A Role for RAD51B in Breast Cancer

Institution: Lawrence Livermore National Laboratory
Investigator(s): Joanna Albala, Ph.D. -
Award Cycle: 1999 (Cycle V) Grant #: 5KB-0123 Award: $406,092
Award Type: New Investigator Awards
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (1999)
The development of breast cancer is associated with defects in critical cellular pathways, such as those involved with DNA repair. The hereditary breast cancer genes, BRCA1 and BRCA2, are known to be involved in one such pathway. Another critical member of this DNA repair pathway, RAD51, has been shown to interact with the BRCA1 and BRCA2 proteins. These proteins have also been co-localized in the nuclei of malignant breast cell lines. Taken together, these studies indicate a role for RAD51 in breast cancer. We have discovered a novel gene, RAD51B, whose DNA sequence suggests a function analogous to RAD51 and is therefore believed to be involved in these processes. DNA repair defects in these genes may lead to the accumulation of genetic mutations and chromosome recombination events that characterize advanced breast cancer.

The screening of a public database for RAD51-like genes led to the identification of a partial unknown sequence from a breast cDNA library, which was later characterized as RAD51B. Isolation and molecular analysis of the RAD51B gene showed that this gene is widely expressed and most abundant in tissues active in recombination. In normal tissues, the RAD51 family functions to preserve chromosomal integrity and promote genetic diversity through chromosomal recombination during meiosis in the egg and sperm. It is likely that in cancer cells, disruption of the RAD51B gene within this pathway will lead to enhanced chromosomal instability and therefore accelerate genetic changes leading to defects in cell function and morphology.

The specific aims of this research project are to: (1) express and and purify recombinant RAD51B and define its localization pattern in normal and malignant breast cancer cell lines; (2) characterize the interactions between RAD51B and the breast cancer gene products, BRCA1 and BRCA2; and (3) isolate and characterize novel binding partners of the RAD51B protein. The approach to identify the native RAD51B will be to isolate protein from cellular extracts derived from both normal and malignant breast cell lines. Newly produced and optimized RAD51B antibodies will be used in these studies. Specific interactions between RAD51B and the BRCA1 and BRCA2 proteins will be examined using yeast two-hybrid analysis. We aim to identify and isolate novel binding partners of the RAD51B protein using immunoaffinity chromotography techniques.

The thrust of these studies is to identify protein factors that interact with RAD51B and thus provide insights into the biological role of the RAD51B protein in mediating the genetic defects that underlie breast carcinogenesis.

Final Report (2002)
Breast cancer can result from mutations in critical genes such as BRCA1 and BRCA2 which are known to be involved in several cellular processes, including DNA repair. RAD51, a key protein involved in the repair of DNA double-strand breaks, has been shown to interact with the BRCA1 and BRCA2 proteins. These proteins have also been co-localized in the nuclei of a malignant breast cell line and overexpression of the RAD51 gene has recently been implicated in breast carcinogenesis. Taken together, these studies indicate a role for RAD51 in breast cancer. There are five proteins known as the RAD51 paralogs (human homologs), RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3, whose functions are hypothesized to be similar to RAD51 based on their sequence identity. Individual knockout cell lines, each missing one of the genes which encode these proteins, all have similar ionizing radiation sensitivity and decreased repair of DNA double-strand breaks suggesting these proteins all have a role in double-strand break repair.

