A Role for Id Proteins in Mammary Gland Physiology and Tumorigenesis Paola de Candia, Robert Benezra, and David B. Solit Memorial Sloan-Kettering Cancer Center, Program in Cell Biology and Department of Medicine, New York, New York 10021

I. Introduction II. The Role of ID Proteins in Embryogenesis, Breast Development, and Lactation A. A Potential Role for Id1 and Id3 in Normal Mammary Development B. A Role for Id2 in Mammary Epithelial Differentiation and Lactation C. Id4 Expression Is Restricted Primarily to the Central Nervous System III. The Role of ID Proteins in Breast Tumorigenesis A. Id1 and Id3 Expression in Breast Tumors B. The Role of Id1 and Id3 in Breast Tumor Angiogenesis C. Id4 Involvement in the Regulation of BRCA1 IV. Conclusions References

Id helix-loop-helix (HLH) proteins are regulators of cell growth and differentiation in embryonic and adult tissues. They are members of the basic HLH family of transcription factors but lack a DNA binding domain. By binding to basic HLH transcription factors, Id proteins regulate gene expression. Id1 and Id3 have extensive sequence homology and similar patterns of expression during embryogenesis and in adult tissues. They are also expressed at high levels in the endothelial cells of tumor-infiltrating blood vessels, and breast tumors spontaneously arising in MMTV-neu mice demonstrate impaired angiogenesis when growing in an Id1- and/or Id3-deficient background. These lesions are typically cystic with a small rim of viable tumor cells surrounding an acellular necrotic core. Id2 plays a critical role in breast differentiation and lactation. Id4 regulates BRCA1 expression and may be involved in hormone-dependent regulation of BRCA1 homeostasis. Thus, all four members of the Id protein family play pivotal roles in distinct aspects of normal and malignant breast biology, the subject of this review. ß 2004 Elsevier Inc.

I. INTRODUCTION The Id genes encode HLH proteins that regulate a wide range of cellular processes (Ruzinova and Benezra, 2003). The first Id gene (Id1) was identified over a decade ago and named for its ability to inhibit DNA binding of basic HLH (bHLH) transcription factors (Benezra et al., 1990). The bHLH superfamily consists of 125 genes grouped into 44 families based on their Advances in CANCER RESEARCH 0065-230X/04 $35.00

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tissue distribution, dimerization capabilities, and DNA binding specificities (Massari and Murre, 2000; Murre et al., 1994). Each member of the family has a highly conserved HLH dimerization domain. Ubiquitously expressed bHLH proteins, such as the E12/E47 family members, serve as obligate dimerization partners of tissue-specific bHLH transcription factors such as MyoD and NeuroD. These heterodimers bind to DNA by means of a basic DNA binding region contained within each protein (Fig. 1) and activate the process of cellular differentiation. Id proteins possess an HLH dimerization domain but lack the basic DNA binding domain. Therefore, Id containing heterodimers cannot bind DNA (Fig. 1). By binding to and sequestering the ubiquitously expressed E-proteins, Id proteins serve as negative regulators of gene expression and, consequently, of cellular differentiation in many different cell lineages. Basic HLH transcription factors are not found in prokaryotes but are present in all major subdivisions of eukaryotes, suggesting that they appeared early in eukaryotic evolution. Id-like bHLH members lacking a DNA binding domain are present in several metazoan species and have been characterized in Drosophila melanogaster, where a single locus, extramacrochaetae, encodes an Id-like protein. In the mouse and human genomes, four Id proteins (Id1-4) have been identified. They contain a highly conserved HLH domain that is more similar among them than to the HLH

Fig. 1 (a) In the absence of Id proteins, ubiquitously expressed bHLH proteins heterodimerize with tissue-specific bHLH proteins and activate transcription of tissue-specific genes. (b) Id proteins sequester ubiquitously expressed bHLH proteins, inhibiting their binding to tissue-specific bHLH proteins and thus blocking bHLH-dependent transcription.

