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,2
* Arkansas Children’s Nutrition Center and
Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR 72202
2To whom correspondence should be addressed. E-mail: simmenrosalia{at}uams.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • mammary gland • development • cancer • PTEN • whey proteins
Breast cancer is a debilitating disease that affects 1 of 9 women in their lifetime and is the leading cause of cancer deaths among women in the Western hemisphere, next to lung cancer (1). Epidemiological studies have suggested that the risk of adult diseases, including breast cancer, has origins during early stages of development that can be influenced by nutrition (2). For example, the linkage of a high intake of soy products by Asian women, especially during adolescence (3,4), to low breast cancer incidence has been widely explored (5), albeit methodological differences and limitations in assessing dietary intake have resulted at times in inconclusive and inconsistent data (6). Our group and others have used the 7,12-dimethyl-benz[a]anthracene (DMBA)3 -induced rat mammary model to examine the effects of diet on onset of mammary cancer in adult females (7–10). Our studies demonstrated reduced incidence of mammary carcinoma in rats fed soy protein isolate (SPI) or whey protein hydrolysate (WPH) relative to rats fed control diets made with casein (CAS) as the sole protein source (9). Several mechanisms may be working simultaneously to reduce the incidence of chemically induced cancer with dietary intake of SPI or WPH. One mechanism of protection may involve the ability of SPI and WPH to decrease tissue expression of the cytochrome P450 gene family-1 members, which convert the procarcinogen DMBA to the active carcinogen (10). Another mechanism may involve progesterone receptor signaling (11). In this regard, the stage of mammary gland differentiation at the time of carcinogen insult may determine the susceptibility of mammary epithelial cells to abnormal proliferation leading to tumorigenesis (7,8). Hence, it was posited that dietary factors that induce mammary gland differentiation over proliferation are likely to be protective (12). Dietary factor–induced enhancement of apoptosis of cells containing genetic lesions and mutations that predispose them to tumorigenesis has been suggested as another viable mechanism for protection (13). Recent studies that focused on genistein, the major phytoestrogen present in soy, documented its ability to decrease the circulating levels of ovarian hormones (14), reduce the percentage of estrogen receptor–positive mammary epithelial cells (15), and upregulate epidermal growth factor receptor expression (16) in rat models. Taken together, these findings demonstrate that dietary factors utilize diverse signaling pathways to confer biological effects in target cells.
PTEN (phosphatase and tensin homolog deleted in chromosome ten), a dual protein and lipid phosphatase that dephosphorylates the 3' position of the signaling molecule phosphoinositide-3,4,5-triphosphate, plays a critical role in controlling cell survival through its antagonistic effects on the Akt/PK-B pathway (17,18). Recently, PTEN was also demonstrated, via its protein phosphatase activity, to suppress cell growth through its inhibition of the growth factor–activated ras/mitogen activated protein kinase (MAPK) pathways (19). PTEN is mutated in many types of advanced cancers, and loss or diminished expression of PTEN leads to a high incidence of tumors (20,21). A role for PTEN in the etiology of breast cancer is underscored by the findings that decreased PTEN expression is associated with invasive cancer and poor prognosis (22,23). Mice with mammary-specific deletion of the PTEN gene exhibited abnormal proliferation and apoptosis of mammary tissues, leading to neoplasia (24); conversely, PTEN overexpression in the mammary epithelium resulted in severely reduced mammary epithelial cell proliferation and increased apoptosis, pointing to its role in the functional development of the mammary gland (25). Thus, enhancement of PTEN expression leading to inhibition of the Akt/PK-B and/or MAPK mediated cell survival pathways may constitute a contributory factor to the protective mechanisms of dietary components on mammary tumorigenesis.
