Quick Links to sections within the chapter below

Genetics

Steroid and Growth Factor Pathways of Cellular Regulation

Cell Cycle and Cell Death

Process of Malignant Progression

Implications of Molecular Biology for Tumor Prevention, Early Detection, Prognosis, and Response to Therapy

References

GeneticsNew Methodologies and Genetic Mechanisms The purpose of this chapter is to provide a current perspective on the rapidly evolving, increasingly integrated study of the genetic, molecular, biochemical, and cellular bases of breast cancer. Although the roles of steroid hormones have occupied breast cancer researchers since the 1950s, the roles of growth factors did not begin to emerge until the 1980s. The 1980s also saw the discovery of many of the oncogenes and suppressor genes, driving progression of the disease, and highlighted their connections to cellular adhesion, growth factor, and steroid regulatory pathways. The 1990s witnessed the discovery of genes that cause familial forms of breast cancer. The final decade of the twentieth century also ushered in major advances in our understanding of the cell cycle, DNA repair, and cell death (apoptosis) and their regulation. As the twenty-first century begins, the field is intensely involved in elucidation of the molecular bases of processes involved in treatment failure: primary mechanisms of resistance to chemotherapy, antiestrogens, and radiation and fundamental processes of invasion, angiogenesis, and metastasis. New technologies are available for human mammary cell culture, for regulatable animal models of cancer, and for high-throughput analyses of pathologic material (tissue microarrays, laser capture microdissection, and automated immunohistochemistry readers). Other advances have been made in cytogenetics [fluorescence in situ hybridization (FISH) and bacterial artificial chromosome array/comparative genomic hybridization (BAC array CGH)], in gene expression analysis [complementary DNA (cDNA) chip array and real-time polymerase chain reaction (PCR) assay methods], and in proteomics analysis (a plethora of techniques). These new methods are now being applied to improve tumor diagnosis and to predict response to existing therapies. Such approaches should also result in our discovery of new targets for more biologic-based therapies and prevention strategies for the disease. Onset and progression of breast cancer appear to be intimately, but not exclusively, tied to genetic alterations (Fig. 33.1-1). Although the possibility of direct, DNA-modifying agents from environmental exposure has been studied for many years for links to breast cancer, this etiology of the disease appears to be quite rare. One clear example, however, is the instance of radiation exposure of Japanese women in World War II. More likely to play major current etiologic roles are indirect environmental effects that enhance breast epithelial cell exposure to reactive oxygen species or to estrogenic hormones. The reader is referred elsewhere for an introduction to this large, complex area of study,1 as this chapter focuses primarily on hormones and growth factors as they affect cellular and tissue interaction aspects of the disease. View Figure

Figure 33.1-1Summary of the genetic and phenotypic alterations associated with the onset and progression of familial and sporadic breast cancers.

Cell biologic studies have begun to establish that defects in cell-cycle checkpoint controls are fundamental to the accumulation of genetic damage in the mammary epithelial cell, leading to cancer.2 The four cell-cycle transitions, from G1S, G2M, spindle formation and function (cytokinesis), and daughter cell separation post M (karyokinesis), appear to be important points of vulnerability for genetic damage. In normal cells, DNA damage and replication defects may be recognized at these and other points, resulting in cell-cycle arrest, DNA repair, and/or programmed cell death (apoptosis). Such DNA damage may be produced by environmental exposures (radiation or chemotherapy drugs), by replicative senescence (erosion of telomeres capping and protecting chromosomal ends), and by premalignancy- or malignancy-associated cellular changes. For example, direct DNA damage, aberrations in DNA methylation, mutation, or loss of expression of BRCA1/2 or p53 genes, deregulated expression of c-Myc or cyclin D1 genes, and defects in DNA mismatch repair genes may all cause defects in DNA damage-dependent cell-cycle checkpoint controls, contributing to onset or progression, or both, of breast cancer. These concepts are further described in the next two sections. Familial Disease As noted in the previous section, significant progress has been made (Table 33.1-1) in the identification of inherited defects in somatic genes responsible for hereditary and familial breast cancers. Although several defined hereditary breast cancer syndromes are characterized by a very high penetrance of additional types of cancer, other breast cancer families may also display a lower penetrance of the disease and not be so clearly associated with elevated risk for multiple other types of cancer. In aggregate, it is estimated that 5% to 10% of breast cancer cases occur in families with significant inherited risk. A general hypothesis in the field of hereditary cancer genetics is the "two-hit" hypothesis, that a point mutation might be inherited in one allele of a candidate gene at a putative susceptibility locus and that loss of heterozygosity (LOH) or another genetic alteration might occur in the other allele of that locus later in life, leading to genomic instability and cancer.3 This model has guided the identification of familial breast cancer genes (see Table 33.1-1). The first triumph in identification of a gene, leading to a multicancer syndrome that includes breast cancer, was for the TP53 gene (encoding p53, on chromosome 17p13).3 Inherited mutations in TP53 are responsible for the Li-Fraumeni syndrome of hereditary breast cancers, sarcomas, and other tumor types. At the time of its implication in Li-Fraumeni, mutations in this gene had already been described in the context of progression of sporadic cancers of the breast and other organs. More recently, mutations in the PTEN phosphatase (or MMAC1) gene (on chromosome 10q22-23) were described in the Cowden syndrome of hereditary breast cancers and multicutaneous lesions. The PTEN protein is discussed in Cell Cycle and Cell Death, later in this chapter, in the context of tumor suppressor genes and the cell cycle. In contrast to TP53, PTEN is not commonly mutated in sporadic breast cancers. Other studies on rarer familial diseases have implicated mutations of the STKII/LKBI gene (on chromosome 19 and involved in Wnt pathway signaling) in the Peutz-Jeghers syndrome of hamartomatous polyps, breast cancers, gastrointestinal cancers, and reproductive cancers and the MLH1 or MLH2, or both, mismatch repair gene(s) in the Muir-Torre syndrome of gastrointestinal and genitourinary tumors and breast cancer. Finally, although initially proposed, mutations in the ataxia-telangiectasia gene ATM (ataxia-telangiectasia mutated) do not appear to contribute strongly to the risk of developing breast cancer.3 Two important genes that confer risk for the far more pervasive, familial forms of breast cancer have been identified: BRCA1 and BRCA2.3 Carriers of these mutant genes may display a range of cancer risk, and the mutant genes are quite prevalent in certain populations. King and coworkers first localized BRCA1 (breast cancer and ovarian cancer-1) to chromosome 17q21. The gene was subsequently cloned and found to be novel, containing an amino-terminal, zinc- and DNA-binding "ring finger" motif; a carboxyl-terminal BCRT "domain;" and a nuclear localization sequence. Interestingly, mutations in this gene are particularly prevalent in breast cancers of Ashkenazi Jewish women and other select populations. However, some families carrying BRCA mutations are nearly devoid of multiple afflicted members. In other studies comparing different carrier populations, BRCA1 mutations have also been reported (but rarely) in women with no familial association of the disease. These studies emphasize the variable penetrance of inherited risk conferred by this gene. Although mutations in the BRCA1 gene are widely prevalent in patients with familial breast and ovarian cancer, mutations are rarely detected in sporadic breast cancers. However, the BRCA1 protein is commonly down-regulated in nonfamilial (sporadic) breast cancers due to methylation of the BRCA1 gene and other mechanisms. Consistent with these findings are observations that antisense oligonucleotides directed against the BRCA1 messenger RNA (mRNA) enhance the proliferation of breast tumor cells and of mammary epithelial cells in culture in vitro. Correspondingly, other studies have demonstrated that retroviral transfer of the nonmutated BRCA1 gene selectively inhibits the growth of non-BRCA1mutated breast cancer cells in vitro and in vivo in nude mice.3 A separate gene, termed BRCA2 (on chromosome 13q13), is also associated with familial cancers of the female and male breast and, to a lesser extent, the ovaries. This gene shares homology with BRCA1, and its encoded protein has similar biochemical functions to BRCA1. However, as noted above, although BRCA2 mutation confers risk of female breast cancer, its effects on risk of ovarian cancer appear smaller than the risk conferred by BRCA1. Mutations of BRCA2 confer risk of male breast cancer and (to a more limited extent) several other cancers, such as prostate cancer, pancreatic cancer, non-Hodgkin's lymphoma, basal cell carcinoma, bladder carcinoma, and fallopian tube tumors. Breast cancers of BRCA1/2 carriers have similar prognostic significance when matched for other characteristics to the sporadic cases of breast cancer in noncarriers, although they more often also show high nuclear grade, TP53 mutations, and amplification of the HER2/neu oncogene.3 Notably, as many as two-thirds of families with hereditary breast cancer appear to have non-BRCA1/2 mechanisms of breast cancer initiation.3 Current studies, using CGH and cDNA microarray analysis, suggest a distinct signature of chromosome gains and losses and gene expression for the different classes of familial breast cancers (BRCA1, BRCA2, BRCAX) compared to sporadic breast cancers.4 As noted earlier, the structure and function of the two distinct BRCA proteins appear to be similar. Each appears to serve as an important regulator of cell-cycle checkpoint control mechanisms, involving cell-cycle arrest, cell death (apoptosis), and DNA repair. BRCA1 appears to interact with the p53 protein (leading to induction of the cell-cycle inhibitor p21) directly, with the RNA polymerase holoenzyme, with the transcription factor CREB, with two proteins termed BAP1 and BARD1, with the estrogen receptor- (ER-, suppressing its function), with the promoter