The Role of the Epidermal Growth Factor Receptor Family in Mammary Tumorigenesis and Metastasis

Experimental Cell Research 253, 78 – 87 (1999) Article ID excr.1999.4706, available online at http://www.idealibrary.com on

The Role of the Epidermal Growth Factor Receptor Family in Mammary Tumorigenesis and Metastasis Harold Kim* ,† and William J. Muller* ,‡ ,1 *Institute for Molecular Biology and Biotechnology, †Department of Medical Sciences, and ‡Department of Pathology, McMaster University, Hamilton, Ontario, Canada L8S 4K1

els of expression of the various EGFR family members have been observed in both primary breast cancers and their derived cell lines. For example, amplification and the consequent overexpression of ErbB-2 have been observed in a significant proportion of primary human breast cancers [2, 3]. Moreover, the extent of overexpression of ErbB-2 has been inversely correlated with patient survival in both node-negative and -positive breast cancer cases [4, 5]. More recent studies have also implicated the expression of other EGFR family members including EGFR, ErbB-3, and ErbB-4 in the genesis of human breast cancer [6 – 8]. These observations suggest that the expression of different members of the EGFR family may play a critical role in mammary tumorigenesis. There is also compelling evidence implicating the expression of various EGFR family ligands in the induction of mammary tumors. For example, the expression of EGFR-specific ligands such as TGFa and EGF can be detected in primary breast cancers [9 –11]. Moreover, expression of Neu differentiation factor (NDFs) or heregulins which are ligands for either ErbB-3 or ErbB-4 has been implicated in the pathogenesis of human breast cancer [12]. Although ErbB-2 cannot bind directly the ligands NDF or EGF, its activity can be influenced profoundly by the expression of these growth factors. For example, ErbB-2 is the substrate of the activated EGFR following stimulation of cells with EGF or TGFa. Similarly, ErbB-2 can be transphosphorylated by either ErbB-3 or ErbB-4 following stimulation of mammary tumor cells with NDFs [13]. The ability of these growth factors to modulate the activity of ErbB-2 is believed to be mediated through the formation of specific heterodimers of ErbB-2 and the different EGFR family members [14 – 19]. The importance of heterodimerization between the different family members is further highlighted by the observation that expression of activated forms of the individual family members is not sufficient to transform fibroblasts lacking all endogenous EGFR family members [20]. Conversely, coexpression of ErbB-2 and EGFR or ErbB-2 and ErbB-3 results in the efficient

A number of receptor systems have been implicated to play an important role in the development and progression of many human cancers. The epidermal growth factor (EGF) receptor tyrosine kinase family has been found to consistently play a leading role in tumor progression. Indeed, in human breast cancer cases the prognosis of a patient is inversely correlated with the overexpression and/or amplification of this receptor family. Furthermore, downstream signaling components such as the Src kinases, PI3*K, and the Ras pathway display evidence of deregulation that can accelerate tumor progression. The transgenic mouse system has been ideal in elucidating the biological significance of this receptor family in mammary tumorigenesis. Molecular events involved in mammary tumorigenesis such as ligand binding, receptor dimerization, and the activation of downstream pathways have been addressed using this system. Although there are many molecular steps that appear to drive each stage of tumor development, the EGF receptor family appears to play a causal role in the progression to a transformed phenotype. © 1999 Academic Press Key Words: transgenic; epidermal growth factor receptor; erbB2; neu; mammary; tumorigenesis.

