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Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University, Washington, DC 20057
* To whom correspondence should be addressed. E-mail: clarkel{at}georgetown.edu.
Pregnancy, mammary epithelial differentiation, and breast cancer risk
Our understanding of the proposed protective role of mammary epithelial differentiation against breast cancer is based mainly on the important work by Irma and Jose Russo (1–3). The Russos have shown that terminal end buds (TEBs)4 in a rat mammary gland are the structures that give rise to malignant mammary tumors on exposure to chemical carcinogens, particularly to 7,12-dimethylbenz[a]anthracene, a polycyclic aromatic hydrocarbon. Breast cancers in women originate from a corresponding structure called the terminal ductal lobular unit (TDLU) or Lobule type 1 (Lob 1) (4). TEBs play a central role in mammary gland development in rats. The mammary fat pad is initially formed during the fetal period, and it contains limited stroma and a rudimentary epithelial tree. The epithelium is quiescent until about 3 weeks of postnatal age, when large club-shaped TEBs appear. They are the most actively growing terminal ductal structures and are believed to contain a pluripotent stem cell population that potentially gives rise to breast cancer (3,5,6). TEBs lead the growth of the epithelial ducts through the fat pad and eventually regress to terminal ducts or differentiate to alveolar buds and further to lobules when the mammary fat pad is filled with a highly branched ductal tree. These alveolar structures do not give rise to malignant tumors. The work by the Russos and others (7,8) has shown that a key developmental event, pregnancy, as well as exposures that mimic the hormonal environment of pregnancy induce differentiation of the TEB/TDLU and dramatically reduce the risk of breast cancer.
The idea that differentiation explains the protective effects of pregnancy has been questioned by several investigators (9). Arguing against this idea are findings that induction of differentiation by placental lactogen or the dopamine receptor inhibitor perphenazine (PPZ) does not reduce breast cancer risk in animal models. An alternative hypothesis has been proposed by Siverman et al. (10); according to his "cell fate hypothesis," pregnancy hormones induce a molecular switch in mammary stem cells that leads to changes in cell proliferation and response to DNA damage. The molecular changes then inhibit cell proliferation in response to subsequent exposure to hormones or carcinogens (10,11).
Pregnancy and increased breast cancer risk
In women, pregnancy reduces breast cancer risk, but the reduction is relatively small (20%) and occurs only in women who had their first child before age 20 (12,13). A transient increase in breast cancer risk, lasting for 3–5 y after a pregnancy, is seen in women who were 25 y of age or older during pregnancy (14–16). Pregnancy induces a life-long increase in breast cancer risk in women who are over 30 at the time of first pregnancy (12). The question is: why is pregnancy-induced mammary epithelial differentiation not sufficient to reduce breast cancer in all women, and why does pregnancy induce a short-lasting and/or life-long increase in breast cancer risk in older first-time mothers? It has been suggested that pregnancy fails to induce full mammary differentiation in those women who are at an increased risk of developing breast cancer, for example, because of familial history (17). Another possibility is that the breasts of an older woman may have acquired mutations that lead to breast cancer under the influence of the pregnancy hormonal environment. This is supported by data showing that women exposed to the highest range of estrogens during pregnancy are at an increased breast cancer risk (18–20).
Mammary tumorigenesis is lower in rats that undergo pregnancy after a carcinogen exposure than in rats that remain nulliparous after being exposed to a carcinogen; i.e., pregnancy reduces breast cancer risk in rats. Using rats, we have been investigating the role of diet in modifying pregnancy hormone levels and its effect on subsequent mammary tumorigenesis. These studies indicate that excessive pregnancy weight gain (21) that increases pregnancy leptin levels (21) or an exposure to a high-fat diet that increases pregnancy estradiol (22) increases breast cancer risk. These exposures also increase mammary epithelial cell proliferation and activate the MAPK signaling pathway (21). Because findings in humans show that women who gained more weight than recommended during pregnancy are at 60% increased risk of developing breast cancer (23), it is likely that the pregnancy hormonal environment can modify the risk of developing this disease.
Wagner and Smith have recently identified pregnancy-induced mammary epithelial cells (PI-MECs) in parous female mice (24). These cells originate from differentiating cells during first pregnancy, and they do not undergo apoptosis during postlactational remodeling. The PI-MECs that exhibit key features of multipotent stem cells and are proposed to mediate the long-term breast cancer risk-reducing effects of pregnancy also are cellular targets for pregnancy-enhanced mammary tumorigenesis. A question that remains to be answered is why PI-MECs differentiate in women who had their first child before age 20 but are susceptible for malignant transformation in women who had their first child after age 30?