of c-MYC (suppressing expression of this protooncogene), and with promoters of other genes. BRCA1 and BRCA2 proteins are also found in complexes with Rad51, a protein important for the cellular response to DNA damage.3 In addition, BRCA1 is phosphorylated by the ATM kinase, in response to DNA damage. Thus, the roles of both BRCA proteins are now emerging as central gatekeepers of genomic stability.3 In studies of mice bearing a conditional knockout of the BRCA1 gene, its further functions have emerged: mammary ductal morphogenesis and checkpoint control in the G1/S and G2/M phases of the cell cycle. Perhaps most interesting among BRCA1 proteinprotein interactions in mammary epithelial cells is the one with the ER-.5 This apparently key antiestrogenic effect may place the BRCA proteins on center stage for control of the sex steroidregulated pathways, long suspected to induce breast cancer. Chromosomal and Genomic Instability Breast cancers, like other forms of malignancy, are thought to progress by accumulation of a series of genetic and phenotypic changes in the pathways regulating cellular proliferation, differentiation, death (apoptosis or necrosis), DNA repair, tissue compartmentalization, and responses to therapy. Using classic cytogenetic methodologies and studies of LOH, genetic regions identified as commonly rearranged, amplified, deleted, or otherwise altered have been frequently detected on chromosomes 1, 3, 6, 7, 8, 9, 11, 13, 15, 16, 17, 18, and 20. More recently, CGH and chromosome painting followed by spectral karyotyping (SKY) have implicated additional chromosome regions, including areas of 4, 10, 12, 19, and 22. CGH has been further refined to a high-throughput array method using BACs containing the entire human genome. Using these techniques, the benign lesions termed fibroadenomas are observed to be largely devoid of genetic imbalances (although they do contain some chromosomal defects), premalignant epithelial tissues and near diploid tumors to contain a spectrum of genetic defects, and aneuploid breast tumors to contain large numbers of genetic mutations and chromosomal aberrations. Breast cancer, in contrast to some other common epithelial tumors, such as colon cancer, is primarily characterized by chromosomal instability, as opposed to point mutation (with TP53 mutations, probably not an early event in the natural history of breast cancer, being a notable exception to this generality). These pervasive chromosomal changes in breast cancer may arise from defects in the centrosomes and in the associated spindle apparatus of mammary epithelial cells. However, the exact pathogenesis of these defects remains to be determined.6,7 Suppressor Genes and Oncogenes The most common genetic abnormalities in the progression of sporadic and familial breast cancers (as in many other types of solid tumors) appear to be LOH at multiple loci (see Table 33.1-1). As noted earlier in Familial Disease, an LOH event uncovers the functional consequences of a mutation in an allele of a tumor suppressor gene by removal of the dominant, normal allele. At the present time, in addition to LOH of the TP53 gene and the two BRCA loci noted earlier in Familial Disease, LOH on 13q, 9p, and 16q are known, respectively, to involve specifically the tumor suppressor genes RB-1, CDKN2 (encoding the p16 protein), and CDH1 (encoding the E-cadherin protein). RB-1 and CDKN2 regulate the cell cycle, whereas CDH1 regulates differentiation and tissue compartmentalization. Other suspected tumor suppressor genes involved in the progression of breast cancer have been proposed to reside on 1p, 3p, 6q, 7q, 11p, 11q, 15q, 17q, and 22q.7 For example, a plasma membrane inner leaflet-associated protein termed caveolin-1 (on 7p3.1) has been proposed to be a tumor suppressor gene in breast cancer. It is notable that two types of DNA alterations can lead to suppressor gene inactivation on one allele, before LOH of the other allele. For example, although point mutation may be more common for TP53 and possibly Rb, gene methylation may be more common for CDKN2 and CDH-1. Additional genes that are commonly methylated in breast cancer include the gene encoding 14-3-3 (HME1, a G2M cell-cycle checkpoint control gene on chromosome 1p35), GSTPI (a carcinogen detoxification gene on chromosome 11p13), RAR2 (a retinoid receptor gene on chromosome 3p24), TIMP-3 (a matrix metalloprotease inhibitor on chromosome 22q13.1), the receptor a for estrogen (ER-, on 6q 25.1), and the receptor for PRA progesterone (on 11q13).8 In some cases, genomic areas containing tumor suppressor genes are also susceptible to complete loss or deletion of both alleles. Certain tumor suppressor gene candidates, such as the gene encoding the p27KIP1 cell-cycle regulatory protein, exhibit the characteristic of haploid insufficiency, whereby (in contrast to TP53, BRCA1, BRCA2, PTEN, CDKN2, and CDH1) a single normal copy of the gene is not fully suppressive of cancer. For this type of suppressor gene, mutation, deletion, and LOH are not common in breast cancer.9 Another common type of cytogenetic alteration in breast cancer is gene amplification. The initial step in gene amplification is thought to be the formation of extrachromosomal, self-replicating units termed double-minute chromosomes. These genetic elements later become permanently incorporated into chromosomal regions and are termed homogenous staining regions. An amplified genetic unit (amplicon) is thought to be initially much larger than the actual size of the principal gene(s) of biologic importance to tumorigenesis. Thus, silent or irrelevant genes may be detected coamplified with one or more expressed genes on an amplicon. The principal, best-established amplified and functional genes for tumorigenesis (also called dominant oncogenes) detected to date in breast cancer are the growth factor receptor HER2/neu (c-ERBB 2 ), the nuclear transcription factors c-MYC and AIB-1, and the cell-cycle kinase regulator CCND1. In some cases, multiple genes may be coamplified; for example, GRB-2 and topoisomerase II, coamplified with HER2/neu, may contribute to breast cancer pathogenesis. DNA gains are common on at least 35 distinct loci in breast cancer, including 6q, 8p, 8q, 11q, 12q, 13q, and 20q, although the specific genes involved in driving the chromosome amplification process are still under active investigation. BAC array CGH, coupled with gene expression microarray, has been used to catalog the genes reproducibly, up-regulated in association with their amplification.10 For example, a neural survival factor termed dermocydin (on 12q 3.1) has been proposed as an oncogene in breast cancer.10 Steroid and Growth Factor Pathways of Cellular Regulation Steroid ReceptorsThis chapter focuses on ER and progesterone receptor (PR) because of the large body of evidence supporting their importance in breast cancer. However, considerable interesting current research has been reported on other nuclear receptors that is beyond our ability to cover adequately here (androgen, peroxisome proliferator, and retinoid receptors). The ER and PR are dimeric, gene regulatory proteins. Estrogen and progesterone are well-established endocrine steroid regulators that modulate multiple aspects of mammary gland pathology. These two hormones work together to direct mammary epithelial growth, differentiation, and survival.11,12 Although both steroids are commonly thought to be of primary importance for tumors arising in the reproductively competent years, between puberty and menopause, local aromatization of adrenal androgens provides additional estrogens in the postmenopausal years. Estrogen and progesterone act through their nuclear receptors (ER and PR, respectively, introduced in Suppressor Genes and Oncogenes, earlier in this chapter) to modulate transcription of target genes.1113 Genes encoding the receptors for each class of steroids are members of a single large superfamily of transcription-modulating factors. ERs may exist either in homodimeric or heterodimeric species, composed of and receptors. In contrast, the PR is always a heterodimeric protein (with PRA and PRB subunits). Although ER- is of key importance in the mammary ductal elongation of puberty, PR and ER- appear to be more involved with lactational differentiation of the lobules.1214 Work with ER-, ER-, and PR has defined additional alternately spliced and mutated receptor forms. Steroid receptors associate with other proteins, including heat-shock proteins, and several coactivator and corepressor proteins (CBP/p300, P/CAF, SRC-1, TIF2, AIB-1, N-CoR, SMRT, NSD1), which modulate the acetylation status of receptors and histones.11,13,38 For example, CBP/p300 acetylates ER- at lysine residues in the ER- hinge/ligand-binding domain. Mutation of these residues selectively enhanced ER- transactivation activity but not mitogen-activated protein kinase (MAPK) stimulation by ER- (a newly recognized mode of action of estrogen, described at the end of this section). These results suggest an important role of ER- acetylation for suppression of ER activity.15 Histone acetylation is also a critical process, thought to allow full access of the DNA to steroid receptors. DNA interaction with steroid receptors occurs through zinc finger structures of the receptors and promotes formation of a stable initiation complex to facilitate the transcription of responsive genes. Superimposed on this complexity, each receptor is able to adopt multiple conformations, depending on the characteristics of interaction of the steroid (or nonsteroid ligand) with the receptor-binding pocket. For example, the ER can adopt multiple distinct conformations, depending on the nature of the specific ligand bound. Functional roles of the other kinase effectors are still under study. Estrogen and progesterone are well known for their abilities to modulate directly the expression of growth factor receptor pathways and downstream, cell-cycle regulatory genes known as nuclear protooncogenes. The nuclear protooncoproteins and other cell-cycle regulatory proteins, such as AIB-1, c-Myc, and cyclin D1, represent points of regulatory convergence of steroid and growth factor pathways in cells.12 Of considerable interest is the observation that the cell-cycle regulator cyclin D1 (product of the CCND1 gene) also interacts with the ER- to promote its transcriptional activity.16 Parallel observations have also been made with the AIB-1 protein, for sensitization of ER transactivation (as well as growth factor signal transduction).17 An alternatively spliced variant form of AIB-1 is even more potent for these effects.18 Amplification and expression of AIB-1 and cyclin D1 are associated with ER-positive breast cancer.39 ER- activation and nuclear localization, as well as coactivator interactions, are regulated through its phosphorylation.11,13,19 Signal transduction pathways induced by growth factors and hormones may