Discussion:
One major goal in the field of prevention is the identification of surrogate biomarkers that might rapidly predict the effect of a given agent on the primary end-point of cancer incidence. The most informative markers are those with modulation that is likely to be directly related to the preventive effect, and a compelling argument can be made that TGF-βs may fall into this category. However, the present data in a well-established preclinical model of breast cancer, employing a variety of highly effective chemopreventive regimens, suggest that this is not the case.
Most of the previous studies on the regulation of TGF-βs by tamoxifen and retinoids have been done in tissue culture [12,13,14,17]. The lack of effect on TGF-β expression in the present in vivo study may reflect the dependence of the response on contextual cues that are only present in the artificial in vitro environment. In an in vivo study [16], all-trans-retinoic acid was shown to cause an upregulation of TGF-β isoforms in rats, with kinetics and isoform selectivity that varied with the target tissue. However, the rats were vitamin A-deficient, and it is not known whether the same effects would be seen in vitamin A-replete animals such as were used in the present study. In a small study in humans [23] tamoxifen treatment was shown to cause a consistent induction in extracellular TGF-β in breast cancer biopsies, when compared with pretreatment biopsies from the same patients, and complex effects of tamoxifen on induction of TGF-β2 in the plasma of patients with metastatic breast cancer have been described [24]. It is possible that tamoxifen is only effective in inducing TGF-β in the context of a tumor, and not in the normal or initiated tissue that was the subject of the present study. However, an optimal surrogate end-point biomarker in a prevention setting needs to be modulated in normal or premalignant tissues. Although we cannot eliminate the possibility of more subtle effects of chemopreventive agents on TGF-β bioavailability or cellular responsiveness, in our preliminary analyses we have seen no effects on the expression of type I and type II TGF-β receptors (data not shown).
There is considerable evidence to suggest that, at late stages in tumorigenesis, TGF-βs can actually promote the tumorigenic process, particularly if the epithelial cells have lost responsiveness to the growth inhibitory effects of TGF-β by this time [9]. While the present work was in progress, a study was reported [25] that showed that loss of the type II TGF-β receptor can already be seen in a significant fraction of hyperplasias without atypia in the human breast. Furthermore, loss of the receptor correlated with increased risk of subsequent development of invasive breast cancer. Thus, loss of TGF-β response may be a very early event in the development of human breast cancer. Because locally elevated TGF-β levels could select for TGF-β-resistant cells, and because TGF-βs can have oncogenic effects on the stroma, it may actually be important for the safety profile of chemopreventive agents to demonstrate that they do not increase TGF-β levels in the at-risk breast. In this regard, this demonstration that the expression of TGF-βs in the preclinical rat model is unaffected by tamoxifen, 9cRA, or 4-HPR may actually have positive implications, because all three agents are already in clinical use.
The NMU-induced rat model of mammary tumorigenesis is widely used for chemoprevention studies, and yields rapid development of hormonally responsive mammary tumors with 100% incidence [2,4,18]. To do this, the initiating agent is given at 8 weeks of age and the chemopreventive agent is started a week later, during the period of active development of the mammary gland. We observed that the histology of the tamoxifen-treated mammary glands differed significantly from control glands when examined after 6 weeks of tamoxifen treatment, showing fewer terminal end-buds and less tertiary branching. Part of the chemopreventive efficacy of antiestrogens and retinoids in this model may therefore be due to a generalized decrease in ductal development. Since chemopreventive agents are unlikely to be given to humans during the pubertal period, this form of preclinical model may not accurately reflect the degree of chemopreventive benefit that could be achieved in humans. Although the accelerated time course and high penetrance of disease reduces the costs of this model, it may be advisable to confirm efficacy of promising agents in a model that delays application of the chemopreventive agent until the mammary gland is fully developed.
In conclusion, we have shown that treatment of rats with tamoxifen or retinoids results in effective chemoprevention of mammary tumorigenesis, without any detectable effect on local expression of TGF-βs. Although we cannot rule out more subtle effects on TGF-β activity, such as the activation of latent forms, the data suggest that TGF-βs are not involved in the underlying molecular mechanism of chemoprevention induced by these agents. This agrees with in vitro work [26] that showed that blockade of TGF-β signaling did not abrogate the growth inhibitory effect of tamoxifen on breast cancer cells. Given the very limited breast tissue available in clinical trials, we do not recommend testing for TGF-βs as a surrogate end-point biomarkers at this time.