Departments of Cancer Biology, of Cell and Developmental Biology, and of Medicine, Division of Endocrinology, Diabetes and Metabolism, and Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6160
Address all correspondence and requests for reprints to: Lewis A. Chodosh, Department of Cancer Biology, University of Pennsylvania School of Medicine, 612 Biomedical Research Building II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104-6160. E-mail: chodosh{at}mail.med.upenn.edu.
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ABSTRACT |
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INTRODUCTION |
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Like humans, both rats and mice exhibit parity-induced protection against breast cancer. For example, administration of the carcinogens 7,12-dimethylbenz(a)anthracene or N-methylnitrosourea to nulliparous rats induces mammary adenocarcinomas that are hormone-dependent and histologically similar to human breast tumors (9, 11, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24). In contrast, rats that have previously undergone a full-term pregnancy are highly resistant to the induction of breast cancer by carcinogen administration. Similar to rats, mice that have undergone an early first full-term pregnancy have also been shown to be less susceptible to carcinogen-induced mammary tumors than age-matched nulliparous controls (25). These studies indicate that key epidemiological features of the influence of reproductive history on breast cancer risk in humans are mirrored in rodent model systems. The use of animal models to study parity-induced protection against breast cancer is further facilitated by the many similarities in structure, function, and development that exist between the human and rodent mammary gland (26, 27). Thus, the ability of animal models to recapitulate relevant epidemiological findings, permit critical aspects of reproductive history to be rigorously controlled, reduce genetic variation, and permit the examination of molecular and cellular events at defined developmental stages of interest in normal tissue is critical for understanding this phenomenon.
Despite long-standing evidence for the differential susceptibility of the parous and nulliparous breast to carcinogenesis, a comprehensive analysis of the persistent molecular and cellular changes induced in the breast by early first full-term pregnancy has not been previously reported. Such information would not only define a protected state of the mammary gland at the molecular level but could also provide insight into the pathways that underlie parity-induced protection. In addition, identifying a panel of molecular markers whose expression is reproducibly altered by early first full-term pregnancy would provide candidate intermediate molecular endpoints by which to monitor the efficacy of pharmacological interventions designed to mimic this naturally occurring protective event.
In this report, we have used high-density oligonucleotide microarrays to analyze the impact of early first full-term pregnancy on global gene expression profiles within the murine mammary gland. This approach has led to the identification of a panel of genes whose expression in the mammary gland is persistently altered as a consequence of parity in multiple strains of mice as well as in two widely used rat models for parity-induced protection against breast cancer. Our findings demonstrate that parity induces the persistent down-regulation of multiple genes encoding epithelial growth factors as well as the persistent up-regulation of the growth-inhibitory molecule, TGF-ß3, and several of its downstream targets. In addition, our findings indicate that parity results in a persistent increase in the differentiated state of the mammary gland as well as permanent changes in the hematopoietic cell types resident within the gland. These findings provide a global molecular description of a developmental state of the mammary gland that is refractory to carcinogenesis and suggest novel hypotheses for the mechanistic basis by which parity may modulate breast cancer risk.
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RESULTS |
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After mating at an age corresponding to the onset of puberty, rats and mice were allowed to undergo a single round of pregnancy, 21 d of lactation, and 28 d of postlactational involution. Examination of carmine-stained whole mounts prepared from these animals demonstrated that the epithelial trees of both the rat and mouse parous involuted mammary gland are more highly branched than those of age-matched nulliparous littermates (Fig. 1). Thus, architectural differences between the nulliparous and parous mouse mammary gland are easily distinguishable, occur in multiple strains, and are conserved among rodent species that exhibit parity-induced protection against breast cancer. Notably, early first full-term pregnancy also results in permanent alterations in the architecture of the human mammary gland, although the specific features of these morphological changes are not necessarily related to those observed in rodents (28).
