Abstracts of Funded Pilots 2009

Timothy Jorgensen, PhD: "Nucleotide Excision Repair and Breast Cancer Risk"

Genetic evidence is accumulating that a particular DNA repair pathway – nucleotide excision repair (NER) – may have an important role in breast cancer risk. These findings suggest that certain genetic variants of NER genes increase risk by compromising the efficiency of NER in the target tissue (i.e., breast epithelial cells), but this conjecture has yet to be substantiated. Our hypothesis is that common variants of a NER gene, ERCC4, will impair NER and result in a DNA repair phenotype in mammary epithelial cells that is intermediate between the wild-type and the null. Proving this hypothesis would be of tremendous value, because it would validate findings demonstrating an association between the NER genotypes and breast cancer risk, and provide a potentially modifiable target for prevention studies.

Specific Aims include:

  1. To employ a DNA incision assay as a means to measure NER repair capacity in cell extracts from mammary cells that have been engineered to express either wild-type, variant, or mutated alleles of ERCC4.
  2. To show that variant alleles of ERCC4 can significantly modify NER capacity.

Success of this project would validate NER capacity as a significant phenotypic endpoint in breast epithelial cells, and could be a valuable biological marker of increased breast cancer risk. It may also provide the means to validate the potential for different variant alleles to affect breast cancer risk, not only for ERCC4, but also for other genes in the NER pathway (27 NER genes). In addition, measuring NER repair in patient breast biopsy tissue or peripheral blood mononuclear cell (PBMCs) may be a useful biomarker of breast cancer risk in a clinical setting and could also be used as an intermediate marker of breast cancer risk in early phase prevention trials. A future goal would be to adapt these NER assays either directly to breast biopsy tissue, or even to PBMCs, if a sufficiently high correlation between the two cell types for NER capacity can be demonstrated. It should also be possible to use this biomarker to determine the proportion of non-BRCA1/2 familial cancers that could be attributed to genes within the NER pathway. This would significantly inform our understanding of the importance of NER to the risk of familial breast cancer not attributed to known inherited mutations.

Tapas Saha, PhD:  "Role of BRCA1 on Nitric Oxide Regulation in Familial Breast Cancer"

Background: Accumulation of oxidized proteins and its improper removal are thought to be one of the major causes of many cancers and it might affect breast cancer too. Nitric Oxide (NO) is consistently detected in tumor microenvironment that promotes tumorigenesis. Conversely, high concentrations of NO, stimulates apoptosis in tumor cells. Reaction between nitric acid radical and superoxide generate a highly toxic peroxinitrate that nitrates several internal proteins forming nitrotyrosine (nTyr). S-Nitrosylation, another modification of proteins, generated by nitric oxide is also toxic to the cells if not removed immediately.

Rationale: In familial breast cancer, expression of BRCA1 is often found to be decreased, or absent. Our preliminary data demonstrate that ectopic expression of BRCA1 reduce protein carbonyl footprint, reduce nTyr formation and induce the production of iNOS (inducible Nitric Oxide Synthase) that synthesize NO. We therefore propose a novel function of BRCA1 as a key modulator of nitrative stress.

Hypothesis: BRCA1 regulate the synthesis of NO and subsequently regulate nitrosylation and nitration of the proteins responsible for breast tumor progression. Phytochemical DIM will stimulate the function of BRCA1 and antioxidant Tempol will maintain protein homeostasis by reducing protein nitration products.

Research Aims and Design: We will manipulate the expression of BRCA1 by over and under expression and investigate the presence of nitrotyrosine, iNOS, s-Nitrosylation via ELISA and activity assay. Next, we will treat the cells with physiological concentrations of DIM and Tempol, a super oxide scavenger, and subsequently measure the same parameters to demonstrate its effects. Finally, tissue microarray will be performed to determine the expression of the proteins under study in different grades of breast cancer with respect to BRCA1.

Clinical Impact: Tumor invasion and metastasis are probably associated with several oxidatively stressed proteins. Phytochemicals that up regulate the expression of BRCA1 and reduces the formation of reactive nitrogen species could in turn enhance protein homeostasis by suppression of the tumor. This proposal will foster the development of novel approaches to restore normal protein function in the treatment of human malignancies.

Luciane Cavalli, PhD:  "BP1 Amplification and Protein Expression in Familial Breast Cancer Cases."

The BP1 homeobox gene, located at 17q21, is an oncogene amplified and overexpressed in breast tumors with poor clinical outcome. BP1 expression was found to correlate with established prognostic factors associated with clinical aggressiveness, including high histological grade, lymph node positivity and estrogen and progesterone receptor (ER and PR) negativity and HER2/NEU positivity.  In addition, BP1 may affect the regulation of genes involved in the DNA repair system pathway, in which BRCA1/2 tumor suppressor genes play active roles. The function of these proteins is critical for homologous recombination repair of DNA double-strand breaks. Tumor cells that are defective in double-strand break repair by homologous recombination become sensitive to DNA cross-linking agents such as platinum analogues and to poly(ADP-ribose) polymerase (PARP) inhibitors. Interestingly, PARP is up-regulated in breast cancer cells that overexpress BP1.

Taken these previous studies together, and based on the established association of BP1 amplification and protein expression with relevant genes in breast cancer, such as ER, PR, HER2/NEU and PARP, we propose to investigate the amplification and protein status of the BP1 gene in familial breast cancer cases (+ or – for BRCA1/2 mutations). There are no studies investigating the role of the BP1 gene in BRCA1/2 mutated breast tumors or in familial breast cancer cases with/without mutations in these genes.

The main objective is to assess whether amplification and/or protein expression of the BP1 homeobox gene play a role in patients with familial breast cancer.

Specific Aims:

Aim 1: To determine the incidence of BP1 amplification in familial breast cancer cases.
Approach: We will analyze tissue microarrays (TMAs) composed of tissue cores of familial breast tumors from patients with long-term follow-up and outcome data using Fluorescence In Situ Hybridization (FISH) for the BP1 gene. In addition, and because of the co-localization of HER2/NEU oncogene on chromosome 17q21, which is amplified in 20-30% of invasive breast carcinomas, we will also analyze the relation between BP1 and HER2/NEU amplification and protein expression in the same specimens. We have previously constructed both of these gene-specific probes and optimized the conditions for their use in formalin-fixed paraffin embedded (FFPE) breast cancer tissues (Cavalli et al, 2008).

Aim 2: To determine the protein expression status of the BP1 gene in familial breast cancer cases.
Approach: This aim will be achieve using Immunohistochemistry (IHC) analyses, using a validated polyclonal antibody against the BP1 and HER2/NEU peptides, which we have previoulsy tested for its specificity and sensitivity (Cavalli et al, 2008). The same TMA used in Aim1 will be analyzed by IHC.

Aim 3: To determine the correlation between BP1 amplification and protein expression. 
Approach: The same TMA will be used for the FISH and IHC analysis, which will allow a direct correlation of gene copy number and protein expression for each case. Appropriate statistical analysis will be used to test this correlation.