Whey proteins from milk have been shown to exert multiple health benefits including the enhancement of immune function, modulation of adiposity [see review (26)], and, from our own studies with rat models, protection from DMBA-induced carcinogenesis (9). Because whey has many bioactive components, it is anticipated that these diverse health benefits of whey may be mediated through multiple signaling pathways. In the present study, we tested the hypothesis that WPH reduces chemically induced carcinogenesis by advancing the maturation and differentiation status of mammary cell, in part through regulation of PTEN expression and, hence, cell survival at an early developmental window. Toward this end, we evaluated: 1) the differentiation, proliferation, and apoptotic status of mammary glands from rats fed CAS or WPH, at weaning [postnatal day (PND) 21–28] and at an early adult (PND 50–53) stage; 2) PTEN expression in mammary glands from rats fed CAS or WPH diets at these same developmental time points; and 3) the apoptotic sensitivity and PTEN expression of mammary glands from rats fed CAS or WPH diets when acutely challenged with DMBA.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Experiment 1. At PND 21 and 50, female pups fed CAS- or WPH-based diets (n = 8 per diet group) were killed and the inguinal mammary gland (gland 4) pair was removed. The left gland was processed for whole mounts, whereas sections of the right gland were fixed for paraffin embedding, immediately homogenized in TriZol for RNA extraction, or frozen at –80°C for later protein extraction (see below). The rest of the pups at PND 50 (n = 48 for CAS, n = 44 for WPH) were subjected to DMBA-induced tumorigenesis following previously described protocols (9). Pups were orally gavaged with sesame seed oil (Sigma) containing DMBA (Sigma) at a dose of 0.08 g/kg body wt. Rats were weighed weekly and beginning at 3 wk after DMBA administration were palpated twice weekly for tumors. The initial detection date of tumor for each rat and subsequent appearance of new tumors and their locations were recorded. Rats from both diet groups were killed at 70 d post-DMBA. Tumors were analyzed for pathology, as described in previous studies from this group (9).
Experiment 2. At PND 28 and 53, female pups (n = 12) from each diet group were killed, and the inguinal mammary gland (gland 4) pair was removed. The left and right mammary glands were analyzed, as described in Expt. 1 (above). The remainder of the PND 53 pups fed the lifetime CAS- or WPH-based diets (n = 16 pups for each diet) were orally gavaged with DMBA (0.08 g/kg rat). At 24 h and 7 d after DMBA administration, female pups (n = 8) from each diet group were killed and the mammary gland pair 4 was removed for analyses, as described above (Expt. 1).
Whole-mount, immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick-end labeling (TUNEL). Mammary gland whole mounts were prepared following previously described protocols from this laboratory (11). For immunohistochemical analysis, tissues were fixed overnight in 10% neutral buffered formalin and embedded in paraffin. Five micrometer–thick sections were mounted on poly-lysine–coated microscope slides (Fisher Scientific), deparaffinized, and rehydrated in graded alcohols (11). Endogenous peroxidase activity was quenched in 3% hydrogen peroxide. For antigen retrieval, sections were sequentially microwaved for 105 s at power 10 and then for 10 min at power 1 in Citra Plus (Biogenex). The sections were then placed in a blocking solution (Cas Block; Zymed) for 20 min. Incubations with anti-PTEN antibody [mouse monoclonal IgG raised against the carboxy-terminus of human PTEN (A2B1, Santa Cruz Biotechnology)] and anti-proliferating nuclear antigen (PCNA) antibody [mouse monoclonal IgG raised against rat PCNA (clone PC10, Dako)] diluted at 1:100 and 1:600, respectively, were carried out overnight (PTEN) or for 1.5 h (PCNA) at 4°C in a humidity chamber. Sections were sequentially incubated with biotinylated goat anti-mouse IgG secondary antibody for 30 min and streptavidin-linked horseradish peroxidase (Bio-Rad Laboratories) for 30 min at room temperature. Immunoreactive proteins were detected by incubation with the chromagen 3,3'-diaminobenzidine tetrahydrochloride (Dako), and sections were counterstained with hematoxylin, dehydrated, cleared, and coverslipped for examination under a microscope. Four randomly chosen fields (200X magnification) per slide per rat were counted for the presence of immunostaining. Only cells showing a dark-brown color were scored for positive expression. The proliferation index was calculated as the percentage of PCNA-positive cells relative to the number of cells counted.
