Judith Clancy Keen, Ph.D. Nancy E. Davidson, M.D.
The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, Maryland.
Nancy E. Davidson, M.D., The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins School of Medicine, 1650 Orleans Street, Baltimore, MD 21231; Fax: (410) 6144073; E-mail: davidna@jhmi.edu.
Abstract
The biology of breast carcinoma is complex, with multiple factors contributing to its development and progression. The current review focuses on the role of several critical genes including estrogen receptor, progesterone receptor, retinoic acid receptor-β, epidermal growth factor receptor family members, p53, BRCA1, and BRCA2 as risk factors for the development of disease, predictors of prognosis and response to therapy, and as therapeutic targets. Studies of the biology of these and other genes that contribute to the development and progression of breast carcinoma have had and will continue to have great impact on all aspects of disease management.
Introduction
Breast carcinoma is a leading cause of cancer mortality among women in the Western hemisphere, second only to lung carcinoma. The American Cancer Society estimates that 203,500 new cases in women will be reported and 40,000 women will die of breast carcinoma in the U.S. in 2002 alone. Current estimates suggest in her lifetime, one in eight American women will be diagnosed with breast carcinoma.[1]
Our growing knowledge regarding breast carcinoma biology is having an ever greater impact on clinical management. Distinct characteristics of breast carcinoma can be exploited to help determine lifetime risk of development of the disease, the overall prognosis after a diagnosis of breast carcinoma, and the likelihood of response to specific therapy. In addition, increased understanding of breast carcinoma pathways may enhance our ability to devise targeted approaches to prevention or therapy. Thus, the biology of breast carcinoma can contribute vital information regarding many aspects of the disease.
It is well established that a myriad of factors including steroid hormones and their receptors, peptide growth factors, oncogenes, and tumor suppressor genes play a crucial role in the transformation of the breast.[2-4] This review will focus on selected biomarkers that play a key role in breast carcinoma, including certain steroid receptors (estrogen receptor [ER], progesterone receptor [PR], and retinoic acid receptor [RAR]-β), members of the HER/erbβ family, and selected tumor suppressor/susceptibility genes (e.g., p53, BRCA1, and BRCA2). Discussion will focus on their function and their possible roles in risk assessment, estimation of prognosis, and prediction of response to therapy, as well as their potential as therapeutic or preventive targets.
Steroid Hormone Receptors
Estrogen receptor
Early menarche, late menopause, and nulliparity are correlated with an increased risk of developing breast carcinoma, suggesting that prolonged exposure to cycling estrogen and progesterone levels contributes to the development of the disease. Removal of endogenous estrogen via oophorectomy decreases the risk of the development of breast carcinoma. Indeed, the earlier the ovaries are removed, the greater the risk reduction.[5,6] In postmenopausal women, the major source of estrogen is androgenic precursors derived from the adrenal glands that are converted into estro-gen by the aromatase enzyme in adipose tissues. Post-menopausal women with increased body fat have in-creased estrogen levels and are more likely to develop breast carcinoma.[4-7] Therefore, increased estrogen ex-posure via a variety of mechanisms appears to be a critical risk factor in the development of breast carci-noma. The effects of estrogen are mediated at least in part by the ER proteins, α and β.
ERα and ERβ; are members of the steroid recep-tor family. In the absence of its ligand, estrogen, ERα or ERβ forms an inactive complex with HSP 90.[8] Upon ligand binding and dissociation from HSP 90, ER is activated, undergoes a conformational change, dimerizes, and autophosphorylates through intrinsic tyrosine kinases. In this active form, ER dimers bind to recognition sequences termed estrogen response ele-ments (ERE), which are found within the promoter of many genes to regulate gene transcription. Activated ER also can activate the mitogen activated protein kinase (MAPK) pathway, which results in the activa-tion of the AP-1 proteins, fos and jun. Studies have shown that ER binds AP-1 consensus sites through protein-protein interactions with AP-1 proteins to reg-ulate gene transcription.[9,11]
Approximately 70- 80% of all breast tumors ex-press ERα protein and therefore are termed ER pos-itive (ER+). These tumors tend to grow more slowly, are better differentiated, and are associated with a slightly better overall prognosis.[4] Thus ER expression is one of a few prognostic factors, along with axillary lymph node status, tumor size, and histologic grade and subtype.[4] More important, the detection of ERα in breast carcinoma cells is an important indicator of potential response to endocrine therapy. A number of endocrine strategies currently exist to deplete the li-gand estrogen (oophorectomy or luteinizing hor-mone-releasing hormone [LHRH] analogues in pre-menopausal women or aromatase inhibitors in postmenopausal women), interfere with ligand-recep-tor interaction (selective ER modulators such as ta-moxifen and raloxifene), or destroy the ER (selective ER destroyers such as fulvestrant or ICI 182, 780). The molecular effects of these strategies are being under-stood more and more. For example, tamoxifen, ralox-ifene, and fulvestrant can reduce the expression of cell cycle proteins including cyclin D1 and cyclin E and inhibit the phosphorylation of the retinoblastoma (Rb) gene, a major target of the cyclin-associated kinases that are critical in cell cycle progression and cellular proliferation.[12,13] Selective ER modulator (SERM) treatment, estrogen withdrawal, or aromatase inhibi-tion results in tumor shrinkage, decreases the num-bers of cells in S-phase, and induces markers of cel-lular apoptosis.[14-17] Clinically, this is manifested by the observation that tumors expressing ERα protein are the most likely to manifest a response to endocrine therapy; those lacking ERα seldom respond.
