Identification of mammary epithelial cells subject to chronic oxidative stress in mammary epithelium of young women and teenagers living in USA

Abstract

Current knowledge of changes in the mammary epithelium relevant to breast carcinogenesis is limited to when histological changes are already present because of a lack of biomarkers needed to identify where such molecular changes might be ongoing earlier during the decades-long latent stages of breast carcinogenesis. Breast reduction tissues from young women and teenagers, representative of the USA's high breast cancer incidence population, were studied using immunocytochemistry and a targeted PCR array in order to learn whether a marker of chronic oxidative stress [protein adducts of 4-hydroxy-2-nonenal (4HNE)] can identify where molecular changes relevant to carcinogenesis might be taking place prior to any histological changes. 4HNE-immunopositive (4HNE+) mammary epithelial cell-clusters were identified in breast tissue sections from most women and from many teenagers (ages 14–30 y) and, in tissues from women ages 17–27 y with many vs. few 4HNE+ cells, the expression of 30 of 84 oxidative stress associated genes represented in SA Bioscience RT2 Oxidative Stress and Antioxidant PCR array was decreased and only one was increased > 2-fold. This is in contrast to increased expression of many of these genes known to be elicited by acute oxidative stress. The findings validate using 4HNE-adducts to identify where molecular changes of potential relevance to carcinogenesis are taking place in histologically normal mammary epithelium and highlight differences between responses to acute vs. chronic oxidative stress. We posit that the altered gene expression in 4HNE+ tissues identified reflects adaptive responses to chronic oxidative stress that enable some cells to evade mechanisms that have evolved to prevent propagation of cells with oxidatively-damaged DNA and to accrue heritable changes needed to establish a cancer.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interests were disclosed.

Acknowledgments

This project was funded in part by a contract with the Pennsylvania Breast Cancer Coalition, which takes no part and is in no way responsible for any analyses, interpretations or conclusions contained herein, and in part under a grant from the Pennsylvania Department of Health using Tobacco Settlement Funds. The Department specifically disclaims responsibility for any analyses, interpretations or conclusions. We thank Dr. Linda Curtiss, Scripps Research Institute, La Jolla, CA, for the antibody against 4-hydroxynonenal lysine adducts used in this study.

Figures and Tables

Figure 1 Photomicrographs of 4HNE immunostained breast tissue sections from nine subjects ranging in age from 14–19 illustrating some of the diversity of staining patterns. Sections were not counterstained. Images were captured via a Nikon Eclipse E600 microscope with Nomarski optics using Spot Digital Camera (Diagnostic Instruments, Inc.) and Image Pro Plus software (version 2). (A1 and A2) Sections from a 14-y-old girl with only a few weakly 4HNE immunopositive mammary epithelial cells at low power in A1 and high power in A2. Insert in lower panel shows a blood vessel with no immunostaining of the endothelium, i.e., no evidence of atherosclerosis. (B1 and B2) Two areas from a section from another 14-y-old girl with intense immunostaining throughout the mammary epithelium: Upper panel shows growing ducts and the lower panel shows at high power a cross section of a duct with 4HNE+ mammary epithelial cells localized mainly in the basal epithelium. (C1 and C2) Upper panel shows at low power a terminal lobular ductal unit (TDLU) from a 19-y-old subject in which mammary epithelial cells are relatively weakly 4HNE immunopositive. Lower panel, the area marked by an arrow in the upper panel, photographed at high power shows localization of immunostaining in luminal epithelial cells. (D1 and D2) Two areas in a tissue section from a 17-y-old girl in which there are only a few intensely 4HNE immunopositive cells. The random localization of these 4HNE+ cells and the granularity of the immunostaining suggest that these might be macrophages, a supposition that needs to be confirmed using a macrophage marker, such as CD68. (E) An essentially immunonegative TDLU surrounded by intensely immunopositive collagenous stroma in a tissue section from a 16-y-old girl. (F1 and F2) 4HNE+ mammary epithelial cells in two growing ducts in a section from a 19-y-old subject. The immunostaining is more intense and widespread in (F1) and less intense and restricted to the tip of the growing duct in (F2). Insert in (F1) shows localization of immunostaining in basal epithelial cells. (G) A portion of a TDLU in a section from an 18-y-old subject in which intense immunostaining is localized in the luminal epithelial cells. (H1 and H2) From a section from an 18-y-old subject in which intensely immunopositive stroma surrounds a TDLU in the upper panel and a blood vessel in the lower panel: the upper panel shows patchy immunostaining of luminal mammary epithelial cells while the lower panel shows immunostaining of the endothelium of the blood vessel indicating atherosclerosis. (I) A section of an aorta, used as a positive control, showing streaks of 4HNE immunostaining indicating atherosclerotic lesions. Control sections incubated with medium from which the primary 4HNE antibody was omitted were uniformly immunonegative.

