Cell Surface Transferrin Receptor Research Articles

High transferrin receptor expression marks tumors with increased immune-effector infiltration and expression of targetable checkpoint molecules: Results of a multi-cancer analysis in solid tumors.

2613 Background: The cell-surface transferrin receptor, TFR1, encoded by TFRC, imports iron-bound transferrin into cells to drive tumor proliferation. We have found worse prognosis with high TFRC expression across multiple tumor types. The mechanism by which TFRC drives tumor cell- intrinsic oncogenesis is multifactorial, but its interaction with the tumor microenvironment (TME) has not been studied. Here, we assessed TME infiltration by immune effector populations and immune checkpoint expression. Methods: Tissue samples underwent comprehensive molecular profiling at Caris Life Sciences: Next-gen sequencing of DNA (592 Gene Panel, NextSeq, or whole exome sequencing, NovaSeq), RNA- seq (NovaSeq, whole transcriptome sequencing, WTS) and immunohistochemistry. RNA deconvolution analysis by the Microenvironment Cell Populations (MCP)-counter method quantified immune populations to assess T cells, B cells, natural killer cells (NK) and myeloid dendritic cells (MDC). Analyses compared quartiles of TFRC mRNA expression between Q4 ( TFRC-H) high and Q1 ( TFRC-L) low expressors. Results: Among 27,607 patients with non-small cell lung cancer (NSCLC), 5,332 with prostate cancer (PC), and 14,287 with colorectal cancer (CRC), TFRC-H tumors were associated with high tumor mutational burden (TMB-high, ≥10 mutations/MB), and TP53 mutation (q<0.05). Multiple immune checkpoint genes showed significantly higher expression in TFRC-H relative to TFRC-L in all tumor types, including CD274 (PDL1), LAG3, PDCD1LG2 (PDL2), and CTLA4 (Table; All values are statistically significant: q<0.05). Moreover, TME assessment by MCP-counter showed significantly increased infiltration by total T cells, CD8+ cells, B cells, NK cells, and MDCs in TFRC-H (Table). Finally, among patients who received Check-point inhibitors (CPI), TFRC-L had improved survival (HR=0.807 (95% CI 0.76 – 0.85) p<0.00001) in NSCLC but no difference was found in CRC or PC. Conclusions: Our study is the first to reveal the TME characteristics associated with TFRC expression in a variety of solid tumor types. We found TFRC-H tumors have increased infiltration by immune effector populations but also express significantly higher levels of immune checkpoint molecules, highlighting immune exhaustion. Altered iron metabolism has been shown to promote tumorigenesis and here we identify a new role for TFRC in remodeling the TME. TFR1- targeting therapeutic agents are in clinical development and warrant further investigation in combination with CPIs. [Table: see text]

G-Quadruplex-Proximized Aptamers (G4PA) Efficiently Targeting Cell-Surface Transferrin Receptors for Targeted Cargo Delivery.

DNA-assembled multiaptamer systems have been demonstrated to significantly promote the aptamer capacity of binding cell-surface-expressed proteins. However, how to conveniently harness them for efficient transmembrane delivery of targets remains a challenge. Toward this goal, here we engineer a G-quadruplex-proximized aptamer (G4PA) system in which a DNA aptamer specific for transferrin receptor (TfR) is guided by a bimolecular G4 and assembles into a dimerized proximity form that well matches homodimeric TfR highly expressed on the cancer cell surface. This system displays a higher capacity for targeting cell-surface TfR than the monomeric aptamer and super transmembrane transportation of nucleic acid cargoes, which is comparable to that of conventional liposome transfection but overcomes the lack of targeting ability of the latter. The G4PA system is then applied to the targeted delivery of siRNA for PLK1 gene silencing in positive cells rather than negative controls, showing great promise for use in precise anticancer therapy.

Pan-cancer association between increased iron utilization and poor prognosis highlights potential of transferrin receptor-targeting therapies in multiple tumor types.

