Conceived and designed the experiments: KP. Performed the experiments: CM GC. Analyzed the data: ID CAO. Wrote the paper: KP.
Current address: Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America
The authors have declared that no competing interests exist.
Iron regulatory proteins, IRP1 and IRP2, bind to mRNAs harboring iron responsive elements and control their expression. IRPs may also perform additional functions. Thus, IRP1 exhibited apparent tumor suppressor properties in a tumor xenograft model. Here we examined the effects of IRP2 in a similar setting. Human H1299 lung cancer cells or clones engineered for tetracycline-inducible expression of wild type IRP2, or the deletion mutant IRP2Δ73 (lacking a specific insert of 73 amino acids), were injected subcutaneously into nude mice. The induction of IRP2 profoundly stimulated the growth of tumor xenografts, and this response was blunted by addition of tetracycline in the drinking water of the animals, to turnoff the IRP2 transgene. Interestingly, IRP2Δ73 failed to promote tumor growth above control levels. As expected, xenografts expressing the IRP2 transgene exhibited high levels of transferrin receptor 1 (TfR1); however, the expression of other known IRP targets was not affected. Moreover, these xenografts manifested increased c-MYC levels and ERK1/2 phosphorylation. A microarray analysis identified distinct gene expression patterns between control and tumors containing IRP2 or IRP1 transgenes. By contrast, gene expression profiles of control and IRP2Δ73-related tumors were more similar, consistently with their growth phenotype. Collectively, these data demonstrate an apparent pro-oncogenic activity of IRP2 that depends on its specific 73 amino acids insert, and provide further evidence for a link between IRPs and cancer biology.
IRP1 and IRP2 are homologous cytoplasmic proteins that post-transcriptionally regulate cellular iron metabolism
IRPs are ubiquitously expressed in mammalian tissues and share at least some degree of functional redundancy. This is evident from the early embryonic lethality at the blastocyst stage, associated with the ablation of both IRP1 and IRP2 in mice
In iron-replete cells, IRP1 assembles a cubane [4Fe-4S] cluster and acquires enzymatic function as cytosolic aconitase, at the expense of its RNA-binding activity
Considering that iron is essential for cell proliferation
Here, we examine the effects of IRP2 in this model. We show that overexpression of IRP2 elicits an opposite phenotype and profoundly stimulates tumor growth, even though both IRPs appear to regulate IRE-containing mRNAs within the tumors in a similar manner. In addition, we provide evidence that the tumor-promoting activity of IRP2 requires its specific insert of 73 amino acids. Finally, we identify distinct gene expression patterns between control tumors and those overexpressing IRP2 or IRP1, that may account for their differential growth phenotypes.
To assess the role of IRP2 in tumorigenesis, we employed H1299 lung cancer cells overexpressing the wild type form of the protein (HIRP2wt) under the control of a tetracycline-inducible promoter. IRP2-transfectants and control parent cells were injected subcutaneously into the flanks of BALB/c (nu/nu) mice to form solid tumor xenografts (
BALB/c nude mice were injected with parent H1299, HIRP2wt or HIRP2Δ73 cells and tumor xenografts were grown for 10 weeks and monitored over time. (A) Representative anesthetized mice before sacrifice; tumor xenografts are shown by arrows. (B–D) Cumulative data from three independent experiments (n = 9 mice for each group) depicting kinetics on tumor xenograft growth (B), mass (C) and volume (D) of isolated tumor xenografts. Data are expressed as mean ± SEM. * p<0.05, ** p<0.01 versus H1299 (Student's t-test).
To exclude the possibility that the observed phenotype is due to a possible clonal effect unrelated to IRP2, further BALB/c (nu/nu) mice were injected with HIRP2wt cells. Half of the animals were receiving tetracycline in their drinking water throughout the experimental period, to turn off the expression of the IRP2 transgene, while the other half were allowed to overexpress IRP2 in the xenograft, without antibiotic (
A total of 6 BALB/c nude mice were injected with HIRP2wt cells to form tumor xenografts. Half of the animals were receiving 2 mg/ml tetracycline in the drinking water throughout the experimental period, starting 4 days before injection. (A) Representative anesthetized mice from the two groups before sacrifice (tumors shown by arrows). (B) Kinetics on tumor xenograft growth. (C) Mass and (D) volume of isolated tumor xenografts. (E) Tumor tissue extracts were analyzed by Western blotting with antibodies against HA, to detect expression of the IRP2 transgene, and β-actin, as loading control. Data are expressed as mean ± SEM. * p<0.05, ** p<0.01 versus HIRP2wt (Student's t-test).