TUNEL assay to detect apoptotic cells was performed following the manufacturer’s instructions (Oncogene). Paraffin-embedded sections (4 µm) were treated with proteinase K (20 mg/L) for 20 min, rinsed in Tris-buffered saline, and then incubated with terminal deoxynucleotidyl transferase and nucleotide mixture for 1 h at 37°C in a humidified chamber. TUNEL-positive cells were counted from 3 randomly selected fields (200X magnification) per slide, and 3 slides were evaluated for each tissue section, with each section representing an individual animal. The total number of TUNEL-positive cells was divided by the number of mammary gland structures (terminal end buds, TEB; ductal epithelium, DE) evaluated. For both immunohistochemical and TUNEL assays, tissue sections representing 4 individual rats per diet were analyzed.
RNA isolation and quantitative RT-PCR. Total RNA was extracted from mammary tissues using TriZol reagent (Invitrogen). Integrity of the isolated RNAs was confirmed using the RNA 6000 Nano LabChip kit with the Agilent 2100 Bioanalyzer System (Agilent Biotechnologies). Approximately 1 µg of total RNA was reverse-transcribed in 10 µL of reaction volume using random hexamers and MultiScribe reverse transcriptase in a 2-step RT-PCR reaction (Applied Biosystems). For PCR, primers that span intron/exon junctions were designed using PrimerExpress software (Applied Biosystems) to prevent amplification of residual genomic DNA; these are summarized in Table 1. Quantitative PCR (QPCR) was performed with the SYBR Green detection system (Applied Biosystems) using an ABI Prism 7000 sequence detector. Thermal cycling conditions included preincubation at 50°C for 2 min, DNA polymerase activation at 95°C for 1 min, and 40 PCR cycles for 15 s at 95°C and for 1 min at 60°C. The transcript levels for each gene were calculated at cycle threshold values (CT) at which each fluorescent signal was first detected above background; these were determined using the ABI Prism 7000 SDS software (Version 1.0) (Applied Biosystems). mRNA levels were normalized to that of the housekeeping gene cyclophilin A to control for input RNA and are reported as overall means ± SEM from 8 (Expt. 1) or 12 (Expt. 2) individual rats per dietary group.
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Statistical analysis. Results are means ± SEM. Significant differences between two groups were determined by Student’s t test or Fischer’s exact test. Proliferation and apoptotic data were subjected to two-way ANOVA using diet, structure, age, and time of DMBA exposure as the main effects in separate analyses, followed by inspection of all differences between pairs of means by Tukey’s test. Differences with P values < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-caseins, PTEN); 2) decreased (cyclin D1, IGF-2); and 3) did not differ (IGFBP2) with age.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We show here that expression of specific genes in mammary glands of rats exposed to dietary WPH varied with developmental stage. While mammary PR and PTEN gene expression at weaning was altered with diet, this difference was not sustained in young adults. On the other hand, there was an increase in ß-casein transcript levels with WPH in mammary tissues of young adults, but not in weanling pups, consistent with greater mammary gland differentiation. The modest degree of changes induced by WPH in a limited number of genes evaluated here is in accord with the absence of drastic modifications in the differentiation status of mammary glands from WPH-fed rats relative to those fed CAS, as measured by changes in TEB numbers and extent of ductal elongation. Nevertheless, it was of interest to note that albeit modest, the altered variables translated to a distinct response of the mammary glands to the procarcinogen DMBA. In particular, whereas mammary tissues from CAS-fed rats showed a significant increase in number of apoptotic cells in TEB and DE structures 24 h post-DMBA administration, those from WPH-fed rats were more resistant to apoptosis and did not exhibit corresponding changes in number of TUNEL-positive cells. Thus, seemingly minor advances in the developmental status of mammary tissues resulting from early life exposure to WPH may confer partial resistance to environmental insults later in life.