3120 Background: The cell-surface transferrin receptor TFR1 imports iron-bound transferrin into cells via clathrin-mediated endocytosis. Tumors require constitutive iron import to drive proliferation, and several studies establish TFR1 as a target able to facilitate intracellular delivery of cytotoxic therapeutic molecules. Our own work previously revealed association between high expression of TFRC, the gene encoding TFR1, and high risk for poor outcome in diffuse large B-cell lymphoma (DLBCL). We showed therapetuic targeting of TFR1 in DLBCL results in significant anti-tumor benefit. Systematic analysis of TFRC expression as a prognostic marker across tumor types, however, has not been investigated. Methods: Tissue samples underwent comprehensive molecular profiling at Caris Life Sciences. Analyses included next generation sequencing of DNA (592 Gene Panel, NextSeq, or whole exome sequencing, NovaSeq), RNA (NovaSeq, whole transcriptome sequencing, WTS) and immunohistochemistry. Overall survival (OS) was calculated from date of tissue collection to last contact from insurance claims data and employed Kaplan-Meier analysis by Wilcoxon statistics, with p < 0.05 defined as significant. Results: Amongst 47 cancer types included, colorectal cancer (CRC) displayed the highest level of TFRC mRNA, followed by gastric cancer. In an all-tumor cohort (n = 93248), patients with higher TFRC expression (cutoff = median) had significantly worse OS (HR = 1.348, 95% CI [1.317-1.38], p < 0.00001). This was statistically significant in 23 individual tumor types. Drilling down further, TFRC adverse prognostic value was mainly driven by cohorts with larger number of samples in the database, including non-small cell lung cancer (n = 17309), CRC (n = 12860), breast cancer (n = 8632), ovarian carcinoma (n = 7998), uterine neoplasms (n = 6097), prostate adenocarcinoma (n = 3411), glioblastoma (n = 2821), gastric cancer (n = 1579), and others. Surprisingly, TFRC overexpression correlated with improved outcome in vulvar squamous cell carcinoma (VSCC, n = 297). TFRC was found to be most prognostic in prostate adenocarcinoma with median OS 1139 days in pts with high vs 3230 days in pts with low TFRC (HR = 2.556, 95% CI [2.213-2.951], p < 0.00001). Conclusions: Our study is the first to combine modern molecular profiling with a large cohort of clinical tissue samples to reveal a prognostic role for TFRC expression in a variety of solid tumor types. We found TFRC overexpression to be prognostic in a large proportion of histologies, though surprisingly association with improved OS in VSCC. Highest expression occured in CRC and gastric cancer, diseases with needs for new therapies. A number of TFR1-targeting therapeutics are currently at various stages of development, and warrant further investigation in disease cohorts identified from our study.

CD71+VISTA+ erythroid cells promote the development and function of regulatory T cells through TGF-β.

Cell-surface transferrin receptor (CD71+) erythroid cells are abundant in newborns with immunomodulatory properties. Here, we show that neonatal CD71+ erythroid cells express significant levels of V-domain Immunoglobulin (Ig) Suppressor of T Cell Activation (VISTA) and, via constitutive production of transforming growth factor (TGF)- β, play a pivotal role in promotion of naïve CD4+ T cells into regulatory T cells (Tregs). Interestingly, we discovered that CD71+VISTA+ erythroid cells produce significantly higher levels of TGF-β compared to CD71+VISTA− erythroid cells and CD71+ erythroid cells from the VISTA knock-out (KO) mice. As a result, CD71+VISTA+ erythroid cells—compared to CD71+VISTA− and CD71+ erythroid cells from the VISTA KO mice—significantly exceed promotion of naïve CD4+ T cells into induced Tregs (iTreg) via TGF-β in vitro. However, depletion of CD71+ erythroid cells had no significant effects on the frequency of Tregs in vivo. Surprisingly, we observed that the remaining and/or newly generated CD71+ erythroid cells following anti-CD71 antibody administration exhibit a different gene expression profile, evidenced by the up-regulation of VISTA, TGF-β1, TGF-β2, and program death ligand-1 (PDL-1), which may account as a compensatory mechanism for the maintenance of Treg population. We also observed that iTreg development by CD71+ erythroid cells is mediated through the inhibition of key signaling molecules phosphorylated protein kinase B (phospho-Akt) and phosphorylated mechanistic target of rapamycin (phospho-mTOR). Finally, we found that elimination of Tregs using forkhead box P3 (FOXP3)-diptheria toxin receptor (DTR) mice resulted in a significant expansion in the frequency of CD71+ erythroid cells in vivo. Collectively, these studies provide a novel, to our knowledge, insight into the cross-talk between CD71+ erythroid cells and Tregs in newborns. Our results highlight the biological role of CD71+ erythroid cells in the neonatal period and possibly beyond.

Blocking transferrin receptor inhibits the growth of lung adenocarcinoma cells in vitro.

BackgroundTransferrin receptor (TfR) is expressed in most lung cancers and is an indicator of poor prognosis in certain groups of patients. In this study, we blocked cell surface TfR to inhibit lung adenocarcinoma (LAC) cell growth in vitro and investigated the associated molecular mechanisms to determine a potential therapeutic target in human LAC.MethodsRNA interference and antibody blocking techniques were used to block the function of TfR in LAC cells, and cell proliferation assays were used to detect the results. Affymetrix microarray analysis was conducted using H1299 cells in which TfR was blocked with an antibody to investigate the molecular mechanisms involved.ResultsThe cell proliferation assay demonstrated that H1299 cell proliferation was significantly inhibited after small interfering RNA knockdown or blocking of TfR. Mechanistic studies found that 100 genes were altered more than two‐fold after TfR was blocked and that blocking TfR was accompanied by decreased expression of the oncogene KRAS. ConclusionOur data provide evidence that blocking TfR could significantly inhibit LAC proliferation by targeting the oncogene KRAS; therefore, TfR may be a therapeutic target for LAC. In addition, our results suggest a new method for blocking the signal from the oncogene KRAS by targeting TfR in LAC.

