Requirement of c-Jun NH(2)-terminal kinase for Ras-initiated tumor formation

Abstract

The c-Jun NH(2)-terminal kinase (JNK) signal transduction pathway causes increased gene expression mediated, in part, by members of the activating transcription factor protein (AP1) group. JNK is therefore implicated in the regulation of cell growth and cancer. To test the role of JNK in Ras-induced tumor formation, we examined the effect of compound ablation of the ubiquitously expressed genes Jnk1 plus Jnk2. We report that JNK is required for Ras-induced transformation of p53-deficient primary cells in vitro. Moreover, JNK is required for lung tumor development caused by mutational activation of the endogenous KRas gene in vivo. Together, these data establish that JNK plays a key role in Ras-induced tumorigenesis.

Fig. 1.

Growth properties of Trp53^−/− Jnk1^−/− Jnk2^−/− embryonic fibroblasts. (A) Loss of contact growth inhibition in Trp53^−/− MEF cultures is prevented by disruption of Jnk1 plus Jnk2. Wild-type (WT), Trp53^−/−, and Trp53^−/− Jnk^−/− MEF were examined using phase-contrast microscopy. Representative images are shown of cells growing in sparse (top) or confluent (bottom) culture conditions. (B) Growth of Trp53^−/− cells in low serum concentrations is prevented by disruption of Jnk1 plus Jnk2. WT, Trp53^−/−, and Trp53^−/− Jnk^−/− MEF were plated on day 0 (D = 0) and cultured for 7 days (D = 7) in medium supplemented with different concentrations of serum. Relative cell numbers were measured by staining with crystal violet. The data are presented as means ± standard deviations (n = 5) and are representative of results from three independent experiments. Statistically significant differences (P < 0.01) between WT MEF and mutant MEF are indicated with an asterisk. (C, D) Soft agar colony formation by Trp53^−/− MEF requires JNK. Cells were grown in soft agar supplemented with 10% serum. Colony formation was assessed after 20 days. (C) Representative images are shown of colonies formed by WT, Trp53^−/−, and Trp53^−/− Jnk^−/− cells. The number of colonies (>10 cells) formed by each cell type was counted and is shown in panel D. Statistically significant differences (P < 0.01) between WT MEF and mutant MEF are indicated with an asterisk.

Fig. 2.

Effect of JNK deficiency on cadherin expression. (A) MEF prepared from Rosa26-Cre^ERT+/− Trp53^LoxP/LoxP KRas^+/LSL-G12D mice (Trp53^−/− Kras^G12D mice) and Rosa26-Cre^ERT+/− Trp53^LoxP/LoxP KRas^+/LSL-G12D Jnk1^LoxP/LoxP Jnk2^−/− mice (Trp53^−/− Kras^G12D Jnk^−/− mice) were treated with 1 μM 4-hydroxy-tamoxifen (24 h). The MEF were examined by phase-contrast microscopy. (B) The expression of E-cadherin, N-cadherin, OB-cadherin, and α-tubulin in Trp53^−/− and Trp53^−/− Jnk^−/− MEF was examined by immunoblot analysis. The effect of activated Ras was examined. (C) E-cadherin and N-cadherin expression was examined by immunofluorescence microscopy (green). DNA was stained with DAPI (blue). (D) The expression of E-cadherin, Snail, and Slug mRNA by KRas^G12D Trp53^−/− MEF and KRas^G12D Trp53^−/− Jnk^−/− MEF was examined by quantitative RT-PCR assays. The data presented are normalized for the amount of Gapdh mRNA in each sample (mean ± SD; n = 3). Statistically significant differences (*, P < 0.05; **, P < 0.001) are indicated.

Fig. 3.

