Research Grants Awarded
Combination Of Low-Dose Antiestrogens With Omega-3 Fatty Acids For Prevention Of Hormone-Independent Breast Cancer
Background/Overall Objective: Tamoxifen and Raloxifene are effective chemopreventive agents in women at high risk of breast cancer. However the clinical impact of these antiestrogens is limited by their toxicity and inability to prevent hormone-independent tumors for which currently there are no known effective chemopreventive interventions.
We hypothesize that upregulation of estrogen receptors, observed in premalignant lesions, contributes to the later development of hormone-independent tumors by crossactivation of other cellular signaling which may eventually operate independently of estrogen action. While these events are known to occur in the transition of established tumors to hormone independence, we propose that similar mechanisms may be operative in mammary carcinogenesis and that a superior tumor preventive effect to include prevention of hormone-independent tumors may be achieved by a combined biological approach targeting the estrogen receptors and other interacting cell signaling pathways. We propose to test a novel strategy by combining antiestrogens with omega-3 fatty acids-induced PPAR? activation since there is experimental evidence in human breast cancer cells that they synergistically downregulate the PI3K/AKT pathway, a relevant oncogenic signal in breast cancer due to its proproliferative and antiapoptotic actions. Use of omega-3 fatty acids is attractive because of lack of toxicity and added health benefits beyond the potential inhibition of breast cancer development. Furthermore, because of the expected synergism of action between the two interventions, it may be possible to reduce the dose of Tamoxifen and Raloxifene and diminish toxicity.
Research Strategy: This proposal includes a preclinical and clinical component involving the co-PIs at Penn State Cancer Institute, Drs. Manni and El Bayoumy with complementary expertise in endocrinology and chemoprevention with nutritional manipulations, Dr. Thompson at Colorado State University who developed the MNU model proposed here, and Dr. Russo at Fox Chase with expertise in genomic aspects of breast cancer prevention.
Specific Aim 1. We will test the chemopreventive effects of escalating doses of Tamoxifen or Raloxifene in the presence of either a control high-fat diet or an isocaloric high-fat diet rich in fish oil using two clinically relevant rodent experimental systems, the MNU-induced rat mammary tumor, developed by the co-investigator, Dr. Thompson and the Polyoma middle T transgenic mouse model system. In both systems, mammary tumor development follows progressive stages of carcinogenesis resembling those of human breast cancer. Furthermore, upregulation of functional estrogen receptors occurs in the premalignant lesions frequently leading to the development of hormone-independent tumors. Therefore, these models will be particularly suited for determining the efficacy of targeting the estrogen receptors in combination with PPAR? activation for prevention of hormone-resistant breast cancer. Endpoints of these experiments include: 1)histopathological analysis of the mammary lesions (hyperplasia, DCIS, and invasive cancer); 2)expression by IHC of biomarkers of hormone dependency (ER and PR) and PI3 kinase signaling (PTEN, phospho-AKT, phospho-mTOR, phospho-S6-kinase, phospho-4E-BP1, Ki67, PCNA) and apoptosis; 3)PPAR? expression and activity by real-time PCR of target genes (ADRP, aP2, FIAF, SCD-1, maspin, K19 and Muc-1) in stroma and normal, benign, and malignant epithelial tissues isolated by laser capture microdissection; 4)genomic profiles assessed by cDNA microarray following isolation of normal, preneoplastic and neoplastic cells by laser capture microdissection; 5)oxidative stress markers (8-OHdG and Etheno adducts) in normal mammary glands of tumor free and bearing animals.
