Transforming growth factors-β are not good biomarkers of chemopreventive efficacy in a preclinical breast cancer model system

Abstract

. Despite demonstrable chemopreventive efficacy in this model, none of these agents, alone or in combination, had any significant impact on the expression of TGF-βs in the mammary ductal epithelium or periductal stroma as determined by immunohistochemistry. These data suggest that TGF-βs are not likely to be useful biomarkers of chemopreventive efficacy in a clinical setting.

Introduction:

]. Because it will not be possible to test many agents in large randomized clinical trials, efforts are underway to develop useful tissue-based surrogate end-point biomarkers that can be used to select only the most promising agents (and doses) for large-scale trials.

]. It is plausible, therefore, that upregulation of endogenous TGF-β could contribute to the chemopreventive efficacy of SERMs and retinoids.

In the present study we used a carcinogen-induced rat mammary carcinogenesis model to test the hypothesis that chemoprevention by tamoxifen and retinoids is associated with local upregulation of TGF-βs in the mammary gland, and that TGF-βs might therefore be useful as potential surrogate end-point biomarkers of chemopreventive efficacy in clinical trials.

Materials and methods:

, beginning 1 week after injection with NMU. The rats were fed 9cRA (Kuraray Company, Osaka, Japan) at 120 mg/kg of diet, tamoxifen (Sigma Chemical Co, St Louis, MO, USA) at 1.0 mg/kg of diet, and 4-HPR (RW Johnson Pharmaceutical Research Unit, Spring House, PA, USA) at 782 mg/kg of diet.

Rats were weighed and palpated for the presence of mammary tumors weekly, and six rats in each experimental group were sacrificed after 6 and 12 weeks of treatment with chemopreventive agent. For experiments to determine the effect of high doses of tamoxifen administered over shorter periods of time, rats were given 10 mg tamoxifen/kg body weight per day intragastrically, or 1 mg tamoxifen/kg in the diet, and were sacrificed after 1 day or 3 weeks of treatment. All palpated tumors were confirmed at necropsy, and mammary glands were fixed in neutral buffered formalin and embedded in paraffin. The number 2 (first thoracic) mammary gland was sectioned for histology and immunohistochemistry.

). Immunohistochemical staining was performed using an indirect immunoperoxidase detection protocol (Vectastain Elite kit, Vector Laboratories, Burlingame, CA, USA). Staining intensity was scored on a scale of 0-4+, using the mouse embryo control section as a reference standard for each run. Ducts and periductal stroma were scored independently. Staining was scored in a blinded manner by two independent observers, and discrepancies were rescored by consensus. Staining intensity was plotted as the mean ± standard deviation for each experimental group.

Results:

).

). They were present both in the ductal epithelium and in the periductal stroma, suggesting that the TGF-βs are synthesized by the epithelial cells, and possibly stromal cells, and are sequestered in the extracellular matrix. This staining pattern is consistent with a role for the TGF-βs in the maintenance of normal mammary homeostasis.

antibodies (data not shown).

). By 12 weeks of treatment, all three chemopreventive agents had a significant effect on glandular histology, with tamoxifen and 9cRA showing the greatest suppression of ductal development and lobule formation, and 4-HPR showing a relatively mild effect.

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.

]. 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).

] 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.

]. 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.

] 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.

Introduction

]. These studies validate the concept of using pharmacologic agents for prevention of human breast cancer in apparently healthy individuals.

]. Since it will not be possible to test many agents in large randomized clinical trials, efforts are underway to develop useful tissue-based surrogate end-point biomarkers that can be used to select only the most promising agents (and doses) for large-scale trials.

]. This strongly suggests that interventions that enhance TGF-β function early in tumorigenesis could delay or prevent the course of the disease.

could be mediated via a local upregulation of TGF-βs, with concomitant enhancement of tumor suppressor activity.

In the present study, we used a carcinogen-induced rat model of mammary carcinogenesis to test whether chemoprevention by tamoxifen and by two different retinoids (4-HPR, also known as fenretinide; and 9-cRA) is associated with local upregulation of TGF-βs in the initiated mammary gland. If this were the case, TGF-βs might be useful as potential surrogate end-point biomarkers in clinical trials. However, the results show that TGF-β levels, as detected immunohistochemically, are not affected by tamoxifen or retinoids in this preclinical model of early-stage breast cancer.