Early life hormonal environment, TEBs, and breast cancer risk
Besides pregnancy, there are 2 other developmental periods when an increase in hormone levels leads to alterations in mammary gland morphology and breast cancer risk. These are the fetal and prepubertal periods. We have shown that in utero exposure to estrogens 1) increases the number of TEBs in the postnatal mammary gland, particularly at the time when the gland is most susceptible to carcinogens; 2) reduces the number of differentiated lobuloalveolar structures; and 3) increases mammary tumorigenesis (25). TEBs themselves are altered: in our preliminary study (our unpublished data), at the time the mammary gland is most susceptible for malignant transformation, no apoptotic cells were detected in the TEBs of rats exposed to estradiol in utero, whereas the cells in the TEBs of the control rats contained apoptotic cells. In contrast, the number of proliferating cells was significantly increased, as was the expression of estrogen receptor-
(ER-) in the TEBs and other epithelial structures in the in utero E2-exposed rats. These findings suggest that the developmental patterns of TEBs, including their number and the level of cell proliferation/apoptosis occurring in the TEBs, may be programmed in utero. Additional support for the idea that in utero hormonal exposures can alter later susceptibility to breast cancer by affecting pathways regulating normal mammary gland cell proliferation, apoptosis, and differentiation comes from our recent observation that these exposures up-regulate genes linked to regulation of stem cell fate, that is, cleaved Notch1 and Notch4 (our unpublished data).
Another period when estrogenic exposures alter mammary gland development is prepuberty. Prepubertal estrogenic exposures initially increase cell proliferation, but after puberty onset, the number of TEBs is reduced (26), the breast epithelium contains fewer proliferating cells and more apoptotic cells than the control glands (our unpublished data), and the risk of developing mammary tumors is reduced (26). We have also been studying whether a prepubertal exposure to dietary estrogens such as the phytoestrogen genistein that activates the estrogen receptor or to (n-3)-polyunsaturated fatty acids (PUFAs) that increase circulating estradiol levels (our unpublished data) alters mammary gland development and susceptibility to develop mammary tumors. The results generated by us and also by others show that prepubertal genistein exposure provides a protection against mammary carcinogenesis and reduces the number of TEBs and increases morphological differentiation of the mammary epithelial tree (26–28). Prepubertal dietary exposure to (n-3)-PUFAs also reduces TEBs, inhibits cell proliferation, and induces apoptosis within the TEBs and other mammary epithelial structures (29). In addition, mammary tumorigenesis is reduced in the rats fed an (n-3)-PUFA–containing diet during the prepubertal period (29). These changes are accompanied by upregulation of genes that repair DNA damage (26) or genes that have antioxidant properties (our unpublished data). These findings are supportive of Siverman's cell fate hypothesis as a mechanism by which postpubescent hormonal exposures provide protection against breast cancer (10). However, in our studies the hormonal exposures have occurred before puberty onset.
Effects of early life estrogenic exposures on gene expression
We have attempted to identify genes that are altered following in utero and prepubertal exposures to explain their effects on mammary gland morphology and tumorigenesis. These attempts have focused on performing gene microarrays as well as studying changes in the expression of caveolin-1. Caveolin-1, a scaffolding protein, is abundantly expressed in many differentiated cells and is the hallmark of caveolae, specialized lipid rafts that perform a number of signaling functions at the cell membrane (30). Within caveolae, caveolin-1 sequesters and regulates the function of various resident proteins associated with initiation of breast cancer or malignant growth. We have shown that in utero E2 exposure reduces caveolin-1 expression and upregulates pAkt and pSrc proteins, whereas prepubertal E2 exposure has an opposite effect on caveolin-1, and it also downregulates Erb-2 and cyclin D1 (our unpublished data). Further, our findings indicate that caveolin-1 controls the expression of putative stem cell marker genes that regulate stem cell self-renewal and differentiation, including Nestin, Sox9, and CXCR4 (our unpublished data). Lisanti and collaborators recently reported that caveolin-1 induces accumulation of mammary stem cells (31). Because breast cancer is likely to be initiated in stem/progenitor cells, caveolin-1 may be an important gene regulating mammary stem cell fate. Studies are under way to determine whether in utero and prepubertal dietary exposures alter caveolin-1 expression.
