Transformation Potential of E2 Exposed Breast Cancer Susceptibility Gene Mutation Heterozygous Epithelial Breast Cells
Transformation Potential of E2 Exposed Breast Cancer Susceptibility Gene Mutation Heterozygous Epithelial Breast Cells
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.
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.
Inclusion Criteria:

 -  healthy

 -  premenopausal

 -  women

 -  BRCA1 mutations

 -  BRCA2 mutations

 Exclusion Criteria:

 -  women with breast or ovarian cancer