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Mechanism for BRCA1 Regulation of Genomic Stability
Background: Inherited mutations of the breast cancer susceptibility gene BRCA1 cause a high risk for breast and ovarian cancers. And absent or reduced BRCA1 expression due to promoter methylation is commonly observed in sporadic breast and ovarian cancers, implicating BRCA1 in non-inherited forms of breast cancer. The tumor suppressor activity of BRCA1 is thought to be due, to a significant degree, to its “caretaker” function in preserving genomic integrity. On the other hand, BRCA1 mutations in mouse and human tumor cells result in the loss of several DNA damage-activated cell cycle checkpoints (intra-S and G2/M) and in genomic instability, characterized by centrosome amplification, aneuploidy, and a “preferred” pattern of chromosomal aberrations.
Preliminary Studies: Using DNA microarray analyses, we found that knockdown of endogenous BRCA1 via small interfering RNA (siRNA) caused significant reductions in the expression of a set of genes involved in ensuring orderly progression through mitosis (and other cell cycle compartments) in human breast (MCF-7) and prostate (DU-145) cancer cell lines. These include genes implicated in the mitotic spindle checkpoint (eg., BUB1, BUB1B, HEC1, SNK, TTK), centrosomal functioning (eg., ASPM, NEK2, CENPF), chromosomal segregation (eg., ESPL1, various mitotic kinesins), and cytokinesis (PRC1). We confirmed several of these findings by RT-PCR analysis. BRCA1 knockdown had no effect on the cell proliferation rate but conferred resistance to apoptosis caused by the DNA damaging agent adriamycin (a topoisomerase IIa inhibitor that causes DNA strand breakage).
Hypotheses: 1) The endogenous BRCA1 transcriptionally regulates genes required for orderly and equal division of genetic material; and 2) Down-regulation of BRCA1 expression will confer genomic instability by inactivating the mitotic spindle checkpoint (the mechanism that blocks mitotic progression when spindle abnormalities occur).
Specific Aims: To test these hypotheses, we will carry out the following aims: 1) To confirm some of the more important microarray findings by independent mRNA and protein analyses in normal human mammary epithelial cells (HMECs) and breast cancer cell lines; 2) To demonstrate that knockdown of endogenous BRCA1 using siRNA causes a reversible defect in the mitotic spindle checkpoint; 3) To determine if the mitotic defect due to loss of BRCA1 expression causes genomic instability, document the pattern of genomic abnormalities, and determine if some of them are reversible; and 4) To identify the BRCA1 target genes responsible for these abnormalities.
Study Design: To achieve these aims, BRCA1 expression will be knocked down using a non-toxic siRNA (and a scrambled-sequence control); and reduced levels of BRCA1 will be maintained by repeated application of siRNA or by an inducible siRNA vector. mRNA and protein levels will be measured by quantitative RT-PCR and Western blotting. The mitotic checkpoint will be assessed by cell cycle analysis after applying a spindle poison (eg., colcemid); and genomic abnormalities will assessed using state-of-the-art cytogenetic techniques (SKY and CGH) [with the assistance of a cytogeneticist]. We will identify BRCA1 target genes responsible for these abnormalities by add-back (expression vector) or deletion (siRNA) experiments to rescue or reproduce the BRCA1 phenotype.
Potential Outcomes and Benefits of Research: These studies will enhance understanding of the contribution of BRCA1 to both hereditary and, particularly, non-hereditary types of breast cancer. They will, for the first time, link BRCA1 to the mitotic spindle checkpoint, which plays a major role in maintenance of genomic stability. And they will, for the first time, identify a set of transcriptional targets of BRCA1 that contribute to its caretaker role in maintaining genomic stability. Some of these genes may be future targets for breast cancer prevention and therapy.
Inherited defects (called mutations) of the breast cancer susceptibility gene-1 (abbreviated “BRCA1”) cause a high risk for breast and ovarian cancers. Although inherited forms of breast cancer account for only a small percentage (5-10%) of all breast cancers, the BRCA1 gene is frequently inactivated in the much larger group of “sporadic” (non-inherited) breast cancers by a process called promoter methylation. This finding indicates a wider role for the normal (non-mutated) BRCA1 gene in suppressing (preventing) sporadic breast cancer development. This “tumor suppressor” activity of BRCA1 is thought to be due to its function as a “caretaker”, ie., a gene required to maintain the integrity of the cell’s genetic material (collectively called the genome). The underlying mechanisms by which BRCA1 maintains genomic integrity are only partially understood, through studies of cells that harbor BRCA1 mutations. The molecular functioning of BRCA1 is due, in large part, to its ability to influence (regulate) the expression of other genes, a process called transcriptional regulation. However, the “target” genes whose expression is regulated by BRCA1 and that are responsible for its caretaker function are not known.
In preliminary studies, we used two state-of-the-art technologies to identify BRCA1 target genes: 1) RNA interference, a technology we used to severely “knock down” (reduce) the levels of BRCA1 in the cell without mutating the BRCA1 gene; and 2) DNA microarray analysis, a technique to compare the expression of a large number of different genes in two cell populations. We found that human breast cancer cells in which BRCA1 levels have been knocked down show significantly decreased expression of a number of genes required for the orderly progression of cells through mitosis, the phase of the cell cycle in which the genetic material is separated equally between the two daughter cells and the cells divide apart. These studies indicate that the BRCA1 gene is required for adequate expression of a series of genes that control different aspects of the mitotic process. We have confirmed some of these findings using independent analyses of gene expression. The implication of these findings (and our hypothesis for this proposal) is that the BRCA1 gene functions to regulate the process of mitosis and ensure that chromosomes segregate equally to the daughter cells. Conversely, when BRCA1 gene is inactivated either because of an inherited mutation or promoter methylation (see above), the normal safeguard that exists during mitosis (called the “mitotic spindle checkpoint”) is lost, resulting in what is termed genomic instability.
In this post-doctoral fellowship application, we propose a series of experiments to confirm and extend our preliminary studies and to test the hypothesis described above. In particular, we will extend our studies to normal mammary epithelial cells, the cell type from which breast cancers develop. We will determine if, as predicted, knock down of the BRCA1 gene using RNA interference causes inactivation of the mitotic spindle checkpoint, a mechanism that is critical for preventing chromosomal imbalances of the type that are common in cancers. We will determine if we can simulate the specific types and patterns of genomic instability that are observed in mouse and human BRCA1 mutant breast cancers by knocking down expression of the normal BRCA1 gene in the absence of a mutation. And we will identify the specific “target” genes through which these functions of BRCA1 are mediated.
The use of RNA interference to knock down BRCA1 expression models the situation in sporadic human breast cancers in which the normal (non-mutated) BRCA1 gene is frequently inactivated. Therefore, these studies will enhance understanding of the contribution of BRCA1 to both hereditary and, particularly, non-hereditary, types of breast cancer. It will, for the first time, link BRCA1 to the mitotic spindle checkpoint, which plays an important role in maintenance of genomic stability. And it will, for the first time, identify a set of targets of BRCA1 that contribute to its caretaker role in genomic maintenance. Some of these BRCA1 target genes may, themselves, be future targets for breast cancer prevention and therapy.