Ongoing gene microarray analysis has identified a number of genes that are differentially expressed in the mammary glands of adult rats exposed to E2 during prepuberty versus in utero. Importantly, most of these genes regulate cell proliferation (cyclins, cdk4, Cyted2, Mapk, TCF4), differentiation (Cited1, TGF-ß, TGFßR2), and apoptosis (Akt, caspases, p53). These findings strongly support the notion that prepubertal estrogenic exposures reduce later breast cancer risk by affecting mammary stem cell division and differentiation.
Research needs
Although pregnancy can reduce breast cancer risk, the protective effect is seen mostly in women who have had their first child before age 20; for a number of reasons, pregnancy is not a suitable cancer preventive approach for women at such young age. In light of the evidence that prepubertal estrogenic exposures induce mammary epithelial differentiation, reduce the expression of genes regulating cell proliferation, and upregulate genes that improve DNA damage repair or induce apoptosis or differentiation, it might be more fruitful to study whether prepubertal dietary exposures that modify the childhood hormonal environment can mimic the effect of pregnancy on mammary epithelial cell fate. Specifically, we need to address whether childhood dietary exposures induce changes in mammary stem cells; that is, is stem cell division reduced and/or differentiation of progenitor cells increased? Are these changes caused by epigenetic modifications of genes that regulate stem cell fate?
In conclusion, our results in rats indicate that early-life hormonal and dietary exposures affect later susceptibility to develop breast cancer. Whether these exposures increase or reduce later breast cancer risk appear to be closely linked to their effects on mammary epithelial cell proliferation and apoptosis, particularly in the TEBs, and epithelial differentiation.
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FOOTNOTES |
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2 This work was supported by grants to Leena Hilakivi-Clarke from American Institute for Cancer Research, Breast Cancer Research Foundation, and NCI (U54 CA00100971, RO1 CA89950). The contents are solely the responsibility of the authors.
3 Author disclosure: no relationships to disclose.
4 Abbreviations used: PI-MECs, pregnancy-induced mammary epithelial cells; PPZ, perphenazine TDLU, terminal ductal lobular unit; TEB, terminal end buds.
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LITERATURE CITED |
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 TOP LITERATURE CITED |
|---|
1. Russo J, Gusterson BA, Rogers AE, Russo IH, Wellings SR, van Zwieten MJ. Comparative study of human and rat mammary tumorigenesis. Lab Invest. 1990;62:244–78.[Medline]
2. Russo J, Russo IH. Biological and molecular bases of mammary carcinogenesis. Lab Invest. 1987;57:112–37.[Medline]
3. Russo J, Russo IH. Toward a unified concept of mammary carcinogenesis. In: Aldaz MC, Gould MN, McLachlan J, Slaga TJ, editors. Etiology of Breast and Gynecological Cancers. New York: Wiley-Liss; 1997. p. 1–16.
4. Russo J, Mailo D, Hu YF, Balogh G, Sheriff F, Russo IH. Breast differentiation and its implication in cancer prevention. Clin Cancer Res. 2005;11:931s–6s.
5. Smith GH, Chepko G. Mammary epithelial stem cells. Microsc Res Tech. 2001;52:190–203.[Medline]
6. Smalley M, Ashworth A. Stem cells and breast cancer: A field in transit. Nat Rev Cancer. 2003;3:832–44.[Medline]
7. Russo IH, Russo J. Hormonal approach to breast cancer prevention. J Cell Biochem. 2000;77:1–6.[Medline]
8. Rajkumar L, Guzman RC, Yang J, Thordarson G, Talamantes F, Nandi S. Short-term exposure to pregnancy levels of estrogen prevents mammary carcinogenesis. Proc Natl Acad Sci USA. 2001;98:11755–9.
9. Medina D. Mammary developmental fate and breast cancer risk. Endocr Relat Cancer. 2005;12:483–95.
10. Sivaraman L, Stephens LC, Markaverich BM, Clark JA, Krnacik S, Conneely OM, O'Malley BW, Medina D. Hormone-induced refractoriness to mammary carcinogenesis in Wistar-Furth rats. Carcinogenesis. 1998;19:1573–81.
11. Sivaraman L, Hilsenbeck SG, Zhong L, Gay J, Conneely OM, Medina D, O'Malley BW. Early exposure of the rat mammary gland to estrogen and progesterone blocks co-localization of estrogen receptor expression and proliferation. J Endocrinol. 2001;171:75–83.[Abstract]
12. MacMahon B, Cole P, Lin TM, Lowe CR, Mirra AP, Ravnihar B, Salber EJ, Valaoras VG, Yuasa S. Age at first birth and breast cancer. Bull World Health Org. 1970;43:209–21.[Medline]
13. Merrill RM, Fugal S, Novilla LB, Raphael MC. Cancer risk associated with early and late maternal age at first birth. Gynecol Oncol. 2005;96:583–93.[Medline]
14. Hsieh C, Pavia M, Lambe M, Lan SJ, Colditz GA, Ekbom A, Adami HO, Trichopoulos D, Willett WC. Dual effect of parity on breast cancer risk. Eur J Cancer. 1994;30A:969–73.