Mammalian IRP2 molecules contain a unique conserved insert of 73 amino acids close to their N-termini, that is absent in IRP1. The function of this sequence remains largely unknown. To better understand the requirements for the apparent pro-oncogenic activity of IRP2, we evaluated the performance of HIRP2Δ73 cells, overexpressing the IRP2Δ73 deletion mutant, in the tumorigenicity assay. These cells formed solid tumor xenografts in nude mice, that exhibited growth characteristics of control tumors derived from parent H1299 cells. Thus, in contrast to wild type protein, IRP2Δ73 failed to drastically stimulate tumor growth and increase tumor mass and size (
Interestingly, this insert also accounts for differential anchorage-independent growth characteristics between HIRP2wt and HIRP2Δ73 cells in soft agar. Thus, the former give rise to fewer but larger colonies compared to parent cells, while the latter form a big number of smaller size colonies (
Histological sections of tumor xenografts were stained with hematoxylin and eosin (
Mitoses are shown by arrows; colored insets indicate eosinophilic cytoplasm, blood vessels, necrosis, nuclear fragmentation, or cytoplasm vacuolization.
Analysis of tumor extracts by Western blotting with a HA antibody (
(A) Extracts from tumor tissue were analyzed by Western blotting with antibodies against HA, TfR1, ferritin, ferroportin, DMT1 and β-actin. (B) Tumor extracts were analyzed for IRE-binding activity by EMSA with a 32P-labeled ferritin IRE probe. (C and D) Analysis of TfR1 and H-ferritin mRNA expression by qPCR.
Tumor extracts containing either IRP2wt or IRP2Δ73 exhibited high IRE-binding activity in an electrophoretic mobility shift assay (EMSA) (
We also performed a biochemical analysis of cultured HIRP2wt and HIRP2Δ73 cells, to explore whether the above findings in the xenograft tissue are due to basic IRP2 overexpression at the cellular level, or possibly reflect more complex responses within the tumor microenvironment. Extracts of both HIRP2wt and HIRP2Δ73 cells were highly active in IRE-binding (
Parent H1299, HIRP2wt and HIRP2Δ73 cells were grown for 3 days in the absence or presence of tetracycline; where indicated, the cells were treated overnight with 100 µM of desferrioxamine (DFO) or hemin. (A) Cytoplasmatic extracts were analyzed by EMSA with a 32P-labeled ferritin IRE-probe. (B) Analysis of TfR1 mRNA expression by qPCR. (C) Western blotting with antibodies against HA, TfR1 and β-actin. (D) The cells were metabolically labeled with 35S-methionine/cysteine and the synthesis of TfR1 was assessed by quantitative immunoprecipitation. (E) Western blotting with antibodies against HA, ferritin and β-actin. (F) The cells were untreated or pretreated for 4 h with 100 µM hemin and, subsequently, metabolically labeled with 35S-methionine/cysteine; the synthesis of ferritin was assessed by quantitative immunoprecipitation.
Further biochemical analysis of the xenografts revealed a consistent and statistically significant 2-fold increase of c-MYC expression and ERK1/2 phosphorylation in tumors derived from HIRP2wt cells (
Extracts from tumor tissue were analyzed by Western blotting with antibodies against c-MYC, phospho-ERK1/2, ERK1/2, CDC14A, VEGF and β-actin. Representative immunoblots and quantification from three independent experiments (n = 9 mice)of (A) c-MYC, relative to β-actin; (B) phospho-ERK1/2, relative to ERK 1/2; (C) CDC14A and VEGF, relative to β-actin. Data are expressed as means of relative band intensity ± SEM. * p<0.05 versus H1299 (Student's t-test).
Duplicate RNA samples (each from different animal) isolated from control, IRP2, IRP2Δ73 or IRP1
(A) Hierarchical clustering of all differentially expressed genes. Red and green colors represent up- and down-regulation. (B) Venn diagram of all differentially expressed genes. (C) Co-expression network of 178 genes shared by all differential groups. Each gene is represented by a node and a Pearson correlation coefficient above 0.90 between any pair of genes is represented by an edge. Genes are colorized according to their distinct MCL cluster which may reflect a shared biological function.