directly or indirectly regulate steroid receptor function through these phosphorylation events. Cyclic adenosine monophosphate, epidermal growth factor (EGF), heregulin (an EGF family member), and insulin-like growth factor-1 (IGF-1) are examples of such ER regulators, in which receptor phosphorylation modulates transactivation of steroid-responsive genes, as well as the steroid specificity of receptors for gene transactivation. The ER- is phosphorylated on serine and threonine residues by cyclin ACDK2, c-SRC, PKA, pp90RSK1, ERK1/2, and p38.11,19,20 Phosphorylation by the latter kinase on Thr311 promotes nuclear localization and coactivator interaction.19 A very exciting discovery has been that the ER may specifically localize to the plasma membrane, bind the p85 submit of phosphoinositide 3 kinase (PI3K), and deliver promitogenic and prosurvival signaling via Akt.21 This represents a novel, "nongenomic" mode of ER action. The nuclear ER may be localized to the cytoplasm for these nongenomic actions via multiple potential mechanisms, including interaction with a shortened, variant inform of a protein termed metastatic tumor antigen 1 (MTA1). MTA1 is up-regulated in several metastatic human cancers and acts to corepress nuclear ER- by a nuclear exclusion mechanism.22 Cytoplasmic ER is more available than nuclear for such nongenomic, plasma membrane protein interactions, as with PI3K. Plasma membrane transfer of ER- from the cytoplasm may involve its specific interaction (via serine 522) with calveolin.23 Caveolin may collaborate in breast tumor suppression, although at present its inactivating mutations have been demonstrated only in the scirrhous class of human breast cancers.24 These findings provide multiple mechanisms and complex roles for growth factor interactions with steroid receptors in the expression of progressively more malignant phenotypes by breast cancer cells and in their escape from normal hormonal control. Because the growth of breast cancer is often regulated by the female sex steroids, determinations of the cellular concentrations of ER and PR in the tumor continue to be used to predict which patients are of good prognosis and may also benefit from antihormonal therapy. Although these assays were originally designed as radioligand techniques, they are more commonly performed today using immunohistochemistry. To improve the value of determinations of the ER for tumor prognosis, the presence of the estrogen-regulated PR protein is routinely performed. In many breast tumor cell lines and in normal, ER-containing tissues, such as the endometrium and brain, PR expression is induced by estrogen.11 It is still not known whether ER regulates PR in normal human mammary epithelium in precisely the same subpopulation of ductal and lobular luminal cells, although this supposition is considered to be likely. It is of interest that the ER and PR appear to be strongly up-regulated in ductal carcinoma in situ and in hormone-dependent breast cancer, relative to normal mammary epithelium. Whereas the ER- and PR-positive epithelial cell populations are distinct from the majority of proliferative cells in the normal gland, in cancer receptor-positive cells grow rapidly. It is of interest that in carcinoma in situ and in histologically normal breast tissue adjacent to cancer, the epithelium also displays an aberrant, proliferative response to estrogen.25 This result suggests that breast tumorigenesis not only involves aberrant ER/PR expression but also aberrant coupling to a proliferative response. As noted earlier, in the section Familial Disease, other studies have made a connection between the ER and the tumor suppressor BRCA1. However, it is not yet known if BRCA1 down-regulation directly contributes to the early deregulation of ER levels or responses, or both, in human breast cancer.7 The relationship between estrogen exposure and onset of breast cancer is thought to relate to aberrant expansion of a mammary epithelial stem cell lineage. However, although mammary epithelial stem cells have been described morphologically by Chepko and Smith, and their cell surface markers described by Dontu et al.,26 it is unknown whether these cells are direct precursors to breast cancer. For example, it is not yet certain how ER-positive mammary epithelial cells arise in the epithelial lineage, and it is not certain how ER-positive cancers relate to their ER-negative counterparts. Several ER-negative breast cancer cell lines do not transcribe the ER mRNA, because of an extensive methylation of the 5' promoter of the gene.8 Treatment of ER-negative breast cancer cells in vitro with azacytidine, an inhibitor of gene methylation, resulted in expression of a functional ER.10 However, the physiologic relevance of this methylation mechanism to silence ER expression during the progression of clinical breast cancer is not yet certain. Central questions in breast cancer research focus on mechanisms of desensitization of the disease to antihormonal therapy and on design of strategies to maintain antihormonal responses in patients. Mechanisms include altered ER- isoforms, coactivators (AIB-1), signaling partners (PI3K), and aromatase expression. Tamoxifen resistance of breast cancer is associated with cellular hypersensitization to the weak estrogenic effects of the drug, perhaps due to expression of receptor-associated regulatory proteins, receptor mutation, alterations in downstream growth regulatory pathways (MAPK, PI3K), or selection for variant ER- receptor isoforms. An exon 5deleted variant of the ER lacks the hormone-binding domain and displays constitutively active, hormone-independent transactivational characteristics. This receptor isoform is elevated in tamoxifen-relapsing breast cancer. Expression of an exon 4deleted ER variant correlates with low histologic grade and high PR. Expression of other variants bearing deletions in exons 2 and 4 or 3-7 are associated with high-grade tumors with high ER content.11 The ER cannot be clearly classified either as an oncoprotein or tumor suppressor protein. ER expressed in ER-negative cell lines functions to suppress cell growth, in spite of its apparently normal action to regulate expression of certain hormonally responsive genes. Thus, the multiple differences between ER-positive and ER-negative breast cancer appear to include incompatible growth regulatory mechanisms. Pathways of estrogen metabolism may be dysregulated in breast cancer. Aromatase, which converts adrenal androgens to estrogen in the postmenopausal breast and in other tissues, may promote breast cancer growth.27 AIB-1 and HER2/neu up-regulation is associated with tamoxifen resistance28; the use of aromatase inhibitors may bypass such effects, although other mechanisms of adaptive hypersensitivity to estrogen, such as up-regulated ER, MAPK, and PI3K activation may also result.11,20,27 Other studies have suggested that receptors for other steroids (potential cancer prevention agents; retinoids and vitamin D) may modulate ER/PR function by modulating their chromatin interactions.29 Growth Factor Pathways in the Normal and Malignant Gland The natural secretory products of the mammary epithelial cell, colostrum and milk, are abundant sources of growth factors.30 Growth factors in the normal gland probably serve multiple purposes in the development of the newborn, in mammary growth, and in mammary carcinogenesis. A large body of literature has shown that estrogen, antiestrogens, progestins, and antiprogestins strongly regulate certain growth factors and receptors of the EGF and transforming growth factor- families, as well as growth factors, receptors, and secreted binding proteins for the IGF family (Table 33.1-2). Most recently, the powerful methodology of gene expression profiling has reconfirmed earlier literature, now bringing the total to more than 400 robustly estrogen-regulated genes involved in cell proliferation, signal transduction, and survival (see Table 33.1-2).31 EGF, apparently the most abundant milk-derived growth factor, is an important regulator of the proliferation and the differentiation of the mouse mammary gland in vivo and of mouse mammary explants in vitro. Circulating, mouse salivary glandderived EGF potentiates spontaneous mammary tumor formation and growth in the mouse models. EGF, or other members of this growth factor family, is a required supplement for clonal anchorage-dependent growth in vitro of normal human mammary epithelial cells. In contrast, human breast cancer cells in culture are largely independent of this exogenous requirement. However, most breast cancer cell lines retain EGF receptors (EGFRs) and appear to be stimulated in their growth by either autocrine or paracrine production of this family of factors.30 Direct modulation of signal transduction pathways by EGF and its family members, as well as their indirect regulation by other unrelated growth factors (described in Signal Transduction and Nuclear Oncogenes, later in this chapter), are critical during mammary development. The EGF family consists of four receptors and nearly two dozen growth factors in mammals and is encoded by certain mammalian viruses. Transforming growth factor- (TGF-) and amphiregulin, close structural and functional homologues of EGF, can produce qualitatively the same proliferative effects as EGF in mouse mammary explants and in cultured human and mouse mammary epithelial cell lines. Each of these factors is produced in proliferative early ductal development and in the later lobuloalveolar development of pregnancy. However, the detailed localization patterns and functions of each family member differ. For example, an immunohistochemical study of the mouse gland has revealed that expression of TGF- is highest in the basal epithelial, proliferative end-bud cap cells, whereas expression of EGF is in scattered ductal luminal secretory cells.31 Hepatocyte growth factor (HGF), a non-EGF family factor that interacts with its cognate receptor/oncoprotein c-Met, and growth hormone/IGF-1 also appears to be involved in ductal morphogenesis (described later in this section). The EGF-related neuregulin subfamily of isoforms (including heregulin) is expressed primarily in the mammary stroma and appears to modulate the lobuloaveolar development of pregnancy. TGF-, the heparin-binding family member termed amphiregulin, and its common receptor, the EGFR, are both detected in vitro in proliferating human mammary epithelial cells in culture. TGF- mRNA levels are relatively low in explanted, primary cultures of resting human mammary epithelial organoids; the entire system appears tightly coupled to proliferation in the normal gland. TGF- and amphiregulin are known to act as autocrine autostimulatory growth factors in normal and immortalized human mammary epithelial cells in mass culture; an anti-EGFR antibody or heparin (respectively) reversibly inhibited proliferation. Although the majority of