15. Bruzzi P, Negri E, La Vecchia C. Short term increase in risk of breast cancer after full term pregnancy. BMJ. 1988;297:1096–8.[Medline]
16. Williams EM, Jones L, Vessey MP, McPherson K. Short term increase in risk of breast cancer associated with full pregnancy. BMJ. 1990;300:578–9.[Medline]
17. Lagiou A, Lagiou P, Vassilarou DS, Stoikidou M, Trichopoulos D. Comparison of age at first full-term pregnancy between women with breast cancer and women with benign breast diseases. Int J Cancer. 2003;107:817–21.[Medline]
18. Titus-Ernstoff L, Hatch EE, Hoover RN, Palmer J, Greenberg ER, Ricker W, Kaufman R, Noller K, Herbst AL, et al. Long-term cancer risk in women given diethylstilbestrol (DES) during pregnancy. Br J Cancer. 2001;84:126–33.[Medline]
19. Wohlfahrt J, Melbye M. Maternal risk of breast cancer and birth characteristics of offspring by time since birth. Epidemiology. 1999;10:441–4.[Medline]
20. Richardson BE, Hulka BS, Peck JL, Hughes CL, van den Berg BL, Christianson RE, Calvin JA. Levels of maternal serum alpha-fetoprotein (AFP) in pregnant women and subsequent breast cancer risk. Am J Epidemiol. 1998;148:719–27.
21. de Assis S, Wang M, Goel S, Foxworth A, Helferich WG, Hilakivi-Clarke L. Excessive weight gain during pregnancy increases carcinogen-induced mammary tumorigenesis in Sprague-Dawley and lean and obese Zucker rats. J Nutr. 2006;136:998–1004.
22. Hilakivi-Clarke L, Onojafe I, Raygada M, Cho E, Clarke R, Lippman M. Breast cancer risk in rats fed a diet high in n-6 polyunsaturated fatty acids during pregnancy. J Natl Cancer Inst. 1996;88:1821–7.
23. Kinnunen TI, Luoto R, Gissler M, Hemminki E, Hilakivi-Clarke L. Pregnancy weight gain and breast cancer risk. BMC Womens Health. 2004;4:7–17.[Medline]
24. Wagner KU, Smith GH. Pregnancy and stem cell behavior. J Mammary Gland Biol Neoplasia. 2005;10:25–36.[Medline]
25. Hilakivi-Clarke L, Clarke R, Onojafe I, Raygada M, Cho E, Lippman ME. A maternal diet high in n-6 polyunsaturated fats alters mammary gland development, puberty onset, and breast cancer risk among female rat offspring. Proc Natl Acad Sci USA. 1997;94:9372–7.
26. Cabanes A, Wang M, Olivo S, de Assis S, Gustafsson JA, Khan G, Hilakivi-Clarke L. Prepubertal estradiol and genistein exposures up-regulate BRCA1 mRNA and reduce mammary tumorigenesis. Carcinogenesis. 2004;25:741–8.
27. Cotroneo MS, Wang J, Fritz WA, Eltoum IE, Lamartiniere CA. Genistein action in the prepubertal mammary gland in a chemoprevention model. Carcinogenesis. 2002;23:1467–74.
28. Hilakivi-Clarke L, Onojafe I, Raygada M, Cho E, Skaar T, Russo I, Clarke R. Prepubertal exposure to zearalenone or genistein reduces mammary tumorigenesis. Br J Cancer. 1999;80:1682–8.[Medline]
29. Olivo SE, Hilakivi-Clarke L. Opposing effects of prepubertal low and high fat n-3 polyunsaturated fatty acid diets on rat mammary tumorigenesis. Carcinogenesis. 2005;26:1563–72.
30. Williams TM, Lisanti MP. The caveolin genes: from cell biology to medicine. Ann Med. 2004;36:584–95.[Medline]
31. Sotgia F, Williams TM, Cohen AW, Minetti C, Pestell RG, Lisanti MP. Caveolin-1-deficient mice have an increased mammary stem cell population with upregulation of wnt/beta-catenin signaling. Cell Cycle. 2005;4:1808–16.[Medline]
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