To identify putative functional relationships, all-against-all PCCs were calculated for all common 178 genes across the dataset. Pairwise gene co-expressions were used to construct an undirected unweighted network such that genes are represented by nodes and co-expression with PCC≥0.90 by edges. The final network consisted of 156 genes (
Further analysis of each MCL cluster for overrepresented GO-BP terms uncovers that IRP1 and IRP2 elicit substantially distinct downstream responses. Moreover, the overexpression of IRP2, but not of IRP2Δ73, promotes differential expression of a wide range of genes primarily involved in signal transduction, transcriptional regulation and G-protein coupled receptor signaling, but also in metabolic processes, cell adhesion and growth (
Prompted by the inhibitory effects of IRP1 in tumor xenograft growth in nude mice
In contrast to the wild type protein, the deletion mutant IRP2Δ73 that lacks an IRP2-specific insert of 73 amino acids, failed to stimulate tumor growth in the xenograft model (
Tumors derived from IRP2-overexpressing cells, as well as these cells in culture, maintained high levels of IRP2 at the experimental endpoint and exhibited an upregulation of TfR1 mRNA and protein (
Surprisingly, the presence of the IRP2wt (or IRP2Δ73) transgene did not affect the expression of ferritin neither in xenografts, nor in cultured cells (
The data in
We noted that the expression of the IRP2 transgene in tumor xenografts was associated with increased levels of the c-MYC oncogene, as well as with increased ERK1/2 phosphorylation (
The IRP2-dependent increase in c-MYC levels deserves particular attention, considering that IRP2 is a direct transcriptional target of c-MYC
Taken together, our data provide strong evidence that the apparent pro-oncogenic activity of IRP2 is unrelated to the established function of this protein as regulator of several known IRE-containing mRNAs. The cDNA microarray analysis (
Human H1299 lung cancer cells and clones expressing wild type IRP2 (HIRP2wt), wild type IRP1 (HIRP1wt) or the IRP2 deletion mutants Δ73 (HIRP2Δ73), ΔD4 (HIRP2ΔD4) or ΔD4/-73d (HIRP2ΔD4/-73d) in a tetracycline-inducible fashion (tet-off) were grown in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin (Wisent Inc, St-Bruno, QC). The generation of these cell lines has been described elsewhere
The capacity of HIRP2wt and HIRP2Δ73 cells to form colonies in soft agar was evaluated as earlier described
All experimental procedures were approved by the Animal Care Committee of McGill University (Protocol 4966). Female BALB/c (nu/nu) mice were obtained from Charles River Laboratories (Cambridge, MA). The animals were housed in macrolone cages (up to 5 animals/cage, 12:12 h light-dark cycle: 7 am – 7 pm; 22±1°C, 60±5% humidity) according to standard guidelines, and had free access to water and food. Tumor xenografts were formed as described in
Tissue staining with hematoxylin and eosin, and Perl's Prussian blue was performed as in
Cells were washed twice in cold PBS and lysed in a buffer containing 20 mM Tris-Cl pH 7.4, 40 mM KCl, 1% Triton X-100, an EDTA-free protease inhibitor cocktail (Roche) and a Halt phosphatase inhibitor Cocktail (Thermo Scientific). Frozen tumor tissue aliquots were suspended in the same lysis buffer and homogenized in a 1 ml glass homogenizer. Cell debris was cleared by centrifugation and the protein concentration was measured with the Bradford reagent (BioRad).
Protein extracts (25–50 µg) were resolved by SDS-PAGE on 8%, 10% or 14% gels and the proteins were transferred onto nitrocellulose filters (BioRad). The blots were saturated with 10% non-fat milk in PBS containing 0.1% (v/v) Tween-20 (PBS-T) and probed with hemagglutinin (HA) (Roche), TfR1 (Zymed), ferritin (Novus), ferroportin (raised in a rabbit against an affinity-purified GST-fusion protein antigen containing 4 tandem copies of the C-terminal 32 amino acid domain of mouse ferroportin), DMT1
Cytoplasmic lysates were analyzed for IRE-binding activity by EMSA with a 32P-labeled ferritin IRE probe
Cells were metabolically labeled with 50 µCi/ml Trans-[35S]-label, a mixture of 70∶30 35S-methionine/cysteine (ICN). After 2 h, the cells were lysed in a buffer containing 50 mM Tris-Cl pH 7.4, 300 mM NaCl and 1% Triton X-100. Cell debris was cleared by centrifugation and cell lysates were subjected to quantitative immunoprecipitation with ferritin or TfR1 antibodies
Total RNA was isolated from frozen tumor tissue using the RNeasy Midi kit (Qiagen). The quality of RNA was assessed by determining the 260/280 nm absorbance ratios and by agarose gel electrophoresis.
Total RNA (5 µg) was reverse transcribed using the Fairplay III Microarray Labeling kit (Stratagene) and labeled with Cy3-dCTP or Cy5-dCTP. Following clean-up using Fairplay columns (Stratagene), labeled cDNA and universal reference (Stratagene) were combined and hybridized to a 4×44K two-color gene expression array (Agilent Technologies). After washes according to the manufacturer's protocol, the array was scanned on the Agilent DNA Microarray scanner at a resolution of 5 microns. All images were extracted and normalized with Feature Extracter 9.5. The microarray data are MIAME compliant and have been deposited in the ArrayExpress database (accession number E-MEXP-2524).