SERMs also have been used prophylactically in women with a high risk of breast carcinoma to reduce the risk of development of disease. Indeed 5 years of tamoxifen use has been shown to reduce the risk of developing breast tumors by up to 50% in high-risk women. It is interesting to note that this strategy re-portedly is beneficial only in reducing the develop-ment of ER+ tumors. Tamoxifen does not appear to have an impact on the development of ER-negative (ER-) tumors.[18]
Given the central role of ERα in defining response to endocrine therapy for breast carcinoma, there is great interest in determining mechanisms for its absence of expression in some breast carcinoma pa-tients. A number of studies have shown that the loss of ER expression in ER- tumors seldom is the result of mutations, deletions, loss of heterozygosity, or poly-morphisms within the gene. Instead, it has been shown that ER gene expression occasionally is si-lenced through reversible epigenetic modifications in-cluding histone deacetylation and DNA methylation. The presence or absence of acetyl groups on histone tails (primarily H3 and H4) can govern chromatin structure and gene transcription. However, histone protein modification is not the only method of gene silencing but likely interacts with a second mecha-nism, DNA methylation. CpG dinucleotides are dis-persed throughout the genome, but are more highly clustered within gene promoter regions. Cytosine res-idues located 5' of guanine residues can be modified by the addition of methyl groups mediated by the DNA methyltransferase (DNMT) proteins. DNA meth-ylation within the promoter and first exon of genes is correlated with gene silencing and a lack of gene ex- pression.[19-23] The ERα promoter contains a CpG is-land within its promoter and first exon that is meth-ylated in ER- human breast carcinoma cell lines. Furthermore, histones isolated from these cell lines are deacetylated, suggesting a dual mechanism of methylation and histone deacetylation for ER silenc-ing in these cells.[21,24-26] Because these posttranscrip-tional modifications are reversible, the treatment of cells with epigenetically silenced genes with histone deacetylase (HDAC) inhibitors including Trichostatin A (TSA) or DNMT inhibitors (such as 5-Aza-2'-deoxy- cytidine) should result in expression from the intact gene. As predicted, the treatment of ER- human breast carcinoma cell lines with TSA or 5-Aza-2'-deoxycyti- dine results in reexpression of ER mRNA and func-tional protein, suggesting that epigenetic mechanisms may be important mechanisms for the absence of hormone response.[21,24-26]
For several decades, it was believed that there was a single ER gene. In 1996, a second ER, termed ERβ, was cloned first from the rat and subsequently from the human.[27,28] ERα and ERβ are structurally similar, sharing key features of the steroid receptor family. Although their overall sequence homology is only ap-proximately 30%, there is high homology within the DNA and hormone-binding domains at 95% and 53%, respectively. This domain-specific homology suggests that ERα and ERβ are likely to share similar DNA and ligand-binding function, but the low overall homology may indicate that their global effects differ.
Similar to ERα, ERβ is expressed in a variety of tissues including mammary gland, uterus, ovary, prostate, epididymus, testis, pituitary, kidney, thymus, bone, and central nervous system.[29] Within normal mammary tissues, ERβ is highly expressed in the epithelial and stromal layers. Investigation into ERβ regulation and its role in breast carcinoma remains in its early stages. Preliminary studies suggest that ERβ expression is readily detectable in ductal carcinomas in situ and lobular carcinomas in situ but drops dra-matically in late-stage tumors.[30-38] Studies published to date have to our knowledge relied primarily on reverse transcriptase-polymerase chain reaction- based techniques because reliable antibodies against ERβ are not yet widely available. The role of ERβ in cancer in general and breast carcinoma in particular still needs to be determined.
Progesterone receptor
The PR gene also is a member of the nuclear receptor superfamily. Two isoforms of the PR, PRA and PRB, are encoded by the same gene, utilizing two distinct transcriptional start sites and yielding proteins that differ with regard to their animo terminal regions and biologic activities.[39] Although both PRA and PRB are highly expressed in normal tissues, PRB protein con-centrations reportedly are elevated in breast carci-noma. This results in a decrease in the PRA:PRB ratio that is believed to be an important parameter for progesterone-mediated functions.[40-42]
Similar to ERα, PR status is a good predictor of tumor responsiveness to therapy. Nearly 50% of all ER+ tumors also are reported to be PR+ and approx-imately 75% of these ER/PR+ tumors respond posi-tively to endocrine therapy.[3] ER+, PR- tumors are reported to be less responsive to therapy, perhaps suggesting that PR may be necessary for positive thERαpeutic outcomes with hormone therapy. Alterna-tively, because ER is a key transcription factor for the activation of PR, lack of PR expression in these ER+ / PR- cells also could suggest that the estrogen response pathway may not be functional in these tumors.[41] To our knowledge only a small fraction of tumors are ER-/PR+ (< 5%) and they demonstrate an intermediate response to endocrine therapy.[3]
Similar to ER, PR expression also is regulated by epigenetic modulation.[25] Methylation and/or acetyla-tion of a CpG island within the PR promoter region also is important for PR expression. However, studies have shown that demethylation alone using 5-Aza-2'- deoxycytidine is not sufficient for reactivation of PR, suggesting that ER-mediated chromatin remodeling of the locus involves several mechanisms in conjunction with demethylation and is critical for the faithful ex-pression of PR.[43] Chromatin reorganization using both HDAC inhibitors and demethylating agents results in the reexpression of PR.[26] It still is unclear whether this is a direct effect on the PR promoter or an indirect effect mediated by enhanced expression of the ER.
