Proper relocation of a tumor suppressor in Breast Cancer

Institution: University of California, Irvine
Investigator(s): Marian Waterman, Ph.D. -
Award Cycle: 1996 (Cycle II) Grant #: 2IB-0220 Award: $50,000
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (1996)
BRCA1 is the first breast cancer susceptibility gene to be identified as a tumor suppressor gene. The BRCA1 gene is either completely absent or mutated in over 80% of families with high rates of early-onset breast and ovarian cancer. While the identification of this gene is a major breakthrough in the study of breast cancer, a role for BRCA1 in sporadic breast tumors, which account for 95% of all breast cancer cases in Europe and North America, has been questionable since these tumors have normal BRCA1 genes. Recently, the BRCA1 protein was found to be completely missing from the nucleus in the majority of breast cancer biopsies examined and in most breast and ovarian cancer cell lines. The nucleus is a compartment of the cell where chromosomes are located and where genes important for cell growth and division are carefully regulated. Although the proper localization of BCRA1 is still under investigation, at least some BCRA1 protein is normally located in the nucleus. This finding, though preliminary, suggests that displacement of BRCA1 from the nucleus results from, or contributes to, the tumorigenic transformation of mammary cells, including sporadic tumors. Its role in this more common form of breast cancer should be reconsidered. Secondly, this observation suggests the possibility that forcing BRCA1 back into the nucleus of breast cancer cells might allow BRCA1 to carry out its normal tumor suppressor function and slow cell growth.

Proteins are guided into the cell nucleus in a multistep process that begins with binding of a nuclear import regulator to small nuclear localization signals within the protein. We have recently identified the genes for the two nuclear import factors known to bind such signals in proteins. Our goal is to use the genes for these import factors as tools to identify and study the nuclear transport pathway that normally guides BRCA1 to the nucleus, to search for a nuclear localization signal in BRCA1 protein, and to study its interaction with the nuclear import pathway in breast cancer cells. Since BRCA1 has only very recently been identified, little is known about the normal structure or function of the protein. Nothing is known about how it is normally transported to the nucleus.

Results from the experiments described here will be important to our understanding of the aberrant localization of BRCA1. It may also provide invaluable insight into the progression of breast cancer, since complete misplacement of BRCA1 is most common in end-stage breast cancer. Finally, our goal is to use the results from these experiments to design a modified BRCA1 protein that can readily move into the nucleus. We may then be able to judge whether forced relocalization of BRCA1 protein to the correct cellular compartment helps to restore normal growth control to these cells - a result that would have direct and important relevance in the search for new strategies to treat breast cancers.

Final Report (1997)
BRCA1 is the first breast cancer susceptibility gene to be identified as a tumor suppressor. The BRCA1 protein (gene product) is a new type of protein, but its normal location and function within mammary cells are not known. Surprising and controversial results have been published that suggest BRCA1 protein is either (i) a secreted factor, which is localized inside vesicles in the cell’s cytoplasm, or (ii) that BRCA1 resides normally in the nucleus where it could act on genes to control cell growth. In addition, it has been reported that the BRCA1 protein is in the nucleus in normal cells, but is found in the cytoplasm (vesicles) in cells from breast cancer biopsies and breast cancer cell lines. Our hypothesis is that BRCA1 is incorrectly distributed in breast cancer cells and not able to express its normal tumor suppressor activity. This would shift the balance of growth control resulting in uncontrolled cell division.

To address these issues of BRCA1 cellular location, our laboratory has been studying two proteins that shuttle regulatory factors such as tumor suppressors into the nucleus. These ‘shuttle’ proteins are called nuclear localization signal (NLS) receptors. Over the past year, we have determined that two types of NLS receptors bind tightly and specifically to BRCA1 protein. Furthermore, we have determined that they bind to separate regions of BRCA1. Other groups have recently shown that these two regions each contain a signal (NLS) that acts to target BRCA1 to the nucleus. Thus our results are consistent with the idea that BRCA1 is normally a tumor suppressor that must be actively transported to the nucleus for its function. Interestingly, we also find that one of the NLS receptors is expressed at low levels in normal breast tissue and at high levels during pregnancy - a time when BRCA1 expression is also elevated and when the rate of cell growth and division is high. This NLS receptor is also highly expressed in all breast cancer cell lines examined.

We are in the process of examining the interaction of NLS receptors with BRCA1 in more detail, and we are looking at the process of nuclear transport in several breast cancer cell lines identified by others as containing cytoplasmic BRCA1 protein. We wish to determine whether there are any differences in the functioning of the nuclear transport pathway in these cells versus normal mammary cells.