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domains of other bHLH proteins (Deed et al., 1994). Outside the HLH domain, Id proteins display extensive sequence divergence. In recent years, binding of Id proteins to and thus regulation of nonbHLH targets has been described (Moldes et al., 1999; Roberts et al., 2001; Shoji et al., 1995). For example, Id proteins bind to several of the key regulators of cell cycle progression such as pRB (Iavarone et al., 1994; Lasorella et al., 1996) and the Ets-family of transcription factors (Ohtani et al., 2001). Though initially described as inhibitors of cellular differentiation, Id proteins have more recently been identified as regulators of cell cycle progression, senescence, and apoptosis (Ruzinova and Benezra, 2003). Evidence also suggests that Id proteins may be key regulators of oncogenic transformation and tumor progression in a subset of tumor types (Benezra, 2001; Israel et al., 1999; Yokota and Mori, 2002). In this review, our current knowledge of the role of Id proteins in normal breast development and in breast cancer transformation and progression will be discussed.

II. THE ROLE OF ID PROTEINS IN EMBRYOGENESIS, BREAST DEVELOPMENT, AND LACTATION A. A Potential Role for Id1 and Id3 in Normal Mammary Development Much of our understanding of the function of the Id proteins in vivo has been derived from the study of Id knockout mice. Id genes are expressed throughout mouse embryogenesis, with Id levels peaking at mid-gestation and declining thereafter (Duncan et al., 1992; Jen et al., 1996, 1997). Id1 and Id3 have nearly overlapping patterns of expression during development; single knockout Id1 and Id3 mice are viable and fertile. In contrast, Id1 and Id3 double knockout mice are embryonic lethal, with death occurring at day 12.5 as a result of premature neuronal differentiation and defective brain angiogenesis (Lyden et al., 1999). By histological examination, Id1
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mice have no detectable phenotype but, when crossed with E2A knockout mice, the loss of Id1 partially rescues the high neonatal lethality observed in these mice (Yan et al., 1997). Id3
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mice display defects in humoral immunity (Pan et al., 1999) and a block in thymocyte maturation during the transition from single- to double-positive thymocytes (Rivera et al., 2000). Neither Id1 nor Id3 separately are essential for breast development, and the single Id1 or Id3 null mice do not show lactation defects. The specific biologic function of Id1 in mammary epithelium was first explored using SCp2 cells, an immortalized line derived from the mammary gland of a midpregnancy mouse (Danielson et al., 1984). When SCp2 cells

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ectopically expressing Id1 are induced to differentiate, they continue to proliferate and are unable to fully differentiate or form stable alveoli in vitro (Desprez et al., 1995). One potential mechanism whereby Id1 regulates mammary cell proliferation and differentiation is by inducing the GTPase-activating zinc finger protein Zfp289 (Singh et al., 2001). Zfp289 has high homology with the yeast GTPase-activating zinc finger protein Gcs-1, which has been identified as a gene required for the reentry of cells into the cell cycle after the stationary growth phase (Ireland et al., 1994). Id1 has also been shown to induce apoptosis in confluent SCp2 cells, a condition resembling mammary epithelial involution in vivo (Parrinello et al., 2001). While these data suggest a function for Id1 in the physiological remodeling of the mammary gland, Id1 null, Id3 wild-type mice, as stated, demonstrate normal breast development, lactation, and involution. The analyses of Id1 and Id3 single and double knockout mice clearly indicate that substantial functional overlap between Id1 and Id3 exists and may thus be relevant in normal breast embryonic development and lactation. Therefore, novel model systems with conditional loss of both Id1 and Id3 in the breast epithelium will be necessary to further elucidate the roles of Id1 and Id3 in normal mammary physiology.

B. A Role for Id2 in Mammary Epithelial Differentiation and Lactation Id2 knockout mice have significant (25%) perinatal lethality (Yokota and Mori, 1999). The surviving Id2-null mice fail to form lymph nodes and Peyer’s patches and exhibit a greatly reduced population of natural killer, Langerhans, and splenic dendritic cells (Hacker et al., 2003; Kusunoki et al., 2003; Yokota and Mori, 1999). Id2 knockout mice have normal embryonic development of breast tissue but show morphological defects during pregnancy and following delivery; a defect in lactation is observed (Mori et al., 2000). The nursing behavior of Id2
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mothers is normal, but their pups die within 2 days of birth. Id2
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virgin females have normal appearing ductal trees. On the day of delivery, though, their mammary glands appear immature and have a decrease in lobulo-alveolar tissue as compared to Id2 wild-type controls (Mori et al., 2000). By immunoblot, Id2 is barely detectable in virgin glands of Id2 wild-type mice. During pregnancy, expression of Id2 increases and peaks by day 18, remaining elevated throughout lactation, during which breast epithelium maintains a fully differentiated phenotype (Parrinello et al., 2001). Transplantation experiments demonstrate that the lactation defect is the result of Id2 deficiency in the breast epithelial cells rather than Id2 deficiency in other