Hscb, a Mitochondrial Iron-Sulfur Cluster Assembly Co-Chaperone, Is a Novel Candidate Gene for Congenital Sideroblastic Anemia

Congenital sideroblastic anemias (CSAs) are uncommon inherited diseases resulting from defects in heme biosynthesis, mitochondrial translation or mitochondrial iron-sulfur cluster (ISC) assembly. CSAs are characterized by pathological mitochondrial iron deposits in bone marrow erythroblasts. Recently, mutations in mitochondrialheat shock protein 70 (HSPA9), a critical chaperone involved in mitochondrial ISC assembly, have been reported as a cause of non-syndromic CSA. Human heat shock cognate protein 20 (HSCB), a highly conserved mitochondrial co-chaperone, is the primary binding partner of HSPA9. HSCB allows the transfer of nascent ISC to HSPA9 and stimulates its ATPase activity, promoting ISC transfer to target proteins.To identify novel genes responsible for CSA, we performed whole exome sequencing on more than 75 CSA probands and their family members. In one patient, a young woman, with pancytopenia characterized by a normocytic anemia with numerous bone marrow ringed sideroblasts, we identified two variants in HSCB : a paternally-inherited promoter variant (c.-134C>A) predicted to disrupt a conserved ETS transcription factor binding site, and a maternally-inherited frameshift (c.259dup, p.T87fs*27). A fibroblast cell-line derived from the proband showed a decrease in HSCB expression, but normal HSPA9 expression compared to healthy, unrelated controls. Impairment of ETS1-dependent transcriptional activation of the promoter variant was demonstrated in K562 cells transfected with an HSCB-luciferase reporter construct.K562 cells were also employed to determine if reduced expression of HSCB could result in impaired erythroid metabolism, maturation, or proliferation. K562 cells infected with shRNA directed against HSCB were deficient in multiple mitochondrial respiratory complexes, had abnormal iron metabolism and a defect of protein lipoylation, all consistent with defective ISC metabolism. In addition, both IRP1 and IRP2 expression were decreased and cell surface transferrin receptor 1 (TFR1) expression was enhanced, suggesting disturbed cellular iron metabolism. Nevertheless, cells lacking HSCB partially retained an ability to respond to iron chelation and iron overload. Cells lacking HSCB lose their ability to hemoglobinize in response to sodium butyrate treatment (Figure 1A). This defect was confirmed in vivo using a morpholino strategy in zebrafish, as fish lacking HSCB are also unable to hemoglobize (Fig 1B).We generated an Hscb conditional mouse to better elucidate the underlying pathophysiology of the disease. Heterozygous (Hscb+/-) animals have no discernable phenotype; however, null animals die prior to embryonic day E7.5. Thus, to avoid this lethality, we employed Vav-cre animals (Tg(Vav1-cre)1Graf) to evaluate the loss of HSCB specifically in the hematopoietic compartment. Hscbc/- Vav-cre+ pups are pale and growth retarded compared to control littermates and die at approximately p10 with severe pancytopenia. To assess the loss of HSCB specifically in the erythroid lineage, we bred conditional animals to EpoR-cre (Eportm1(EGFP/cre)Uk) mice. Hscbc/- EpoR-cre+ mice die at approximately E12.5 due to a complete failure of erythropoiesis (Figure 1C). Finally, temporally inducible, hematopoietic-specific deletion animals were generated by transplantation of fetal livers from Mx-Cre (Tg(Mx1-cre)1Cgn) positive Hscbc/- animals. After polyinosinic:polycytidylic acid (pIpC) induction, global defects of hematopoiesis were observed in Mx-Cre+ animals, leading to their death 3-weeks post-induction from profound pancytopenia. A transient siderocytosis was seen in the peripheral blood between days 6-8 post-pIpC. Flow cytometry using FSC-TER119-CD44 gating strategy confirmed the defect in erythropoiesis.Taken together, these data demonstrate that HSCB is essential for hematopoiesis; both whole animal and in vitro cell culture models recapitulate the patient's phenotype, suggesting that the two patient mutations are likely disease-causing. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Development of a complete human IgG monoclonal antibody to transferrin receptor 1 targeted for adult T-cell leukemia/lymphoma

