Upregulation of BRCA1 as a Cancer Preventive Strategy

Institution: University of Southern California
Investigator(s): Colin Hill, Ph.D. -
Award Cycle: 2000 (Cycle VI) Grant #: 6PB-0126 Award: $610,319
Award Type: Request for Applications
Research Priorities
Etiology and Prevention>Prevention and Risk Reduction: ending the danger of breast cancer

Initial Award Abstract (2000)
Mutations in inherited genes associated with the development of breast cancer, such as BRCA1, have been more frequently identified in young women. However, these mutations account for only 5-10% of all breast cancers. In addition, there is controversy as to which mutations are harbingers of future disease. The need to determine how such genes, mutated or not, may affect the risk of this disease in all women is of vital interest. Clearly, identifying mutations, while a vital link to some diseases, is not the only key to dissecting the interrelationships between susceptibility genes and breast cancer in humans. Female hormone status has been definitively linked to breast cancer risk in women. Pregnancy and lactation are protective while early menarche and childlessness increase risk.

The intent of this proposal is to develop an understanding of how BRCA1 interacts with female hormones in the mammary gland and how these interactions translate into breast cancer risk. The hypothesis driving my research has the goal of focusing attention on how normal BRCA1 helps to prevent cancer and what preventive strategies can be used to increase its tumor suppressor activity during critical periods in a female’s lifetime. This hypothesis is: In individuals whose BRCA1 alleles both encode normal BRCA, 'hormonal' breast cancer risk factors are associated with BRCA1 expression levels. This hypothesis implies that there exists a minimum level of tumor suppressor gene product that the cell must accumulate to be effective against tumorigenesis. If the levels of tumor suppressors could be increased during susceptible periods in life, then it may be possible to protect the females from developing this disease.

Direct studies to investigate this question in humans are difficult for practical and ethical reasons. One alternative is to utilize animal models to analyze the interaction between BRCA1, hormone status and breast cancer. Past studies in this laboratory indicate that rat strain susceptibility to mammary cancer is proportional to BRCA1 RNA expression within the mammary gland and dependent upon the hormonal status of the animal. We propose to use surgical techniques to alter the hormonal environment of the female rat. We will utilize RNA and protein measuring techniques to assay BRCA1 activity and then determine how sensitive our rats' mammary cells are to becoming cancerous under these various hormonal conditions.

Finally, we propose to retrospectively analyze human breast tumor tissue, and where available, normal breast tissue, for BRCA1 activity as well as the activity of several other genes associated with breast cancer risk including p53, and RAD51. This specific aim will take advantage of an ongoing USC study of white patients diagnosed with cancer prior to age 50 with a detailed history of menstrual status and oral contraceptive use. The results could lend critical insight into BRCA1-hormone interactions in the mammary gland and associated breast cancer risk.

Final Report (2005)
Female hormone status has been linked to breast cancer risk. The intent of this study was to develop an understanding of how the breast cancer suppressor genes BRCA1 and BRCA2 interact with female hormones in the mammary gland. This project has the goal of focusing attention on how normal BRCA1/BRCA2 help to prevent cancer and what strategies can be used to increase their tumor suppressor activity during critical periods in a female's lifetime. The hypothesis is: In individuals with normal BRCA1 genes, 'hormonal' breast cancer risk factors are associated with how much BRCA1 is produced in breast cells.

This hypothesis implies there is a minimum level of tumor suppressor the cell must express to protect normal breast cells from forming tumors. It may be possible to increase the levels of tumor suppressor proteins, especially during the susceptible early years of development, to delay or prevent the occurrence of breast cancer. This study utilized the rat mammary model. Previously this lab has shown that genetically susceptible Wistar Furth rat produces 2-5X less BRCA1 RNA than other more resistant rat strains. In the third year of the study we confirmed our earlier preliminary results that showed BRCA1 levels rise as the young rat enters puberty, and that rats susceptible to inducible mammary cancer (Wistar Furth) have much lower levels of BRCA1 than resistant rats (Cop). We also completed a study looking at the RNA expression level of BRCA 1 and BRCA2 at different stages of the estrous cycle going from cycle 1 through cycle 6.