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breast cell types or alteration in the circulating hormonal milieu. Breast epithelial cells from Id2 wild-type mice transplanted into wild-type recipients develop large alveoli with expanded alveolar lumina showing active milk secretion at parturition, as judged by the presence of large lipid droplets in the cytoplasm. In contrast, breast epithelium derived from Id2
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mice and transplanted into wild-type recipients demonstrate disorganized alveolar development with small central lumens and disrupted cellular contacts (Miyoshi et al., 2002). In the Id2 null transplanted tissue, alveolar cells show limited secretory activity, which is not directed toward the apical membrane (i.e. the secreting side) and lumen. There is evidence of only small lipid droplets, and they are often found in the basal region of secretory cells. A defect in myoepithelial cells is also observed. While in wild-type animals, these cells are flat and closely attached to the secretory cell layer; in Id2 null animals they have a round shape and appear detached from the central cells (Miyoshi et al., 2002). At a molecular level, the absence of Id2 expression in the epithelial cells of Id2
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mice causes a proliferation defect in the early stage of pregnancy (7 days), accompanied by up-regulation of p21WAF1 and p27Kip1. An increase in apoptosis is observed by day 14, accompanied by an elevation of p53 and the proapoptotic gene bax (Mori et al., 2000). Expression profile studies using a ‘‘mammochip,’’ a cDNA microarray enriched in mammary specific sequences, indicate that Id2 null tissues at term exhibit an expression profile similar to wild-type tissues at earlier stages of pregnancy (Miyoshi et al., 2002). Differentiation markers such as -casein, -lactalbumin, and whey acidic protein, which are expressed sequentially in the mammary glands of pregnant mice, are underexpressed, while -, -, and -caseins are completely absent in Id2 null pregnant mice (Miyoshi et al., 2002). Rab18, a small guanosine triphosphatase involved in the regulation of vesicular transport, is up-regulated in wild-type mammary glands at term, but is not expressed in the mammary glands of Id2
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mice at the same stage of development (Miyoshi et al., 2002). This finding could explain the lack of directed milk protein secretion in Id2 null mammary epithelium. While this may seem paradoxical to the role of Id proteins as inhibitors of differentiation, loss of Id2 may propel cells into the wrong differentiation pathway, resulting in the loss of physiological differentiation markers. A correlation between Id2 expression and mammary epithelial differentiation is also observed in vitro. Proliferating SCp2 cells lack detectable levels of Id2, but when induced to differentiate, Id2 expression is increased (Parrinello et al., 2001). In differentiated SCp2 cells, induced overexpression of Id2 leads to increased levels of -casein, suggesting that Id2 is able to accelerate differentiation. On the other hand, when the same cells are infected with a viral vector containing Id2 in the antisense orientation, -casein expression remains almost undetectable, suggesting that depletion

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of Id2 alone can inhibit differentiation (Parrinello et al., 2001). As stated, this may be due to the loss of Id2 propelling cells into a differentiation compartment where these physiological markers are not expressed.

C. Id4 Expression Is Restricted Primarily to the Central Nervous System In contrast to the overlapping embryonic expression patterns of Id1, Id3, and Id2, Id4 has a distinct pattern of gene expression during embryogenesis, and its expression is primarily restricted to the central nervous system (Jen et al., 1997). Id4 knockout mice exhibit growth retardation and significant embryonic and early postnatal mortality. The surviving adult Id4
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mice have alterations in brain morphology, such as fewer neurons and glia, an overall reduction in brain size, and abnormally enlarged ventricles (Yokota, 2001). No phenotypic abnormalities in breast physiology have been reported to date.