This study has focused on the RAD51B protein and the objective of the proposal was to determine if RAD51B interacts with either BRCA1 or BRCA2 in a similar manner to RAD51 or alternatively in a protein complex involved in double-strand break repair. RAD51B was shown to be a nuclear protein by immunocytochemical techniques in normal MCF10A breast epithelial cells as well as several breast cancer cell lines. There are however some breast cancer cell lines that appear to have RAD51B mislocalized to the cytoplasm as well as present in the nucleus. Unlike RAD51, which requires BRCA2 for its nuclear localization, RAD51B localization was not found to correlate with any known breast cancer factors. Further experiments are necessary to determine whether the cytoplasmic localization of RAD51B in some breast cancer cell lines is significant. Investigation of a putative interaction between RAD51B and the BRCA1 and BRCA2 proteins showed that these proteins do not interact directly by yeast two hybrid analysis, co-immunoprecipitation studies and immunocytochemical localization. Furthermore, in vivo immunoprecipitation showed that RAD51B is not localized with RAD51. Previous studies have suggested interactions between the RAD51 paralogs themselves. Using a variety of biochemical techniques, our studies have demonstrated the in vitro complex of RAD51B with RAD51C as well as an in vivo complex containing RAD51B, RAD51C, RAD51D and XRCC2 (BCD2) from several human breast cancer cell lines. A second complex containing RAD51C and XRCC3 was also shown. None of the paralogs appear to interact with RAD51 in vivo. The cell lines examined included normal mammary epithelial cells, breast cancer cells, BRCA1 mutant cells and BRCA2 mutant cells, which were all found to contain the BCD2 complex.

Our results indicate that there are no changes in RAD51B binding partners in breast cancer cells and more importantly suggests that the BCD2 complex function independently of the RAD51/BRCAl/BRCA2 complex in double strand break repair. Further studies to examine the in vivo function of RAD51B are needed to better understand the role of RAD51B and its complex partners in DNA repair and carcinogenesis.

Symposium Abstract (2005)
Our research aims to understand the how the process of DNA repair impacts the initiation of breast cancer. Our lab has identified a protein, Rad51B, which is important in the repair of DNA double-strand breaks in conjunction with a number of other proteins, including Rad51 and BRCA2, a protein known to be mutated in familial breast cancer. Using several human breast cell lines, we have examined whether Rad51B and BRCA2 co-localize in these cell types. Examination of fluorescently-labeled Rad51B protein in these cells has shown that Rad51B localizes to the nucleus and we have identified the signal sequence for this nuclear localization on the Rad51B protein. Using a cell line mutant for BRCA2 protein, CAPAN-1, we have shown that Rad51B localizes to the nucleus independent of BRCA2 in contrast to Rad51 which is dependent on BRCA2 for its subcellular localization. In addition, Rad51B and BRCA2 do not appear to directly associate although both proteins are clearly involved in the repair of DNA double-strand breaks. The impact of mutations in Rad51B on breast carcinogenesis is the subject of future study.

Interactions involving the Rad51 paralogs Rad51C and XRCC3 in human cells
Periodical:Nucleic Acids Research
Index Medicus: Nucleic Acids Res
Authors: Wiese C, Collins DW, Albala JS, Thompson LH, Kronenberg A, Schild D
Yr: 2002 Vol: 30 Nbr: Abs: Pg:1001-1008

RAD51C interacts with RAD51B and is central to a larger protein complex in vivo exclusive of RAD51
Periodical:Journal of Biological Chemistry
Index Medicus: J Biol Chem
Authors: Miller KA, Yoshikawa DM, McConnell IR, Clark R, Schild D, Albala JS
Yr: 2002 Vol: 277 Nbr: 10 Abs: Pg:8406-8411

Mediator function of the human Rad51B-Rad51C complex in Rad51/RPA-catalyzed DNA strand exchange
Periodical:Genes and Development
Index Medicus: Genes Dev
Authors: Sigurdsson S, Van Komen S, Bussen W, Schild D, Albala JS, Sung P
Yr: 2001 Vol: 15 Nbr: Abs: Pg:3308-3318

The rad51 paralog RAD51B promotes homologous recombinational repair
Periodical:Molecular and Cellular Biology
Index Medicus: Mol Cell Biol
Authors: M. Takata, M. Sasaki, E. Sonoda, T. Fukushima, C. Morrison, J. Albala, et al.
Yr: 2000 Vol: 20 Nbr: 17 Abs: Pg:6476-82