In these studies, we examined the tumor suppressor PTEN as a possible mediator of WPH action in developing mammary glands. Although basal PTEN expression in these tissues showed only a tendency (P = 0.08) to increase between weaning and the early adult stage, WPH significantly increased mammary PTEN expression (RNA and protein) at weaning, although this effect of WPH did not persist in young adults. The time lag between the observed increase in PTEN expression at weaning and the changes in proliferation and apoptotic status, as well as in the expression of the differentiation marker ß-casein only in young adults, suggests a lack of a direct effect of PTEN on the proliferative and apoptotic status of mammary cells. We posit that the delayed consequence of enhanced PTEN expression may more reflect the limited number of mammary cells targeted by WPH, whose negative regulation by PTEN results in apoptosis and their subsequent removal from developing mammary tissues. It has been suggested that cancer arises from a series of sequential mutations occurring as a result of genetic instability and environmental insults (36,37); hence, tumors could initiate from only a few hundred breast cancer cells (38). Moreover, some tumors express early developmental markers, coincident with loss of PTEN expression, suggesting a functional correlation among differentiation status, PTEN, and tumorigenesis (39). Thus it is possible that the enhanced expression of the pro-apoptotic PTEN in cells targeted by WPH could favor the removal of a population of undifferentiated cells with high tumorigenic potential early in development, leaving a population of mammary cells with phenotypes that are less susceptible to genetic mutations at a later life stage. The latter would likely manifest as cells with less proliferative, and hence enhanced, differentiation status. Indeed, our data provide support to this possibility because mammary glands from WPH-fed rats at PND 50–53 exhibit: 1) decreased PCNA immunoreactivity; 2) increased numbers of apoptotic cells; 3) increased ß-casein gene expression; 4) decreased numbers of TEB; 5) increased ductal extension into the fat pad; and 6) less responsiveness to chemical carcinogen (DMBA) challenge, subsequent to enhanced PTEN expression at weaning. Although we did not directly test the above possibility, subsequent studies of mammary glands from CAS- and WPH-fed rats at or prior to weaning for expression of epithelial progenitor cell markers such as keratin 6 and Sca-1 (39,40), whose levels we anticipate to decrease with WPH as a function of neonatal development, should support this hypothesis.
Two signaling pathways involving PTEN could potentially mediate the protective effects of WPH on chemically induced tumorigenesis. Our results showing the coincident decrease in PR transcript levels with increased PTEN expression at weaning suggest a role for progesterone-mediated PR signaling. Ligand-bound PR has been shown to inhibit PTEN activity in the rat uterus (41), and endometrial adenocarcinoma expressing high levels of PR has low or no PTEN expression (42). Because mice null for the PR gene are less susceptible to DMBA-induced tumorigenesis, suggesting a critical role for PR in tumor development (29), it is possible that decreased PR activity at an early developmental window results in increased PTEN signaling, leading to inhibition of protein kinase (e.g., Akt/PK-B and MAPK) activation that promotes cell survival and proliferation. In this regard, constitutive activation of Akt/PK-B is a characteristic of breast cancer cell lines and primary invasive breast cancers (43). The signaling pathway mediated by the epidermal growth factor receptor family member Her-2/neu may also be involved in the tumor-protective effects of WPH, because overexpression of Her-2/neu occurs in 
30% of breast cancers (44) and is associated with constitutive activation of PI3-and Akt/PK-B kinases (45), both of whose actions are inhibited by PTEN (46). Moreover, PTEN and EGFR signaling pathways also interact, albeit in an opposing manner, via PTEN inhibition of the EGFR-mediated activation of the Ras/Raf/ERK pathway of MAP kinase signaling (47). The relevance of PTEN/EGFR "cross-talk" in the genesis of breast cancer is underscored by the findings that sensitivity to the EGFR-selective tyrosine kinase inhibitor "Iressa" is lost in PTEN-mutant cells (48,49). Further analysis of the functional interactions between PTEN and components of PR and of EGF signaling pathways, respectively, will likely provide insights into the mechanisms of cellular transformation and cell cycle regulation affected by dietary factors.