III. THE ROLE OF ID PROTEINS IN BREAST TUMORIGENESIS A. Id1 and Id3 Expression in Breast Tumors Id1 is the Id family member most extensively studied in human cancer. Id1 overexpression has been observed in endometrial (Takai et al., 2001), ovarian (Schindl et al., 2003), cervical (Schindl et al., 2001), medullary thyroid carcinomas (Kebebew et al., 2000), and melanoma (Polsky et al., 2001), as well as other malignancies (Norton et al., 1998). In the breast cancer cell line T47D, Id1 has been reported to be a downstream effector of estrogen in promoting cell proliferation (Lin et al., 2000). In addition, constitutive expression of Id1 renders these cells refractory to growth inhibition by progesterone. Expression of Id1 correlates with the secretion of a 120-kDa gelatinase, having the characteristics of a matrix metalloproteinase, and with invasive potential in a variety of human breast cancer cell lines (Desprez et al., 1998). Moreover, Id1 expression is found to be dysregulated in highly invasive metastatic mammary carcinoma cell lines (Lin et al., 2000). A complex, containing SP-1, NF-1, Rb, and HDAC-1 proteins, binds a 31 bp sequence within the Id1 promoter, located 200 bp upstream of the initiation of transcription. Dysregulation of this complex in highly invasive cell lines may be responsible for the constitutive expression of Id1 in these cells (Singh et al., 2002).

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While these studies strongly suggest that Id1 may play a role in breast cancer development and progression, studies of Id1 expression in human tumor samples have yielded conflicting results. In a study by Lin (2000), 10 ductal carcinomas in situ (DCIS) and 12 infiltrating breast carcinomas were evaluated for Id1 expression by immunohistochemistry (IHC). In this study, Id1 was found to be expressed more frequently in infiltrating ductal carcinomas as compared to DCIS [Id1 was expressed in 8/12 (66%) infiltrating carcinomas [versus 2/10 (20%) DCIS samples] (Lin et al., 2000). A second larger study by Schoppmann (2003), using IHC, analyzed the frequency of Id1 expression in the tumor cells of 191 breast cancer patients. In this study, 15 (7.9%) specimens had strong expression, 75 (39.3%) moderate expression, 55 (28.8%) weak expression, and 46 cases (24.1%) had no Id1 expression. Furthermore, Id1 expression was a negative predictor of overall and disease-free survival (Schoppmann et al., 2003). In contrast to these findings, a recent analysis of Id1 expression in human mammary tumors demonstrated high levels of Id1 expression in the endothelial cells of tumor-infiltrating blood vessels but not in the breast tumor cells (De Candia et al., 2003). In 30/30 and 29/30 invasive breast carcinoma samples, Id1 and Id3 mRNAs were expressed in the tumor vasculature as determined by in situ hybridization. In contrast, Id1 and Id3 mRNA expression was observed in tumor cells in only a subset of the samples evaluated (33% for Id1, 43% for Id3). These RNA probes for Id1 and Id3 have been shown to be highly specific (Lyden et al., 1999). By IHC, Id1 protein expression, however, was restricted to the vascular endothelial cells and was never observed in breast tumor cells (De Candia et al., 2003). The discrepancy between the in situ hybridization and IHC findings regarding Id1 expression in at least a subset of tumor samples may be due to differences in sensitivity of the two techniques or posttranscriptional regulation of Id1. A similar pattern of Id1 and Id3 expression has also been reported in a murine model of breast cancer (De Candia et al., 2003). In tumors arising in MMTV-neu transgenic mice, which spontaneously develop mammary adenocarcinomas, high levels of Id1 expression by IHC were observed in tumor endothelial cells but never in the tumor cells themselves (Fig. 2). This result is consistent with the expression pattern of Id1, described in human tissue samples by De Candia et al., (2003) as outlined above. The disparate profiles of Id1 protein expression in breast tumors reported by these groups is likely due to differences in tissue processing and the grading systems employed. Antibody specificity may also be variable. Even though the same antibody was used in all the studies reported to date, significant batch-to-batch variability has been observed with this antibody (De Candia, unpublished observations). The use of tissues from Id1 null mice as negative controls has proved to be critical in ensuring specificity and minimizing the problem of nonspecific binding. For example, the Id1