work on the biology of the EGF family has focused on regulation of proliferation, it is now clear that many other aspects of mammary biology may be under its control, such as cell differentiation and survival. The complex heterodimerization pattern of the EGFR family may play a role in governing the multiple responses of this system.30 Transgenic expression of most members of the EGF family (including TGF- and the neuregulins) in the mouse mammary gland have been shown to lead to mammary tumorigenesis.30,31 Although observations of overexpression of EGF family growth factors have led to significant biologic insights into the disease, the greatest clinical impact has come from the study of EGF family receptors. Gene amplification and resultant overexpression of EGFR-related HER-2/neu protein occur in approximately 25% of human breast cancer cases (see Table 33.1-1; discussed in Implications of Molecular Biology for Tumor Prevention, Early Detection Prognosis, and Response to Therapy, later in this chapter), and overexpression of the EGFR occurs in the absence of gene amplification in 40% of breast tumors. Expression of each protein signifies poor prognosis for the patient. Activational mutations in the extracellular domain of the Her-2/neu oncoprotein were commonly detected in transgenic mouse mammary carcinomas. Although this type of mutation seldom occurs, an alternate splice form of extracellular domain has been described in human breast cancer. Stimulation of cells through the EGFR family may also sensitize cells to other carcinogenic insults. For example, expression of a TGF- transgene accelerates the progression of carcinogen-initiated mouse mammary tumors. In summary, there is compelling evidence from human pathologic studies and from transgenic mouse models for key roles of EGF family growth factors and their receptors in mammary tumorigenesis, combining many types of data.30,31 In humans, autocrine and paracrine functions of the EGFR ligand system may be most critical in normal gland growth and early stages of breast tumorigenesis. In more advanced disease, paracrine, tumor-host interactive functions of this family of factors (discussed in Process of Malignant Progression, later in this chapter) may dominate. Strategies using humanized, anti-HER-2/neu (Herceptin, or trastuzumab) or humanized anti-EGFR antibodies (IMC-225), or small molecule kinase inhibitors, are in various stages of clinical trial testing at present, because a large portion of hormone-independent breast cancers express significant levels of these receptors. Antitumor action of trastuzumab appears to depend on inhibition of PI3K/Akt for effects to increase p27, to decrease cyclin D1, to slow cell-cycle progression, and to induce apoptosis.33 A second growth factor family of potential importance in cancer is the fibroblast growth factor (FGF) family.30 Members of this family have also been implicated in mammary gland growth and malignant transformation. These growth factors bind to heparin, require a heparin cofactor for proper presentation to and interaction with receptors, and accumulate in the extracellular matrix after their release from cells. Some FGF isoforms do not possess a signal sequence for secretion, but all forms appear to be released by one or more mechanisms from cells. Although FGF-1 and FGF-4 are expressed during the ductal growth phase of the mouse mammary gland in luminal ductal epithelial cells, expression of FGF-2 and FGF-7 is primarily stromal. FGF-1, FGF-2, and FGF-7 have been detected in mammary preneoplasias, tumors, and cell lines but not in levels significantly elevated over normal. FGF-3 (also known in the mouse as int-2) is a well-known oncogenic growth factor activated in the mouse mammary gland by mouse mammary tumor virus insertional mutagenesis. FGF-4 also has been associated with metastasis of certain mouse mammary tumors. Amplification of the genes encoding FGF-3 and FGF-4 in human breast cancer is not associated with increased protein levels. This finding appears to be the result of their coamplification with other more important genes located nearby on the chromosomes (genes encoding HER-2/neu and cyclin D1, respectively). In the human mammary gland, FGF-1 and FGF-2 have been localized to myoepithelial and to epithelial cells. Although in vitro studies have implicated FGF-2 as an autocrine growth factor in immortalized human mammary epithelial cells, expression of FGF-2 in clinical human breast cancer is correlated with good-prognosis disease. FGF-1 has been detected in human breast cancer, localized primarily to macrophages, and may thereby contribute to inflammation and angiogenesis. FGF receptors 1 to 4 (and the gene encoding FGFR1, occasionally amplified) are overexpressed in human breast cancer. Several of the FGF family members have been shown to promote tumor growth and dissemination of metastasis in xenograft models of human breast cancer, at least partially through effects on the tumor vasculature.30 TGF-, a family of at least three growth factors distinct from FGF and EGF families, is also present in the normal and malignant mammary epithelium and in human milk.33,34 Receptors for TGF- comprise a family of heterodimeric serine-threonine kinases, signaling through interaction with Smad family proteins. TGF- has antiproliferative effects on the mouse mammary gland ductal epithelium in vivo and on most other epithelial cell types. Expression of TGF- family members is suppressed by estrogen and progesterone. TGF- proteins are detected at the mRNA level in the developing mouse mammary epithelium. All isoforms of TGF-, once produced, are retained in the stromal matrix surrounding the mammary ducts, although they are absent from the matrix of growing end buds and lateral branches. Glandular production of TGF- decreases, along with its stromal accumulation around alveoli, during midpregnancy and lactation; TGF- suppresses lactation. However, TGF- is again elevated as postlactational glandular regression occurs. The roles of TGF- have also been examined by using a transgenic mouse approach. Mammary-targeted expression of TGF- inhibits alveolar development and lactation. All three TGF- isoforms have been detected in the human gland, with a similar distribution in the mouse. Although TGF- serves a growth inhibitory role in the normal gland, progression to cancer may be associated with epithelial desensitization to this growth factor. Paradoxically, TGF- production increases with malignant progression in breast cancer, perhaps promoting the fibrous desmoplastic stroma of the disease, for tumor angiogenesis and for immune suppression. Overproduction of TGF- may contribute to aberrant tumor-host interactions in breast cancer. At least in some cell lines in vitro, TGF- may stimulate tumor cell invasion. Loss of TGF- receptor, by mutation or by loss of expression, has been observed in colon cancer and retinoblastoma, respectively. In breast cancer, interruption of signaling occurs more distally. Thus, TGF- signals through heterodimeric types I and II receptors and phosphorylates Smad transcription factors. In early-stage human breast cancer, lack of Smad2P expression (an element in TGF- receptor signaling) correlated with shortened overall survival from the disease. In addition, loss of Smad4 correlated with auxiliary lymph node metastases in higher-stage breast cancers. Interestingly, type III TGF- receptor expression, previously thought simply to present the growth factor to its functional RI/II heterodimeric receptors, may serve to modulate TGF- responses. Loss of its expression during breast cancer progression may serve to convert TGF- from a tumor suppressor to a tumor promoter.35 A complex regulatory system is also emerging from studies of the insulin-like growth factors.36 Although the IGFIR is a heterodimeric tyrosine kinase, closely related to the insulinR, the IGFIIR is an unrelated binding protein capable of interacting with TGF- and with cathepsin D. IGF-II production, as well as cellular responsiveness to IGFs, is stimulated by estrogen and inhibited by antiestrogens in some hormone-dependent breast cancer cell lines. Cellular responsiveness is complex; IGF extracellular growth factorbinding proteins and intracellular signal transductioncoupling proteins (IRS-1 and -2) are regulated by multiple factors.30,36 IGF-II is thought to be a potential autocrine growth factor in breast cancer. IGF-I, in contrast, is synthesized in the tumor stroma and has important paracrine growth and survival-stimulatory actions in the disease. Transgenic mouse models have demonstrated that mammary expression of IGF-I causes ductal hypertrophy and suppression of postlactional involution and that IGF-II expression can lead further, to mammary cancer. The cellular responsiveness to IGFs appears to be modulated by estrogens and antiestrogens, as a result of regulation of both receptors (type I being induced and type II repressed); IGF-binding proteins 2, 4, and 5 (each of which is estrogen induced) and IGF-binding protein 3 (which is estrogen inhibited); and the signal transduction docking phosphoprotein IRS-1 (which is estrogen induced). The biologic functions of the IGF-binding proteins are not fully understood, although BP-1 appears inhibitory of the actions of IGF-1, even with in vivo models. Although BP-3 may contribute to poor prognosis of breast cancer, BP-4 and the IGFIR correlate with good prognosis. IRS-1 (but not IRS-2) is up-regulated by estrogen and down-regulated by tamoxifen. The IGFRII is under active investigation as a tumor suppressor gene in cancers of the breast and other tissues. Its gene is subject to frequent LOH and possibly mutations in breast cancer.36 Several dozen other growth factors have been identified in breast cancer, but their consideration is beyond the scope of the current chapter because of limitations in availability. This information is reviewed elsewhere.30,37 In brief, prolactin might be an autocrine positive factor in breast cancer, and mammary-derived growth inhibitor and mammostatin may serve negative growth functions. HGF/scatter factor (SF) is a stromal-derived paracrine-acting stimulator of epithelial growth and of tumor angiogenesis. HGF/SF, its oncogenic receptor c-Met, the proteolytic activator of HGF/SF termed matriptase, and the cognate inhibitor of matriptase, HAI-1, are all correlated with poor prognosis of the disease. Finally, the vascular endothelial growth factor (VEGF) family, pleiotrophin, and platelet-derived growth factors may serve as angiogenic, vascularization-inducing factors in the disease. Expression of VEGFA correlates with poor prognosis of human breast cancer because of its proangiogenic effects, whereas VEGFC and VEGFD induce lymphangiogenesis. Many other cytokines (such as osteopontin), Wnt family members, and other growth factors are also expressed in breast cancer. Future investigations should evaluate their