Retinoic-acid receptor
All three RARs (RAR-α, RAR-β, and RAR-γ) are highly expressed in normal mammary epithelial tissues. Similar to other members of the nuclear receptor superfamily including ER and PR, RARs are ligand-activated receptors that regulate gene transcription through interactions with retinoic acid response elements (RAREs) found within gene promoter regions. When activated, RARs form homodimers or heterodimers with the retinoid X receptors (RXR-α, RXR-β, and RXR-γ). They function as tumor suppressor genes inhibiting proliferation and inducing cell differentiation and apoptosis.[44] These antiproliferative and apo- ptotic effects may be regulated by inhibition of the cell cycle, arresting cells in the G1-S-phase.[45] RAR-β expression is high in normal mammary epithelial cells, but is down-regulated at both the mRNA and protein levels in malignant tumors including tumors of the lung, head and neck, esophagus, ovary, prostate, and breast.[46-52]
Mechanisms underlying the loss of RAR-β expression are an active area of research. Loss of heterozygosity at 3p24, the chromosomal region encoding RAR-β, is detected in primary breast tumors, suggesting that one mechanism of loss may be mediated by allelic deletion.[53,54] Recent studies have shown that RAR-β also is under epigenetic regulation by methylation and histone deacetylation of the promoter region, resulting in gene silencing.[44,54-58] In a study of both primary breast carcinomas and breast carcinoma cell lines, TSA induced the reexpression of RAR-β2, even in the absence of retinoic acid, a known inducer of RAR-β2 expression. This suggests that chromatin remodeling is a critical mechanism for the regulation of RAR-β2 expression that can be exploited with novel therapeutic approaches.[59]
Peptide Growth Factors and Their Receptors
A number of peptide growth factors and their recep-tors have been implicated in normal mammary devel-opment and carcinogenesis. These include members of the HER/erbB, tumor growth factor-β (TGF-β), and insulin-like growth factor families. Here we focus on the HER/erbB family to illustrate how these families might contribute to the development of breast carci-noma and be exploited clinically.
Epidermal growth factor receptor family
The HER or erbB proteins are members of the subclass I of the receptor tyrosine kinase (RTK) superfamily. This subgroup of RTKs contains four members: epi-dermal growth factor receptor (EGFR/erbB1/HER-1), erbB2/neu/HER-2, erbB3/HERβ, and erbB4/HER-4. These transmembrane proteins share a similar struc-ture but only 25-30% overall homology. There are at least 25 known ligands that can bind HER family members including epidermal growth factor (EGF), TGF-α, amphiregulin, heparin-binding EGF (HbEGF), β-cellulin, epiregulin, cripto-1, neuregulin, and heregulin.[60] Upon ligand binding and activation, HER proteins form homodimers or heterodimers comprised of different combinations of family members.[60] It is interesting to note that, to our knowledge, a specific ligand for HER-2 has not been identified; rather, HER-2 frequently is the preferred partner for other ligand-bound HER molecules.
In vitro and in vivo, both HER-1 and HER-2 have been shown to play a clear role in neoplastic transfor- mation.[61-64] The HER-2/neu/erbB2 protein is overex-pressed in approximately 25% of invasive breast tu-mors, usually because of gene amplification.[65] In some studies, HER-2 overexpression has been re¬ported to be correlated with poor prognosis, but not with tumor size, degree of differentiation, or meta¬static potential, suggesting that HER family members may play a role in overall outcome, but not in the pathway leading to the transformed state.[66-68] It is unclear what role HER-3 or HER-4 play in normal mammary cells, but HER-3 protein often is overex¬pressed in breast tumors in conjunction with HER-B2. This finding suggests the possibility that HER-2-HER-3 dimers may play a role in these tumors.[69]
Because overexpression of HER-1 and HER-2 characterizes a significant fraction of breast carcinoma cases, there has been great interest in developing therapies targeting the HER family members. ZD1839 is a member of the anilinoquinazoline class of RTK1 that initially was developed as a HER-1 inhibitor; however, in vitro, ZD1839 also is reported to be a very effective HER-2 inhibitor as well.[70] Mechanistically, ZD1839 decreases HER-1 and HER-2 expression by interfering with phosphorylation of PI3K, activation of AKT, and phosphorylation of the MAPK cascade.[71-73] ZD1839 also inhibits cell cycle progression by down-regulating key proteins in cell cycle progression in-cluding cyclin D1, Cdk4, p27Kip1, and Cdk2. This re-sults in inhibition of proliferation and induction of apoptosis in HER-2-positive cell lines.[56,72-75] It is in-teresting to note that the p85 subunit of PI3K associ-ates with HERβ and not with HER-2, suggesting that the functional dimer includes HERβ as well in tumors that overexpress HER-2.[71] Clinical trials of ZD1839 as a single agent currently are underway for women with advanced breast carcinoma.
Trastuzumab is a monoclonal antibody that was raised against the ectodomain of HER-2 that blocks cell proliferation, inhibits cell growth, and induces apoptosis in breast carcinoma cells.[76,77] Therefore, the presence of overexpressed HER-2 serves as a good predictive factor of clinical response to trastuzumab. Trastuzumab inhibits both PI3K activation of the AKT pathway and activation of the MAPK pathway. Cells treated with trastuzumab accumulate in the G1 phase of the cell cycle, suggesting that trastuzumab inhibits cell proliferation via a G1-S-phase block.[78-80] The activity of trastuzumab alone and with cytotoxics has been established in women with advanced breast tu-mors overexpressing HER-2.[75,80] Preclinical studies suggest that ZD1839 and trastuzumab may work syn- ergistically to inhibit tumor progression via inhibition of both the AKT and MAPK pathways. A combination of ZD1839 and trastuzumab enhanced apoptosis and tumor regression in tumor cells overexpressing HER-2 compared with tumor cells that were negative for HER-2.[72,75,81] A Phase I clinical trial of the combination currently is in progress.