Mammary carcinogenesis studies

, beginning 1 week after injection with NMU. Rats were fed 9cRA (Kuraray Company, Osaka, Japan) at 120 mg/kg of diet, tamoxifen (Sigma Chemical Co, St Louis, MO, USA) at 1.0 mg/kg of diet, and 4-HPR (RW Johnson Pharmaceutical Research Institute) at 782 mg/kg of diet.

Rats were weighed weekly and palpated for the presence of mammary tumors. Six rats in each experimental group were sacrificed after 6 and 12 weeks of treatment with chemopreventive agent. The 6-week sacrifice time was chosen for the immunohistochemical studies to represent the period of premalignancy, because the incidence of palpable tumors is less than 20% for all experimental groups at that time. By 12 weeks all rats that have not received a chemopreventive agent have tumors, so the primary purpose of the 12-week sacrifice time was to allow an accurate determination of chemopreventive efficacy for the particular experiment.

For experiments to determine the effect of high doses of tamoxifen administered over shorter periods of time, rats were given 10 mg tamoxifen/kg body weight per day intragastrically or 1 mg tamoxifen/kg in the diet, and were sacrificed after 1 day or 3 weeks of treatment.

All palpated tumors were confirmed at necropsy, and mammary glands were fixed in neutral buffered formalin and embedded in paraffin. The number 2 (first thoracic) mammary gland was sectioned for histology and immunohistochemistry.

Immunohistochemistry of TGF-βs

].

on Western blots.

-CC, and anti-LTBP). In analysis of the full experimental set, for any given antibody all sections were stained at the same time so as to be directly comparable, and a normal mouse embryo section was included as a positive control. A normal rabbit immunoglobulin control was also run for the whole set.

Quantitation of immunostaining

Two different systems were used to grade immunostaining. For all samples, staining of the ducts and periductal stroma were scored independently. For samples after 6 weeks of chemopreventive treatment, staining intensity was scored on a scale of 0–4+, using the mouse embryo control section as a reference standard for each run. Staining was scored in a blinded manner by two independent observers, and scores never differed by more than one point. Discrepancies were rescored by consensus. Staining intensity was plotted as the mean ± standard deviation for each experimental group.

Chemopreventive efficacy

). This suggests that there was minimal toxicity associated with the chemopreventive intervention, except in the tamoxifen + 4-HPR group, in which mild toxicity was observed.

Effect of chemopreventive agents on TGF-β expression in initiated mammary gland

shows the typical immunohistochemical staining pattern for the TGF-βs and the LTBP (part of the naturally occurring latent TGF-β complex) in initiated mammary glands of 15-week-old rats that had not been treated with chemopreventive agents. All three TGF-β isoforms and the LTBP showed broadly similar staining patterns. They were present both in the ductal epithelium and in the periductal stroma, suggesting that the TGF-βs are synthesized by the epithelial cells, and possibly stromal cells, and are sequestered in the extracellular matrix. This staining pattern is consistent with a role for the TGF-βs in the maintenance of normal mammary homeostasis.

). Human clinical material for biomarker analysis in primary chemoprevention studies is also likely to comprise normal and initiated, at-risk epithelium with some early preneoplastic changes, but without evidence of major neoplastic change.

and the invasive carcinomas were in the control group. However, both samples also had histologically normal ducts on the same slide, which were scored for the analysis. There was no difference in staining between the ducts that were proximal to the tumor and those that were more distal, and neither were there any differences in staining observed between histologically normal and hyperplastic ducts in any of the samples analyzed (data not shown). Since the focus of the present study was on TGF-β expression changes in the preneoplastic gland, staining of the tumors was not scored for the analysis, but in the two cases present, the staining did not differ significantly from that of the surrounding normal-appearing ducts (not shown).

antibodies (data not shown).