Specific Aim 2. This includes a two-year trial where normal postmenopausal women with a breast density >25% will be randomly assigned to one of these groups: 1)placebo; 2)Raloxifene 60 mg daily; 3)Raloxifene 30 mg; 4)omega-3 fatty acid preparation (Nutrilite-total fat 1.5 gm/EPA 450 mg/DHA 450 mg, three soft gels), 5)Raloxifene 30 mg+omega-3 fatty acid preparation. Fatty acid composition in the serum will be measured to monitor compliance. The dietary aspects of the study will be closely monitored by the co-investigator, Dr. Terryl Hartman, a nutritionist with extensive background in the area of diet and cancer prevention and control. The primary endpoint of the study is breast density assessed at baseline and years 1 & 2. Total sample size of 372 women will yield at least 80% power for detecting a difference of 6% in breast density between any two groups at the 0.008 significant level (instead of 0.05 to account for the increased type 1 error rate induced by multiple tests). Secondary endpoints include:1)markers of oxidative stress including urinary 8-Isoprostane F-2? and 8OHdG, lymphocyte DNA 8OHdG and Etheno adducts; 2)parameters of estrogen metabolism including urinary 2-hydroxyestrone, 16-alpha-hydroxyestrone and 4-hydroxyestrone; 3)markers of inflammation including highly sensitive C-reactive protein and IL-6; and 4)markers of altered IGFI signaling including serum IGFI and IGFBP3 levels. These biomarkers will be measured semiannually.
In summary, we believe the novelty and impact of our proposal center around these major points: 1)Ability to prevent development of hormone-independent breast cancer in addition to hormone-dependent tumors; 2)Safety of the approach, which may bring additional health benefits besides its chemopreventive effect; 3)Immediate applicability to the clinic if found to be scientifically valid.
Breast cancer is second only to lung cancer as a cause of cancer-related deaths in American women. One in seven women in Western countries like the United States is expected to develop breast cancer in her lifetime. Despite significant improvements in the treatment of this disease, prevention offers the best hope for reducing breast cancer mortality as well as decreasing the physical, psychological and financial burdens associated with this diagnosis. Since preventive measures are offered to normal women (as opposed to treatments which are given to patients who already have cancer), it is critical that such interventions are not only effective, but also easy to administer and, most importantly, without significant side effects. Recently, drugs such as Tamoxifen and Raloxifene, called antiestrogens, which block the action of the female hormone estrogens, have been found to reduce by almost 50% the incidence of breast cancer. Although these results are exciting and groundbreaking, the current use of these drugs for breast cancer prevention is associated with two major limitations: 1) an increased risk of clotting complications such as phlebitis and, most importantly, pulmonary embolism and strokes which can lead to death. The use of Tamoxifen is also associated with an increased risk of cancer of the uterus; 2) both drugs are ineffective in preventing the most aggressive type of breast cancer, called hormone independent, which spreads more readily, leading to the patient?s demise. There are currently no known treatments able to prevent the development of hormone-independent tumors. In this proposal, we will use two animal models of breast cancer which closely resemble the human disease to address these two critical issues. Our studies are designed to provide experimental support for a novel approach to breast cancer prevention which includes the combination of lower doses of either Tamoxifen or Raloxifene with a healthy diet rich in omega-3 fatty acids, such as in fish oil. This strategy will maximize safety, since antiestrogens will be given at lower, less toxic doses and such diet, in addition to being devoid of toxicity, will provide additional health benefits (such as a reduction in cardiovascular risk) besides protecting from breast cancer. If our hypothesis on the mechanism of interaction between this diet and antiestrogens is proven to be correct by our results, we predict that such combined intervention will reduce the development of both hormone-dependent and ?independent tumors. In parallel with the animal studies, we will conduct a clinical trial in normal postmenopausal women to test the feasibility, safety, and efficacy of combined low-dose Raloxifene (less toxic than Tamoxifen) and omega-3 fatty acids. While this trial will not be large enough to demonstrate an effect on breast cancer development itself, it will establish whether the combined approach is superior to the individual administration of Raloxifene and omega-3 fatty acids in reducing breast density (measured by mammogram) which is strongly predictive of breast cancer risk. In addition, we will also investigate the effects of our interventions on a variety of parameters (known as biomarkers) in the blood and in the urine which are associated with breast cancer risk. In summary, the proposed research may identify a new, safer and more effective means to inhibit breast cancer and, hence, reduce mortality from this common malignancy.