Activation of the AP-1 family of transcription factors has previously been implicated in lung tumorigenesis but direct evidence for involvement of this pathway in vivo has been lacking. In the current study, we used B(a)P-induced lung carcinogenesis to confirm the presence of increased AP-1 activity in mouse lung tumors. Using a transgenic mouse line expressing the firefly luciferase reporter gene under control of a consensus AP-1 promoter we detected increased luciferase mRNA in lung tumors, but not normal lung tissue, 40 weeks after injection of the lung carcinogen B(a)P. This data supports the concept that AP-1 target genes are specifically activated in lung tumors during lung tumorigenesis. The use of the AP1-luciferase reporter mice enabled a readout of AP-1 transcriptional activity using a well established AP-1 responsive promoter. While we cannot be certain that the AP-1 consensus promoter directing luciferase expression in this mouse is representative of all AP-1 responsive genes, the data is consistent with an important role for AP-1 activation in mouse lung tumors. Determining AP1 luciferase reporter gene expression at additional time points will increase our understanding of the timing and extent of AP-1 activation during mouse lung tumorigenesis.
Previous studies demonstrated a tumor inhibitory effect of the dominant negative mutant Tam67 during mouse skin and breast tumorigenesis in vivo
). Expression of Tam67
under control of an epidermal specific promoter inhibited skin papilloma formation in a two stage initiation/promotion model (30
) and inhibited UVB-induced squamous cell carcinomas (31
). Interestingly, in both cases early epidermal hyperplasia was unaffected although inhibition of AP-1 activity was confirmed using the AP1-luciferase reporter mouse suggesting that AP-1 transcriptional activation is important during the tumor promotion stage of skin tumors. In Protocol 2, we observed an inhibitory effect of Tam67 on lung tumorigenesis when the transgene was turned on 10 days after carcinogen administration. This result indicates that inhibition of AP-1 during the tumor promotion stage of lung tumorigenesis is sufficient to reduce tumor number. The same Tre-Tam67
transgenic mice used in our study have also been crossed to MMTV-rtta
transgenic mice enabling doxycycline inducible Tam67
expression in mammary epithelial cells (32
). When MMTV/Tam67
mice were crossed to MMTV-ERBB2
transgenic mice that develop mammary tumors, administration of doxycycline reduced mammary tumor incidence and increased survival in trigenic mice. The present study extends to lung the tumor types that can be successfully inhibited by expression of Tam67 in vivo
Examination of lung tumor pathology in carcinogen treated Ccsp/Tam67
mice indicated that Tam67
expression resulted in a 56% (P=0.039) reduction in carcinomas compared to mice in which Tam67
was not induced. There was also a trend for reduction of adenomas in the Tam67
expressing mice, while there was no effect on dysplasias (). These data suggest that Tam67
expression inhibited progression of lung tumors. An increase in lower grade lesions was not observed as might be predicted if Tam67 was acting solely by inhibiting progression from lower to higher grade lesions. In fact, the frequency of hyperplasias was inhibited to a similar extent as carcinomas (). We interpret this result to mean that Tam67 can exert inhibitory effects throughout the process of lung tumorigenesis. It is possible that expression of the Tam67
transgene could be activated at different times in different cells during the tumorigenesis process. Expression of the transgene is ultimately determined by the 2.3 kb rat CCSP promoter driving rtTA
expression and this promoter may be transiently activated or inactivated in subsets of epithelial cells within the lung. This effect may be particularly prevalent in rapidly proliferating tumor cells. In addition, the transgene promoter does not direct rtTA
expression in all epithelial cells (39
) and thus carcinogen-induced tumors can arise in non-expressing cells contributing to the partial inhibition seen. Shen et al. (32
) observed that Tam67
expression was absent in breast tumors that persisted following doxycycline treatment and a similar situation may occur in our model.
The ability of Tam67 to inhibit lung tumor formation indicates this pathway has potential as a target of intervention in this disease. Several classes of chemopreventive agents are reported to inhibit AP-1 activity including glucocorticoids (47
) and tea catechins (19
) and this may mediate part of their chemopreventive effect. Because of the multiple pro-tumorigenic effects of AP-1 signaling it has been suggested to be an attractive target for cancer chemoprevention (48
). Studies to directly address the contribution of AP-1 inhibition in the cancer chemopreventive effect of these agents are needed both to confirm the importance of this transcription factor in tumorigenesis and improve our understanding of the mechanisms used by effective chemopreventive agents. To this end we have preliminary data indicating that a purified green tea extract, a known lung cancer chemopreventive agent, is ineffective in preventing lung tumors in induced Ccsp/Tam67
mice (Y. Yan, Y. Wang, J. Tichelaar, M. You, unpublished data) although additional studies are needed to confirm this result. This approach should be useful in determining the relative contribution of AP-1 towards the efficacy of many chemopreventive agents.
The fact that Tam67 inhibited lung tumorigenesis when doxycycline was given post-initiation (Protocol 2) as well as pre-initiation (Protocol 1) suggests that Tam67 has a direct effect on inhibiting lung tumor progression. However, the ability of Tam67 to affect expression of enzymes mediating carcinogen metabolism and clearance has not been explored. In the present study, Protocol 1 was conducted only with B(a)P while Protocol 2 used urethane. Given that these two carcinogens are metabolized by distinct enzymes it is possible that using both protocols for each carcinogen could yield carcinogen specific results. However, both carcinogens induce Kras mutations which are thought to be a primary oncogenic driver of these tumors. Therefore, it is likely that inhibition of AP-1 activation in developing tumors plays a key role in the inhibitory effect of Tam67.
A key unanswered question is the specific gene targets mediating the lung tumor inhibiting effects of TAM67. Multiple studies have implicated genes involved in control of the G1/S transition of the cell cycle as targets for TAM67 inhibition including Rb, E2F1, E2F2, cyclin A, cyclin D and the CDK inhibitor p27 (38
). Recent studies highlight that a subset of AP-1 responsive genes may mediate the inhibitory effects of TAM67 in a given cell type (51
). mRNA expression profiling indicated that targets of TAM67 during inhibition of skin tumorigenesis were classified as being involved in invasion, metastasis and inflammation to a greater extent than proliferation or cell survival (51
). In addition to inhibition of AP-1, some studies have demonstrated interactions between Tam67 and NFκB transcription complexes (54
). Our results indicate that AP1-dependent reporter gene expression is increased in B(a)P induced lung tumors.
Our results demonstrate the inhibitory effect of Tam67 expression on mouse lung tumorigenesis. The model described here will be useful in determining the key targets mediating the lung tumor inhibitory effects of Tam67. This in turn will enhance our understanding of the molecular alterations that occur during lung tumorigenesis and may lead to the identification of novel targets for chemopreventive and/or therapeutic intervention.