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Oligonucleotide microarray expression profiling was performed in triplicate on pooled mammary gland samples, each of which was derived from 1520 animals to control for sources of biological variation such as the estrus cycle. Additionally, because stromal-epithelial interactions have been clearly shown to affect the behavior of mammary epithelial cells, expression changes were profiled in intact mammary glands with the exception that the lymph node present in the no. 4 mammary gland was removed. Although the morphological changes characteristic of postlactational involution are essentially complete after 14 d (DCruz, C. M., and L. A. Chodosh, unpublished data), we chose to profile the mammary glands of parous animals after 28 d of involution to facilitate the identification of persistent changes in gene expression due to parity rather than acute changes in gene expression due to the processes of pregnancy, lactation, or involution per se.
Three independent, age-matched pools of total RNA derived from nulliparous (15 wk G0P0) and parous (15 wk G1P1) cohorts were hybridized to high-density oligonucleotide microarrays (Affymetrix Mu6500, Sunnyvale, CA) representing approximately 5300 murine genes and expressed sequence tags. Affymetrix comparative algorithms (MAS 4.0) were used to identify genes whose expression levels were significantly altered as a consequence of parity by comparing each of the three independent nulliparous samples with its paired parous sample. A total of 16 genes (of 5300) were identified by Affymetrix algorithms as exhibiting differential expression in each of the three parous-nulliparous sample comparisons that were performed. Fourteen of these 16 genes were tested by Northern hybridization to confirm their parity-dependent patterns of differential expression. Northern hybridization was performed on triplicate, independent sets of parous and nulliparous mammary gland samples, each of which was pooled from 1520 animals and each of which was independent of the original six samples that had been subjected to microarray analysis. This analysis confirmed the consistent differential expression of 14 of 14 genes identified by Affymetrix algorithms as being differentially expressed in each of the three independent microarray comparisons (Fig. 2
and data not shown). Given the 100% true-positive rate for genes exhibiting differential expression by Affymetrix algorithms in three of three comparisons, the remaining two genes in this subclass (
-casein, and Lyzozyme P) were not tested by Northern analysis but were subsequently confirmed by analysis on independent microarrays (see below).
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Finally, the differential expression of this set of genes was subjected to a third round of confirmatory testing by using a different generation of Affymetrix microarrays (MGU74Av2) to analyze a third set of triplicate, independent pooled nulliparous and parous mammary samples from FVB mice. Of note, the probe set sequences used on MGU74A arrays to detect gene expression differ from those used on Mu6500 arrays. As such, this final confirmatory approach used both samples and probe set array sequences that were independent of those used for the initial identification of candidate differentially expressed genes. Statistical significance was assessed by comparing oligonucleotide hybridization intensities using a nonheteroscedastic t test. This analysis revealed that 38 of the 40 originally identified genes were differentially expressed in the predicted manner between the parous and nulliparous glands with P values less than 0.02.
Together, therefore, these three independent methods (Mu6500 array analysis, Northern hybridization analysis, and MGU74A array analysis) resulted in the identification of a total of 38 genes whose differential patterns of expression were reproducibly observed among a total of 18 independent parous and nulliparous pooled mammary samples (Table 1 and Fig. 2
). These genes are listed in Table 1
and include 8 genes that are preferentially expressed in the nulliparous mammary gland and 30 genes that are preferentially expressed in the parous involuted mammary gland. Notably, expression levels of cytokeratins 5, 8, 14, and 18, were not found to differ between the nulliparous and parous mammary gland, suggesting that the observed changes in gene expression are not a consequence of a change in size of the epithelial cell compartment (Fig. 2
and data not shown).
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Because the phenomenon of parity-induced protection against breast cancer is conserved among humans, rats, and mice, we predicted that the molecular changes that underlie this effect would be conserved across species. As Sprague-Dawley and Lewis rats represent the most widely used models of parity-induced protection against breast cancer, we wished to determine whether genes identified as having a parity-dependent pattern of expression in FVB mice would be sufficient to predict correctly the reproductive histories of rats. Accordingly, six independent sets of pooled mammary gland RNA samples from parous and nulliparous Lewis rats (Harlan) were analyzed on RGU34A high-density rat microarrays. To facilitate comparison of mouse data sets to microarray expression data obtained from rats, genes identified as being expressed in a parity-dependent manner in FVB mice were mapped via Homologene to the rat genome. Cluster analysis of rat microarray data revealed that the subpanel of 30 genes identified on the basis of their parity-dependent expression in FVB mice was sufficient to correctly distinguish mammary gland samples harvested from nulliparous and parous rats (Fig. 3C). To extend these findings, we performed Northern analysis on pools of mammary gland total RNA isolated from nulliparous and parous Sprague-Dawley rats, another widely used model for parity-induced protection against breast cancer. Each of the nine genes examined in the rat exhibited a parity-dependent differential pattern of expression identical to that observed in the mouse (Fig. 3G
and data not shown). Together, our findings demonstrate that the expression patterns of the genes that we have isolated are reproducibly and persistently altered as a consequence of parity and are conserved in different mouse strains and rodent species that exhibit parity-induced protection against breast cancer.