The nature of the cross-talk between the ER and HER pathways also is of great interest. Studies of ER, EGF receptor (EGFR), and HER-2 in breast carcinoma suggest that ER expression is inversely correlated with EGFR or HER-2 expression. The possibility that HER-2 overexpression is associated with tamoxifen resistance has been suggested by some but not all studies. For example, serial samples of primary breast tumors during a course of neoadjuvant endocrine therapy suggest that HER-2-overexpressing tumors do not demon- strate the same decrease in Ki-67 as observed in HER- 2-negative tumors.[82] If confirmed, these findings could have implications for the ability of HER-2 to serve as a predictive marker for endocrine therapy and could support clinical trials of combinations of anti- HER and endocrine therapy.
Tumor Suppressor Genes and Breast Carcinoma Susceptibility Genes p53
Somatic cell mutation in the p53 nuclear phosphopro- tein is observed in approximately 20-30% of primary breast carcinoma cases.[83,84] Although these mutations are found scattered throughout the entire gene, the majority of mutations are confined to a 200-amino acid span containing 1 of 4 conserved core domains and result in decreased DNA binding affinity and de-creased gene transactivation.[83,85-88] In the majority of p53-negative tumors, a missense mutation of one al-lele is associated with deletion of the second allele. Tumors with p53 mutations are more likely to be highly invasive, poorly differentiated, high-grade breast tumors. It is hypothesized that p53 mutations may precede the development of tumors with fully malignant and invasive phenotypes.[89] Therefore, mu-tant p53 has been suggested to be a biomarker pre-dicting risk for subsequent breast carcinogenesis.[89 93] The ER has been shown to physically associate with the amino terminus of p53 to form complexes con-taining p53 and MDM2.[93-96] It is interesting to note that ERα protects p53 from MDM2-mediated degradation, suggesting that ERα signaling results in the up-regulation of p53 mRNA and protein and stabilizes expression to mediate G1 cell cycle arrest.[93,95,97-99] However, overexpression of ERα has been reported to mediate the overexpression of MDM2 and decrease p53 transcriptional activity.[96,100] This may be a potential mechanism leading to neoplastic transformation of the cell and suppression of p53 with increased cellular proliferation through lack of control at critical cell cycle checkpoints.[96,100]
Germline p53 mutation also serves as a risk factor for breast carcinoma development as part of the Li- Fraumeni syndrome. Although quite rare, Li-Fraum- eni is a dominant inherited cancer syndrome that manifests itself with a high rate of early-onset breast carcinoma as well as multiple other tumor types.[101] p53 mutations have been identified in nearly 60% of families with this disease, suggesting that loss of p53 may be a critical parameter in the development of multiple carcinomas. Fibroblasts isolated from pa-tients with Li-Fraumeni syndrome have not been re-ported to exhibit permanent G1 or G2 cell cycle arrest, suggesting that a loss of p53 results in the loss of cell cycle checkpoint control, which may be responsible for the increased cellular proliferation.[102,103]
BRCA1/BRCA2
Hereditary breast carcinoma is reported to account for a small proportion of all breast carcinoma cases. Germline mutations in two breast carcinoma suscep-tibility genes, BRCA1 and BRCA2, have been impli-cated in a fraction of these via an autosomal dominant inheritance mechanism. It is interesting to note that although these genes are important in hereditary breast carcinoma, they have not been found to be associated with the development or progression of sporadic breast carcinoma. Tumorigenesis in women with BRCA1 or BRCA2 mutations requires the loss or inactivation of the remaining wild-type allele, result-ing in expression of a nonfunctional protein and a loss of cell cycle control and DNA repair mechanisms.[104] BRCA1 and BRCA2 apparently function to regulate DNA repair and gene transcription and maintain ge-nome integrity. Women with a mutation in 1 of these genes are reported to have an approximately 60- 80% risk of developing breast carcinoma in their lifetime.[105] Although hundreds of mutations are found scattered throughout these genes, some mutations are more prevalent and have a higher penetrance than others. Some of these “hotspot” mutations are more highly expressed in particular ethnic groups. For example, three mutations (BRCA1 185delAG, BRCA1 5382insC, and BRCA2 6174delT) are found to have a high penetrance in the Ashkenazi Jewish population.[106,107] Screening for and detection of BRCA1/ BRCA2 mutations may be helpful in determining the overall risk for the development of breast carcinoma, especially in families with hereditary cases. Individu-als who are mutation carriers may wish to undertake different surveillance strategies, chemoprevention in-terventions, or surgical prophylaxis for carcinomas of the breast and ovary.
Conclusions
The molecular mechanisms and changes therein lead-ing to the development and progression of breast car-cinoma are extremely complex. The biology of breast carcinoma can be exploited to determine risk, overall prognosis, and response to specific therapy. BRCA1, BRCA2, and p53 are genes that are reported to be involved in hereditary breast carcinoma. Individuals with mutations in these genes, usually leading to a truncated and nonfunctional protein, are reported to be at a higher risk of developing breast carcinoma in their lifetime. Some of these mutations are correlated with early onset of disease, whereas others are asso-ciated with increased overall lifetime risk. Therefore, testing for mutations in these genes can contribute critical information regarding the risk of developing breast carcinoma. The evaluation of certain molecular markers such as ER and PR expression in individual tumors also may contribute to the determination of prognosis in patients with breast carcinoma. Several genes also are reported to be predictors of clinical outcome with current therapy. For example, the pres-ence of ER and/or PR is reported to predict response to endocrine therapies such as SERMs and selective estrogen down-regulators (SERDs), whereas HER-2 overexpression predicts the response to trastuzumab. Finally, knowledge of the biology of these and other genes and their molecular changes can lead to the development of novel agents for the treatment and prevention of breast carcinoma.