pathophysiologic roles and possible targeting for therapy.30,37 Signal Transduction and Nuclear Oncogenes Unifying, mechanistic links between the proliferative actions of growth- and survival-modulatory steroids, growth factors, and integrins in diverse tissues are represented by the multiple classes of nuclear protooncogenes and other transcription factors (see Table 33.1-1). These transcription-regulating proteins mediate convergent pathways of regulatory stimuli, directly through steroid action, through growth factorinduced MAPK, through other cytoplasmic tyrosine kinases (Fak, Src) or phospholipase C-PKC, through cytokine-induced JAK-STAT pathways, through TGF- familyinduced Smad molecules, and through integrin-induced Fak/Src pathways. Steroid and growth factor pathways ultimately regulate gene expression through specific acetylation of histones and transcription factors. Activation of different members of the histone acetyltransferase family of enzymes causes these key acetylation reactions to proceed; specific histone deacetylases are also recruited to sites of active, regulated transcription, to allow for tight control on these processes. Components of the cell-cycle regulation apparatus are under control by acetylation and deacetylation, making this mode of regulation key to the onset, progression, and potential treatment of breast cancer.38,39 The MAPK pathways are central to proliferative and survival stimuli, exerted through the EGFR, HER-2/neu, and insulinR type I families. Receptors trigger this pathway through autophosphorylation and subsequent binding to SH2/SH3 or PTB domains of signal transduction adaptor proteins. After mitogenic growth factor treatment of many types of cells, including normal and malignant breast epithelial cells, a cascade of protein phosphorylations occurs. Expression of early-response genes is induced, including c-Myc, AP-1 (activator protein 1) acting (c-Fos, c-Jun, and Jun B), c-Myb, and Ets protooncogenes and ATF, EIK, SRF, and NFKB transcription factors are commonly observed to be induced. The protein products of multiple nuclear protooncogenes, c-Myc, c-Fos, c-Jun, and cyclin D1 are also induced by estrogen and by progesterone in breast cancer. Progestins additionally induce JunB. Not surprisingly, tamoxifen down-modulates c-Myc expression, followed by cyclin D1 expression, during treatment-induced regression of patient tumors. c-Myc, c-Fos, c-Jun, and cyclin D1 induction have also been shown to occur in human mammary epithelial cells in vitro and in the rat uterus in response to estrogen treatment in vivo. Cyclin D2 is a nuclear oncoprotein downstream of AP-1 and other mitogenic transcriptional controls. Not only does it regulate the G1 phase of the cell cycle, but it also enhances ER transactivational activity.39 c-Fos and c-Jun proteins contain specific domains that allow them to form a heterodimeric complex that can interact with gene promoter consensus sequences termed AP-1. In an analogous manner, the c-Myc protein, of central importance to estrogenic stimulation of breast cancer cells, dimerizes with another protein termed Max to modulate genes through a different consensus sequence, termed an E-Box (and possibly other sequences). The cellular supply of Max that is available for productive dimerization with c-Myc depends on its interaction with its other family members, termed Mad and Mxil. The interaction of Max with either of these proteins serves to reduce its availability for interactions with Myc and may exert negative transactivation through E-box sites. Myc-Max dimers are known to induce proliferation, apoptosis, and chromosomal instability, depending on the cellular context and degree of expression. Myc-Max interaction with TATA binding protein stimulates basal transcription. Secondly, Myc-Max complexes bind promoters of genes, such as those encoding the CAD, DHFR, and ODC enzymes, through their E-boxes. However, multiple c-Mycregulated genes do not contain E-box sequences, and these sequences are not required for c-Myc effects on proliferation and apoptosis. Several other c-Mycinteractive proteins exist in addition to TATA binding protein and Max, including TRRAP, BIN1, DAM, p107, YYI, MIZ1, and TFII-1, which contribute to transcriptional effects of c-Myc. Estrogen and progesterone induce c-Myc and cyclin D1. c-Myc may modulate the mammary epithelial cell cycle by inducing the synthesis of CDK4, triggering the degradation of p27kip1, and suppressing p21 and p15ink4B transcription, possibly through interaction with Miz-1, inactivating CDK2. Activation of CDK2 inhibits pRb phosphorylation, promoting G1/S cell-cycle progression. c-Myc induces cyclin A to activate CDK2 and induces E2F1 to promote S-phase cell-cycle progression. However, c-Myc can induce apoptosis through p53-dependent and possibly independent mechanisms. Thus, effects of c-Myc are highly complex and likely to depend on a multitude of environmental and other cellular factors.39,40 Antisense c-Myc oligonucleotides block estrogen-induced proliferation in breast cancer cells, and amplification of the c-MYC gene is a common genetic alteration in breast cancer; approximately one-fifth of breast cancers contain this genetic change. A putative suppressor gene (possibly HME1, encoding 14-3-3) on chromosome 1p32-pter is proposed to interact with c-MYC to suppress its gene amplification in breast cancer.39,40 c-MYC protein has a very short half-life, and few suitable monoclonal antibodies are capable of specifically staining paraffin sections. c-MYC amplification is associated with poor prognosis, high S phase, and postmenopausal disease, although the latter has not been confirmed.40 Based on several investigations in various epithelial malignancies, including those of the ovary and liver, c-MYC amplification is thought to cooperate with TGF-, with EGFR overexpression, and with downstream signaling pathways (notably Ras) to activate the cell cycle and suppress apoptosis. Thus, dual stimulation of the EGFR pathway and c-Myc may serve a general cooperative function in epithelial transformation.40 The c-Myc protein promotes cell proliferation, inhibits differentiation, modulates cell adhesion, effects immune recognition, regulates initiation of DNA replication, and modulates DNA and energy metabolism, perhaps in part through activation of expression of telomerase.40 Induction of apoptosis by c-Myc in mouse models depends on p19ARF (p14ARF in humans; see Cell Cycle and Cell Death) stabilization of p53 function, although a role for this mechanism in mammary cancer remains to be demonstrated. Expression of c-Myc increases with aging in multiple tissue types and has been proposed to contribute to aberrant mitogenic responses of the tissue in postmenopausal breast cancer, although increased expression of Mxil during aging may attenuate such effects.41 Cell Cycle and Cell Death Tumor Suppressor Genes and the Cell Cycle Seminal studies by Broca in the nineteenth century established that breast cancer can have a familial pattern of onset in 5% to 10% of cases. Subsequently, in studies of the inherited childhood cancer syndrome retinoblastoma, of familial cancers, Knudson42 proposed the "two-hit model" of tumor suppressor gene function. In addition, Harris39 demonstrated that certain chromosomes could suppress malignancy in vitro in cell hybrid studies. These three concepts led to the discovery of several tumor suppressor genes in breast cancer, some encoding proteins regulating the cell cycle (p16, Rb, p53) or cell death (p53, Pten), or both, and others regulating other aspects of the progression of the disease (E-cadherin). A variety of tumor suppressorlike proteins have also been described, which are down-modulated but seldom mutated in nonfamilial breast cancer (such as the gene encoding p27, BRCA1, Syk, KAI-1, Kiss1, and nm23).39,43,44 During the malignant progression of breast cancer to its fully metastatic state, mutation, inactivation, loss, or down-regulated expression of tumor-suppressing genes commonly occurs. Estimated incidences of these processes for the relevant, known suppressor genes are as follows: TP53 (30% to 40%), RB-1 (15% to 20%), CDKN2 (20% to 30%), and CDH1 (20% to 30%). Tumor suppressor genes appear to function in at least four major ways: as antiproliferative or antisurvival factors, as DNA-repair inducers (all in DNA damage-response pathways), and as differentiation-promoting agents. BRCA1 and BRCA2 serve roles to direct repair of damaged DNA; the ATM protein (described earlier in Familial Disease as a suspected tumor suppressor) detects the damage and transmits the signal to the BRCA proteins, whereas multiple other proteins, such as Rad51 and p53, serve roles downstream of the BRCAs.45 E-cadherin sequesters -catenin (a proliferation-promoting protein that regulates the T-cell factor class of transcription factors) and strengthens homotypic interactions of mammary epithelial cells to maintain their differentiated status.46 p53, by inducing the p21 protein (Waf-1/CIP-1), also inhibits proliferation, whereas the p16 protein also serves to inhibit the cell cycle; p53 and p16 suppressor proteins ultimately promote phosphorylation and inactivation of pRb to block G1 and G1/S transit of the cell cycle. Although pRb is thought to be a central tumor suppressor protein in breast cancer, its inactivation is likely to occur through cyclin D1 overexpression, and pRb mutation status remains to be fully assessed. Pten serves to suppress cell survival and proliferation by dephosphorylating phosphoinositides to prevent their activation of the three AKT signal transduction kinases.47 Dozens of additional candidate tumor suppressor genes are reviewed elsewhere.43 Study of the TP53 gene has provided remarkable insights into multiple areas of cancer biology. TP53 is a tumor suppressor gene, but, when mutated in one of several sensitive regions, its conformation changes, its stability increases, and its regulatory properties are radically altered. Mutation can confer a loss of tumor suppressor activity or gain, or both, of tumor promotion function. The nonmutated p53 gene product is an oligomeric DNA-binding protein that functions to trigger cellular responses to DNA damage; it has been termed guardian of the genome. p53 functions by proteinprotein interactions and by regulation of transcription. As noted under New Methodologies and Genetic Mechanisms, earlier in this chapter, the p53 protein appears to function in the context of DNA damage as a G1/S and G2/M checkpoint controller, to slow cell growth and induce DNA repair; cell death is triggered in a process termed apoptosis if damage is too severe for repair. It is of particular importance for the progression of many cancers, including breast cancer, that mutation of p53 is associated with enhanced genetic instability. Certain viral proteins, although they are probably not relevant to breast cancer, are known to inactivate