Functional Gene Categories Accurately Predict Reproductive History
Examination of the 38 genes that we identified as being differentially expressed as a consequence of parity revealed several distinct functional categories (Table 1). These include: growth-promoting molecules such as amphiregulin (Areg), pleiotrophin (Ptn), and insulin-like growth factor 1 (Igf1); molecules related to epithelial differentiation such as milk proteins; molecules expressed by hematopoietic cells such as B cells, T cells, and macrophages; and molecules involved in the TGF-ß signaling pathway. In light of this observation, we asked whether smaller subsets of genes representing each of these individual functional gene categories would be sufficient to correctly distinguish nulliparous and parous mammary gland samples in a blinded manner.
To address this question, the 18 nulliparous and parous mammary samples described above were clustered based on expression profiles for genes within each of four functional gene categories (growth-promoting; differentiation-related; immune-related; and TGF-ß pathway). Strikingly, even when considered in isolation, cluster analysis performed in a blinded manner demonstrated that gene expression patterns within any one of these four subgroups were sufficient to correctly distinguish parous and nulliparous mammary samples derived from FVB mice, Balb/c mice or Lewis rats (Fig. 3, D and E, and data not shown). For example, gene expression patterns for the growth factors Areg, Ptn, and Igf1 were themselves sufficient to accurately identify all 18 mammary samples from FVB mice, Balb/c mice and Lewis rats as being nulliparous or parous (Fig. 3E
and data not shown). Similarly, expression patterns for TGF-ß3 and three of its downstream targets, clusterin, Eta-1 and Id-2, were sufficient to correctly determine the reproductive histories of the animals from which these same samples were taken (Fig. 3D
and data not shown). Finally, expression patterns for as few as seven differentiation-related genes (ß-casein,
-casein, WAP, lactotransferrin,
-lactalbumin, LPS-binding protein, and carboxyl ester lipase), or eight genes expressed by immune cells [Macrophage metalloelastase (MME), Mpeg1, Eta-1, IgA heavy chain, IgG1 heavy chain, IgG2b heavy chain, IgM heavy chain, and Ig
light chain], were sufficient to correctly distinguish parous and nulliparous samples in both mice and rats (data not shown). These findings demonstrate that expression changes within each of these four functional gene categories robustly and independently distinguish between the parous and the nulliparous states of the mammary gland in multiple rodent strains and species that exhibit parity-induced protection against breast cancer. As such, our findings suggest that the down-regulation of specific genes involved in epithelial proliferation, and the up-regulation of genes involved in epithelial differentiation, immune regulation, and TGF-ß-signaling represent cardinal features of parity-induced changes in the mammary gland.
Differentiation Markers Are Preferentially Expressed in the Parous Mammary Gland
Consistent with our previous report (31), a prominent functional category of genes whose expression was persistently elevated in the parous involuted mammary glands of both mice and rats included markers for mammary epithelial differentiation such as -casein, ß-casein,
-casein,
-casein, whey acidic protein, lactoferrin, and
-lactalbumin (Table 1
, Fig. 3
, and data not shown). These observations indicate that the parity-dependent up-regulation of markers for mammary epithelial differentiation is conserved among different mouse strains and rodent species that exhibit parity-induced protection against breast cancer. These findings provide additional molecular evidence supporting prior reports that parity results in a persistent increase in the expression of epithelial differentiation markers within the mammary gland (31, 32). In addition, the preferential expression of adipocyte differentiation-related protein, which is up-regulated in differentiated adipocytes (33), in the parous involuted gland suggests that stromal compartments of the mammary gland may also become more differentiated as a consequence of parity.