References
Jemal A, Thomas A, Murray T, Thurn M. Cancer statistics. CA Cancer J Clin. 2002;52:23-47.
Clarke R, Dickson RB, Lippman ME. Hormonal aspects of breast cancer. Crit Rev Oncol Hematol. 1992;12:1.
Elledge RM, Fuqua SA. Estrogen and progesterone receptors. In: Harris JR, editor. Diseases of the breast, volume 2. Philadelphia: Lippincott Williams & Wilkins, 2000:471-488.
Clark G. Prognostic and predictive factors. In: Harris JR, editor. Diseases of the breast, volume 2. Philadelphia: Lippincott Williams & Wilkins, 2000:489-514.
Trichopoulos D, MacMahon B, Cole P. Menopause and breast cancer risk. J Natl Cancer Inst. 1972;48:605-613.
Hulka BS, Moorman P. Breast cancer: hormones and other risk factors. Maturitas. 2001;38:103--116.
Maehle B, Tretli S. Pre-morbid body-mass-index in breast cancer: reversed effect on survival in hormonal receptor negative patients. Breast Cancer Res Treat. 1996;41:123- 130.
Nicholson R, McClelland R, Robertson J, Gee J. Involvement of steroid hormone and growth factor cross-talk in endocrine response in breast cancer. Endocr Relat Cancer. 1999;6:373-387.
Maruyama S, Fujimoto N, Asano K, Ito A. Suppression by estrogen receptor f of AP-1 mediated transactivation through estrogen receptor a. J Steroid Biochem Mol Biol. 2001;78:177-184.
Paech K, Webb P, Kuiper GGJM, et al. Differential ligand activation of estrogen receptors ER a and ER f at AP1 sites. Science. 1997;277:1508-1510.
BarhemT, Carlsson B, Nilsson Y, EnmarkE, Gustafsson J-A, Nilsson B. Differential response of estrogen receptor a and estrogen receptor f to partial estrogen agonists/antago- nists. Mol Pharmacol. 1998;54:105-112.
Osborne CK, Boldt D, Clark G. Effects of tamoxifen on human breast cancer cell cycle kinetics. Cancer Res. 1983; 43:3583.
Sweeney K, Musgrove E, Watts C. Cyclins and breast cancer. In: Dickson RB, Lippman ME, editors. Mammary tumor cell cycle, differentiation and metastasis. Boston: Kluwer Academic Publishers, 1996:141.
Pang H, Faber L. Estrogen and rapamycin effects on cell cycle progression in T47D breast cancer cells. Breast Cancer Res Treat. 2001;70:21-26.
Foster J, Wimalasena J. Estrogen regulates activity of cy- clin-dependent kinases and retinoblastoma protein phos-phorylation in breast cancer cells. Mol Endocrinol. 1996;10: 488-498.
Prall O, Sarcevic B, Musgrove E, Watts C, Sutherland R. Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased Cyclin D1 expression and decreased Cyclin-dependent kinase Inhibitor association with Cyclin E-Cdk2. J Biol Chem. 1997;272: 10882-10894.
Zafonte B, Hulit J, Amanatullah D, et al. Cell-cycle dysregu-lation in breast cancer: breast cancer therapies targeting the cell cycle. Front Biosci. 2000;5:D938-D961.
Chlebowski RT, Collyar DE, Somerfield MR, Pfister DG. American Society of Clinical Oncology Technology assessment on breast cancer risk reduction strategies: tamoxifen and raloxifene. J Clin Oncol. 1999;17:1939-1955.
Cheung W, Briggs S, Allis C. Acetylation and chromosomal functions. Curr Opin Cell Biol. 2000;12:326-333.
Bird A. Methylation talk between histones and DNA. Science. 2001;294:2113-2115.
Ferguson AT, Lapidus R, Baylin S, Davidson NE. Demeth- ylation of the estrogen receptor gene in estrogen receptornegative breast cancer cells can reactivate estrogen receptor gene expression. Cancer Res. 1995;55:2279-2283.
Ottaviano Y, Issa J, Parl F, Smith H, Baylin S, Davidson N. Methylation of the estrogen receptor gene CpG island marks loss of estrogen receptor expression in human breast cancer cells. Cancer Res. 1994;54:2552-2555.
Bird A, Wolffe A. Methylation-induced repression-belts, braces, and chromatin. Cell. 1999;99:451-454.
Ferguson AT, Vertino P, Spitzner J, Baylin S, Muller M, Davidson NE. Role of estrogen receptor gene demethyl- ation and DNA methyltransferase-DNA adduct formation in 5-Aza-2'-deoxycytidine-induced cytotoxicity in human breast cancer cells. J Biol Chem. 1997;272:32260-32266.
Lapidus R, Ferguson AT, Ottaviano Y, et al. Methylation of estrogen and progesterone receptor 5' CpG islands correlates with lack of estrogen and progesterone receptor gene expression in breast tumors. Clin Cancer Res. 1996;2:805- 810.
Yang X, Phillips DL, Ferguson AT, Nelson WG, Herman JG, Davidson NE. Synergistic activation of functional estrogen receptor (ER)- a by DNA methyltransferase and histone deacetylase inhibition in human ERα-negative breast cancer cells. Cancer Res. 2001;61:7025-7029.
Kuiper GGJM, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson J-A. Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA. 1996;93:5925-5930.
