Combined deficiency for MAP kinase-interacting kinase 1 and 2 (Mnk1 and Mnk2) delays tumor development

Abstract

MAP kinase-interacting kinase 1 and 2 (Mnk1 and Mnk2) are protein-serine/threonine kinases that are activated by ERK or p38 and phosphorylate eIF4E, which is involved in cap-dependent translation initiation. However, Mnk1/2 double knockout (Mnk-DKO) mice show normal cell growth and development despite an absence of eIF4E phosphorylation. Here we show that the tumorigenesis occurring in the Lck-Pten mouse model (referred to here as tPten(-/-) mice) can be suppressed by the loss of Mnk1/2. Phosphorylation of eIF4E was greatly enhanced in lymphomas of parental tPten(-/-) mice compared with lymphoid tissues of wild-type mice, but was totally absent in lymphomas of tPten(-/-); Mnk-DKO mice. Notably, stable knockdown of Mnk1 in the human glioma cell line U87MG resulted in dramatically decreased tumor formation when these cells were injected into athymic nude mice. Our data demonstrate an oncogenic role for Mnk1/2 in tumor development, and highlight these molecules as potential anticancer drug targets that could be inactivated with minimal side effects.

Fig. 1.

Mnk1 and Mnk2 are not essential for steady-state cell growth or responses to culture stress. (A) Growth curves of primary MEFs of the indicated genotypes under standard culture conditions. Data shown are the mean ± SD of three cultures/genotype. (B) L-glutamine starvation. Primary MEFs of the indicated genotypes (1 × 10⁵ cells/well) were cultured at the indicated concentrations of L-glutamine for 7 d and stained with 0.5% crystal violet dye in 20% ethanol. (C) Glucose starvation-induced apoptosis. Primary MEFs of the indicated genotypes were cultured in the absence of D-glucose for the indicated time points, stained with Annexin V/PI, and analyzed by flow cytometry. Percentages of positive cells within a quadrant are indicated. (D) Hypoxia. Spontaneously immortalized MEFs of the indicated genotypes were cultured in 0.2% oxygen for 24 h. The induction of Vegf and Glut1 mRNAs was measured by quantitative real-time PCR and normalized to values observed under normoxia. For all figures, results presented are representative of at least two independent trials.

Fig. 2.

Primary MEFs from Mnk1^−/−Mnk2^−/− mice are resistant to oncogenic transformation. (A) Growth curves under standard conditions. Primary MEFs from mice of the indicated genotypes were infected with H-RasV12 (Ras) plus SV40LT antigen (T-Ag). Transformed cells (5 × 10⁴/well) were plated in six-well plates. (B and C) Reduced anchorage-independent cell growth. The transformed MEFs of A and empty vector-transduced control MEFs were allowed to form colonies for 21 d in soft agar. Colonies were stained with crystal violet (B) and colony numbers were counted (C). Results shown in C are the mean colony number ± SE of triplicate samples and are representative of two independent experiments. *P = 0.003 (Student's t test).

Fig. 3.

Combined Mnk1/2 deficiency delays tumorigenesis in T-cell-specific Pten-deficient mice. (A) Kaplan–Meier survival plots for tPten^−/−; Mnk-DKO mice and tPten^−/−; non-Mnk-DKO (Mnk1^+/−Mnk2^+/−, Mnk1^−/−Mnk2^+/−, and Mnk1^+/−Mnk2^−/−) mice. Mice were killed when tumors measured 1 cm across or at the first signs of mouse morbidity (abdominal distension with hunched back, etc.). Median tumor-free survival: tPten^−/−; non-Mnk-DKO = 75 d (n = 11; male:female = 5:6), tPten^−/−; Mnk-DKO = 94 d (n = 13; male:female = 6:7). P = 0.005 (log-rank test). (B and C) Characterization of tPten^−/− T-cell lymphomas. (B) Photographs of the thymus, spleen, and lymph nodes (LN) from a representative mouse of the indicated genotypes. Enlarged spleens (splenomegaly; long diameter > 2 cm) were confirmed for all tPten^−/− mice in A. (Scale bars, 1 cm.) (C) Flow cytometric profiles of CD4/CD8 expression by cells isolated from lymphomas in the indicated tissues in B. (D) Loss of eIF4E phosphorylation in tPten^−/−; Mnk-DKO lymphomas. WT thymocytes (thymus) and cells isolated from thymic lymphomas in mice of the indicated genotypes were subjected to immunoblotting to detect the indicated proteins. Actin, loading control.

Fig. 4.

Combined Mnk1/2 deficiency abolishes eIF4E phosphorylation in lymphomas of T-cell-specific Pten-deficient mice. Sections of WT thymus and spleen and lymphomas from mice of the indicated genotypes were stained with H&E (A–C and G–I) or with specific antibody to detect phosphorylated eIF4E (P-eIF4E; D–F and J–L). (Scale bars, 50 μm in A–F; 100 μm in G–L.) Black arrows, representative phospho-eIF4E⁺ cells.

Fig. 5.

Mnk1 knockdown reduces tumor growth in a glioma xenograft model. (A) Confirmation of Mnk1 knockdown and reduced eIF4E phosphorylation. U87MG cells were infected with virus expressing control shRNA (C), Mnk1 shRNA#1 (1), or Mnk1 shRNA#2 (2). Four days after puromycin selection, lysates were immunoblotted to detect the indicated proteins. (B) Reduced focus formation. The cells in A were plated at high density (7.5 × 10⁴ cells/well; upper panel) or low density (1 × 10⁴ cells/well; lower panel) and focus formation was examined at 4 or 2 d postplating, respectively. (C) Decreased S-phase population. The cells in A were seeded at 3 × 10⁵/10-cm plate, cultured for 4 d, and then pulsed with BrdU for 40 min. Cells were stained with APC-anti-BrdU for 20 min to detect BrdU incorporation (vertical axis), and with 7-AAD to detect total DNA content (horizontal axis). Upper box, cells incorporating BrdU (S phase); lower left box, G₀/G₁ cells; lower right box, G₂/M cells; upper right number, percentage of total BrdU⁺ cells. (D) Decreased tumor formation. Nude mice were injected with control shRNA-expressing U87MG cells in the left flank (L), and with Mnk1 knockdown cells (either sh#1 or sh#2) in the right flank (R). (Upper) Gross appearance of mice. (Lower) Dissected tumors at day 30 postinjection. U87MG cells expressing Mnk1 shRNA#1 did not form detectable tumors. (E) Decreased tumor mass. The dissected tumors from D were weighed. (Left) Each circle represents the tumor mass from one mouse. Horizontal line, mean total mass of all tumors originating from the indicated cells. (Right) Weights of control and sh#2-expressing tumors dissected from the same mouse are connected with a line to facilitate comparison. Data from three mice are shown. *P = 0.008 (Student's t test), P = 0.0006 (Fisher's exact test).