Alterations of gene expression in the development of early hyperplastic precursors of breast cancer - PubMed (original) (raw)

Alterations of gene expression in the development of early hyperplastic precursors of breast cancer

Sangjun Lee et al. Am J Pathol. 2007 Jul.

Abstract

Enlargement of normal terminal duct lobular units (TDLUs) by hyperplastic columnar epithelial cells is one of the most common abnormalities of growth in the adult female human breast. These hyperplastic enlarged lobular units (HELUs) are important clinically as the earliest histologically identifiable potential precursor of breast cancer. The causes of the hyperplasia are unknown but may include estrogen-simulated growth mediated by estrogen receptor-alpha, which is highly elevated in HELUs and may be fundamental to their development. The present study used DNA microarray technology and RNA from microdissected pure epithelial cells to examine changes in gene expression and molecular pathways associated with the development of HELUs from TDLUs. The results suggest that HELUs evolve from TDLUs primarily by reactivation of pathways involved in embryonic development and suppression of terminal differentiation. Changes in ERBB genes were particularly prominent, including a uniform switch in ligands for the ERBB1 receptor (14-fold decrease in epidermal growth factor and 10-fold increase in amphiregulin, respectively) in HELUs compared with TDLUs. Epidermal growth factor regulates terminal differentiation in adult breast and amphiregulin is critical to normal embryonic breast development. Because HELUs are such early potential precursors of breast cancer, targeting some of these alterations may be especially promising strategies for breast cancer prevention.

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Figures

Figure 1

Figure 1

A and B: HELUs are much larger (up to 100-fold increased volume and number of cells) than the normal TDLUs they evolve from (H&E stain). This study used DNA microarray technology and RNA isolated from nearly pure (>95%) populations of epithelial cells isolated by laser capture microdissection to compare gene expression profiles between paired TDLUs (C–E) and HELUs (F–H). Original magnifications, ×40 (A, B).

Figure 2

Figure 2

An unsupervised hierarchical clustering using 6023 filtered genes resulted in near-perfect separation of paired normal TDLUs and HELUs. In a supervised comparison (more than or equal to twofold; P < 0.05), 825 genes showed significantly different levels of expression.

Figure 3

Figure 3

Selected results from pathway analyses using CGAP GO Summary (A), BRB Array Tools (B), and Ingenuity software (C), highlighting the potential importance of pathways involving ERBB genes (red type or box) and estrogen/ER-regulated genes (blue type or box) in the development of HELUs from TDLUs.

Figure 4

Figure 4

Hierarchical clustering and gene list from a supervised comparison (more than or equal to twofold; P ≤ 0.05) between paired samples of normal TDLUs and HELUs of expressed genes in the ERBB pathway as defined by Affymetrix.

Figure 5

Figure 5

An unsupervised hierarchical clustering using 385 genes filtered from a total list of 584 estrogen-regulated genes resulted in near-perfect separation of paired normal TDLUs and HELUs. In a supervised comparison (more than or equal to twofold; P ≤ 0.05), 52 genes were expressed at significantly different levels in TDLUs and HELUs, including AREG and EGF in a manner consistent with their known regulation by estrogen.

Figure 6

Figure 6

qRT-PCR was used to confirm the differential expression of selected interesting genes, including epidermal growth factor receptor (EGFR), EGF, AREG, annexin 8 (ANXA8), prominin 1 (PROM1), and v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT). Changes (direction and magnitude) in all six genes were confirmed using total RNA from the original eight paired samples. AREG and EGF were further evaluated by qRT-PCR using total RNA from 10 additional paired samples of TDLUs and HELUs, with the same results as the original eight samples. Five of these 10 breasts also contained foci of ADH arising within HELUs, and the same patterns of expression in HELUs were observed in ADH.

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