Parity Results in a Decrease in Growth Factor Gene Expression
Interestingly, genes that encode growth regulatory molecules constituted more than half of genes that were found to be persistently down-regulated by parity (Table 1). These include Areg, Ptn, Igf1, leptin (Ob), and thyroid-stimulating hormone receptor (TshR) (Table 1
and Figs. 3
and 4A
). The consistent decreases in gene expression observed for these genes in the mammary glands of parous FVB mice, Balb/c mice, and Lewis rats indicates that their down-regulation is a characteristic feature of parity-induced changes in the rodent mammary gland (Fig. 3
).
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Parity Results in Increased Mammary Expression of TGF-ß3 and Its Targets
In addition to the above findings, our microarray and Northern hybridization data indicated that parity resulted in a persistent increase in steady-state mRNA levels for the growth-inhibitory cytokine, TGF-ß3, in the mammary glands of FVB mice, Balb/c mice, Lewis rats and Sprague-Dawley rats (Table 1 and Figs. 3
, 5
, and 7
). In addition, clusterin, Eta-1, and Id-2, each of which has previously been implicated as a downstream transcriptional target of TGF-ß3 (35, 36, 37, 38, 39), were also found to be persistently up-regulated by parity in rats and mice (Table 1
and Figs. 3
, 5
, 6
, and 7
). Northern and in situ hybridization analysis confirmed maximal expression of TGF-ß3 and clusterin by epithelial cells at d 2 of involution as well as the persistently elevated levels of expression of these genes at d 28 of involution (Fig. 5
, B and C). These developmental expression profiles are consistent with a role for these molecules in cell death during mammary gland involution (36, 38, 40). The coordinate elevation in expression of TGF-ß3 along with several of its transcriptional targets suggests that the up-regulation of TGF-ß3 mRNA in the parous mammary gland may be accompanied by a bona fide increase in activity of the TGF-ß3 pathway.
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To investigate this hypothesis further, we examined the expression patterns for several immune-related genes during stages of postnatal mammary development representing puberty, pregnancy, lactation, and involution. Northern analysis revealed that expression of the B cell-specific gene, -LC, is first detected during lactation with elevated levels of expression persisting throughout postlactational involution (Fig. 6B
). The increase in
-LC expression during lactation is partially masked by the dilutional effects that result from the massive increase in milk protein gene expression that occurs during this period, as evidenced by the apparent decrease in ß-actin expression that occurs during this same period (Fig. 6B
). In situ hybridization confirmed the up-regulation of
-LC expression during lactation and further revealed that this up-regulation is due to an increase in the number of
-LC expressing cells (Fig. 6C
and data not shown). The spatio-temporal pattern of
-LC expression is consistent with the reported influx of lymphocytes into the breast that occurs during lactation (45). Surprisingly, however, our findings suggest that this lymphocyte population persists in the fully involuted gland.
MME or matrix metalloprotease-12 was also found to be persistently up-regulated in the mammary gland as a consequence of parity (Fig. 6A). MME is a secreted metalloprotease that cleaves plasminogen to generate angiostatin, a potent inhibitor of endothelial cell proliferation (46). Northern hybridization demonstrated that MME expression levels increase dramatically at d 7 of postlactational involution and remain elevated compared with age-matched nulliparous controls after 28 d of involution (Fig. 6B
). In situ hybridization revealed that MME expression is restricted to isolated cells within the mammary stroma at d 7 of involution (Fig. 6C
and data not shown). This finding is consistent with this genes reported expression in macrophages and with previous evidence that macrophages are recruited to the breast during involution where they participate in the clearance of postapoptotic debris (38). Interestingly, by d 28 of involution foci of MME expression became tightly associated with the epithelial compartment reflecting either a persistent population of macrophages residing within the parous epithelium or a subset of epithelial cells expressing MME (Fig. 4C
and data not shown).