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Fig. 2 Id1 expression is restricted to the nuclei of endothelial cells of breast tumors that develop spontaneously in MMTV-neu, Id1 wild-type mice. Endothelial cells of tumor-infiltrating blood vessels from Id1 knockout mice do not stain for Id1 and serve as a negative control.

antibody stains the cytoplasm of both myoepithelial and smooth muscle cells in Id1 knockout breast tissues, and thus this staining is attributable to nonspecific antibody binding (Uehara et al., 2003, unpublished observations). Using Id1
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tissues as negative control, it has also been established that the high level of Id1 expression in tumor-infiltrating endothelial cells is indeed specific (De Candia et al., 2003); Fig. 2. Different patterns of Id1 subcellular localization have also been reported by various groups. While previous reports showed Id1 to be localized to the cytoplasm of human tumor cells with clear exclusion of the nuclei (Schoppmann et al., 2003), more recent reports indicate that the Id1 present in the endothelial cells of both human and murine breast tumors is confined primarily to the nucleus (De Candia et al., 2003; Fig. 2), where it would be predicted to be found as an inhibitor of bHLH transcriptional factors. Further studies and multiple antibodies against Id1 will be needed to clarify the expression pattern of this protein in normal and pathological breast tissue.

B. The Role of Id1 and Id3 in Breast Tumor Angiogenesis As discussed earlier, Id1
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Id3
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mice are not viable and die during early embyogenesis due to forebrain hemorrhage (Lyden et al., 1999). This phenotype highlighted for the first time a role for Id1 and Id3 in developmental angiogenesis. Further experiments also established an important function for these factors in the formation of tumor blood vessels. When mice missing from one to three copies of the Id1 and Id3 genes were challenged

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with tumor xenografts, a dramatic inhibition or a significant delay of tumor growth was observed. As an example, implantation of the murine breast cancer cell line (B-CA) in Id1þ/
Id3
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mice resulted in a small peak in tumor growth on day 5, followed by complete regression by 20 days post transplant. Histological examination of tumor xenografts demonstrated a clear impairment in tumor neovascularization, with stunted and occluded vessels in the Id1- and Id3-deficient mice (Lyden et al., 1999). To further study the role of Id1 and Id3 in the development and progression of breast cancer in a more physiologic tumor model than xenografts, MMTV-neu transgenic mice, previously cited, were bred with Id1- and Id3deficient mice. Surprisingly, in contrast to the phenotype observed with the transplantable tumors, Id1 and Id3 deficiency did not prevent or delay tumor formation, but it did alter the tumor histopathology dramatically (De Candia et al., 2003). Tumors arising in Id-deficient mice had a cystic morphology with a viable rim of tumor cells surrounding a nonviable core of cellular debris (Fig. 3). This hollow core may have been the result of necrosis or apoptosis of the central tumor cells or vascular leakage and extravasation of fluid from the abnormal Id-deficient vasculature. These tumors progressed at a similar rate as compared to the solid tumors in Id wild-type mice, and survival was similar in the Id wild-type and Id-deficient populations. As tumors arising in Id-deficient mice frequently demonstrate only a small rim of viable tissue surrounding a nonviable hemorrhagic core, it was hypothesized that drugs targeting the viable rim may be more effective in this setting. 17-AAG, an Hsp90 inhibitor, causes the degradation of client proteins that depend on this molecular chaperone for maturation or stability.

Fig. 3 Breast tumors arising in MMTV-neu, Id1- or Id3-deficient mice are frequently cystic with a small rim of viable tumor cells surrounding a fluid filled cyst. Breast lesions arising in Id1 wild-type mice show a solid phenotype being composed of a homogeneous sheet of tumor cells.

