Targeted Chemoprevention in a Mouse Model for DCIS

Institution: University of California, Davis
Investigator(s): Jeffrey Gregg, M.D. -
Award Cycle: 2005 (Cycle 11) Grant #: 11IB-0123 Award: $135,326
Award Type: IDEA
Research Priorities
Etiology and Prevention>Prevention and Risk Reduction: ending the danger of breast cancer

Initial Award Abstract (2005)
Introduction: Current strategies for chemoprevention and adjuvant therapy for women at high risk for breast cancer (estrogen receptor positive) for the most part rely on anti-estrogen therapy. Anti-estrogen therapy is an effective strategy for chemoprevention, but it is evident that such therapy is not effective in all patients and could be improved. In turn, combination therapy has shown to be effective for invasive carcinomas, and there is no reason to doubt that the use of combination therapy for chemoprevention may offer improved efficacy and duration. The studies detailed in this proposal will explore this paradigm in order to prove that the combination of different therapeutics can be synergistic and improve efficacy for chemoprevention.

Hypotheses: We hypothesize that anti-estrogen therapy (anastrozole/Arimidex) combined with the concurrent or intermittent addition of clinically available therapeutics (rapamycin and celecoxib) that have synergistic mechanisms will yield improved therapeutic efficacy and have the potential to eradicate preneoplastic disease in the breast.

Methodology: For these studies, we will utilize a mouse model that we have developed that mimics the biology, pathology, and behavior of human ductal carcinoma in situ (DCIS). Mice will be treated with different dosages and combinations of agents, and the efficacy and toxicity of each treatment regimen will be assessed. These data will be used to additionally treat and compare mice using an intermittent combinational therapeutic regimen, with the theory that intermittent therapy would have a more benign side effect profile and thus be more amenable to future human clinical trials. For all treatments, preneoplasias and tumors will be snap-frozen for molecular studies to confirm the therapeutic mechanisms of action and to correlate the clinical endpoints with molecular data.

Innovative elements: The use of combinational therapy for chemoprevention has not been utilized or discussed in earnest as a treatment option for women at high risk of breast cancer. This project aims to determine if this therapeutic regimen can offer improved outcomes in one of the only mouse models for ductal carcinoma in situ (DCIS) or preneoplastic breast disease. Because this model recapitulates most of the aspects of preneoplastic disease of the breast in women, these results will have direct impact on the evaluation of this strategy as a therapeutic option in women. Therefore, if the results of these studies support the hypothesis that combinational therapy is more effective than current therapies, they will provide a genesis for new clinical trials.

Advocacy involvement and human issues: New therapeutic strategies for treating women at high risk for breast cancer are required. The use of combinational therapy, with agents that can act synergistically and target complementary mechanisms, whether given continuously or intermittently, offers a new paradigm with significant potential for improved efficacy. The grant proposal detailed here describes a method of testing this strategy in a unique mouse model for DCIS. If these studies prove that this strategy is superior to that of single-agent therapy, new therapeutic strategies for chemoprevention using multi-modality therapy can be explored. Specifically, clinical trials utilizing combinational therapy comprised of clinically available therapeutics can be initiated.

Final Report (2008)
Introduction: Current strategies for chemoprevention and adjuvant therapy for treating women at high risk for estrogen receptor-positive breast cancer rely predominantly on anti-estrogen therapy. However, studies show that anti-estrogen therapy is not effective in all patients and may have limited duration of efficacy. As combination therapy has been shown to be effective for treating invasive carcinomas, the use of combination therapy may also offer improved efficacy and duration for chemoprevention in high risk, preneoplastic breast lesions, or human ductal carcinoma in situ (DCIS). Early studies suggest that anti-estrogen therapy combined with agents that inhibit proliferation and promote apoptosis may have improved efficacy over the single agents.

Progress Towards Specific Aims: In the proposal, we utilized single agents, anastrozole, rapamycin, and then the used these agents in combination. The hypothesis is that anastrozole (an aromatase inhibitor) would decrease proliferation, rapamycin (an mTOR inhibitor) would induce apoptosis, and the use of both together would yield improved efficacy in our mouse model for DCIS. In the first experiments, tamoxifen, an estrogen blocker, with a related mechanism of action to anastrozole, was utilized. Tamoxifen reduced the pre-neoplastic growth and decreased the tumor incidence. The mechanism of action of tamoxifen was shown to be related to an effect of decreased proliferation. This data was published in Breast Cancer Research (2005;7(6):R881-9). In a second experiment, rapamycin was utilized in the model. It was shown that it also reduced the pre-neoplasia growth and reduced the tumor incidence. The primary mechanism of action was an induction of apoptosis. These results were published in Clinical Cancer Research (2006;12(8):2613-21). In both cases the therapeutics did not completely inhibit the progression to invasive carcinoma. With microarray gene profiling technology, we have identified a cell signaling pathway that we believe is induced with rapamycin to promote resistance. Central to this pathway is an increase in PPARgamma, a nuclear receptor protein that function as a transcription factor.