In conclusion, dietary WPH can lead to transient induction in PTEN expression at a sensitive developmental window (weaning), possibly resulting in long-term consequences on mammary gland differentiation. The latter is manifested by higher basal apoptotic index in TEB and resistance to DMBA-induced cell damage in TEB and DE. Studies using rats exposed to WPH and purified fractions thereof, at specific windows of early development, should help delineate the signaling pathways mediating the protective effects of early diet intervention on adult onset of breast cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: CAS, casein; DE, ductal epithelium; DMBA, 7,12-dimethyl-benz[a]anthracene; IGF, insulin-like growth factor; MAPK, mitogen activated protein kinase; PCNA, proliferating cell nuclear antigen; PND, postnatal day; PR, progesterone receptor; PTEN; phosphatase and tensin homolog deleted in chromosome ten; QPCR, quantitative real-time polymerase chain reaction; SPI, soy protein isolate; TEB, terminal end buds; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick-end labeling; WPH, whey protein hydrolysate.
Manuscript received 12 May 2004. Initial review completed 4 July 2004. Revision accepted 27 September 2004.
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  R. Singhal, K. Shankar, T. M. Badger, and M. J. Ronis Estrogenic status modulates aryl hydrocarbon receptor--mediated hepatic gene expression and carcinogenicity Carcinogenesis, February 1, 2008; 29(2): 227 - 236. [Abstract] [Full Text] [PDF]
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Y. Su, R. R. Eason, Y. Geng, S. Till, T. M. Badger, and R. C.M. Simmen In utero exposure to maternal diets containing soy protein isolate, but not genistein alone, protects young adult rat offspring from NMU-induced mammary tumorigenesis Carcinogenesis, May 1, 2007; 28(5): 1046 - 1051. [Abstract] [Full Text] [PDF]
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 R. Singhal, T. M. Badger, and M. J. Ronis Reduction in 7,12-Dimethylbenz[a]anthracene-Induced Hepatic Cytochrome-P450 1A1 Expression Following Soy Consumption in Female Rats Is Mediated by Degradation of the Aryl Hydrocarbon Receptor J. Nutr., January 1, 2007; 137(1): 19 - 24. [Abstract] [Full Text] [PDF]
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 S. E. Fenton Endocrine-Disrupting Compounds and Mammary Gland Development: Early Exposure and Later Life Consequences Endocrinology, June 1, 2006; 147(6): s18 - s24. [Abstract] [Full Text] [PDF]
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B. Dave, R. R. Eason, Y. Geng, Y. Su, T. M. Badger, and R. C. M. Simmen Tp53-Associated Growth Arrest and DNA Damage Repair Gene Expression Is Attenuated in Mammary Epithelial Cells of Rats Fed Whey Proteins J. Nutr., May 1, 2006; 136(5): 1156 - 1160. [Abstract] [Full Text] [PDF]
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B. Dave, R. R. Eason, S.R. Till, Y. Geng, M. C. Velarde, T. M. Badger, and R. C.M. Simmen The soy isoflavone genistein promotes apoptosis in mammary epithelial cells by inducing the tumor suppressor PTEN Carcinogenesis, October 1, 2005; 26(10): 1793 - 1803. [Abstract] [Full Text] [PDF]
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 M. C. Velarde, Y. Geng, R. R. Eason, F. A. Simmen, and R. C.M. Simmen Null Mutation of Kruppel-Like Factor9/Basic Transcription Element Binding Protein-1 Alters Peri-Implantation Uterine Development in Mice Biol Reprod, September 1, 2005; 73(3): 472 - 481. [Abstract] [Full Text] [PDF]
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