Mosselman S, Polman J, Dijkema R. ER f: Identification and characterization of a novel human estrogen receptor. FEBS Lett. 1996;392:49-53.
Kuiper GGJM, Carlsson B, Grandien K, et al. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors a and f. Endocrinology. 1997; 138:863-870.
Fuqua SA, Schiff R, Parra I, et al. Expression of wild-type estrogen receptor f and variant forms in human breast cancer. Cancer Res. 1999;59:5425-5428.
Speirs V, Parkes A, Kerin M, et al. Coexpression of estrogen receptor a and f: poor prognostic factors in human breast cancer? Cancer Res. 1999;59:525-528.
Jensen E, Cheng G, Palmieri C, et al. Estrogen receptors and proliferation markers in primary and recurrent breast cancer. Proc Natl Acad Sci USA. 2001;98:15197-15202.
Dotzlaw H, Leygue E, Watson PH, Murphy LC. Estrogen receptor p messenger RNA expression in human breast tumor biopsies: relationship to steroid receptor status and regulation by progestins. Cancer Res. 1999;59:529-532.
Iwao K, Miyoshi Y, Egawa C, Ikeda K, Noguchi S. Quantitative analysis of estrogen receptor p mRNA and its variants in human breast cancers. Int J Cancer. 2000;88:733- 736.
Rutherford T, Brown W, Sapi E, Aschkenazi S, Munoz A, Mor G. Absence of estrogen receptor p expression in metastatic ovarian cancer. Obstet Gynecol. 2000;96:417-420.
Leav I, Lau K-M, Adams J, et al. Comparative studies of the estrogen receptors p and a and the androgen receptor in normal human prostate glands, dysplasia, and in primary and metastatic carcinoma. Am J Pathol. 2001;159:79-92.
Roger P, Sahla M, Makela S, Gustafsson J-A, Baldet P, Rochefort H. Decreased expression of estrogen receptor p protein in proliferative preinvasive mammary tumors. Cancer Res. 2001;61:2537-2541.
Jarvinen TAH, Pelto-Huikko M, Holli K, Isola J. Estrogen Receptor p is coexpressed with ER a and PR and associated with nodal status, grade, and proliferation rate in breast cancer. Am J Pathol. 2000;156:29-35.
Kastner P, Krust A, Turcotte B, et al. Two distinct estrogen- regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B. EMBO J. 1990;9:1603-1614.
Ariga N, Suzuki T, Moriya T, et al. Progesterone receptor A and B isoforms in the human breast and its disorders. Jpn J Cancer Res. 2001;92:302-308.
Graham JD, Roman SD, McGowan E, Sutherland RL, Clarke CL. Preferential stimulation of human progesterone receptor B expression by estrogen in T-47D human breast cancer cells. J Biol Chem. 1995;270:30693-30700.
Bamberger A-M, Milde-Langosch K, Schulte H, Loning T. Progesterone receptor isoforms, PR-B and PR-A, in breast cancer: correlations with clinicopathologic tumor parameters and expression of AP-1 factors. Horm Res. 2000;54:32-37.
Ferguson AT, Lapidus R, Davidson NE. Demethylation of the progesterone receptor CpG island is not required for progesterone receptor gene expression. Oncogene. 1998;17: 577-583.
Widschwendter M, Berger J, Muller H, Zeimet A, Marth C. Epigenetic downregulation of the retinoid acid receptor p2 gene in breast cancer. J Mammary Gland Biol Neoplasia. 2001;6:193-201.
Seewaldt V, Dietze E, Johnson B, Collins S, Parker M. Retinoic acid mediated G1-S phase arrest of normal human mammary epithelial cells is independent of the level of p53 protein expression. Cell Growth Differ. 1999;10:49-59.
Zhang X, Liu Y, Lee M, Pfahl M. A specific defect in the retinoic acid receptor associated with human lung cancer cell lines. Cancer Res. 1994;54:5663-5669.
Houle B, Rochette-Egly C, Bradley WE. Tumor suppressor effect of the retinoic acid receptor p in human epidermoid lung cancer cells. Proc Natl Acad Sci USA. 1993;90:985- 989.
Xu X, Ro J, Lee L, Shin D, Hong W, Lotan R. Differential expression of nuclear retinoid receptors in normal, prema-lignant, and malignant head and neck cancers. Cancer Res. 1994;54:3580-3587.
Qiu H, Zhang W, El-Naggar A, et al. Loss of retinoic acid receptor p expression is an early event during esophageal carcinogenesis. Am J Pathol. 1999;155:1519-1523.
Sabichi A, Hendricks D, Bober M, Birrer M. Retinoic acid receptor p expression and growth inhibition ofgynecologic cancer cells by the synthetic retinoid N-(4-Hydroxyphenyl) retinamide. J Natl Cancer Inst. 1998;90:597- 605.
Lotan Y, Xu X, Shalev M, et al. Differential expression of nuclear retinoid receptors in normal and malignant prostates. J Clin Oncol. 2000;18:116-121.
Swisshelm K, Ryan K, Lee X, Tsou H, Peacocke M, Sager R. Downregulation of retinoic acid receptor b in mammary carcinoma cell lines and its upregulation in senescing normal mammary epithelial cells. Cell Growth Differ. 1994;5: 133-141.
Deng G, Lu Y, Zlotnikov G, Thor AD, Smith HS. Loss of heterozygosity in normal tissue adjacent to breast carcinomas. Science. 1996;274:2057-2059.
Yang Q, Mori I, Shan L, et al. Biallelic inactivation of retinoic acid receptor p2 gene by epigenetic change in breast cancer. Am J Pathol. 2001;158:299-303.