Finally, we examined the developmental basis for the persistent parity-dependent up-regulation of Eta-1 expression (Fig. 6A). Northern analysis revealed that Eta-1 expression, which has been reported in macrophages and T-lymphocytes as well as in mammary epithelial cells, is dramatically up-regulated at d 12 of pregnancy and remains high through d 7 of involution (Fig. 6B
) (47). Though declining somewhat by d 28 of postlactational involution, Eta-1 expression remains markedly elevated compared with age-matched nulliparous animals. In situ hybridization analysis demonstrated that Eta-1 expression in the parous involuted gland is restricted to a subset of mammary epithelial cells (Fig. 6C
and data not shown). In aggregate, our data suggest that parity induces persistent increases in populations of hematopoietic cells present within the mammary gland, as well as changes in cytokine expression within the mammary epithelium itself.
Parity-Induced Changes in Gene Expression Persist in the Mammary Gland
Epidemiological observations suggest that whichever parity-induced changes in the mammary gland are responsible for protection against breast cancer are likely to be permanent. Accordingly, changes in gene expression that are involved in this protective effect would be predicted to persist for periods of involution greater than 4 wk. To determine whether the parity-dependent changes in gene expression identified in this study persist for longer periods of involution, we analyzed cohorts of mice that were mated at 4 wk of age, underwent 21 d of lactation, and were then allowed to undergo either 4, 16, or 30 wk of postlactational involution. Northern analysis revealed that expression levels of lactoferrin, -LC, TGF-ß3, clusterin, and Eta-1, were each consistently up-regulated in the mammary glands of parous animals compared with age-matched nulliparous controls even after 30 wk of postlactational involution (Fig. 7
). These findings indicate that, for at least a subset of the genes identified in this study, parity-induced changes in gene expression are essentially permanent.
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DISCUSSION |
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For the majority of genes investigated in this study, parity-induced changes in gene expression were found to be independent of the length of postlactational involution, indicating that the altered patterns of expression that we have identified are persistent, if not permanent. Furthermore, we have confirmed that parity-induced changes in gene expression are conserved in three stains of mice as well as in two well-characterized rat models for parity-induced protection against breast cancer. Beyond indicating that many of the molecular changes that we have identified are conserved among rodents, our findings further suggest that similar parity-induced changes in gene expression may be seen in the human breast.
One of the most striking findings of our microarray analysis was the observation that parity induces a persistent down-regulation in the expression of multiple genes involved in the regulation of cell growth and proliferation. Specifically, the reduced expression of Areg, Ptn, Igf1, TshR, and Ob suggest that multiple mitogenic pathways may be down-regulated in the mammary gland as a consequence of parity. Of note, elevated expression of Areg, Ptn, and Igf1 have each been implicated in the pathogenesis of human breast cancer (48). AREG, a ligand for the epidermal growth factor receptor, has been shown to be overexpressed in 3550% of primary human breast cancers (49, 50, 51). Consistent with this, Areg is overexpressed in hyperplastic stages of mammary tumor development in mouse mammary tumor virus-polyoma middle T antigen and metallothionein-TGF transgenic mice (52), and is a potent stimulator of anchorage-dependent growth in nontransformed MCF-10 cells (53). Moreover, targeted inactivation of Areg in mice causes a marked delay in ductal elongation during puberty and down-regulation of Areg expression in transformed mammary epithelial cell lines results in growth inhibition and reduced tumorigenicity (54, 55, 56). These findings indicate that Areg plays an important role in promoting mammary epithelial cell proliferation and predict that the parity-dependent down-regulation of Areg could contribute to a decrease in the susceptibility of the parous mammary gland to cancer.
Similar to Areg, the heparin-binding growth factor, pleiotrophin, was also found to be down-regulated in the parous mammary gland. A majority of human breast cancers, as well as carcinogen-induced rat mammary tumors, display high levels of PTN mRNA expression (57, 58). Moreover, Ptn overexpression in NIH 3T3 cells induces cellular transformation, whereas overexpression of a dominant negative PTN mutant in human breast cancer cell lines reverses their transformed phenotype (59, 60). As such, like Areg, the down-regulation of Ptn represents a biologically plausible mechanism that could contribute to parity-induced protection against breast cancer.