Iron is an essential nutrient for normal cell growth, and reprogramming of iron metabolism is essential to tumor cell survival and progression. HTLV-1-associated adult T-cell leukemia/lymphoma (ATLL) has no effective therapy and high levels of cell surface transferrin receptor 1 (TFR1) expression have been reported in ATLL by us and other groups. In this study, to develop a novel molecular-targeted therapy against TFR1 to modulate iron metabolism, we initially determined the expression pattern of several iron-related genes along with TFR1 and found that ATLL cells presented characteristic of an iron-deficiency state such as high expression of iron-regulatory protein 2 (IRP2) and low expression of its E3 ubiquitin-ligase, FBXL5. Therefore, we developed human IgG monoclonal antibodies to human TFR1 using a phage display method (ICOS method) to block the incorporation of the transferrin (TF)-iron complex into ATLL cells for inhibiting cell growth. One of the mAbs, JST-TFR09, presented its greater affinity to TFR1 on ATLL cells in flow cytometry (FCM) analysis than those of commercially available anti-TFR1 antibodies and identified high expression of TFR1 in most of the acute-type ATLL cells. Moreover, JST-TFR09 could interfere with binding between TFR1 and TF, which resulted in effective blockade of TFR1 internalization and induction of cell apoptosis by the treatment of ATLL cells with JST-TFR09. JST-TFR09 showed dual activities through direct cell cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC), and the treatment of JST-TFR09 significantly suppressed cell growth of ATLL cells with induction of apoptosis in in vitro and in vivo experiments. Thus, JST-TFR09 described here may become a promising therapeutic antibody for the treatment of ATLL.

An epirubicin-peptide conjugate with anticancer activity is dependent upon the expression level of the surface transferrin receptor.

Epirubicin (EPI) is one of the most widely used anticarcinogens; however, serious side effects, including cardiomyopathy and congestive heart failure, limit its long‑term administration. To overcome this problem, the HAIYPRH peptide ligand was used with EPI in the synthesis of a HAIYPRH‑EPI conjugate. The anticancer activity and cellular uptake of the conjugate were measured and evaluated. The results of the present study indicated that the cytotoxicity of HAIYPRH‑EPI was correlated with the expression of the cell surface transferrin receptor (TfR). The conjugate exerted high cytotoxicity and proapoptotic function when in an LN229 glioma cell line, which overexpresses surface TfR. It was hypothesized that transferrin (Tf) can promote cytotoxicity. Conversely, the conjugate exhibited very low cytotoxicity and proapoptotic function in a U87 glioma cell line, in which surface TfR expression was undetectable. In addition, fluorescence microscopy and flow cytometry methods were used to evaluate cellular uptake, and the results of these methods were consistent with the present hypotheses. The conjugate cellular uptake of the conjugate in LN229 cells was markedly higher compared with that in U87 cells, and it was hypothesized that Tf can enhance the uptake in LN229 cells. The cytotoxicity of HAIYPRH‑EPI was dependent upon the expression of surface TfR. Considering that the majority of cancer cells have high rates of iron uptake and surface TfR is generally overexpressed on cancer cells, it was speculated by the authors that HAIYPRH‑EPI may form part of an effective strategy for increasing the selectivity of EPI for cancer cells, as well as reducing its systemic toxicity. To confirm the hypothesis, the effects of HAIYPRH‑EPI on non‑cancerous cell lines were investigated. A future study will examine the side effects of HAIYPRH‑EPI, using a suitable delivery system in an animal model.

Glutaminolysis and Transferrin Regulate Ferroptosis

Ferroptosis has emerged as a new form of regulated necrosis that is implicated in various human diseases. However, the mechanisms of ferroptosis are not well defined. This study reports the discovery of multiple molecular components of ferroptosis and its intimate interplay with cellular metabolism and redox machinery. Nutrient starvation often leads to sporadic apoptosis. Strikingly, we found that upon deprivation of amino acids, a more rapid and potent necrosis process can be induced in a serum-dependent manner, which was subsequently determined to be ferroptosis. Two serum factors, the iron-carrier protein transferrin and amino acid glutamine, were identified as the inducers of ferroptosis. We further found that the cell surface transferrin receptor and the glutamine-fueled intracellular metabolic pathway, glutaminolysis, played crucial roles in the death process. Inhibition of glutaminolysis, the essential component of ferroptosis, can reduce heart injury triggered by ischemia/reperfusion, suggesting a potential therapeutic approach for treating related diseases.

