Role of JNK in a Trp53-Dependent Mouse Model of Breast Cancer

The cJun NH2-terminal kinase (JNK) signal transduction pathway has been implicated in mammary carcinogenesis. To test the role of JNK, we examined the effect of ablation of the Jnk1 and Jnk2 genes in a Trp53-dependent model of breast cancer using BALB/c mice. We detected no defects in mammary gland development in virgin mice or during lactation and involution in control studies of Jnk1−/− and Jnk2−/− mice. In a Trp53−/+ genetic background, mammary carcinomas were detected in 43% of control mice, 70% of Jnk1−/− mice, and 53% of Jnk2−/− mice. These data indicate that JNK1 and JNK2 are not essential for mammary carcinoma development in the Trp53−/+ BALB/c model of breast cancer. In contrast, this analysis suggests that JNK may partially contribute to tumor suppression. This conclusion is consistent with the finding that tumor-free survival of JNK-deficient Trp53−/+ mice was significantly reduced compared with control Trp53−/+ mice. We conclude that JNK1 and JNK2 can act as suppressors of mammary tumor development.


Introduction
The cJun NH 2 -terminal kinase (JNK) group of signaling enzymes are activated by cytokines/growth factors and also by exposure to environmental stress [1]. Targets of the JNK pathway include members of the activator protein 1 (AP1) group of transcription factors (e.g. cJun, JunB, and JunD). JNK is therefore a major regulatory mechanism of AP-1 dependent gene expression [1]. In addition, JNK can regulate many cytoplasmic and nuclear processes [2]. These studies have implicated the JNK signaling pathway in the regulation of cell growth and cell death [1]. Dysregulation of the JNK pathway may therefore contribute to the development of cancer [3].
The purpose of this study was to test the requirement of JNK1 and JNK2 in a mouse model of mammary carcinoma. Somatic mutation of the human p53 gene (TP53) is common in sporadic breast cancer [11]. Furthermore, mammary carcinoma is the most common form of cancer in women with heritable mutations in TP53 (Li-Fraumeni syndrome) [12]. Initial studies using mouse models demonstrated that Trp53 2/2 animals develop lymphoma with high frequency and that Trp53 2/+ animals display a moderately broader tumor spectrum with slower onset of disease [13,14]. Subsequent studies using Trp53 2/+ mice on a BALB/c strain background demonstrated that, like humans with Li-Fraumeni syndrome, mammary carcinomas were frequently observed, together with some lymphomas and sarcomas [15]. The BALB/c mouse model can therefore be used to examine Trp53-dependent formation of mammary carcinoma.
We report that JNK1 and JNK2 are not required for the development of mammary carcinoma in the Trp53 2/+ BALB/c mouse model. In contrast, the tumor-free survival of JNK-deficient Trp53 2/+ mice was reduced compared with control Trp53 2/+ mice. These data suggest that JNK may partially contribute to tumor suppression.

Mice
We have described Jnk1 2/2 mice [16] and Jnk2 2/2 mice [17] on a C57BL/6J strain background [18], and mice with Trp53 gene ablation [13] on a BALB/cMed strain background [19]. The mice used in this study were backcrossed (ten generations) to the BALB/ cJ strain (Jackson Laboratories) and were housed in a facility accredited by the American Association for Laboratory Animal Care (AALAC). The Institutional Animal Care and Use Committee (IACUC) of the University of Massachusetts Medical School approved all studies using animals (Docket A-1032).

Genotype analysis
Genotype analysis was performed by PCR using genomic DNA as the template. The wild-type Jnk1 (460 bp) and knockout Jnk1

