Tumorigenic properties of iron regulatory protein 2 (IRP2) mediated by its specific 73-amino acids insert

Abstract

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 IRP2(Delta73) (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, IRP2(Delta73) 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 IRP2(Delta73)-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.

Figure 1. IRP2-dependent accelerated tumor growth, mediated by its specific 73 amino acids insert.

BALB/c nude mice were injected with parent H1299, HIRP2_wt or HIRP2_Δ73 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).

Figure 2. Tetracycline-dependent repression of the IRP2 transgene abolishes accelerated tumor growth.

A total of 6 BALB/c nude mice were injected with HIRP2_wt 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 HIRP2_wt (Student's t-test).

Figure 3. Hematoxylin and eosin staining of tumor xenografts derived from H1299 (left), HIRP2_wt (middle) or HIRP2_Δ73 (right) cells (40× magnification).

Mitoses are shown by arrows; colored insets indicate eosinophilic cytoplasm, blood vessels, necrosis, nuclear fragmentation, or cytoplasm vacuolization.

Figure 4. Effects of wild type or mutant IRP2 transgenes in the expression of known downstream targets within the tumor xenograft.

(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.

Figure 5. Effects of wild type or mutant IRP2 transgenes in the expression of known downstream targets in cultured H1299 cells.

Parent H1299, HIRP2_wt and HIRP2_Δ73 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.

Figure 6. Tumor xenografts derived from HIRP2_wt cells display an increase of c-MYC expression and ERK1/2 phosphorylation.

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).

Figure 7. Analysis of gene expression profiles in tumor xenografts with altered expression of IRPs.

(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.