The Structural Basis of Iron Sensing by the Human F‐box Protein FBXL5

Iron is an essential chemical element for all forms of life. It serves as a cofactor for many proteins and enzymes involved in oxygen transport, energy metabolism and DNA synthesis[1, 2]. Mammalian cells need to maintain a sufficient amount of iron to support the synthesis of proteins that require iron as a co-factor. Iron is imported into the cells through the circulating iron transporter transferrin[3, 4]. Iron-loaded transferrin binds to cell surface transferrin receptor, resulting in the endocytosis of transferrin and delivery of the iron cargo into the cells[4]. Excess iron in the cells is stored in the cytosolic protein ferritin[4]. Iron regulatory proteins IRP1 and IRP2 maintain the homeostasis of iron through post-translational regulation of the expression of transferrin receptor and ferritin[2, 5]. In iron-replete cells IRP2 is ubiquitinated and degraded by the proteasome. The F-box and leucine-rich repeat containing protein FBXL5 serves as a cytosolic iron sensor that regulates the ubiquitination of IRP2 by the SKP1-CUL1-FBXL5 (SCF) E3 ubiquitin ligase complex[6, 7]. FBXL5 also plays critical roles in sensing oxygen in the cytosol[6, 7]. Knock out of FBXL5 in mice result in embryonic mortality due to excess accumulation of iron[8].

Glycine Transporter 1 Plays a Crucial Role in Hemoglobinization

Abstract 345Vertebrate heme synthesis requires three substrates: succinyl-coenzyme A, which regenerates in the tricarboxylic acid cycle, iron and glycine. It is well recognized that inadequate delivery of iron to immature erythroid cells leads to a decreased production of heme, but virtually nothing is known about the impact of restricted transport of glycine on the process of hemoglobinization. Two ATP- and Na+-dependent glycine membrane transport systems have been identified in reticulocytes and shown to decrease during maturation to erythrocytes (Weigensberg & Blostein, J Membr Biol 86:37, 1985). However, it is unknown whether the reticulocyte glycine transporters are related to glycine transporter 1 (GlyT1) and 2 (GlyT2) identified more recently in the brain (rev. in Zafra & Giménez UBMB Life 60:810, 2008) and how relevant they are for proper hemoglobinization during erythroid differentiation. To address these issues, we exploited mice in which the gene encoding GlyT1 was disrupted (Tsai et al., PNAS 101:8485, 2004). Since the GlyT1 knockout (GlyT1−/−) mice die during the first postnatal day, we conducted analysis of blood parameters on newborn pups. As shown in the table below GlyT1−/− animals develop microcytic hypochromic anemia. Additionally, we observed that GlyT1−/− fetuses were typically paler than their GlyT1+/+and heterozygous (GlyT1+/−) counterparts. We next isolated erythroid cells from fetal livers (E 13.5) of GlyT1+/+, GlyT1+/−and GlyT1−/− mice and studied their hemoglobinization using a two-phase liquid culture method. Heme levels and biosynthesis rate were measured spectrophotometrically and by incorporation of [2-l4C]glycine into heme, respectively. Erythroid cells from GlyT1−/− mice exhibited a substantial decrease in both heme levels and production rate, as compared to those derived from GlyT1+/− or GlyT1+/+ mice. These observations are congruent with a strong decrease in total uptake of [2-l4C]glycine by erythroid cells from GlyT1−/− mice. Moreover, both total and cell surface transferrin receptor levels were decreased in GlyT1−/−erythroid cells. This was accompanied by a severe decrease in cellular iron acquisition from transferrin and its incorporation into heme. Furthermore, treatment of GlyT1+/+ cells with specific inhibitors for GlyT1 resulted in decreased expression of transferrin receptors, diminished cellular iron uptake and reduced iron incorporation into heme. In contrast, a specific inhibitior of GlyT2 had no effect on the expression of transferrin receptors or iron incorporation into heme. Finally, during erythroid differentiation, β-globin mRNA levels correlated with mRNA for GlyT1 but not for GlyT2. These results provide the first evidence that GlyT1 is essential for proper hemoglobinization of developing erythrocytes in vivo and in vitro. Our finding that curtailed cellular acquisition of glycine restricts heme synthesis suggests that Glyt1 may be a rate limiting step of heme synthesis in erythroid cells. It may be of interest to point out that heme inhibits the acquisition of both iron (Ponka & Neuwirt, Blood 33:690, 1969) and glycine (Ponka & Schulman, Blood 65:850, 1985) by reticulocytes. Hence, it is tempting to speculate that a similar mechanism is involved in coordinating the acquisition of both of these heme precursors by erythroid cells. It also needs to be stressed that inhibitors of GlyT1 are currently being considered for the treatment of schizophrenia (Javitt, Curr Opin Drug Discov Devel 12:468, 2009). However, our study provides a warning that such a therapy could lead to the development of hypochromic microcytic anemia.Table:Red blood cell parameters in newborn mice containing both GlyT1 alleles (+/+), only one allele (+/−) or none (−/−)RBC (x106/ mm3)HGB (g/dL)HCT (%)MCV (μm3)MCH (pg)RDW (%)GlyT1+/+ (n=10)3.60 ± 0.1813.06 ± 0.5339.24 ± 1.66109.20 ± 5.4536.33 ± 1.6516.66 ± 0.36GlyT1+/− (n=18)3.42 ± 0.3012.34 ± 0.96*36.94 ± 2.60*108.39 ± 4.4236.16 ± 1.6217.06 ± 0.90GlyT1−/− (n=12)3.32 ± 0.21*9.61 ± 0.59***29.52 ± 1.83***89.00 ± 2.37***28.94 ± 1.05***18.54 ± 0.78****=p<0.05;***=p<0.0001; Statistical analysis was done by using one way ANOVA followed by Dunnett’s multiple comparison test. Disclosures:No relevant conflicts of interest to declare.