Our finding that Igf1, Ptn, Ob, and TshR, are down-regulated by parity raises the possibility that parity-induced alterations in other growth promoting pathways may also contribute to protection against breast cancer. This hypothesis is intriguing given the wealth of experimental evidence implicating the IGF-I pathway in human breast cancer. These include the observations that IGF-I levels are elevated in women with breast cancer, that IGF-I receptor levels and activity are elevated in human breast cancer cells, and that expression of the IGF downstream signaling molecule, IRS-1, is correlated with decreased disease-free survival (61, 62, 63, 64). Finally, the strong positive correlation that exists between circulating IGF-I concentrations and breast cancer risk among premenopausal women (65) suggests that the persistent down-regulation of this pathway could contribute to parity-induced protection against breast cancer.
In aggregate, our studies demonstrate that parity induces a persistent down-regulation in the expression of multiple genes involved in the regulation of cell growth and proliferation. Nevertheless, while decreased epithelial proliferation rates in parous animals have been reported by some investigators, other studies have failed to find a consistent difference in cellular proliferation rates between parous and nulliparous animals (23, 29, 30, 66, 67). Interestingly, Sivaraman et al. (68) have recently shown that mimicking the protective effect of parity by treatment with estradiol and progesterone results in a block to mammary epithelial proliferation following a carcinogen challenge, and p53 has been implicated as a potential mediator of this effect. As such, it is possible that the down-regulation of growth-promoting molecules that we have observed in this study may only result in differences in epithelial proliferation in the context of a response to a specific carcinogenic challenge. Further studies will therefore be required to determine the significance of these findings.
In addition to decreases in growth factor gene expression, we have also identified a parity-dependent increase in the expression of TGF-ß3 and several of its downstream transcriptional targets in the mammary gland. Overexpression of TGF-ß isoforms inhibits mammary epithelial proliferation, enhances mammary epithelial apoptosis, and can suppress tumorigenesis in the mammary gland in response to carcinogens or oncogenic stimuli (40, 69, 70). Conversely, down- regulation of TGF-ß activity results in increases in epithelial proliferation as well as in spontaneous and carcinogen-induced tumorigenesis in the mammary gland (71, 72). These properties make up-regulation of the TGF-ß3 pathway a biologically plausible contributor to parity-induced protection against breast cancer. While our data do not directly demonstrate activation of the TGF-ß3 signaling pathway in the parous mammary gland, the coordinate regulation of several downstream transcriptional targets of TGF-ß3 lends support to this model. Further investigation of TGF-ß signaling molecules would provide additional evidence for the up-regulation of this pathway.
It has previously been proposed that parity-induced protection against breast cancer may be mediated by an increased state of differentiation of the parous mammary gland (9, 10). Together with our previous findings, our data provide molecular evidence to support the contention that the epithelial compartment of the parous mammary gland is more differentiated than that of the nulliparous gland, as well as evidence to suggest that parity may also increase the differentiated state of the stromal compartment of the mammary gland (31). Consistent with this, Ginger et al. (32) recently reported the use of suppression subtractive hybridization-PCR to identify genes that are persistently up-regulated in the mammary glands of Wistar-Furth rats after treatment with estrogen and progesterone. Several of the differentially expressed genes isolated in this study encode proteins that are markers for mammary epithelial differentiation, such as -casein, ß-casein, and
-casein, or are involved in regulating cellular proliferation. Nevertheless, the recent finding in rats that the dopamine antagonist, perphenazine, induces epithelial differentiation yet does not protect against carcinogen-induced mammary tumorigenesis casts doubt on a central role for differentiation in parity-induced protection against breast cancer (14). As such, while a persistent increase in the differentiated state of the mammary gland remains a biologically plausible mechanism for reducing cancer susceptibility, differentiation may nevertheless be unrelated to the mechanism by which parity reduces breast cancer risk. However, since the nature or extent of epithelial differentiation induced by perphenazine may differ from that induced by parity, further studies will be required to conclusively rule out differentiation as a contributing factor.
While differentiation per se may not protect against breast cancer, protection could be conferred by the persistent up-regulation of differentiated gene products. For example, the milk protein gene lactoferrin, which is up-regulated approximately 5-fold by parity, has been shown to inhibit the proliferation of human breast cancer cells in vitro and in vivo, to potentiate lymphokine-activated killer cell and NK cell-mediated cytotoxicity against breast cancer cell lines, and to inhibit the growth of transplantable tumors and experimental metastases in mouse models (73, 74, 75, 76, 77). These findings suggest that differentiation-induced changes in gene expression may contribute to parity-induced protection against breast cancer by mechanisms that are unrelated to differentiation itself.
