Identification of the Transformation Potential of Normal Estrogen Exposed BRCA1 (Breast Cancer Susceptibility Gene 1) and BRCA2 (Breast Cancer Susceptibility Gene 2) Heterozygous Epithelial Breast Cells Due to Irradiation
Overview
- Phase
- N/A
- Intervention
- Not specified
- Conditions
- BRCA1 Gene Mutation
- Sponsor
- Hadassah Medical Organization
- Enrollment
- 30
- Locations
- 1
- Primary Endpoint
- The transcriptional profile of heterozygous epithelial breast cells
- Last Updated
- 12 years ago
Overview
Brief Summary
Susceptibility to breast cancer is related to the combination of genetic, hormonal and multiple other environmental risk factors, such as mutations in the BRCA gene and excess exposure to exogenous estrogen, respectively. BRCA is a nuclear protein that maintains genome stability, by acting as a key player in the DNA repair complex. Recently, evidence has emerged that BRCA mutation heterozygosis itself enhances aborted DNA repair and can contribute to breast cancer initiation after exposure to irradiation. In our preliminary results on short-term lymphocyte cultures, we found additional evidence that healthy heterozygous BRCA1 and BRCA2 mutation carriers have a different response to DNA damage than do non-carriers.
The main aim of our ongoing project is to identify the transcriptional modulation and transformation potential of normal BRCA1 and BRCA2 mutation heterozygous epithelial breast cells following irradiation and to examine how it is affected by exposure to estrogen. Our hypotheses will be investigated by RNA-seq and microRNA-seq in order to identify a unique molecular expression profile of the estrogen exposed cells following ionizing irradiation.
Understanding the role of BRCA heterozygosity in cell response to exposure to estrogen and to irradiation may facilitate the development of more appropriate diagnostic and therapeutic strategies for these individuals.
Detailed Description
To study the different cell lineages, we plan to isolate and propagate luminal progenitors from a normal human breast tissue. To this end, 10 human breast tissue samples will be obtained from risk-reducing mastectomy in healthy premenopausal women with BRCA1 mutations. Tissues from 10 premenopausal women undergoing esthetic breast surgery with no family history of breast or ovarian cancers will serve as a control group. In order to isolate primary epithelial cells, human mammary tissue will be minced and enzymatically digested overnight in collagenase and hyaluronidase to yield suspension of epithelial organoids. These organoids will be collected and further digested with trypsin, dispase and deoxyribonuclease 1 (DNAse), will be filtered to generate a single cell suspension, resuspended in Hank's + 2% fetal bovine serum (FBS) and 0.1 mg/mL DNAse, and also incubated with a blocking antibody for 15 minutes on ice. Cells will be treated with estrogen (E2) (10 nM) for 48 h and then will be irradiated for inducing double-strand break. Cells will be irradiated with 8 Gray (Gy) using a Co60 source. To accomplish our study, estrogen exposed and unexposed BRCA mutation heterozygous epithelial breast cells will be irradiated and 1 h later RNA will be extracted from the cells using Tri-reagent (Sigma). The RNA will be converted to a library of cDNA (complementary DNA) fragments and will be sent for deep sequencing in the illumina Hi-Seq platform.
Investigators
Asher Salmon
Asher Salmon
Hadassah Medical Organization
Eligibility Criteria
Inclusion Criteria
- •premenopausal
- •BRCA1 mutations
- •BRCA2 mutations
Exclusion Criteria
- •women with breast or ovarian cancer
Outcomes
Primary Outcomes
The transcriptional profile of heterozygous epithelial breast cells
Time Frame: human breast tissue samples will be obtained from premenopausal women undergoing breast surgery. cells will be isolated immediatly and 48h later the transcriptional profile will be identified.
we will perform a RNA-seq expression profiling analysis of normal estrogen exposed BRCA heterozygous epithelial cells following irradiation.