Hepcidin and Anemia of the Critically Ill Patient

A NEMIA occurs frequently in critically ill patients, with 60–66% of them having anemia when admitted to the intensive care unit (ICU), and is associated with worse outcomes (such as increased length of stay and increased mortality). Anemia is only partially corrected during ICU stay because recent recommendations have led to a decrease in transfusion triggers. Today the prevalence of anemia in patients being discharged from the ICU is very high, being found in at least 75% of the patients upon final hemoglobin measurement. This common, and severe anemia at ICU discharge may also be prolonged after the ICU stay. The median time to recovery of anemia is 11 weeks. One observational study reports that more than half of such patients are still anemic at 6 months. Although the impact of anemia on rehabilitation after ICU discharge or quality of life has not been fully evaluated, it is well accepted that anemia is associated with worse outcomes for postoperative rehabilitation and for patients with chronic diseases such as cancer and thus should retain our interest at the bedside and in the research unit. Because blood transfusion is not an option for complete correction of this anemia, one may advocate the use of other treatments, such as erythropoiesis-stimulating agents (ESA) or iron. ESA efficacy has been studied in critically ill patients and has not proven to be indicated, at least according to the design of the studies. Iron is needed for erythropoiesis and thus may be indicated for the treatment of this anemia, but iron is also a toxic compound with the ability to induce oxidative stress or promote bacterial growth. A better understanding of iron metabolism regulation may help to define the place of iron in these indications. The recent discovery of hepcidin (i.e., the master regulator of iron metabolism) has shed new light on the regulation of iron homeostasis and has increased understanding of the physiopathology of anemia in complex clinical situations where several regulatory circuits interfere with iron metabolism, such as anemia of inflammation and anemia of critically ill patients. This article reviews the link between iron metabolism and anemia in critically ill patients and discusses therapeutic perspectives in this area.

Regulation of cellular iron metabolism

Iron is an essential but potentially hazardous biometal. Mammalian cells require sufficient amounts of iron to satisfy metabolic needs or to accomplish specialized functions. Iron is delivered to tissues by circulating transferrin, a transporter that captures iron released into the plasma mainly from intestinal enterocytes or reticuloendothelial macrophages. The binding of iron-laden transferrin to the cell-surface transferrin receptor 1 results in endocytosis and uptake of the metal cargo. Internalized iron is transported to mitochondria for the synthesis of haem or iron–sulfur clusters, which are integral parts of several metalloproteins, and excess iron is stored and detoxified in cytosolic ferritin. Iron metabolism is controlled at different levels and by diverse mechanisms. The present review summarizes basic concepts of iron transport, use and storage and focuses on the IRE (iron-responsive element)/IRP (iron-regulatory protein) system, a well known post-transcriptional regulatory circuit that not only maintains iron homoeostasis in various cell types, but also contributes to systemic iron balance.

ZRT/IRT-like Protein 14 (ZIP14) Promotes the Cellular Assimilation of Iron from Transferrin

ZIP14 is a transmembrane metal ion transporter that is abundantly expressed in the liver, heart, and pancreas. Previous studies of HEK 293 cells and the hepatocyte cell lines AML12 and HepG2 established that ZIP14 mediates the uptake of non-transferrin-bound iron, a form of iron that appears in the plasma during pathologic iron overload. In this study we investigated the role of ZIP14 in the cellular assimilation of iron from transferrin, the circulating plasma protein that normally delivers iron to cells by receptor-mediated endocytosis. We also determined the subcellular localization of ZIP14 in HepG2 cells. We found that overexpression of ZIP14 in HEK 293T cells increased the assimilation of iron from transferrin without increasing levels of transferrin receptor 1 or the uptake of transferrin. To allow for highly specific and sensitive detection of endogenous ZIP14 in HepG2 cells, we used a targeted knock-in approach to generate a cell line expressing a FLAG-tagged ZIP14 allele. Confocal microscopic analysis of these cells detected ZIP14 at the plasma membrane and in endosomes containing internalized transferrin. HepG2 cells in which endogenous ZIP14 was suppressed by siRNA assimilated 50% less iron from transferrin compared with controls. The uptake of transferrin, however, was unaffected. We also found that ZIP14 can mediate the transport of iron at pH 6.5, the pH at which iron dissociates from transferrin within the endosome. These results suggest that endosomal ZIP14 participates in the cellular assimilation of iron from transferrin, thus identifying a potentially new role for ZIP14 in iron metabolism.