Pairwise similarity in gene expression vectors was expressed by the Pearson correlation coefficient (PCC). Gene pairs that correlated above a predefined PCC threshold were represented in the form of an undirected unweighted network
Total RNA (1 µg) was reverse transcribed using the Quantitech Reverse Trancription Kit (Qiagen). Gene-specific primers (
Results are presented as mean ± SEM. Comparisons were made using unpaired student's t test. A value of P<0.05 was considered statistically significant.
Deletion of the 73 amino acids insert of IRP2 alters growth properties in soft agar. (A) Schematic representation of wild type IRP2 and the IRP2Δ73 deletion mutant, depicting the 4 domains of the protein, the 73 amino acids insert within domain 1, the hinge linking domains 3 and 4, and the C-terminal HA tag. (B) Tetracycline-inducible expression of wild type IRP2 or IRP2Δ73. Extracts of parent H1299, HIRP2wt and HIRP2Δ73 cells, grown for 48 h without (−) or with (+) 2 mg/ml tetracycline, were analyzed by Western blotting with antibodies against HA (top) and β-actin (bottom). (C) Anchorage-independent growth of the cells in soft agar. Representative images of colonies derived from a total of 2×104 plated cells (100× magnification) are shown on top and colony formation efficiency at the bottom. Media didn't contain tetracycline to allow expression of transfected IRP2 or IRP2Δ73. * p<0,001 versus H1299 (Student's t-test).
(2.83 MB TIF)
The 73 amino acids insert of IRP2 is not sufficient to promote tumor growth. (A) Schematic representation of wild type IRP2 and the deletion mutants lacking domain 4, either in the presence (IRP2ΔD4) or absence of the 73 amino acids insert (IRP2ΔD4/−73d). (B) Growth of tumor xenografts derived from HIRP2ΔD4 and HIRP2ΔD4/−73d cells (n = 3 nude mice per group). (C) Detection of mutant IRP2 expression in tumor extracts by Western blotting with antibodies against HA and control β-actin. (D) Mass and (E) volume of isolated tumor xenografts. Data are expressed as mean ± SEM. The graphs are in the same scale as those in
(0.70 MB TIF)
The low pro-oncogenic activity of IRP2Δ73 is not due to reduced expression levels of this mutant in tumors. The graph depicts the ratio of tumor volume values (derived from HIRP2wt and HIRP2Δ73 cells) by the relative band intensities of HA-tagged IRP1wt and IRP2Δ73 (obtained by densitometric analysis of Western blots). Data are from three independent experiments (n = 9 mice); ** p<0.01 versus HIRP2wt (Student's t-test). (B) Quantification of TfR1, ferritin, ferroportin and DMT1 expression in tumor xenografts derived from HIRP2wt and HIRP2Δ73 cells. Western blots from three independent experiments were quantified by densitometry. Values of protein band intensities (mean ±SEM) were normalized to β-actin; ** p<0.01 versus H1299 (Student's t-test).
(0.91 MB TIF)
Box plot of the normalized TFRC (TfR1) expression values in tumor xenografts derived from parent H1299, HIRP1wt, HIRP2wt and HIRP2Δ73 cells, generated by the cDNA microarray analysis.
(0.45 MB TIF)
Functional annotations of pairwise (“IRP2 vs control”, “IRP2 vs IRP1” and “IRP2 vs IRP2Δ73”) differentially regulated genes in tumor xenografts derived from parent H1299, HIRP2wt, HIRP1wt [ref. (19)] and HIRP2Δ73 cells.
(0.79 MB TIF)
Principal component analysis. Experiments were plotted by mapping values of 1st and 2nd principal components to X- and Y- axis respectively. The distance of separation between samples corresponding to “control” and “IRP2Δ73” tumors is insignificant, suggesting a common signal intensity pattern.
(0.88 MB TIF)
Gene specific primers used for qPCR experiments.
(0.06 MB PDF)
Full list of pairwise (“IRP2 vs control”, “IRP2 vs IRP1” and “IRP2 vs IRP2Δ73”) differentially expressed genes in tumor xenografts derived from parent H1299, HIRP2wt, HIRP1wt [ref. (19)] and HIRP2Δ73 cells.
(0.69 MB XLS)
Annotation and network statistics for common differentially expressed genes between “IRP2 vs control”, “IRP2 vs IRP1” and “IRP2 vs IRP2Δ73”.
(0.15 MB XLS)
We thank Abdel Hosein and Dr. Mark Basik for assistance with the microarray experiments.