p53 as a critical event in viral carcinogenesis.50,51 Mutation of p53 not only abrogates the G1/S checkpoint but also G2/M and a post M spindle assembly checkpoint.48 Two family members of p53, termed p73 and p63, are under current study of their p53-like properties.48 It is not yet fully clear what molecular events induce (through protein stabilization) the p53 protein. However, it is well known that ultraviolet irradiation and double-strand DNA breaks are strong inducers of p53 stabilization through the DNA-dependent protein kinase and the ATM gene product. ATM is a signal transduction protein with high homology to PI3K. Inhibitors of protein kinase C, or serine-threonine phosphatases, and cyclic adenosine monophosphate can prevent p53-mediated responses. p53 induces growth arrest, in part through induction of p21, and thereby inhibition of cyclin ECDK2catalyzed phosphorylation of pRb (discussed in Cyclins, Cyclin-Dependent Kinases, and Inhibitors, later in this chapter). p53 regulates pRb and directly binds a partner of pRb termed p107. p53 induces transcription of DNA repair genes, including cyclin G, ERCC, and Gadd 45. A third general process triggered by p53 is apoptotic death. Negative regulators of p53 include phosphorylations by several kinases, acetylation by histone acetylase, its nuclear exclusion, and, most importantly, its degradation through a ubiquitin ligase called MDM-2 (murine double-minute gene-2). MDM-2 is itself a p53-inducible protein that can function as a collaborative oncogene (at least in some cancers other than breast cancer).48 The nuclear tumor suppressor protein pRb is inactivated by phosphorylation and catalyzed by several cyclin-dependent protein/kinases and through binding growth regulatory proteins, including c-Myc, SP1, C/EBP, ID2, histone deacetylases, c-Abl, and many other transcription factors. pRb restricts entry into the S phase of the cell cycle. Hypophosphorylated Rb binds E2F-1 and DP-1 family members to restrict access of these transcription factors to the chromatin. The result is a blockade of transcription from genes involved in G1S progression and S phase in the cell cycle.30,38,48,49 Cyclins, Cyclin-Dependent Kinases, and Inhibitors As discussed earlier, growth factors, oncogenes, and tumor suppressors function to a large extent in the G1 phase of the cell cycle. The cell cycle is directly controlled by an ordered series of cyclin-dependent kinases (CDKs), their positive regulatory subunits (cyclins), and their inhibitors (CDK inhibitors). Early G1 is driven by the three cyclin D family members bound to CDK4 and CDK6. In the next portion of the cycle, the G1S transition is driven by cyclin ECDK2. The S phase is driven by cyclin ACDK2, and then the G2M transition is driven by cyclin B/ACDC2 (CDK1).30,38 As important regulators of the epithelial cell cycle in breast cancer, tyrosine kinase receptoracting growth factors and sex steroids function to induce c-Myc and cyclin D1. Thus, HER2/neu may exert its proliferative effects through a required induction of c-Myc and cyclin D1. HER2/neu (in contrast to c-Myc) fails to induce mammary tumors in mice rendered nullizygous for cyclin D1. Cyclin D1 knockout mice are also defective for lobuloalveolar development in the mammary gland. Cyclin D1CDK4 is inhibited by the CDKI termed p16 (INK4a), by the tumor-suppressive CDKI p16 (MTS1 or INK4b), and by p18 and p19, other members of the same CDKI family. Cyclin DCDK4 and cyclin ECDK2 are also each inhibited by the CDKI termed p21 (Waf-1/CIP1, which is induced by p53), by p27 (kip1), and by p57 (kip2), which may be involved in mammary epithelial cell senescence. c-Myc has been shown to induce or inhibit cyclin D1 expression in cycling cells, to induce cyclin E (and possibly CDC 25A), and to trigger the proteosome-mediated destruction of the CDKI p27 (kip1) and activation of CDK2. c-Myc also dysregulates S phase by inducing cyclin A (to activate CDK1) and by inducing E2F-1.30,38,40,49,51 The synthesis of c-Myc and CDK4 is inhibited by TGF- and CDK4; overexpression of CDK4 leads to TGF- resistance. p27 is induced by TGF-. As noted in the previous section, pRb is phosphorylated and inactivated by the combined actions of cyclin DCDK4 and cyclin ECDK2 kinases. The result of all of the multitude of common oncogenic and tumor suppressor aberrations in breast cancer seems to be similar: defective G1S transitions in the mammary epithelial cell cycle.52 In addition, DNA damage-induced checkpoints of G1/S, G2/M, and post M are commonly abrogated; cells lose proliferative requirements for estrogen, growth factors, and cell substrate adhesion; and they lose inhibition by TGF- family growth factor.39 Apoptotic Mechanisms and Survival Signaling The mammary gland undergoes cycles of proliferation followed by programmed cell death. The menstrual cycle generates such cycles on a limited scale (with cell death occurring just after the luteal phase), and significant apoptotic death results at the end of pregnancy (involution). The latter effect has been proposed to contribute to the breast cancerprotective effects of early pregnancy in women. Regulation of members of the Bcl family of proteins is central to these cell mechanisms. Estrogen and activation of the HER-2/neu, EGFR, and insulin receptor all induce the antiapoptotic Bcl-2 and Bcl-XL family members. These growth factors activate PI3K, creating the phospholipid IP3, which activates the Akt kinase. Akt phosphorylates the forkhead transcription faction [controlling proapoptotic Fas ligand, caspase 9, the antiapoptotic Bad protein, and the IB kinase (I, I), which activates NFB, -catenin, and Bcl-XL/Bcl-2]. In contrast, p53 and c-Myc up-regulate proapoptotic Bax. Thus, Bcl-2 and Bcl-XL, induced by growth factors and other stimuli, block apoptosis; Bcl-2 is commonly expressed in p53-mutated breast cancer cells. The balance of pro- and antiapoptotic Bcl family members leads to control of mitochondrial membrane permeability to cytochrome c and other regulators of the caspase cascade (initiated by caspase 9), a protease system serving to execute death pathway signals. Bcl-2 is localized in the mitochondria, nuclear membrane, and endoplasmic reticulum. It functions, along with Bcl-XL, to suppress the function of Bax, a death-inducing protein; the entire Bcl family acts to integrate pro- and antiapoptotic stimuli by forming mitochondrial pores of differential ionic permeability. Several other stimulatory and inhibitory family members exist, including Bcl-Xs, a promoter of apoptotic death. The apoptotic system is thought to be triggered by hypoxia or by a shift in the redox potential of the cell. A distinct set of proapoptotic signals may be initiated by ligation of death receptors on the cell surface by tumor necrosis factor, FasL, and other cytokines to initiate caspase 8dependent death pathway signals to the same executor caspases (such as caspase 3).53,54 Although quite complex, the balance of life/apoptosis is critically regulated in cancer progression and response to therapy. Estrogen, progesterone, TGF-, EGF, and insulin all appear to suppress apoptosis and promote survival of breast cancer model systems. Antiestrogens, antiprogestins, TGF-, and the overexpressed c-Myc oncoprotein can induce apoptosis unless countered with a survival-promoting, environmental influence. Approximately 80% of breast cancers express Bcl-2, and expression is correlated with the ER, whereas Bcl-XL predominates in ER-negative breast cancers. Bax expression is generally low in breast cancer, and its expression has been correlated in some studies with better survival and responsiveness to chemotherapy. However, a large study has failed to associate any of these effectors of apoptosis with responsiveness of advanced human breast cancer to chemotherapy.54 Roles of Estrogen and Progesterone in Cell Cycle and Cell Death The well-orchestrated program of proliferation and apoptosis in the normal mammary gland depends on the regulated cycles of estrogen and progesterone. In breast cancer, these two steroids remain key regulators of a significant fraction of cases. In terms of cell-cycle regulation in breast cancer, estrogen induces c-Myc and cyclin D1, and cyclin D1 activates CDK4/6, and estrogen and c-Myc then activate cyclin E/CDK2. Cyclin E/CDK2 is not inhibited by p21 due to effects of estrogen (possibly through c-Myc) to block synthesis of p21. Estrogen synergizes with insulin for cell-cycle progression, potentially through p21 interactions.55 Estrogen induces c-Myc55 and cyclin D1 transcription to promote S-phase entry. ER interaction with a complex containing the Src tyrosine kinase and p85-PI3K/Akt appear to be required for cyclin D1 induction.56 Estrogen also induces Bcl-2 transcription, providing a strong antiapoptotic signal.57 Antiestrogen suppresses transcription of c-Myc and cyclin D1, and antisense c-Myc mimics the effect of antiestrogens. Antiestrogen also promotes apoptosis of mammary tumors in vivo.55 In contrast to the actions of estrogen, progestins first transiently stimulate breast epithelial and breast cancer proliferation but then suppress proliferation. This antiproliferative effect is due to induction of p27 to block cyclin D-Cdk4/6 and p18 (INK4c) to block cyclin ECDK2 complexes.58 Process of Malignant Progression Malignant progression of breast cancer involves the conversion of "benign proliferative lesions" and carcinomas in situ to early (stages I and II) disease, to locally advanced (stage III) disease, and then to metastasis to bone, brain, lungs, and other sites (stage IV). Fundamental to malignant progression are the heterotypic processes regulating epithelial mesenchymal transition, hypoxia, desmoplasia, and angiogenesis.30,43,59 The development of cancer involves suppression of apoptosis and senescence, dysregulation of proliferative signaling factors, activation of oncogenes, dysregulation of growth inhibitory factors, and loss of tumor suppressor genes (see Fig. 33.1-1).30,39 Genetic damage is minimal in benign breast disease or premalignant atypical ductal or lobular lesions, and expression of hTERT (catalytic submit of the DNA replication-associated enzyme telomerase) is induced, suggesting that suppression of cell senescence may occur early in disease.60 High levels of telomerase expression lead to cell immortalization, and the catalytic subunit of this enzyme (hTERT), together with three oncogenesSV40T (a viral oncogene, which inactivates p53 and Rb), Sv40 small t (which inactivates the PP2A phosphatase), and a mutant c-RasHconvert human mammary epithelial cells to cancer.61 Human and mouse cells critically differ in a signal transduction pathway activated by RasH that is required for transformation: The Raf/MAPK pathway is dominant in mouse cells, whereas in human cells, the key pathway is that of Ras GPS/PI3K. c-MYC was commonly amplified in these cells. Telomerase overexpression has been detected in