In the course of our experiments, we were surprised to find the parity-induced up-regulation of genes whose expression mark distinct classes of hematopoietic cells. These observations indicate that parity results in a persistent increase in the number of macrophages, B-lymphocytes, and T-lymphocytes that reside within the mammary gland. In addition, we have shown that parity induces changes in the expression of specific cytokines within the mammary gland, including that of Eta-1/Osteopontin within the mammary epithelium. These findings suggest a model for paracrine signaling mechanisms by which parity-induced changes in epithelial gene expression may influence the function of immune cells within the gland and thereby create an environment that is refractory to tumorigenesis. Consistent with this general hypothesis, we have found that the secreted antiangiogenesis factor, MME, is highly expressed by a subset of epithelial cells in the involuted parous mammary gland. MME cleaves plasminogen to produce angiostatin, a potent inhibitor of endothelial cell proliferation, angiogenesis, and primary tumor growth (46). More broadly, our findings suggest that parity may induce widespread changes in the immunological environment of the mammary gland that could plausibly affect cancer susceptibility either by tumor surveillance mechanisms, or by the parity-dependent creation of an environment that is less conducive to transformation, tumor establishment or tumor progression.
Finally, the recognition that particular reproductive endocrine events alter breast cancer risk in a predictable manner raises the possibility that naturally occurring events known to decrease breast cancer risk might be mimicked pharmacologically. The desire to pursue this objective is heightened by the fact that, while it is now possible to identify women at increased risk for breast cancer, few interventions currently exist. As such, reducing breast cancer risk via chemopreventive approaches designed to mimic naturally occurring endocrine events could represent an attractive alternative. It is to this end that early first full-term pregnancy has been proposed as a logical paradigm on which to model the hormonal chemoprevention of breast cancer. The achievement of this goal, however, has been hampered by our lack of understanding of the mechanisms by which reproductive events alter breast cancer risk. Understanding these mechanisms will ultimately facilitate the design of safe and effective hormonal chemoprevention regimens. Moreover, the development and testing of such regimens will be facilitated by the identification and use of intermediate molecular endpoints that accurately detect changes in the breast associated with changes in breast cancer risk. We have chosen to exploit the defined relationship between parity and carcinogenesis in the breast to generate surrogate endpoint biomarkers for changes in the breast associated with a reduction in breast cancer risk. While our findings do not address whether the molecular and cellular alterations identified in this study are causally related to parity-induced protection against breast cancer, they do suggest promising new avenues for investigation. We believe that such biomarkers will ultimately prove essential for understanding the molecular and cellular basis of parity-induced protection against breast cancer, and for the rational design and testing of hormonal chemoprevention regimens aimed at mimicking this naturally occurring protective event.
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MATERIALS AND METHODS |
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Whole Mounts and Histology
Number 4 mammary glands were mounted on glass slides, fixed overnight in neutral buffered formalin, and transferred to 70% ethanol. For whole mounts, glands were rinsed in water for 5 min and stained in a filtered solution of 0.2% carmine (Sigma, St. Louis, MO) and 0.5% aluminum potassium sulfate for 13 d. Glands were then dehydrated sequentially through 70%, 90%, and 100% ethanol for 15 min each, then de-fatted and stored in methyl salicylate. For histological analysis, fixed glands were blocked in paraffin, sectioned, and stained with hematoxylin and eosin.
RNA Isolation and Northern Analysis
Snap-frozen tissue was homogenized in guanidine thiocyanate supplemented with 7 µl/ml 2-mercaptoethanol, and RNA isolated by centrifugation through cesium chloride as previously described (78). Equal amounts of RNA from each of 1520 mice or 10 rats were combined for each independent pool. Total RNA was separated on a 1% LE agarose gel, and passively transferred to Gene Screen (NEN Life Science Products, Boston, MA). Northern hybridization was performed per manufacturers instructions using PerfectHyb Plus Hybridization Buffer (Sigma) and 32P-labeled cDNA probes corresponding to GenBank sequences represented on the Affymetrix oligonucleotide microarray Mu6500 Gene Chip.
