Project 6. Development of Nuclear Receptors as Targets for Prevention

PPARg Ligands

PPARg was initially identified as a regulator of fat cell differentiation and was also shown to inhibit cell growth associated with adipogenesis. Recently, the Spiegelman lab and others have demonstrated that PPARg is also expressed at significant levels in other tissues and in cancers of the breast, colon and prostate. When activated by ligands, PPARg can effectively inhibit cancer cell proliferation and is able to revert the gene expression profile of tumor cells into a more benign phenotype. As further evidence for an important role of this receptor in cancer, heterozygous somatic mutations of PPARg in 10% of patients with colon cancer and deletions in 40% of prostate cancer samples have been found. All the mutations identified so far lead to a loss of PPARg function. In addition follicular thyroid carcinomas frequently have a chromosomal translocation involving PPARg, leading to the formation of the PAX8-PPARg fusion oncoprotein. Furthermore, preliminary data in the colon suggests that PPARg heterozygous mice treated with a carcinogen have a higher rate of colon cancer compared to wild-type animals, demonstrating that PPARg acts as a bona fide tumor suppressor gene in vivo.

Other Nuclear Receptor Ligands

Retinoid X receptors (RXRs) are heterodimeric partners for a number of nuclear receptors including PPARg. In the case of PPARg, certain RXR ligands or "rexinoids" modulate the activity of PPARg and have been suggested as potential selective PPAR modulators or (SPARMs). These ligands are in clinical development for the treatment of type II diabetes and pre-clinical studies of their effectiveness in cancer chemoprevention are underway.

In addition several other members of the nuclear receptor family that remain as "orphans" lacking known ligands have been implicated in the regulation of cell growth. These include members of the Nurr/NGFI-B subfamily of orphan nuclear receptors. These receptors have been identified as immediate early response genes for a number of growth factor signaling pathways and may prove to be interesting targets for chemoprevention.

Ductal Lavage

In an effort to improve upon the Nipple aspirate fluid (NAF) and fine needle aspiration (FNA) concepts, a ductal lavage system has been devised. This method initially involves suction of the nipple in order to localize NAF-yielding duct(s). NAF-producing ducts can then be cannulated by a small, very flexible catheter (Induct catheter by ProDuct Health) and lavaged with saline in order to retrieve duct epithelial cells. In a study recently presented at the San Antonio Breast Conference, 507 high-risk women were enrolled on a trial examining ductal lavage. In 84% of women, NAF was obtained and they went onto ductal lavage. The mean cellular yield was 40,000 cells with an increasing cellular yield depending on the expertise of the operator. Abnormal cytology was found in 24% of cases: mild atypia in 17% and suspicious or malignant cells in 7%. Of these 7% (n=26), 11 have gone on to a further procedure, either ductogram, ductectomy or mastectomy. 4 or these 11 have had DCIS identified while 5 had papillomas and 2 had only fibrocystic disease. There were few complications of this procedure. There were 2 cases of mild cellulitis treated successfully with oral antibiotics. In addition, pain score measurements were low, with 50% of patients reporting that this was more comfortable than a mammogram. Although this data is exciting, many unanswered questions remain such as the reproducibility of the procedure, the sensitivity and specificity of the procedure, and the feasibility of using this procedure to assess the cellular material obtained with more sophisticated research tools.

In this project we will test the hypothesis that nuclear receptors can be targeted for the prevention of ER- breast cancers. Specifically we will investigate the possible roles of PPARg and various other nuclear receptors in ER- breast cancer. This will involve both the development of preclinical models for the action of these receptors in mammary epithelial cells and the translation of these findings into pilot clinical trials. The specific aims of this project are:

1. Transcription profiling of PPARg ligand and rexinoid activation in human breast cancer cells
   
   
2. Genetic analysis of PPARg function in the mouse breast
   
   
3. Therapeutic models of PPARg ligands in breast cancer
   
   
4. Mine the gene expression profiling data obtained in Project 2 for nuclear receptors and their targets expressed in ER- breast cancers

Further we hypothesize that it is feasible to measure a variety of biological markers in ductal lavage specimens, and we are specifically interested in genes altered by rosiglitizone and other nuclear receptor ligands. We will:

1. Assess the feasibility of obtaining breast duct epithelial cells using ductal lavage in premenopausal women with a prior diagnosis of ER- breast cancer before and three months after the initiation of a chemoprevention agent.
   
   
2. Examine biomarkers in ductal lavage specimens before and after 3 months of a prevention agent. Specifically, pilot projects will be performed examining the impact of rosiglitizone and in the future other nuclear receptor ligands. Quantitative RT-PCR will be performed for growth regulatory genes found to be regulated by these agents in the pre-clinical models.

Myles A. Brown, M.D.-Dr. Brown Associate Professor of Medicine, Dana-Farber Cancer Institute and Harvard Medical School. He is a member of the Executive Committee of the Dana-Farber Cancer Institute Women's Cancer's Program and the DF/HCC Breast Cancer Program. His lab focus is on the role of coregulators in nuclear receptor function. He will serve as Principal Investigator of the COE. In addition he will serve as a co-investigator on Project 6 and a collaborator on Projects 2 and 3. Email: Myles_Brown@dfci.harvard.edu

Judy E. Garber, M.D., M.P.H.-Dr. Garber is an Associate Professor of Medicine, Dana-Farber Cancer Institute and Harvard Medical School. She is the Director of the Cancer Risk and Prevention Clinic, Dana-Farber Cancer Institute. She is Chair of the CALGB Prevention Committee. She will serve as a co-investigator on P6 directing the clinical trials and a collaborator to Project 4. Email: Judy_Garber@dfci.harvard.edu

Bruce M. Spiegelman, Ph.D.-Dr. Spiegleman is Professor of Cell Biology, Dana-Farber Cancer Institute and Harvard Medical School. His lab cloned PPARg and in addition to describing its critical role in adipogenesis has made major contributions to discovering a role for PPARg in cancer. He will serve as co-investigator on Project 6. Email: Bruce_Spiegelman@dfci.harvard.edu

DATA

Project Update 2003

Effects of PPARg ligands

Based on preclinical data showing an antiproliferative effect of PPARg ligands, we have performed two small clinical trials, one in liposarcoma and one in prostate cancer patients. The first trial shows that ligand activation of PPARg causes differentiation of mixoid/pleiomorphic liposarcomas and decreases in the expression of Ki-67, a marker of cell growth. In the prostate cancer trial, one third of patients show prolonged PSA stabilization. These results suggest that targeting PPARg may have a therapeutic effect in tumors in vivo and support the exploration of this pathway in breast cancer

Effects of rexinoids on IMECs

Rexinoid signaling in immortalized mammary epithelial cells (IMECs) has been analyzed using four different assays: transient transfection analysis, regulation of cell cycle progression and apoptosis, gene expression analysis of RXR, and regulation of p63 expression. These studies indicate that the IMECs are responsive to signaling through RXR and that rexinoid signaling in IMECs is both cytostatic and pro-apoptotic.

Treatment of the IMECs with RXR-specific ligands leads to a G1 arrest at 48 h and by 96h causes the induction of apoptosis. This was in contrast to the RAR ligand all-trans RA which was not pro-apoptotic. This suggests that RXR specific ligands, rexinoids, differ from the retinoids that have been tested previously in breast cancer prevention trials and merit of further study.

NucleaRDB

[back to top]