DNA replication licensing and progenitor numbers are increased by progesterone in normal human breast

Abstract

Proliferation in the nonpregnant human breast is highest in the luteal phase of the menstrual cycle when serum progesterone levels are high, and exposure to progesterone analogues in hormone replacement therapy is known to elevate breast cancer risk, yet the proliferative effects of progesterone in the human breast are poorly understood. In a model of normal human breast, we have shown that progesterone increased incorporation of 5-bromo-2'-deoxyuridine and increased cell numbers by activation of pathways involved in DNA replication licensing, including E2F transcription factors, chromatin licensing and DNA replication factor 1 (Cdt1), and the minichromosome maintenance proteins and by increased expression of proteins involved in kinetochore formation including Ras-related nuclear protein (Ran) and regulation of chromosome condensation 1 (RCC1). Progenitor cells competent to give rise to both myoepithelial and luminal epithelial cells were increased by progesterone, showing that progesterone influences epithelial cell lineage differentiation. Therefore, we have demonstrated that progesterone augments proliferation of normal human breast cells by both activating DNA replication licensing and kinetochore formation and increasing bipotent progenitor numbers.

Figure 1

Morphology, lineage markers, and ER and PR expression in matrix-embedded culture of normal breast acini. PAS staining with diastase pretreatment of normal breast cells in matrix-embedded culture (A) and normal breast tissue (B and C) showing distinct apical glycocalyx and intraluminal secretions. Triple-immunofluorescence staining of the progenitor marker CK5/6 (blue), myoepithelial marker p63 (red), and luminal epithelial marker Muc-1 (green) in sections prepared from primary cells grown in matrix-embedded cultures (D) and in primary cells grown in matrix-embedded culture after initial passage in monolayer (F; yellow or white arrows demonstrate committed premyoepithelial and preluminal cells, respectively; white arrowhead shows mature luminal cells; asterisks represent uncommitted progenitor cells). E, Ratio of luminal and myoepithelial markers in primary cells in matrix-embedded and monolayer culture. Dual-immunofluorescence staining of matrix-embedded cultures: (G and H) showing colocalization within the luminal compartment of ER (G) or PR (H; red) with the luminal epithelial marker Muc1 (green); (I–K) showing colocalization of PRA and PRB, PRA expression (I); PRB expression (J); merge (K). PR expression in normal breast cells in matrix-embedded culture; without (L) and with estrogen treatment (M). Scale bars, 20 μm.

Figure 2

Enriched functional categories in differentially expressed gene sets from normal breast cells in monolayer and matrix-embedded culture. Vector graphic representation of enrichment in specific functional categories organized into gene ontology hierarchies. Gene expression was compared on cDNA arrays of 7425 genes. Functional categories enriched in matrix-embedded cultures (A) and monolayer culture of primary normal breast cells (B) when compared with the original tissue. Significance of enrichment of individual categories (two sided Fisher exact test) is indicated by the black circles at each enriched node, with size inversely proportional to P value. Labels are included only for functional categories with significant P values for enrichment or which represent branching points in the gene ontology tree.

Figure 3

Progesterone responsiveness and proliferation of normal breast cells in matrix-embedded culture. PR expression and proliferation (BrdU incorporation) of normal breast cells in matrix-embedded culture determined by dual-immunofluorescence staining. Progesterone (P) down-regulated its own receptor as shown by fewer PR+ acini (shaded column) (A) and fewer cells per acinus expressing PR (B). C, Progesterone treatment increased the total number of cells per acinus incorporating BrdU. D, Progesterone treatment increased total cell numbers in cultures. E, Progesterone preferentially increased BrdU incorporation within PR+ acini. *, P < 0.05 Mann Whitney test; bars, sem. V, Vehicle.

Figure 4

Progesterone increased proliferation and bipotent progenitor cell numbers. Progesterone increased proliferation (BrdU incorporation) of normal breast cells in matrix-embedded culture; BrdU incorporation without (A) and with progesterone treatment (B). Dual-immunofluorescence staining of matrix-embedded cultures: (C–E) showing colocalization of BrdU and PR; BrdU incorporation (C); PR expression (D); merge (E); arrow shows proliferating PR+ cell. Progesterone increased progenitor cells: progesterone treatment induced a mean 3.1-fold increase (F) in mammosphere numbers in primary suspension cultures (n = 2; normalized data, t test: paired two sample for means, one tail, P = 0.06), and a significant increase in aldefluor-positive progenitor cells by flow cytometry (G) (n = 4; t test: paired two sample for means, two tail, P = 0.005). *, P < 0.05; bars, sem. H–J, Triple immunofluorescence staining for uncommitted progenitor cells after primary suspension culture. H, Myoepithelial marker p63 (red) and luminal marker CK18 (green). I, Progenitor marker CK5 (blue). J, Merge, arrows show uncommitted progenitor cells. Scale bars, 20 μm.

Figure 5

Distinct profiles of progesterone-regulated gene expression in normal and malignant breast cells. A, Primary normal breast epithelial cultures (Bre) from five individuals (25, 27, 52, 62, 64) were treated with 100 nm progesterone or vehicle for 6 h. Gene expression profiles were determined using Illumina whole-genome arrays. Enrichment analysis of differentially expressed genes demonstrated overrepresentation only of the functional categories shown. B, Primary normal breast epithelial cells or T-47D breast cancer cells were grown in matrix-embedded culture and treated (6 h) with 100 nm progesterone. RNA was isolated and gene expression profiling was performed on Illumina whole-genome expression arrays. Numbers of progesterone regulated genes in normal breast and T-47D breast cancer cell matrix-embedded cultures, and their overlap, are indicated. The number genes detected and not regulated by progesterone in either cell type are indicated in the rectangle.

Figure 6

Progesterone activates a network of genes controlling DNA replication licensing and cell cycle progression. A, Functional analysis of genes regulated by progesterone in normal breast cultures revealed an enriched network of genes involved in regulation of DNA replication licensing, G₁/S transition, G₂/M progression, and kinetochore function. Filled arrows indicate increased and decreased expression. ORC, Origin recognition complexes. B, DNA replication licensing and cell proliferation genes increased by progesterone on expression profiling were confirmed. Histograms indicate relative mRNA transcript levels estimated by quantitative real-time RT-PCR. Open bars, vehicle; shaded bars, 6 h progesterone (100 nm). C, Images show immunodetection of the indicated protein products in normal breast cultures, after 48 h treatment with 100 nm progesterone (P) or vehicle (V).