Analysis of tissue morphology
Mammary gland development was examined in virgin female mice (8 to 10 weeks of age), lactating mice (1 week post partum), and mice with mammary gland involution (pups removed at 1 week post-partum). The fourth inguinal mammary gland pair was dissected from each mouse; one gland was analyzed by whole mount and the other was formalin-fixed and paraffin-embedded.
Whole mounts were performed by spreading the gland on a glass slide and incubation (2-4 hrs.) with Carnoy's fixative (60% ethanol, 30% chloroform, 10% glacial acetic acid). The glands were then incubated with a graded series of 70%, 50% and 25% ethanol (15 mins each), followed by 5 minutes in water and stained with carmine alum overnight. The glands were washed in 70%, 90% and 100% ethanol (15 mins each), two changes of xylene (30 mins), and then mounted with Permount (Fisher Scientific).
Analysis of tissue sections was performed using tissue fixed in 10% formalin for 24 h, dehydrated, and embedded in paraffin. Sections (7 mm) were cut and stained using hematoxylin and eosin (Biocare Medical). Immunofluorescence analysis was performed using deparafinized sections treated with the endogenous Biotin-Blocking kit (Invitrogen), staining (4uC, 12 h) with biotin-conjugated anti-PCNA (Invitrogen), and the incubation (25uC, 1 hr) with AlexaFluor633conjugated Streptavidin (Invitrogen). The sections on coverslips were washed and mounted on slides using VectaShield medium containing DAPI (Vector Labs.). Images were examined using a Leica TCS SP2 confocal microscope.

Effect of JNK-deficiency on mammary gland development
We backcrossed Jnk1 2/2 mice [16] and Jnk2 2/2 mice [17] to the BALB/cJ strain background. To test whether JNK-deficiency altered mammary gland development, we examined Jnk1 2/2 and Jnk2 2/2 BALB/c mice. No defects were detected in whole mount preparations of fourth inguinal mammary glands of JNK-deficient virgin female mice compared with control mice ( Figure 1A). Sections prepared from these mammary glands confirmed that JNK-deficiency did not cause major defects in virgin mammary gland development ( Figure 1B).
Pregnancy causes major changes in mammary gland development, including the formation of alveoli. Sections prepared from the fourth inguinal mammary glands of JNK-deficient lactating mice and control lactating mice were similar ( Figure 2). Indeed, sections stained for proliferating cell nuclear antigen (PCNA) indicated that JNK-deficiency did not alter epithelial cell proliferation in the lactating mammary gland (Figure 2).
Involution of the lactating mammary gland occurs after weaning pups. We compared sections of the fourth inguinal mammary glands prepared on day 2 and day 3 following weaning. No defects in involution were detected in JNK-deficient mice compared with control mice (Figure 3).
Together, these data demonstrate that JNK1-deficiency and JNK2-deficiency did not cause detected changes in mammary gland development. Similarly, no developmental defects caused by JNK1-deficiency or JNK2-deficiency were detected in Trp53 2/+ mice.
Together, these data indicate that JNK1 and JNK2 are not required for mammary carcinoma development in the Trp53 2/+ BALB/c mouse model of breast cancer. However, both JNK1 and JNK2 can influence breast cancer development. It appears that JNK can contribute to tumor suppression.

Discussion
JNK1 and JNK2 are not required for the development of mammary carcinoma in the Trp53 BALB/c mouse model JNK plays a critical role in the development of some forms of cancer [1]. Thus, carcinogen-induced hepatocellular carcinoma [5] and BcrAbl-induced lymphoma [4] are strongly suppressed in Jnk1 2/2 mice and carcinogen-induced skin cancer is suppressed in Jnk2 2/2 mice [6]. Moreover, studies of glioblastoma, prostate cancer, and lung carcinoma cell lines have identified important roles for JNK2 [7][8][9][10]. Together, these data confirm that JNK plays an important role in cancer development.
The results of this study suggest that JNK may play a different role in mammary carcinogenesis because neither JNK1-deficiency nor JNK2-deficiency in the Trp53 BALB/c mouse model caused a reduction in the incidence of mammary carcinoma. This observation strongly contrasts with the finding that JNK-deficiency can markedly suppress hepatocellular carcinoma, lymphoma, and skin cancer [4][5][6].
Although JNK1-deficiency and JNK2-deficiency did not suppress mammary carcinogenesis in the Trp53 BALB/c mouse model, we cannot exclude the possibility that deficiency of both JNK1 plus JNK2 might reduce the formation of mammary carcinoma. Indeed, the Jnk1 and Jnk2 genes may have partially redundant functions [18,[20][21][22]. Studies of compound mutants with disruption of Jnk1 plus Jnk2 are required. The early embryonic lethal phenotype of Jnk1 2/2 Jnk2 2/2 mice [23] makes such studies difficult. Nevertheless, the effect of compound JNKdeficiency on mammary carcinoma development needs to be tested in future studies.