Oligonucleotide Microarray Hybridization and Analysis
Approximately 40 µg of total pooled RNA from each sample were used to generate cDNA and biotinylated cRNA as described (79). Hybridization to a set of Affymetrix Mu6500K microarrays was performed per manufacturers instructions. After washing and staining with streptavidin-phycoerythrin, chips were scanned using a Hewlett-Packard Co. (Palo Alto, CA) Gene Array Scanner. Grid alignment and raw data generation was performed using Affymetrix GeneChip 3.1 software. Raw gene expression levels were scaled and normalized data sets were compared using Affymetrix algorithms to identify differentially expressed genes.
The genes identified in this study as being differentially expressed were selected an confirmed by multiple overlapping methods. Briefly, 16 genes were identified by Affymetrix comparative algorithms (MAS 4.0) as having expression levels that differed significantly between nulliparous and parous samples in each of the three independent comparisons that were performed. The parity-dependent patterns of differential expression for 14 of these 16 genes were tested and confirmed by Northern hybridization of triplicate, independent pooled sets of parous and nulliparous mammary gland samples that were independent of the original 6 samples used for microarray analysis. Two of the 16 genes in this subclass were not tested by Northern analysis but were subsequently confirmed by analysis on independent microarrays. An additional 59 genes identified by Affymetrix algorithms as being differentially expressed in 2 of 3 independent Mu6500 microarray comparisons were subsequently tested by Northern hybridization in a manner identical to that outlined above. Twenty-four of the 59 genes tested in this class demonstrated consistent differential expression by Northern analysis. Finally, the differential expression of the 40 genes that had been confirmed by Northern analysis was tested using a different generation of Affymetrix microarrays (MGU74Av2) to analyze a third set of triplicate, independent pooled nulliparous and parous mammary samples from FVB mice. Statistical significance was assessed using a nonheteroscedastic t test to compare oligonucleotide hybridization intensities. The number of significantly (P < threshold) differing oligonucleotide hybridizations within an individual probe set measuring a given transcript was tabulated, and an empirical P value of expression difference for the entire transcript was calculated using a reference data set generated from six independent, identical control samples. This analysis revealed that 38 of the 40 originally identified genes were differentially expressed in the predicted manner between the parous and nulliparous glands with P values less than 0.02. For clustering analysis, probe sets for differentially expressed genes identified in the Mu6500 array analysis were mapped to corresponding probe sets on Affymetrix MGU74 microarrays via the Unigene and LocusLink databases. A list of orthologous probe sets on the Affymetrix RGU34 chip was generated using matches obtained by querying each differentially expressed gene identified in the mouse against the Homologene database (http://www.ncbi.nlm.nih.gov/HomoloGene/). Of the resulting matches, only curated and calculated reciprocal best match Homologene hits were selected for further analysis. Data were scaled such that the mean signal intensity was equivalent across all array samples excluding the top and bottom 2% of data points. Samples were standardized before cluster analysis such that the median expression level and standard deviation for each gene equaled 0 and 1, respectively, across the set of samples being clustered. The data was then filtered to include only probe sets for genes shown to be differentially expressed between parous and nulliparous mouse samples (Table 1), or the orthologues for these genes in the rat. Cluster software was used to generate hierarchical clustering trees, which were visualized using Treeview (Eisen, M.; http://www.microarrays.org/software).
In Situ Hybridization
In situ hybridization was performed as described (78). Antisense and sense probes were synthesized with the Promega Corp. (Madison, WI) in vitro transcription system using 35S-UTP and 35S-CTP from the T7 and SP6 RNA polymerase promoters of a PCR template containing the same sequences used for Northern hybridization analysis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 C.M.D. and S.E.M. contributed equally.
Abbreviations: Areg, Amphiregulin; -LC,
-light chain; MME, macrophage metalloelastase; Tshr, thyroid-stimulating hormone receptor.
Received for publication February 15, 2002. Accepted for publication June 10, 2002.
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REFERENCES |
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