Summary: We have now shown that PPARgamma provides a survival mechanism and/or resistance mechanism for rapamycin/mTOR therapy. Therefore, we believe that combination therapy as proposed in the grant and that combination therapy that engages this PPARg and/or genes in its pathway, in conjunction with rapamycin, will provide better efficacy.

Symposium Abstract (2005)
We have used a transplantable mouse model for premalignant mammary hyperplasia that progress to invasive carcinoma to test the effectiveness of therapeutics in preclinical settings. These mouse Mammary Intraepithelial Neoplasia-Outgrowth (MIN-O) lines were derived from the premalignant mammary hyperplasia of MMTV- Polyoma Virus middle T (PyVmT) transgenic mice (Maglione, et al 2004). The PyVmT is a potent oncogene that is capable of transforming cells by the same signaling pathways activated by receptor tyrosine kinases, such as ErbB2, which has been dysregulated in human breast cancer. Mammary tumors in MMTV-PyVmT animals are similar to human breast cancer in terms of their biological progression (Lin, et al 2003), histology and molecular changes (Desai, et al 2002). The MIN-O lines have been maintained by serial transplantation over 3 years and preserve the biological characteristics of original MMTV-PyVmT tumor progression. The MIN-Os progress to tumors at a predictable latency and are associated with dysregulation of genes and pathways that are similarly altered in human DCIS (Maglione, et al 2004, Namba, et al 2004). We have started to utilize this model for chemoprevention studies by taking advantage of the short and consistent tumor latencies of the MIN-Os. The MIN-O tissues and the tumors derived from them have activated PI3-K/Akt pathway due to the PyVmT transgene. Rapamycin, an inhibitor of mTOR, which is a downstream effector of Akt, was used to study its effect on the growth of the MIN-Os. In vivo rapamycin treatment effectively inhibited the activation of targets of mTOR, P70S6K and 4E-BP1. Significant difference in the MIN-O size as well as global gene expression profiles were observed after one-week of treatment. Rapamycin treatment inhibited cell growth by the down-regulation of gene expression through genes involved in transcription and translation. Immunohistochemistry study showed that rapamycin treated MIN-Os also had reduced cell proliferation (p=0.0016) and angiogenesis (p<0.0001). After 5-weeks of treatment, differences in MIN-O size were more pronounced. While the untreated MIN-Os had increased their size and developed tumor foci within them, the treated MIN-Os were 30% smaller than the control MIN-Os and none of them had developed tumors. Since the transplanted MIN-O tissues are not palpable until tumor formation, micro-Positron Emission Tomography (uPET) imaging was used to image and follow the growth of the MIN-O tissues during the course of rapamycin treatment. Animals were imaged once a week, starting just prior to the start of the rapamycin treatment. MIN-O volume and FDG uptake decreased dramatically and remained low during the treatment, while the MIN-Os in the untreated animals continued to grow and transformed into tumors. When the treatment was ended after 2-weeks, however, the MIN-O growth resumed at the pre-treatment level, suggesting that the inhibitory effect is reversible. This model provides a platform that is amenable for the molecular dissection of tumor progression and for preclinical studies with easily measurable end-points.

References Lin, et al. Am J Pathol. 2003:163(5)2113-26. Desai, et al. Proc Natl Acad Sci USA. 2002:99(10)6967-72. Maglione, et al. Molecular Cancer Therapeutics 2004;3(8) pp.941-953. Namba, et al. Molecular Cancer Research 2004;2(8) pp.453-463.

Selective estrogen receptor modulators inhibit growth and progression of premalignant lesions in a mouse model of ductal carcinoma in situ
Periodical:Breast Cancer Research
Index Medicus: Breast Cancer Res
Authors: Ruria Namba, Lawrence JT Young, Jeannie E Maglione, Erik T McGoldrick, Stephenie Liu,
Yr: 2005 Vol: 7 Nbr: 6 Abs: Pg:881-889

Rapamycin inhibits growth of premalignant and malignant mammary lesions in a mouse model of ductal carcinoma in situ.
Periodical:Clinical Cancer Research
Index Medicus: Clin Cancer Res
Authors: Namba R, et al, and Gregg JP
Yr: 2006 Vol: 12 Nbr: 8 Abs: Pg:2613-2621

Preclinical imaging of mammary intraepithelial neoplasia with positron emission tomography.
Periodical:Journal of Mammary Gland Biology and Neoplasia
Index Medicus: J Mammary Gland Biol Neoplasia
Authors: Abbey CK, Borowsky AD, Gregg JP, Cardiff RD, Cherry SR
Yr: 2006 Vol: 11 Nbr: 2 Abs: Pg:137-49

Heterogeneity of mammary lesions represent molecular differences.
Periodical:BMC Cancer
Index Medicus: BMC Cancer
Authors: Namba R, et al., and Gregg JP
Yr: 2006 Vol: 6 Nbr: Abs: Pg:275