Insulin-stimulated Phosphorylation of the Rab GTPase-activating Protein TBC1D1 Regulates GLUT4 Translocation

Insulin stimulates the translocation of the glucose transporter GLUT4 from intracellular locations to the plasma membrane in adipose and muscle cells. Prior studies have shown that Akt phosphorylation of the Rab GTPase-activating protein, AS160 (160-kDa Akt substrate; also known as TBC1D4), triggers GLUT4 translocation, most likely by suppressing its Rab GTPase-activating protein activity. However, the regulation of a very similar protein, TBC1D1 (TBC domain family, member 1), which is mainly found in muscle, in insulin-stimulated GLUT4 translocation has been unclear. In the present study, we have identified likely Akt sites of insulin-stimulated phosphorylation of TBC1D1 in C2C12 myotubes. We show that a mutant of TBC1D1, in which several Akt sites have been converted to alanine, is considerably more inhibitory to insulin-stimulated GLUT4 translocation than wild-type TBC1D1. This result thus indicates that similar to AS160, Akt phosphorylation of TBC1D1 enables GLUT4 translocation. We also show that in addition to Akt activation, activation of the AMP-dependent protein kinase partially relieves the inhibition of GLUT4 translocation by TBC1D1. Finally, we show that the R125W variant of TBC1D1, which has been genetically associated with obesity, is equally inhibitory to insulin-stimulated GLUT4 translocation, as is wild-type TBC1D1, and that healthy and type 2 diabetic individuals express approximately the same level of TBC1D1 in biopsies of vastus lateralis muscle. In conclusion, phosphorylation of TBC1D1 is required for GLUT4 translocation. Thus, the regulation of TBC1D1 resembles that of its paralog, AS160.

Targeting Hypoxia-inducible Factor-1α With Tf–PEI–shRNA Complex via Transferrin Receptor–mediated Endocytosis Inhibits Melanoma Growth

Malignant melanoma (MM) is a major public health problem. The development of effective, systemic therapies for MM is highly desired. We showed here that the transferrin receptor (TfR) was a suitable surface marker for targeting of gene therapy in MM and that the hypoxia-inducible factor-1α (HIF-1α) was an attractive therapeutic molecular target in MM. We observed that inhibition of HIF-1α blocked cell proliferation and induced cell apoptosis in vitro. We then showed that a transferrin–polyethylenimine–HIF-1α–short-hairpin RNA (Tf–PEI–HIF-1α–shRNA) complex could target MM specifically and efficiently both in vivo and in vitro, exploiting the high expression of the TfR in MM. The systemic delivery of sequence-specific small-interfering RNA (siRNA) against HIF-1α by the Tf– PEI–HIF-1α–shRNA complex dramatically inhibited tumor growth in the A375 MM xenograft model. The underlying concept of transfecting a HIF-1α shRNA expression vector complexed with Tf–PEI to block HIF-1α holds promise as a clinical approach to gene therapy for MM.

The metal-binding domain of IGFBP-3 selectively delivers therapeutic molecules into cancer cells

Conventional chemotherapy for cancer has limited specificity for cancer cells. Here, we investigate the possibility of improving the selectivity of chemotherapy by coadministering targeted biological modifier peptides. We show that the 22-amino acid metal-binding transporter domain (MBD) derived from insulin-like growth factor-binding protein-3 selectively targets cancer cells. The rate of MBD uptake by cells was measured using a panel of 54 human cancer cell lines and correlated with MBD cross-linking to cell surface transferrin receptor, caveolin 1, and integrin beta. Gene array data show that MBD uptake correlates with the expression of genes associated with cellular stress-coping mechanisms commonly upregulated in cancer (nuclear factor-kappaB, Hsp-70B). MBD-tagged peptides designed to inhibit such mechanisms have cytotoxic effects on a broad range of human cancer cell lines. The discriminant validity of these peptides as potential cotherapeutic agents was investigated by comparing their cytotoxicity to cancer cell lines versus normal human cell counterparts. Synergies between these peptides and marginally cytotoxic levels of 5-fluorouracil were demonstrated. Biodistribution data from in-vivo experiments in mice and rats confirm that MBD-tagged peptides and proteins preferably localize to specific tissues, such as kidney and pancreas. Intracardial injection of CCRF-CEM T-cell leukemia or MDA-MB-435 cells into Rag-2 mice establishes disseminated disease within 7 days. Twenty-five-day subcutaneous administration of a three-peptide cocktail (3 mg/kg) in combination with 5-fluorouracil in Rag-2 mice with established CCRF-CEM leukemia significantly reduces splenomegaly and bone marrow cancer cell burden. In a similar experiment using MDA-MB-435 cells, MBD-tagged peptides reduced human cell burden in bone marrow. Taken together, these data suggest that MBD-tagged molecules can be used as highly selective chemosensitizers in the treatment of hematological and disseminated malignancies.