Liu Y, Lee M, Wang H, et al. Retinoic acid receptor beta mediates the growth-inhibitory effect of retinoic acid by promoting apoptosis in human breast cancer cells. Mol Cell Biol. 1996;16:1138-1149.
Arapshian A, Kuppumbatti Y, Mira-y-lopez R. Methylation of conserved CpG sites neighboring the beta retinoic acid response element may mediate retinoic acid receptor beta gene silencing in MCF-7 breast cancer cells. Oncogene. 2000;19:4066-4070.
BovenziV, MomparlerR. Antineoplastic action of5-aza-2'- deoxycytidine and histone deacetylase inhibitor and their effect on the expression of retinoic acid receptor p and estrogen receptor a genes in breast carcinoma cells. Cancer Chemother Pharmacol. 2001;48:71-76.
Sirchia S, Ferguson AT, Sironi E, et al. Evidence of epigenetic changes affecting the chromatin state of the retinoic acid receptor b2 promoter in breast cancer cells. Oncogene. 2000;19:1556-1563.
Sirchia SM, Ren M, Pili R, et al. Endogenous reactivation of the RARp2 tumor suppressor gene epigenetically silenced in breast cancer. Cancer Res. 2002;62:2455-2461.
Olayioye M, Neve R, Lane H, Hynes N. The ErbB signaling network: receptor heterodimerization in development and cancer. EMBO J. 2000;19:3158-3167.
Kokai Y, Myers J, Wada T, et al. Synergistic interaction of p186c-neu and the EGF receptor leads to transformation of rodent fibroblasts. Cell. 1989;58:287-292.
Guy C, Webster M, Schaller M, Parsons T, Cardiff R, Muller W. Expression of the neu protooncogene in the mammary epithelium of transgenic mice induces metastatic disease. Proc Natl Acad Sci USA. 1992;89:10578-10582.
Muller H, Sinn E, Pattengale P, Wallace R, Leder P. Singlestep induction of mammary adenocarcinoma in transgenic mice bearing the activated c-neu oncogene. Cell. 1988;54: 105-115.
Matsui Y, Halter S, Holt J, Hogan B, Coffey R. Development of mammary hyperplasia and neoplasia in MMTV-TGF a transgenic mice. Cell. 1990;61:1147-1155.
Slamon D, Clark G, Wong S, Levin W, Ullrich A, McGuire W. Human breast cancer: correlation of relapse and survival with amplification of the HER2/neu oncogene. Science. 1987;235:177-182.
Slamon D, Godolphin W, Jones L, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science. 1989;244:707-712.
Thor AD, Liu S, Edgerton S, et al. Activation (Tyrosine Phosphorylation) of ErbB-2 (HER-2/neu): a study of incidence and correlation with outcome in breast cancer. J Clin Oncol. 2000;18:3230-3239.
Emi Y, Kitamura K, Shikada Y, Kakeji Y, Takahashi I, Tsutsui S. Metastatic breast cancer with HER2/neu-positive cells tend to have a morbid prognosis. Surgery. 2002;131:S217- S221.
Siegel PM, Ryan ED, Cardiff RD, Muller WJ. Elevated ex-pression of activated forms of Neu/ErbB-2 and ErbB-3 are involved in the induction of mammary tumors in transgenic mice: implications for human breast cancer. EMBO J. 1999;18:2149-2164.
Chan K, Knox W, Gee J, et al. Effect of epidermal growth factor receptor tyrosine kinase inhibition on epithelial proliferation in normal and premalignant breast. Cancer Res. 2002;62:122-128.
Moasser M, Basso A, Averbauch S, Rosen N. The tyrosine inhibitor ZD1839 (“Iressa”) inhibits HER2-driven signaling and suppresses the growth of HER2-overexpressing tumor cells. Cancer Res. 2001;61:7184-7188.
Moulder S, Yakes F, Muthuswamy S, Bianco R, Simpson J, Arteaga C. Epidermal growth factor receptor (HER1) tyrosine kinase inhibitor ZD1839 (Iressa) inhibits HER2/neu (erbB2)-overexpressing breast cancer cells in vitro and in vivo. Cancer Res. 2001;61:8887- 8895.
Lane H, Beuvink I, Motoyama A, Daly J, Neve R, Hynes N. ErbB2 potentiates breast tumor proliferation through mod-ulation of p27Kip1-Cdk2 complex formation: receptor overexpression does not determine growth dependency. Mol Cell Endocrinol. 2000;20:3210-3223.
Arao Y, Yamamoto E, Miyatake N, et al. A synthetic oestrogen antagonist, tamoxifen, inhibits oestrogen-induced transcriptional, but not post-transcriptional, regulation of gene expression. Biochem J. 1996;313(Pt 1):269-274.
Vogel CL, Cobleigh MA, Tripathy D, et al. Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol. 2002;20:719-726.
Kita Y, Tseng J, Horan T, et al. ErbB receptor activation, cell morphology changes, and apoptosis induced by anti-HER2 monoclonal antibodies. Biochem Biophys Res Commun. 1996;226:59-69.
Kunisue H, Kurebayashi J, Otsuki T, et al. Anti-HER2 antibody enhances the growth inhibitory effect of anti-oestrogen on breast cancer cells expressing both oestrogen receptors and HER2. Br J Cancer. 2000;82:46-51.
Mayfield S, Vaughn J, Kute T. DNA strand breaks and cell cycle perturbation in Herceptin treated breast cancer cell lines. Breast Cancer Res Treat. 2001;70:123-129.
Cobleigh M, Vogel C, Tripathy D, et al. Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. J Clin Oncol. 1999;17: 2639-2648.