Role of E-cadherin in KRas-mediated transformation of control and JNK-deficient MEF. (A) KRas^G12D Trp53^−/− MEF and KRas^G12D Trp53^−/− Jnk^−/− MEF were transduced with recombinant retroviruses that express E-cadherin (lane 3) or dominant negative (dn) E-cadherin (lane 4). The expression of E-cadherin, dn-E-cadherin, JNK, and α-tubulin was examined by immunoblot analysis. (B) The morphology of MEF grown to confluence was examined by phase-contrast microscopy. Representative images are illustrated. (C) MEF (5 × 10⁴ cells) were plated in 24-mm dishes and cultured for 6 days. The effect of expression of E-cadherin and dn-E-cadherin was investigated. The relative number of cells was examined by staining with crystal violet (mean ± SD; n = 4). (D) Soft agar colony assays were performed using KRas^G12D Trp53^−/− MEF and KRas^G12D Trp53^−/− Jnk^−/− MEF. The effect of expression of E-cadherin and dn-E-cadherin was investigated. The efficiency of colony formation (%) is presented (mean ± SD; n = 4). Statistically significant differences are indicated (*, P < 0.001; **, P < 0.0001).

Fig. 4.

JNK suppresses stress-induced apoptosis in p53-deficient MEF. (A) MEF were exposed to the indicated doses of UV radiation. Lysates were prepared at 4 h postirradiation, and genomic DNA fragmentation was measured by ELISA. Statistically significant differences (P < 0.01) between WT MEF and mutant MEF are indicated with an asterisk. (B) MEF were exposed to UV radiation (60 J/m²). Relative cell numbers were measured by staining with crystal violet. The data are presented as means ± standard deviations (n = 3) and are representative of results from three independent experiments. Statistically significant differences (P < 0.01) between WT MEF and mutant MEF are indicated with an asterisk. (C) MEF were exposed to UV (60 J/m²). At 6 h postirradiation, the cells were replated at the indicated dilutions. Colony formation was assessed after 10 days in culture by staining with crystal violet. Representative images are shown. The mean number of colonies per dish (n = 5) is indicated. (D) Total cell lysates were prepared from MEF at the indicated times after exposure to UV radiation (60 J/m²). The amount of PARP and caspase 3 was assessed by immunoblot analysis using antibodies that detect both the cleaved (designated with an asterisk) and full-length forms of these proteins. The expression of α-tubulin was examined to confirm equal loading of each lane.

Fig. 5.

Effect of activated Ras on Trp53^−/− MEF and Trp53^−/− Jnk^−/− MEF. (A) MEF were exposed to 0 or 60 J/m² UV-C, harvested at 60 min postirradiation, and examined by immunoblot analysis using antibodies to phospho-JNK (pJNK), JNK, and α-tubulin. (B) MEF cultures were examined by phase-contrast microscopy. Representative images are illustrated. (C) The saturation density of MEF cultured in medium (10 days) with different concentrations of serum was examined by staining with crystal violet (mean ± SD; n = 3). (D, E) Cell proliferation in medium supplemented with 1% (D) or 10% (E) fetal calf serum. Relative cell number was measured by staining with crystal violet (mean ± SD; n = 3).

Fig. 6.

Ras-mediated signal transduction in Trp53^−/− MEF and Trp53^−/− Jnk^−/− MEF. (A) MEF were exposed to 0 or 60 J/m² UV-C and harvested at 60 min postirradiation. The amounts of phospho-p38 MAP kinase (p-p38), phospho-ERK (pERK), phospho-JNK (pJNK), and phospho-Ser⁴⁷³-AKT (pAKT) were measured by multiplexed ELISA (mean ± SD; n = 3). (B) ROS accumulation was examined by measurement of fluorescence intensity in studies of MEF stained with H₂DCFDA. The relative fluorescence intensity is indicated in the lower right corner of each panel. (C) The effect of rotenone and diphenyleneiodonium chloride (DPI) on ROS production by Ras-transformed Trp53^−/− MEF was examined by measurement of mean fluorescence intensity (mean ± SD; n = 3). Statistically significant differences (P < 0.05) are indicated with an asterisk. (D) The expression of Nox4 and p22^phox mRNA by Ras-transformed Trp53^−/− MEF and Trp53^−/− Jnk^−/− KRas^G12D MEF was examined by quantitative RT-PCR assays. The data presented are normalized for the amount of Gapdh mRNA in each sample (mean ± SD; n = 3 samples). Statistically significant differences (P < 0.01) are indicated with an asterisk.

Fig. 7.

