A dominant-negative c-jun mutant inhibits lung carcinogenesis in mice

Abstract

Lung cancer is the leading cause of cancer mortality in the United States and worldwide. The identification of key regulatory and molecular mechanisms involved in lung tumorigenesis is therefore critical to increase our understanding of this disease and could ultimately lead to targeted therapies to improve prevention and treatment. Induction of members of the activator protein-1 (AP-1) transcription factor family has been described in human non-small cell lung carcinoma. Activation of AP-1 can either stimulate or repress transcription of multiple gene targets, ultimately leading to increased cell proliferation and inhibition of apoptosis. In the present study, we show induction of AP-1 in carcinogen-induced mouse lung tumors compared with surrounding normal lung tissue. We then used a transgenic mouse model directing conditional expression of the dominant-negative c-jun mutant TAM67 in lung epithelial cells to determine the effect of AP-1 inhibition on mouse lung tumorigenesis. Consistent with low AP-1 activity in normal lung tissue, TAM67 expression had no observed effects in adult mouse lung. TAM67 decreased tumor number and overall lung tumor burden in chemically induced mouse lung tumor models. The most significant inhibitory effect was observed on carcinoma burden compared with lower-grade lesions. Our results support the concept that AP-1 is a key regulator of mouse lung tumorigenesis, and identify AP-1-dependent transcription as a potential target to prevent lung tumor progression.

Figure 1. AP1-dependent luciferase expression in B(a)P-induced mouse lung tumors

Lung tumorigenesis was induced with B(a)P in AP1-luciferase mice on the A/J strain background, tumor formation allowed to progress for 40 weeks and frozen lung tissue obtained. Lung cryosections were used for in situ hybridization with digoxigenin-labeled riboprobes recognizing luciferase mRNA followed by colorimetric detection with anti-digoxigenin antibodies. Sections were counterstained with hematoxylin. (A) H&E stain showing representative adenocarcinoma at 40 weeks post-B(a)P. (B) The same lung tumor hybridized with anti-sense luciferase riboprobe. Note the lack of staining in normal appearing lung and strong signal from the tumor. (C) A serial section hybridized with a sense riboprobe gave no signal.

Figure 2. Doxycycline-inducible expression of TAM67 in mouse lung

Bitransgenic Ccsp/Tam67 mice were generated by breeding previously characterized Tre-Tam67 and Ccsp-rtta transgenic lines (see Materials and Methods). (A) RT-PCR analysis showing doxycycline induced Tam67 mRNA in lung tissue within 2 days and transgene expression was maintained with continued doxycycline administration. Days on doxycycline is indicated above each lane. Actb: β-actin mRNA. (B) Protein lysates from lung tissue were immunoprecipitated with a mouse monoclonal anti-Flag antibody followed by immunoblotting with the same antibody. Days on doxycycline is indicated above each lane. 7* indicates 7 days of doxycycline was initiated 7 days after injection of urethane. All other lanes represent mice that did not receive urethane. 7/-6 indicates mice that were fed doxycycline for 7 days followed by removal of doxycycline for 6 days. In both A and B, each lane represents a separate lung sample. Flag: Flag epitope tagged Tam67 protein; LC: immunoglobulin light chain. (C and D) H&E stains of lung tissue from Ccsp/Tam67 mice receiving doxycycline (D) were indistinguishable from littermates that did not receive doxycycline (C).

Figure 3. TAM67 inhibited lung carcinogenesis when induced pre-initiation

(A). Experimental design (Protocol 1) to determine the effect of TAM67 induction prior to carcinogen administration. 6-8 week old Ccsp/Tam67 mice were started on doxycycline diet and lung carcinogenesis initiated by i.p. injection of B(a)P five days later. Doxycycline diet was continued throughout the experiment until termination 40 weeks post-B(a)P. Induction of the TAM67 transgene with doxycycline (Dox) significantly decreased tumor multiplicity (B) and total tumor burden per animal (C) compared to uninduced age-matched animals (No dox). Bars represent mean ± SD; n = 13 mice/group. *, p = 0.007 for multiplicity and p = 0.012 for tumor burden.

Figure 4. TAM67 inhibited lung tumorigenesis when induced post-initiation

(A). Experimental design (Protocol 2) to determine the effect of TAM67 induction after initiation of lung carcinogenesis with urethane. 6-8 week old mice were injected i.p. with urethane to initiate lung carcinogenesis and doxycycline diet started 10 days later. (B). Doxycycline had no effect on tumor multiplicity in Ccsp single transgenic animals (C Dox versus C No Dox) but significantly inhibited tumor multiplicity in Ccsp/Tam67 bitransgenic mice (C/T Dox versus C/T No Dox, p = 0.034). (C). Paraffin embedded, formalin fixed lung tissue sections from Ccsp/Tam67 mice were stained with H&E and tumors graded as adenomas, dysplasias or adenocarcinomas as described in Materials and Methods. Digitized images of H&E stained sections containing all five lung lobes (3 slides per mouse separated by 200 μm) were used to determine the area occupied by each tumor grade. Induction of TAM67 significantly inhibited carcinoma burden and the total tumor burden (all grades). Bars represent mean ± SD; n = 4-6 mice/group. *, p = 0.039 for carcinoma burden and p = 0.034 for total tumor burden.