TfR1 than their normal counterparts [11] and take up Fe at a higher rate [12], [13], [14]. Considering the essential role of iron, several genes involved in cell cycle progression and proliferation have been shown to be regulatedby intracellular Fe levels (for review see [15]) and the molecular mechanisms of iron carcinogenesis have been investigated.

Many components of the cell division machinery, such as cyclin and cyclin-dependent kinases (cdk), require iron [16]. Although none of the cell cycle regulatory proteins is known to bind iron directly, Sanchez et al. [17] recently identified a novel iron-responsive element (IRE) located in the 3′-untranslated region of mRNA encoding the cell division cycle 14A (CDC14A) tumor suppressor gene. CDC14A has been shown to dephosphorylate cdk substrates that are critical for the G₁ to S phase progression, such as cyclin E [18], and alteration of CDC14A expression has been found to cause abnormal chromosome segregation, arguing that CDC14A plays an important role in cell division [18], [19]. The newly identified CDC14A IRE was able to bind to IRP1 and IRP2 and the CDC14A IRE mRNA was specifically regulated by iron deficiency [17], demonstrating that this molecule belongs to the IRE/IRP regulatory network and suggesting a possible new link between iron metabolism and the cell cycle.

The cell cycle is regulated further by checkpoint mechanisms that cause growth arrest and/or apoptosis in the event of cellular damage. These multiple cell cycle checkpoints are regulated by the tumor suppressor gene p53 [20], which is expressed at low levels in normal cells and is frequently mutated or inactive in human tumors [21]. Iron chelation causes an increase in the protein levels and DNA-binding activity of p53 [22], [23], which is therefore susceptible to cellular Fe levels.

Apart from the effect of iron on the cell cycle, the correlation between Fe levels and the role of proto-oncogenes such as c-myc in controlling cellular proliferation has also been investigated. Studies by Wu et al. [24] have demonstrated that genes controlling intracellular Fe levels, namely IRP2 and H-ferritin, are coordinately regulated targets of c-myc. Indeed, c-myc inhibits the expression of H-ferritin while inducing the expression of IRP2, leading to free iron accumulation and oxidative stress. Furthermore, O'Donnell et al. [25] showed that TfR1 is a critical downstream target of c-myc in a human B lymphocyte model system, that TfR1 expression correlates with inducible c-myc expression in an in vivo murine model of lymphoma and that enforced expression of TfR1 confers a growth advantage to cells and greatly enhances the rate of c-myc -mediated tumorigenesis in nude mice. These findings indicate that TfR1 plays a pivotal role in the c-myc target gene network and likely explain while this receptor is commonly over-expressed in human tumors.

Recently, several investigations have shown that expression of the metastasis suppressor Ndrg-1, a downstream target of tumor suppressor genes such as p53 and oncogenes such as c-myc and N-myc, is linked to intracellular iron levels [26] and induced by Fe depletion [27]. Furthermore, a role of Fe(III) in the modulation of caspase-3 activity and in the regulation of apoptosis was established [28]. These new results support previous observations that iron chelators can induce apoptosis in various cancer cell lines and provide a direct evidence of the correlation between iron and tumor growth and proliferation.

For the investigations described above, it is clear that iron is an attractive target for chemotherapeutic agents and several cell permeable iron chelators are currently studied on cancer cells in vitro, in vivo and in a number of clinical and preclinical trials. For instance, the aggressive ribonucleotide reductase inhibitor Triapine®, a tridentate Fe chelator, has been shown to be effective in tumor models in animals [29] and has entered phase I and phase II clinical trials [30], while other compounds showing a great anti-proliferative activity in vitro, such as tachpyridine, require evaluation in animal models. A new class of recently identified iron chelators displayed in vivo in animal models a wide spectrum of potent antitumor activity that overcomes resistance to chemotherapeutics [31].

Numerous studies concerning Fe metabolism of cancer cells have attempted to understand the function, regulation of expression and clinical relevance of TfR1. In contrast, very little is known about TfR2. High levels of TfR2 mRNA have been detected in HepG2 (human hepatoma), K562, and HEL-R (both human erythroleukemic) cell lines [1], [2]. The human lymphoid cell lines Raji and MOLT-16 and the human myeloid cell lines U937, NB-4, HL-60, KCL22 and KG-1 expressed low or undetectable levels of TfR2 mRNA [2]. An analysis of TfR2 expression in patients showed that myeloid and nonerythroid leukemia cases displayed significantly elevated TfR2 levels compared to non-malignant bone marrow cells and that this up-regulation contributed to a better prognosis [2], [32].

Importantly, all aforementioned studies on neoplastic cells only tested TfR2 expression at mRNA level, while data at the protein level are completely lacking. Therefore, the aim of our study is to investigate TfR2 protein expression by flow cytometry and Western blot analysis in a wide variety of human cell lines derived from solid tumors and leukemic cell lines.