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HER2/neu is one of the most sensitive targets of this agent and is potently degraded and thus inactivated in murine tumors at nontoxic doses (Solit et al., 2002). Treatment of MMTV-neu, Id wild-type mice resulted in only a modest delay in tumor growth. However, in mice with Id deficiency, 17-AAG was dramatically more effective (De Candia et al., 2003), perhaps due to the ability of 17-AAG to target for degradation both HER2 (the tumor initiating oncogene) and HIF1 (a mediator of the hypoxic stress response; Mabjeesh et al., 2002). These data suggest that breast tumor progression can occur in a background of defective angiogenesis such as that observed in Id1- and Id3deficient mice, but that tumors developing in such an environment may be especially sensitive to drugs such as 17-AAG. Thus, it has been proposed that targeting both the tumor neovascularization and the Hsp90 stress response may be effective in patients with advanced breast cancers. Moreover, the fact that the Id1 and Id3 proteins are not essential for breast physiological remodeling, but play crucial roles in breast tumor vascularization, makes them ideal targets for breast cancer drug development.

C. Id4 Involvement in the Regulation of BRCA1 A link has recently been established between Id4 and the breast and ovarian cancer susceptibility factor BRCA1 (Beger et al., 2001). This gene functions as a tumor suppressor, and loss of heterozygosity of the wild-type copy is observed in breast tumors arising in BRCA1 mutation carriers (Miki et al., 1994). BRCA1 mRNA levels are reduced or undetectable in sporadic breast and ovarian carcinomas and breast cancer cell lines (Thompson et al., 1995; Wilson et al., 1999). CpG methylation of the promoter may contribute to this decreased expression (Dobrovic and Simpfendorfer, 1997), but it is unlikely to be the sole mechanism (Catteau et al., 1999). By using a ribozyme library, an ‘‘inverse genomics’’ approach led to the identification of cellular factors regulating BRCA1 expression. Ribozymes are catalytic RNA molecules that recognize RNA targets based on sequence complementarity, and enzymatically cleave these RNA targets, thereby inhibiting the expression of the corresponding gene (Haseloff and Gerlach, 1988). When the ribozyme library is expressed in cells, the subsequent selection for a given phenotype enables the identification of ribozymes capable of inducing that particular phenotype. The target recognition sequence of those ribozymes is then used to identify the corresponding target gene(s). By using this approach, Id4 was identified as a gene involved in repressing BRCA1 expression and increasing anchorage-independent growth and colony formation in soft agar (Beger et al., 2001). Following estrogen stimulation, BRCA1 expression increases while Id4 RNA expression is significantly reduced, suggesting that Id4 may be involved in hormone-dependent regulation of BRCA1 expression

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(Beger et al., 2001). The precise mechanism whereby Id4 regulates BRCA1 remains to be elucidated. Microarray analysis comparing transcription profiles of epithelial cells with low endogenous levels of BRCA1 versus transcription profiles of cells with two- to four-fold higher induced levels of BRCA1 expression show that Id4 expression is activated in the latter cells (Welcsh et al., 2002). This reciprocal regulation, by which BRCA1 induces Id4 and Id4 negatively regulates BRCA1, may establish a regulatory loop that functions to maintain appropriate levels of expression of both genes during normal cell division. The expression of BRCA1 and Id4 has also been evaluated by IHC in primary infiltrating ductal breast tumors, and the staining intensities of the two proteins have been found to be highly correlated (Welcsh et al., 2002). This result suggests that the BRCA1-Id4 regulatory loop may be disrupted in many breast cancers.

IV. CONCLUSIONS Since their discovery more than 10 years ago, Id proteins have been identified as regulators of cell growth, apoptosis, cell survival, senescence, oncogenesis, and tumor progression in a variety of cell types. A role for all four Id proteins has been proposed in either normal breast physiology or tumor development and progression. Id1 and Id3 have significant functional overlap and similar patterns of expression. Breast development is normal in Id1 and Id3 single-knockout mice, but these mice demonstrate abnormalities in tumor angiogenesis. Id2 plays a critical role in breast differentiation and lactation, while Id4 may be a critical regulator of BRCA1. Further studies will be required to fully elucidate the different functions of Id proteins in normal and malignant breast development.

ACKNOWLEDGMENTS We thank Dr. M. Ruzinova for critically reading the manuscript. This work was supported by the American Italian Cancer Foundation and by grants from the Breast Cancer Research Foundation and the National Institutes of Health.

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