Slamon DJ, Leyland-Jones B, Shak S, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med. 2001;344:783-792.
Normanno N, Campiglio M, De L, et al. Cooperative inhibitory effect of ZD1839 (Iressa) in combination with trastuzumab (Herceptin) on human breast cancer cell growth. Ann Oncol. 2002;13:65-72.
Dowsett M, Harper-Wynne C, Boeddinghaus I, et al. HER-2 amplification impedes the antiproliferative effects of hormone therapy in estrogen receptor-positive primary breast cancer. Cancer Res. 2001;61:8452-8458.
Kern SE, Kinzler K, Bruskin A, et al. Identification of p53 as a sequence-specific DNA-binding protein. Science. 1991; 252:1708-1711.
Sullivan A, Yuille M, Repellin C, et al. Concomitant inactivation of p53 and Chk2 in breast cancer. Oncogene. 2002; 21:1316-1324.
Cho Y, Gorina S, Jeffrey P, Pavletich N. Crystal structure of a p53 tumor suppressor-DNA complex: understanding tu- morigenic mutations. Science. 1994;265:346-355.
Baker S, Markowitz S, Fearon E, Willson J, Vogelstein B. Suppression of human colorectal carcinoma cell growth by wild-type p53. Science. 1990;249:912-915.
Kern SE, Peitenpol J, Thiagalingam S, Seymour A, Kinzler K, Vogelstein B. Oncogenic forms ofp53 inhibit p53-regulated gene expression. Science. 1992;256:827-830.
Keshava C, Frye B, Wolff M, McCanlies E, Weston A. Waf-1 (p21) and p53 polymorphisms in breast cancer. Cancer Epidemiol Biomarkers Prev. 2002;11:127-130.
Bosari S, Roncalli M, Viale G, Bossi P, Coggi G. p53 immu- noreactivity in inflammatory and neoplastic diseases of the uterine cervix. J Pathol. 1993;169:425-430.
Fabian C, Kamel S, Zalles C, Kimler B. Identification of a chemoprevention cohort from a population of women at high risk for breast cancer. J Cell Biochem. 1996;25(Suppl): 112-122.
Rohan T, Hartwick W, Miller A, Kandel R. Immunohisto-chemical detection of c-erbB-2 and p53 in benign breast disease and breast cancer risk. J Natl Cancer Inst. 1998;90: 1262-1269.
Levesque M, Yu H, Clark G, Diamandis E. Enzyme-linked immunoabsorbant assay-detected p53 protein accumulation: a prognostic factor in a large breast cancer cohort. J Clin Oncol. 1998;16:2641-2650.
Fabian C, Kimler B, Zalles C, et al. Short-term breast cancer prediction by random periareolar fine-needle aspiration cytology and the Gail risk model. J Natl Cancer Inst. 2000; 92:1217-1227.
Yu C-L, Driggers P, Barrera-Hernandez G, Nunez S, Segars J, Cheng S-y. The tumor suppressor p53 is a negative regulator of estrogen receptor signaling pathways. Biochem Biophys Res Commun. 1997;239:617-620.
Liu G, Schwartz J, Brooks S. Estrogen receptor protects p53 from deactivation by human double minute-2. Cancer Res. 2000;60:1810-1814.
Kato K, Horiuchi S, Takahashi A, et al. Contribution of estrogen receptor a to oncogenic K-Ras-mediated NIH3T3 cell transformation and its implication for escape from senescence by modulating the p53 pathway. J Biol Chem. 2002;277:11217-11224.
Hurd C, Khattree N, Dinda S, Alban P, Moudgil V. Regulation of tumor suppressor protein, p53 and retinoblastoma, by estrogen and antiestrogens in breast cancer cells. Oncogene. 1997;15:991-995.
Hurd C, Khattree N, Alban P, et al. Hormonal regulation of the p53 tumor suppressor protein in T47D human breast carcinoma cell line. J Biol Chem. 1995;270:28507- 28510.
Zheng W, Lu JS, Zheng J-M, Hu F-x, Ni C-r. Variation of ER status between primary and metastatic breast cancer and relationship to p53 expression. Steroids. 2001;66: 905-910.
Hori M, Shimazaki J, Inagawa S, Itabashi M, Hori M. Over-expression of MDM2 oncoprotein correlates with possession of estrogen receptor alpha and lack of MDM2 mRNA splice variants in human breast cancer. Breast Cancer Res Treat. 2002;71:77-84.
Varley J, Evans D, Birch J. Li-Fraumeni syndrome — a molecular and clinical review. Br J Cancer. 1997;76:1-14.
Boyle J, Spreadborough A, Greaves M, Birch J, Scott D. Chromosome instability in fibroblasts derived from Li- Fraumeni syndrome families without TP53 mutations. Br J Cancer. 2000;83:1136-1138.
Boyle J, Spreadborough A, Greaves M, Birch J, Varley J, Scott D. The relationship between radiation-induced G(1) arrest and chromosome aberrations in Li-Fraumeni fibroblasts with or without germline TP53 mutations. Br J Cancer. 2001;85:293-296.
Welcsh P, King MC. BRCA1 and BRCA2 and the genetics of breast and ovarian cancer. Hum Mol Genet. 2001;10:705-713.
Lee W-H, Boyer T. BRCA1 and BRCA2 in breast cancer. Lancet. 2001;358:s5.
Struewing JP, Abeliovich D, Peretz T, et al. The carrier frequency of the BRCA1 185delAG mutation is approximately 1 percent in Ashkenazi Jewish individuals. Nat Genet. 1995;11:198-200.
Ellisen L, Haber DA. Hereditary breast cancer. Annu Rev Med. 1998;49:425-436.