Figure 2 Photomicrographs of representative 4HNE immunostained breast tissue sections from nine subjects between the ages 17–27 y whose tissues were used for transcriptional profiling. Sections were not counterstained. Images were captured via a Nikon Eclipse E600 microscope with Nomarski optics using Spot Digital Camera (Diagnostic Instruments, Inc.) and Image Pro Plus software (version 2). Upper six panels (A–E), tissues with many strongly 4HNE+ terminal lobular ductal units (TDLU), (G–I) tissues with only few at most weakly 4HNE+ TDLUs. (A–E) Upper and lower portion of each of the panels show images captured from the same tissue section at high and low magnification, respectively. (F1) and (F2) images from sections from different tissue blocks from the same subject: In (F1) the mammary ducts are surrounded by lipocytes and in (F2) by strongly immunopositive collagenous stroma. Note: (1) in (C2), localization of 4HNE immunostaining in luminal epithelial cells in; (2) in (E2), some immunopositive nuclei in mammary epithelial cells and; (3) in (G), immunonegative TDLU is surrounded by strongly 4HNE immunopositive collagenous stroma and fat cells. Control sections incubated with medium from which the primary 4HNE antibody was omitted were uniformly immunonegative.

Figure 3 Immunoblot of protein isolated from breast parenchyma from reduction mammoplasty surgical specimens from seven subjects ages 14–19 y (lanes 2, 4–9) and from an invasive breast cancer (lane 3). Lane 1, molecular weight (MW) marker. Ages of subjects indicated below the image. Proteins were isolated, separated under reducing conditions and visualized by immunostaining with an antibody specific for 4HNE lysine adducts as described under Methods. The one prominent immunopositive band identified in all samples corresponds closely in size to that identified in the laboratory of Esterbauer in immunoblots of plasma from children with Systemic Lupus Erythematosus and from atherosclerotic plaques.61,62 In the sample from one 19-y-old subject there is a second prominent immunopositive band of a slightly higher MW. The only distinguishing immunocytochemical feature of sections from this subject was the presence of an unusually large number of growing ducts with immunopositive termini. The variable amount of 4HNE+ and 4HNE− adducts in cells in the mammary epithelium and stroma contributing to the 50 µg protein loaded onto the gels, and the difficulty of extracting proteins from the collagenous stroma, precluded being able to correlate the intensity of the protein bands with extent of immunostaining in tissue sections.

Figure 4 Fold-difference in relative expression of 84 genes represented in the Oxidative Stress Antioxidant PCR Array (upper six panels) and of 12 genes represented in the Housekeeping PCR Array (bottom left panel) in breast tissues from subjects at either end of the spectrum of immunostaining for 4HNE protein adducts [designated 4HNE-(controls) and 4HNE+ (unknown), respectively]. The average age of the 4HNE− and 4HNE+ subjects was 21.6 and 20.2 y, respectively. Tissues in the 4HNE− and 4HNE+ categories were selected based on criteria described in Material and Methods and illustrated in Figure 2. The assays were carried out using SABioscience RT2 Profiler Human Oxidative Stress, Antioxidant Defense (panels A–E) and Housekeeping PCR Arrays on a minimum of two (duplicate) samples from each subject. Results from the Oxidative Stress Array are show with the genes grouped according to the broad categories suggested by the manufacturer. The two genes used for data-normalization, GAPDH and HTRP1, were ones identified in the Housekeeping Array not to differ significantly in their relative expression between the 4HNE− and 4HNE+ tissues. Vertical axis, fold differences in average gene expression in 4HNE+ (unknown) tissues compared to 4HNE− (control) tissues. The numbers above or below the bars correspond to the rank-order of fold differences in genes listed in the side panels. Black bars show the genes that differed 2-fold or more in 4HNE+ tissues vs. 4HNE− tissues. Bottom right panel shows comparison of values obtained by qRT-PCR and by Oxidative Stress and Antioxidant PCR Array for four genes (SOD3, PRDX3, PRG3 and GPX5).

Table 1 List of genes represented in the SABioscience Human RT2 Profiler Oxidative Stress and Antioxidant PCR Array the expression of which differed 1.5-fold or more between 4HNE+ (experimental) and 4HNE− (control) tissues, listed in rank order

Table 2 Sequences of PCR primers and probes used in Quantitative PCR