There is strong evidence from this RNA data that BRCA 1 levels are hormonally controlled. It appears that both BRCA1 and 2 expression is initiated by the onset of estrous with a maxima just after the prolactin level reaches a maxima during the estrous cycle. However, the expression of BRCA1 and 2 takes another estrous cycle to reach a minimum. Thus we found three complete BRCA1 and 2 cycles in the first six estrous cycles. The protein analysis was more difficult to perform. The data suggest there is a cyclic variation in BRCA1 protein level but the maxima maybe be sometime after the maxima seen in the RNA expression study. We are attempting to verify this observation with further hormonal manipulation experiments in the young Wistar Furth rat to see if the expression and protein level of BRCA1 and BRCA2 can be altered directly by changing hormone level artificially. In Aim 3, the carcinogenesis study, we exposed Wistar Furth rats to a chemical or physical carcinogen when BRCA1 is at a high level and also at its lowest level in the estrous cycle to see if the tumor burden produced can be modulated. This study, recently completed, showed that rats exposed to radiation at high levels of BRCA1 produced lower numbers of tumors than those exposed at times of lower levels of BRCA.

In the human study we are retrospectively analyzing BRCA1 levels in human breast tumor tissue, from pre and post-menopausal women to simulate low hormone and high hormone activity. Digital-image analysis is ongoing.

Symposium Abstract (2005)
We have been studying the role of hormones in the developing rat mammary gland using the Wistar Furth rat. This rat has three known susceptibility genes and although it does not produce mammary tumors spontaneously it can be induced to do so by exposure to a carcinogen within 150 to 200 days of exposure. Our earlier studies have shown that BRCA1 is expressed at a lower level in Wistar Furth rats than in non-susceptible and resistant rat strains. We have also shown that the mammary cancer induction and time to tumor appearance can be modified in the Wistar Furth rat by regular exercise suggesting a physiological change can reduce the carcinogen effect. In this study we present data from experiments showing that BRCA 1 and BRCA 2 RNA expression and protein expression in the mammary gland resting mammary epithelial cells (RMEC) changes with the position of the rat in the estrous cycle. We then exposed female rats to radiation or DMBA at specific times in the estrous cycle that correspond to high or low levels of BRCA1. Rat mammary glands are harvested from rats of appropriate ages and the RMEC cells are harvested and counted. Total RNA is extracted using the Trizol method and used to make cDNA of the genes of interest using PCR techniques. We have developed probes for the rat BRCA1, BRCA2, TNF alpha, Beta-Actin and a ribosomal gene. Quantitative analysis of the level of RNA expression is done using ABI-Prism automated PCR machines. Protein is analyzed by western blots using fluorescent antibodies for the proteins provided by Pierce Co. In all three sets of experiments there appears to be three maximum peaks of BRCA1 expression that coincides either with an estrogen peak or the prolactin peak during an estrous cycle. These two peaks run close together. It appears from the data that hormones released during the estrous cycle initiate the rise in BRCA1 and 2, but that expression continues past the end of one estrous cycle. We speculate it follows a proliferation wave in the RMEC. Once the level falls to a low value again the next wave of BRCA1/2 expression is initiated by the next estrous cycle estrogen or prolactin peak that occurs. In the first six estrous cycles we see three peaks of BRCA1 and BRCA2 activity. Rats exposed at the peak of BRCA1 RNA expression or the nadir of RNA expression but the probable peak of protein expression show a delay in onset of tumor appearance and it takes much longer for all the tumors to appear. We postulate this delay could be due to either the BRCA1 activity reducing the load of un-repaired damage in individual cells and thus slowing the time to tumor initiation or the BRCA1/2 activity reduces the number of damaged cells that can produce a tumor.