JNK is required for colony formation in soft agar and tumor formation in vivo. (A, B) Ras-transformed MEF and (Trp53^−/− Jnk^−/− MEF were treated with 1 μM 4-hydroxy-tamoxifen (24 h). The MEF were plated in soft agar and incubated for 2 weeks. (A) Representative images of colonies formed by the MEF are illustrated. The number of soft agar colonies per plate was counted (mean ± SD; n = 6) (B). Statistically significant differences (P < 0.001) are indicated with an asterisk. (C, D) MEF were injected subcutaneously in the flank of host mice. (C) Palpable tumor nodules were measured. (D) The mice were euthanized at day 21 postinjection, and the tumor mass (mean ± SD; n = 10) was measured. Statistically significant differences (P < 0.001) are indicated with an asterisk.

Fig. 8.

JNK is required for Ras-induced lung tumorigenesis. (A) KRas^+/LSL-G12D mice (control KRas^G12D mice) and KRas^+/LSL-G12D Jnk1^LoxP/LoxP Jnk2^−/− mice (KRas^G12D Jnk^−/− mice) were exposed to adenovirus-Cre by nasal instillation. Lung tumors were detected by examination of the lungs at 12 weeks postinfection. (B) Lung tissue sections prepared from control KRas^G12D mice and KRas^G12D Jnk^−/− mice at 4 weeks and 8 weeks postinfection were stained with hematoxylin and eosin. (C) The number of hyperplastic lesions per tissue section was measured at 4, 8, and 12 weeks postinfection. Significant differences between control and JNK-deficient tumors are indicated with an asterisk (P < 0.05; n = 10). (D) The number of adenomas (>0.5 mm) per tissue section was measured at 4, 8, and 12 weeks postinfection. Significant differences between control and JNK-deficient tumors are indicated with an asterisk (P < 0.05; n = 10). (E) Lung sections were stained with an antibody to the proliferation marker PCNA (red). DNA was stained with DAPI (blue). (F) Lung extracts prepared from mice treated without adenovirus-Cre (control and Jnk^LoxP/LoxP) and with adenovirus-Cre (KRas^G12D and KRas^G12D Jnk^−/−) at 12 weeks postinfection were examined by immunoblot analysis using antibodies to detect PCNA, cleaved PARP, caspase 3 (Casp. 3), cleaved caspase 3 (Casp. 3*), and α-tubulin.

Fig. 9.

Analysis of lung tumors detected in JNK-deficient mice. Nasal instillation of adenovirus-Cre was performed using control KRas^+/LSL-G12D mice and JNK-deficient Jnk1^LoxP/LoxP Jnk2^−/− KRas^+/LSL-G12D mice. Cre-mediated ablation of the LoxP-Stop-LoxP cassette causes expression of KRas^G12D in the lung epithelium. (A) Genomic DNA was isolated from individual lung tumors in Jnk1^LoxP/LoxP Jnk2^−/− KRas^+/LSL-G12D mice and examined by PCR analysis to detect the KRas⁺, KRas^G12D, and KRas^LSL-G12D alleles (top) and the Jnk1^LoxP and Jnk1^Δ alleles (bottom). Control (Con) studies were performed using equal mixtures of genomic DNA isolated from normal lung and from lung tumors of Jnk1^LoxP/LoxP Jnk2^−/− KRas^+/LSL-G12D mice. These data confirm efficient deletion of the LoxP-Stop-LoxP cassette from the KRas^G12D gene and retention of the Jnk1^LoxP allele in the JNK-deficient tumors. (B) The structures of the wild-type KRas (WT), KRas^G12D, Jnk1^LoxP, and Jnk1^Δ genes are illustrated schematically. Amplimers employed for PCRs designed to distinguish between these genes are indicated. (C) Sections of lung tumors were stained using an antibody to phospho-Ser⁶³-c-Jun and DAPI and examined by fluorescence microscopy (left). The fluorescent images were merged with images obtained using differential interference contrast microscopy (right). Ser⁶³ is a phosphorylation site of c-Jun by JNK. These data demonstrate that lung tumors in JNK-deficient mice retain a functional JNK signaling pathway.