Effect of chemopreventive agents on the histology of the mammary gland

). By 12 weeks of treatment, all three chemopreventive agents had a significant effect on glandular histology, with tamoxifen and 9cRA showing the greatest suppression of ductal development and lobule formation, and 4-HPR showing a relatively mild effect.

TGF-βs as candidate biomarkers

]. This suggested that the chemopreventive action of these agents against breast cancer could be mediated in part through enhancing the tumor suppressor activity of the endogenous TGF-β system, and thus that changes in TGF-β expression might serve as useful surrogate end-point biomarkers of chemopreventive efficacy. However, here we used the NMU-induced rat model of mammary carcinogenesis to show that the chemopreventive effect of tamoxifen and two retinoids is not associated with any consistent changes in TGF-β levels, at least as determined immunohistochemically.

Comparison with earlier studies

-retinoic acid upregulated TGF-β expression 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, or whether the response would vary with the specific retinoid used.

]. It is possible that tamoxifen is only effective in inducing TGF-β in the context of a tumor, and not in normal or initiated tissue, which was the subject of the present study. This issue could be reassessed in preclinical models using the same agents to treat established proliferative intraepithelial neoplasia. However, for ease of tissue acquisition, an optimal surrogate end-point biomarker in a prevention setting needs to be modulated in normal or premalignant tissues.

Alternative levels of regulation of the bioefficacy of TGF-βs

]. To date, expression of TGF-β receptors and downstream signaling components such as the Smads have not been well-characterized in this rat model, but in our preliminary analyses we saw no effect of retinoids on type I and type II TGF-β receptor expression in the mammary gland (data not shown). At this time, however, we certainly cannot rule out the possibility that tamoxifen and retinoids may be having subtle effects on the TGF-β system at levels other than the regulation of TGF-β expression.

Lack of effect of chemopreventive agents on TGF-β expression may have positive implications

].

] showing 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 subsequently developing invasive breast cancer. Thus, unlike in the colon, loss of TGF-β response may be a very early event in the development of human breast cancer.

] to be associated with an increase in TGF-βs and concomitant immunosuppressive effects on natural killer cells. In this regard, our demonstration that the expression of TGF-βs in the preclinical rat model is unaffected by tamoxifen, 9cRA, and 4-HPR may actually have positive implications, because these agents are already in clinical use.

Limitations of the NMU-induced rat model of mammary carcinogenesis

]. To do this, the initiating agent is given at 8 weeks of age, during early puberty, and the chemopreventive agent is typically given continuously, starting 1 week later. Since sexual maturity is achieved at approximately 11 weeks of age in rats, this means that the chemopreventive agent is given during a period of active development of the mammary gland.

]. Thus, part of the chemopreventive efficacy of antiestrogens and retinoids in this model may be due to a generalized decrease in ductal development. Because 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.

Conclusion

]. Given the very limited breast tissue available in clinical chemoprevention trials, we do not recommend testing for TGF-βs as surrogate end-point biomarkers at this time.

Acknowledgements

We thank Drs Calle Heldin and Kohei Miyazono for the generous gift of Ab39, and Dr Clint Grubbs for the rat tissues treated with high levels of tamoxifen for short durations. We are grateful to Dr Donald Gardner for veterinary pathology expertise, to Dr Michael Sporn for many inspiring discussions on the concept and practice of chemoprevention, and to Dr Anita Roberts for continued support and guidance. This study was funded in part by an American Society of Clinical Oncology Young Investigator Award to Dr Zujewski.

Figures and Tables

Mean weight of rat. The open arrow indicates the start of the chemopreventive intervention.

normal rabbit immunoglobulin. The brown stain indicates a positive immunoperoxidase reaction. Images were shot at 630× original magnification.

.

Initiation with NMU at 8 weeks, followed by treatment with tamoxifen from 9 to 15 weeks of age; scant numbers of atrophic primary and secondary mammary gland ductules are noted, with no alveolar bud development evident. (a, c, e) Shot at 100×; and (b, d, f) shot at 400× original magnification.

Keywords

• antiestrogens
• breast cancer
• chemoprevention
• retinoid
• transforming growth factor-βs