Early Effects on T lymphocyte Response to Iron Deficiency in Mice. Short Communication

Iron deficiency is a common nutritional disorder, affecting about 30% of the world population. Deficits in iron functional compartments have suppressive effects on the immune system. Environmental problems, age, and other nutrient deficiencies are some of the situations which make human studies difficult and warrant the use of animal models. This study aimed to investigate alterations in the immune system by inducing iron deficiency and promoting recuperation in a mouse model. Hemoglobin concentration, hematocrit, liver iron store, and flow cytometry analyses of cell-surface transferrin receptor (CD71) on peripheral blood and spleen CD4+ and CD8+ T lymphocyte were performed in the control (C) and the iron-deficient (ID) groups of animals at the beginning and end of the experiment. Hematological indices of C and ID mice were not different but the iron stores of ID mice were significantly reduced. Although T cell subsets were not altered, the percentage of T cells expressing CD71 was significantly increased by ID. The results suggest that iron deficiency induced by our experimental model would mimic the early events in the onset of anemia, where thymus atrophy is not enough to influence subset composition of T cells, which can still respond to iron deficiency by upregulating the expression of transferrin receptor.

New liver cell mutants defective in the endocytic pathway

To isolate mutant liver cells defective in the endocytic pathway, a selection strategy using toxic ligands for two distinct membrane receptors was utilized. Rare survivors termed trafficking mutants (Trf2–Trf7) were stable and more resistant than the parental HuH-7 cells to both toxin conjugates. They differed from the previously isolated Trf1 HuH-7 mutant as they expressed casein kinase 2 α″ (CK2α″) which is missing from Trf1 cells and which corrects the Trf1 trafficking phenotype. Binding of 125I-asialoorosomucoid (ASOR) and cell surface expression of asialoglycoprotein receptor (ASGPR) were reduced approximately 20%–60% in Trf2–Trf7 cells compared to parental HuH-7, without a reduction in total cellular ASGPR. Based on 125I-transferrin binding, cell surface transferrin receptor activity was reduced between 13% and 88% in the various mutant cell lines. Distinctive phenotypic traits were identified in the differential resistance of Trf2–Trf7 to a panel of lectins and toxins and to UV light-induced cell death. By following the endocytic uptake and trafficking of Alexa488-ASOR, significant differences in endosomal fusion between parental HuH-7 and the Trf mutants became apparent. Unlike parental HuH-7 cells in which the fusion of endosomes into larger vesicles was evident as early as 20 min, ASOR endocytosed into the Trf mutants remained within small vesicles for up to 60 min. Identifying the biochemical and genetic mechanisms underlying these phenotypes should uncover novel and unpredicted protein–protein or protein–lipid interactions that orchestrate specific steps in membrane protein trafficking.

Evidence for a sequential transfer of iron amongst ferritin, transferrin and transferrin receptor during duodenal absorption of iron in rat and human

To elucidate the sequential transfer of iron amongst ferritin, transferrin and transferrin receptor under various iron status conditions. Incorporation of 59Fe into mucosal and luminal proteins was carried out in control WKY rats. The sequential transfer of iron amongst ferritin, transferrin and transferrin receptor was carried out in iron deficient, control and iron overloaded rats. The duodenal proteins were subjected to immunoprecipitation and quantitation by specific ELISA and in situ localization by microautoradiography and immunohistochemistry in tandem duodenal sections. Human duodenal biopsy (n = 36) collected from subjects with differing iron status were also stained for these proteins. Ferritin was identified as the major protein that incorporated iron in a time-dependent manner in the duodenal mucosa. The concentration of mucosal ferritin was significantly higher in the iron excess group compared to control, iron deficient groups (731.5 +/- 191.96 vs 308.3 +/- 123.36, 731.5 +/- 191.96 vs 256.0 +/- 1.19, P < 0.005), while that of luminal transferrin which was significantly higher than the mucosal did not differ among the groups (10.9 +/- 7.6 vs 0.87 +/- 0.79, 11.1 +/- 10.3 vs 0.80 +/- 1.20, 6.8 +/- 4.7 vs 0.61 +/- 0.63, P < 0.001). In situ grading of proteins and iron, and their superimposition, suggested the occurrence of a sequential transfer of iron. This was demonstrated to occur through the initial binding of iron to luminal transferrin then to absorptive cell surface transferrin receptors. The staining intensity of these proteins varied according to the iron nutrition in humans, with intense staining of transferrin receptor observed in iron deficient subjects. It is concluded that the intestine takes up iron through a sequential transfer involving interaction of luminal transferrin, transferrin-transferrin receptor and ferritin.