the earliest stages of breast cancer, as noted earlier; it is known to be induced by estrogen and by c-Myc. Thus, telomerase dysregulation may serve a subtle, early function in breast tumorigenesis: replicative immortality.60,61 The subsequent steps in tumorigenesis almost entirely involve spontaneous gene amplification, LOH, methylation, and mutations, which arise as a result of overactive cell cycles, defective cell cycle checkpoints, or defective cell death responses.62 Multiple oncogenes and tumor suppressor genes (BRCA1, p53, c-Myc, Akt, Rb, p21, p27) are involved. Once these types of genetic alterations begin to occur, a cascade of further genetic changes occurs, resulting from overall genomic and chromosomal instability.68,62 Familial disease may bypass one or more steps in this cascade.3 The mechanisms for genetic instability and accumulation of further mutations in other cancers, such as colon cancer, have been proposed to depend initially on overexpression of a mutator gene termed MSH2. To date, however, these mechanisms have not been fully evaluated for breast cancer, and mismatch repair defects appear to be associated primarily with later stages of progression metastasis of breast cancer. In contrast to colon cancer, in breast cancer centrosomal defects appear to predominate in early disease, potentially causing the chromosomal type of instability.7,62 Almost certainly, some of the most important consequences of these changes in genomic stability initially relate to therapeutic responses. As discussed earlier in Steroid and Growth Factor Pathways of Cellular Regulation, the mechanisms of antihormonal therapy failure in hormone receptorpositive disease may potentially relate to sensitization of the tumor to very low levels of sex hormones and to the partially estrogenic properties of the clinically used hormonal antagonists, such as tamoxifen. Chemotherapy resistance in breast cancer is also not fully understood but probably involves altered cell death responses and altered metabolism of the drugs. Resistance to radiation therapy may involve defects in the ATM pathway (including BRCA proteins and p53), leading to DNA damageinduced death. During malignant progression after treatment, additional amplifications, LOHs, and mutations occur. The cancer cells with the greatest capacity for growth, invasion, or survival undergo positive selection. Other cells may be more susceptible to necrotic or apoptotic death. Studies have detected genetic changes in histologically normal tissue surrounding breast tumors, consistent with this clonal evolution hypothesis.62 Certain common patterns of cytogenetic alterations and gene expression exist in breast tumors, reflecting signatures of distinct initiating lesions and pathways of progression. Acquired genetic changes may interact unfavorably with residual growth regulatory pathways operating in the normal tissue. For example, an unfavorable interaction occurs between cyclin D1 overexpression and the ER.16 Amplified HER-2/neu or c-MYC genes synergize with the presence of an autocrine- or paracrine-activated EGFR system. HER-2/neu amplification associated with activation of c-RASH gene is also an unfavorable interaction. The tyrosine kinase signal transduction pathways (such as those downstream of c-RasH) that govern proliferation or survival, or both, enhance oncogenesis induced by c-Myc in cellular models in vitro and in transgenic mouse models of mammary cancer. c-Mycinduced tumors are enhanced by overexpression of apoptosis-suppressing Bcl-2. Finally, mutant p53 enhances HER-2/neuinduced tumors by blocking apoptotic pathways.30,39 An important approach for the future understanding of this phenomenon involves generation of the transgenic and gene knockout mouse models, which more faithfully recapitulate human disease. Investigators could harness these models to understand mechanisms of breast cancer onset and progression. Such studies may identify the biochemical and cellular bases of various patterns of malignant progressions. Future studies aimed at detailed characterization of the interaction of ER, PR, growth factors, protooncogenes, and suppressor genes with therapeutic approaches in animal models may determine molecular genetic interactions governing onset and progression of breast cancer.30,31 The ultimate event that leads to mortality from breast cancer is metastasis. Two separate, but apparently interactive, cellular processes seem to occur to allow metastasis of the disease: tumor angiogenesis and loss of proper tissue compartmentalization (invasion).63 The process of tumor-stromal interactions becomes disrupted in multiple aspects in malignant progression. Loss of cell-cell attachment, altered cell substratum attachment, and altered cytoskeletal organization play a role in regulating cellular invasion. In addition, cell locomotion, proteolysis, and the ability to survive and proliferate at distant sites also must contribute. Although acquisition of this group of characteristics is responsible for a cancer to invade host tissue locally, the ability of a tumor to distribute itself to distant sites also requires the development of a tumor vasculature: the complex process of angiogenesis.30,37 Some studies have shown that metastatic alterations may have at least some genetic basis and that distant metastases are more likely to exhibit dominance of a malignant clone than are primary tumors. In human breast cancer, a well-established precursor of invasive disease is carcinoma in situ (either ductal or lobular varieties, DCIS and LCIS, respectively). DCIS and LCIS may be characteristic of hypoxic areas, indicating oxygen starvation of epithelial cells as they proliferate beyond a normal pseudostratified layer to fill the ductal and lobular structures. A normal response of such cells to hypoxia is to trigger a stress response program governed by hypoxia-inducible factor 1.64 This factor controls metabolic adaptation to hypoxia through regulation of glycolysis, angiogenesis (through VEGF), and proliferation/survival (through IGF-2 and c-Met transcription).64,65 Thus, carcinomas in situ may be under selective pressure to use these growth factor/receptor pathways for their survival. In the case of c-Met function, cleavage of its cognate ligand HGF is required. This may involve expression of uPA/PAI-1 and matriptase/HAI-1 serine protease systems, which have been shown to be expressed in poor-prognosis, early-stage breast cancers.66,67 As growth factors, such as VEGF and HGF, are expressed in early development of invasive breast cancers, the stroma becomes more fibrotic (desmoplastic), expressing characteristic extracellular matrix proteins (such as tenacin C and versican).68 Angiogenesis also is a response of these developing lesions, contributing to their progression toward tumorigenesis.69 The E-cadherin cell-cell adhesion protein plays a central role in maintaining breast epithelial tissue structure, suppressing invasive behavior, and suppressing proliferation. In addition, it maintains a characteristic cytoskeletal organization. Loss of E-cadherin, through its genetic mutation or methylation, its expression, or its protein modification, is of central importance to progression of breast cancer. Not only does loss of E-cadherin serve to promote cell motility and invasion, but it also serves to release -catenin, an oncogenic-transcription factor.46 The E-cadherin gene promoter is under complex regulation by multiple factors, including negative regulation by the Snail zinc finger transcription factor. Interestingly, estrogen appears to down-regulate expression of E-cadherin in breast cancer cells, potentially contributing to the progression of the disease.70 However, estrogen also may function in some respects to suppress Snail expression (through a protein termed MTA3), to maintain epithelial morphology of breast tumors. Loss of functional E-cadherin contributes to an "epithelial-mesenchymal transition," characterized by expression of mesenchymal cadherins (-N and 11), more fibroblastic morphology, increased motility, and increased tissue invasiveness.71 Experimental studies targeting Rho family guanosine triphosphatase (involved in cell motility) have successfully suppressed metastatic dissemination of mammary tumor cells without modulating primary tumor growth.72 Malignant progression involves colonization of distant organs by the spreading tumor cells. cDNA gene expression microarray studies suggest the key genes involved in these final steps. For example, a study in bony metastasis has identified a characteristic multigenic program in such cells, involving interleukin-11, matrix metalloproteinase 1 (MMP-1), connective tissuederived growth factor, and bone-homing chemokine receptor.73 Implications of Molecular Biology for Tumor Prevention, Early Detection, Prognosis, and Response to Therapy A major hope in the study of genetic changes in breast cancer is that they will lead to development of new prevention and early-detection strategies, therapies, and prognostic tools.13 More rapid, accurate, and cost-effective assays of determining mutations in BRCA1 and BRCA2 are needed to better identify women with a familial propensity for breast cancer. In addition, the gene(s) responsible for a significant number of breast cancer families (more than half) remain to be identified, and the bases (genetic and/or environmental) for the variable penetrance of the BRCA genes remain to be determined. Women at high risk will undoubtedly be an important population of emphasis for future prevention trials. Although the benefits of prophylactic mastectomy and oophorectomy are now established, tamoxifen is also now known to be an effective prevention strategy. However, in the specific context of BRCA carriers, tamoxifen appears to be effective for prevention of breast cancer selectively initiated by BRCA2 (but not BRCA1).74 A major current trial is comparing tamoxifen to raloxifene (an antiestrogen thought to produce fewer endometrial cancers and to provide other benefits).13 However, new pharmacologic or dietary strategies are needed, particularly to prevent ER-negative breast cancer. For example, a novel approach could use the dietary compound indole-3-carbinol to block the effects of the ER to promote breast tumorigenesis.75 In the area of screening and early detection, the authors now know that overproduction of growth factors such as TGF- and HGF is very common, but they have not yet been shown to distinguish benign proliferative disease from malignant disease. It is possible that detection of telomerase expression, or the ability to detect subtle genetic alterations, will provide useful new approaches for marking the onset of cancer. Development of new nipple aspirate methodologies (such as ductal lavage), blood and urinary assays for growth factors, growth factor receptors, autoantibodies to oncoproteins, and tumor DNA are also currently under