Altered proliferation and differentiation properties of primary mammary epithelial cells from BRCA1 mutation carriers

Abstract

Female BRCA1 mutation carriers have a nearly 80% probability of developing breast cancer during their life-time. We hypothesized that the breast epithelium at risk in BRCA1 mutation carriers harbors mammary epithelial cells (MEC) with altered proliferation and differentiation properties. Using a three-dimensional culture technique to grow MECs ex vivo, we found that the ability to form colonies, an indication of clonality, was restricted to the aldehyde dehydrogenase 1-positive fraction in MECs but not in HCC1937 BRCA1-mutant cancer cells. Primary MECs from BRCA1 mutation carriers (n = 9) had a 28% greater ability for clonal growth compared with normal controls (n = 6; P = 0.006), and their colonies were significantly larger. Colonies in controls and BRCA1 mutation carriers stained positive for BRCA1 by immunohistochemistry, and 79% of the examined single colonies from BRCA1 carriers retained heterozygosity for BRCA1 (ROH). Colonies from BRCA1 mutation carriers frequently showed high epidermal growth factor receptor (EGFR) expression (71% EGFR positive versus 44% in controls) and were negative for estrogen receptor (ERalpha; 32% ER negative, 44% mixed, 24% ER positive versus 90% ER positive in controls). Expression of CK14 and p63 were not significantly different. Microarray studies revealed that colonies from BRCA1-mutant PMECs anticipate expression profiles found in BRCA1-related tumors, and that the EGFR pathway is up-regulated. We conclude that BRCA1 haploinsufficiency leads to an increased ability for clonal growth and proliferation in the PMECs of BRCA1 mutation carriers, possibly as a result of EGFR pathway activation. These altered growth and differentiation properties may render BRCA1-mutant PMECs vulnerable to transformation and predispose to the development of ER-negative, EGFR-positive breast cancers.

Figure 1

The ability to form colonies ex vivo is restricted to the ALDH1-positive subpopulation in HMECs but not in breast cancer cells. ALDH1 was measured in the absence (dark line) or presence (light line) of DEAB. A, ALDH1-positive subpopulations in immortalized HMECs (HMEC htert), (B) HCC 1937 breast cancer cells, (C) HMECs isolated from a reduction mammoplasty, and (D) HMECs isolated from a BRCA1 mutation carrier. E, clonal growth in three-dimensional culture was restricted to sorted ALDH1+ in nonmalignant HMECs (top) but not in HCC1937 cancer cells (bottom).

Figure 2

Progenitor cell colonies from BRCA1 mutation carriers have distinct morphologies. A, isolated HMECs from BRCA1 mutation carriers (1st and 2nd row) or reduction mammoplasties (3rd and 4th row) were grown for 12 to 14 d, photographed, and subjected to H&E stain (3rd column).C, progenitor cell colonies from BRCA1 mutation carriers mostly retain heterozygosity for wild-type BRCA1 (ROH). DNA was extracted from single or pooled (–10) colonies and sequenced. B and D, progenitor cell colonies from BRCA1 mutation carriers are more numerous and larger than normal controls. B, total colony counts are displayed. Each column represents triplicate or quadruplicate results from one breast. Adjacent columns represent results from the left and right breast from one individual. Columns are arranged from left to right in order of ascending age. D, the mean colony count for individuals from each cohort; BRCA1 mutation carriers (filled symbol) versus normal controls (open symbol) is displayed as a function of colony diameter. The mean colony number (Y axis) for each volume channel (X axis) was determined from quadruple assays from PMECs of 9 BRCA1 mutation carriers and six controls.

Figure 3

Representative IHC images of colonies grown ex vivo (days 12–14). A, expression of BRCA1, lineage markers CK14 and p63, ERα, and EGFR in ex vivo colonies from BRCA1 mutation carriers (top) or normal controls (bottom). B, colonies were either positive (>80%), mixed, or negative (<10%). For EGFR, staining intensity was graded. Colonies were counted and the significance levels were calculated using a two-sided t test. Neg, negative; pos, positive; N.s., not significant.