way. The new technique, made available by the rapid advancement of the field of proteomics, now renders these realistic avenues for exploration.76,77 In the area of prognosis and response to therapy, much hope has been vested in development of more accurate and rapid methods for immunohistochemical and fluorescent in situ methodologies for characterization of oncogenes, suppressor genes, and related proteins. Serum and plasma assays for growth factors are also of interest, in addition to more classic tumor markers of CA15.3 and CEA (carcinoembryonic antigen) for determining prognosis and response to therapy. Sensitive assays to detect tumor-derived cells and DNA in the blood are also under development. Again, advances in proteomic analysis of blood and tissue biopsies may lead the way for these studies in the next decade.78 Detection of ER and PR in tumors has established the field of integration of molecular markers into clinical decisions regarding prognosis and response to therapy. Gene expression microarray studies now provide a new way to classify breast cancers, as a basal epithelial class, an HER-2/neuoverexpressing class, an ER-positive luminal epithelial class, and an ER-luminal epithelial class have been distinguished in this manner.79 High-risk, early-stage breast cancers are also distinct in their gene expression profile from lower-risk early-stage cancers.80 These gene expression array methods will need to be converted to methods compatible with archival tissue analysis (such as RNA extraction/real-time PCR) for general application to breast cancer diagnosis and prognosis. As described in Growth Factor Pathways in the Normal and Malignant Gland, earlier in this chapter, other studies have identified specific growth factor receptor systems (Met/HGF) and protease inhibitor systems (uPA/PAI-1 and matriptase/HAI-1) that can be applied as poor-prognosis markers for early-stage disease using antibody/histochemistry methodology.66,67 Pure antiestrogens and aromatase inhibitors are now in clinical trials for ER-positive relapsing tumors. Studies to detect the co-expression of cyclin D1 and AIB-1 with ER have provided new insights into mechanisms for resistance to tamoxifen.28,87 A hope is that resistance to tamoxifen may be known prospectively and an alternate, ultimately more successful mode of hormonal therapy prescribed. The HER-2/neu oncoprotein also indicates poor-prognosis tumors and poor response to adjuvant hormonal therapy and chemotherapy. Although technically more difficult to measure, the EGFR is also of interest for future study of tumors that are likely to have a poor prognosis and poor response to hormonal therapy. Because of common expression or overexpression of these two receptors in breast cancer, they are now targets of new experimental therapies using tyrosine kinase inhibitors, antibodies to their extracellular domains, and coupling or gene-fusing these antibodies to toxic moieties.33,81 A large group of studies have confirmed that 20% to 30% of breast tumors contain an amplification of the HER-2/neu gene and overexpress the encoded receptor protein. HER-2/neu is also overexpressed in a very high portion of ductal carcinoma in situ. Expression of the HER-2/neu protein is associated with an elevated mitotic rate; it correlates with poor clinical response to certain chemotherapeutic and antihormonal drugs [5-fluorouracil, methotrexate, cyclophosphamide (Cytoxan), and tamoxifen-containing regimens] and insensitivity to tamoxifen in vitro. HER-2/neu expression is also associated with poor prognosis in patients who do not receive treatment with chemotherapeutic or antihormonal drugs. The HER-2/neu gene amplification association with poor prognosis may relate to response to treatment with chemotherapy and hormonal therapy.82,83 Gene expression profiling is now yielding new insights into the determinants of resistance of breast cancer to chemotherapy.84,85 The HER-2/neu protein also holds significant interest for breast tumor immunology. Certain antibodies to the extracellular domain of this protein seem to sensitize cells to killing by cis-platinum, carboplatinum, and doxorubicin in vivo. It is thought that the mechanism of this effect is interference with DNA repair mechanisms. As noted above, the shed extracellular domain of HER-2/neu may represent a useful antigenic blood-borne marker of breast cancer burden and the protein itself a new target of immunotherapy of cancer.33,86 Specifically, the humanized, antiHER-2/neu antibody termed trastuzumab was effective in clinical trials and is now part of standard therapy regimens.33 As noted in Growth Factor Pathways in the Normal and Malignant Gland, earlier in this chapter, results also suggest the possibility of active immunotherapy targeting the HER-2/neu protein86; a lymphoplasmocytic infiltrate in breast cancer was initially shown by Pupa and coworkers to indicate good prognosis for HER-2/neupositive patients. This study also noted production of growth-inhibitory antibodies by peripheral lymphocytes from these patients. The EGFR is also a target for therapy, with drugs such as tyrosine kinase inhibitors and humanized antibodies. However, clinical results with these approaches have not been as successful, to date, as with HER-2/neudirected approaches.81 As has been emphasized, nuclear protooncogenes are common mechanistic links between the actions of growth-promoting steroids and growth factors in diverse tissues. Study of c-MYC gene expression in breast cancer, using new fluorescence in situ and PCR methodologies, is likely to improve c-Myc studies in human breast cancer. The c-Myc protein has a very short half-life, and, until quite recently, no high-quality monoclonal antibodies were available that are capable of staining paraffin sections. Amplification of c-MYC gene is associated with poor prognosis. EGF family growth factors enhance c-Myc oncoprotein-induced tumorigenesis. Thus, targeting the EGFR or the HER-2/neu together with c-Myc may be useful therapeutic strategies. Other nuclear oncogenes, cyclin D1 and AIB-1, have already been discussed in association with ER function in ER-positive disease.28 However, with ER-positive disease, a good-prognosis category, the additional presence of expression of cyclin D1 is of poor prognostic consequence.81 More research is needed to more fully understand these complexities. p53 protein mediates apoptotic death induced by virtually all forms of adjuvant therapy and is easily measured by immunohistochemical methodology. However, wild-type and mutant proteins are difficult to distinguish immunologically. TP53 overexpression confers poor prognosis and likelihood of a poor response to endocrine therapy and chemotherapy. p53 is a potential agent for gene therapy trials in cancer,48 as are the downstreams of p53. First, loss of pRb, detected by immunohistochemistry, appears to indicate poor prognosis,88 as does expression of Bcl-2 and down-regulation of Bax.54 However, more studies need to be carried out on RB-1 mutation analyses and on detection of other Bcl family members. Finally, a new area of study is the identification of poor prognosis and response to therapy genes by use of high-throughput cDNA chip assay analyses. Such studies have the potential to improve tumor diagnosis and prognosis.84,85 Studies of metastasis have suggested that quantification of tumor angiogenesis and deposition of specific extracellular matrix proteins may be of supplementary value in prognostication with traditional lymph node biopsy measurements.68,69 Promising new drugs are in clinical trial for blockade of angiogenesis.69 Metastasis itself seems to depend on the elaboration of proteases, the most promising of which, for prognostic significance, are PAI-1, cognate inhibitor of urokinase, and HAI-1, cognate inhibitor of matriptase.66,67 In addition, when tumors express uPA/PAI-1, they are more sensitive to chemotherapy.89 Although anti-MMP drugs, such as marimastat, have not shown much promise in clinical trials,90 blockade of other classes of proteases (such as these serine proteases) will be a very active area of future drug development. Finally, the adhesive changes that metastatic cells undergo are of major interest. Loss of expression of E-cadherin (and acquisition of a mesenchymal phenotype marked by the intermediate filament vimentin), loss of 21 integrin, overexpression of a 67-kD laminin-binding protein, and overexpression of a variant form of the hyaluronic acid receptor (CD44) are all of poor prognosis or correlated with poor tumor grade in early clinical studies.91 Much larger studies are needed to fully evaluate the clinical significance of these metastasis-associated cellular changes. Antiangiogenic drugs are now undergoing extensive testing as anticancer agents. Although angiostatin and endostatin received early attention, probably one of the most promising antiangiogenic targets now is VEGF.69 Recent work has also found active agents to target breast cancer in the metastatic site of bone. Specifically, bis-phosphorates inhibit osteoclast-mediated bone resorption and may selectively induce through antideath growth factor effects in the metastatic site cells.92 Thus, significant promise for antimetastatic approaches to therapy exist. In summary, although a very large number of genetic and phenotypic alterations have been suggested in breast cancer, only a handful have been fully identified and brought to clinical study. It is quite encouraging that study of each of these genes and phenotypic changes has provided its own unique perspective to the biology of the disease. The challenge for the future, however, is to take advantage of this knowledge to improve detection of familial risk; develop prevention strategies; improve early detection, clinical diagnosis, and prediction of therapeutic outcome; and develop therapies and rapidly apply more novel biologic therapeutics. It is the authors' prediction that the next decade of discovery in breast cancer will focus on the molecular basis of treatment failure: invasion, angiogenesis, metastasis, and resistance to therapy. Central to these hopes is the development of new technologies for high-throughput analyses of pathologic material, such as laser capture microdissection techniques, FISH, and other molecular cytogenetic methods; cDNA chip array assay methods; SAGE (serial analysis of gene expression); and other cutting-edge RNA and protein analysis techniques. It is essential that new technologies be developed to improve tumor diagnosis and prediction of response to existing therapies. These approaches should also result in our discovery of new targets for more biologic-based therapies and prevention strategies for the disease. Prospects for clinical translation of basic molecular biologic results continue to be bright. References1. Seifried HE, McDonald SS, Anderson DE, et al. 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