INTRODUCTION

The progression of a primary mammary epithelial cell to a malignant phenotype is believed to involve multiple genetic events including the activation of dominant-acting oncogenes and the loss of specific tumor suppressor genes. In this regard, the activation of the epidermal growth factor receptor tyrosine kinases (EGFR RTKs) has been strongly implicated in the development and progression of human cancers. The EGFR family comprises four closely related type 1 RTKs that include the EGFR, ErbB-2 (Neu, HER2), ErbB-3 (HER3), and ErbB-4 (HER4) [1]. Elevated lev1

To whom correspondence and reprint requests should be addressed. Fax: (905) 521-2955. E-mail: [email protected]. mcmaster.ca. 0014-4827/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

78

ROLE OF EGFR FAMILY IN MAMMARY TUMORIGENESIS AND METASTASIS

transformation of both fibroblasts and mammary epithelial cells in vitro [21–24]. Taken together, these observations suggest that the efficient transformation of these cells requires the concerted action of ErbB-2 and another member of the EGFR family. The central importance of ErbB-2 in the hierarchy of EGFR interactions has been further demonstrated by ablating ErbB-2 function in breast cancer cells by use of single-chain ErbB-2 antibodies. Expression of the single-chain antibody in these cells ablates the ability of these breast cancer cell lines to respond to mitogenic stimulation of both EGFs and NDFs [25, 26]. Consistent with this observation, a kinase-deficient as well as a truncated form of Neu can suppress transformation and tumorigenicity via the inability to activate the receptor heterodimer formed between Neu and the EGFR [27–29]. In contrast, the generation of a kinasedeficient EGFR [30] or a carboxyl-terminal truncated EGFR [31] coexpressed with wild-type ErbB-2 results in heterodimerization, transphosphorylation of ErbB-2, substrate activation, mitogenesis, and ErbB2-mediated association of Grb2 and Shc [31] suggesting the importance of ErbB-2/Neu in heterodimer formation and receptor activation. The molecular basis for the cooperative ability of the different EGFR family members to transform cells may reflect the distinct signaling specificity of the different heterodimers. For example, activation of ErbB-3 by NDFs is believed to recruit phosphatidylinositol 39kinase (PI-39K) to the inner face of the cytoplasmic membrane through the formation of an ErbB-2/ErbB-3 heterodimer [23, 32]. Indeed, the ErbB-3 RTK possesses six consensus tyrosine autophosphorylation sites for the SH2 domain of the p85 subunit of PI-39K [23, 32] suggesting that ErbB-3 mediates the majority of PI-39K signaling. In addition, EGF stimulation has been observed to correlate with an increase in Src activity. Indeed, the ability to respond to EGF is augmented by Src overexpression in fibroblasts [33, 34] as well as in established tumor cell lines where EGF stimulation induces a rapid and sustained increase in Src family kinase activity [35]. Moreover, profiles of tyrosine hyperphosphorylation on the EGF receptor have identified a number of sites that correlate with the overexpression of Src [36, 37] suggesting synergy between Src and the EGF receptor in mediating a biological response [38, 39]. However, evidence also suggests strongly that among the EGFR family the ErbB-2 RTK appears to be the preferred binding partner for the Src family of tyrosine kinases both in vitro and in vivo. Therefore, EGF stimulation may initiate the formation of an ErbB-2/EGFR heterodimer which then permits Src to bind directly and specifically to ErbB-2 thus providing a further mechanism for signaling specificity [40 – 42]. Another signaling component discovered to play a role in EGFR family signaling is

79

the signal transducer and activator of transcription (STAT) factors. With regard to ligand-induced signaling specificity, treatment of cells with NDF results in the association of STAT5 with a specific ErbB-4/EGFR heterodimer, in contrast to the specific association of STAT5 with only the EGFR in EGF-stimulated cells [43] suggesting that differential STAT activation is modulated by ligand binding to specific heterodimers. Recently, the molecular mechanism of receptor specificity mediated by various combinations of ligands and receptor heterodimers was addressed. Specifically, both Shc and Grb2 appear to associate to the EGFR upon EGF stimulation; however, in the presence of NDF the EGFR only associates with Shc while Grb2 association is not detectable. Furthermore, the rate of receptor internalization as well as temporal differences in substrate activation varied depending on the ligand used. For example, stimulation of the EGFR with EGF results in a strong biphasic association with the p85 subunit of PI-39K that correlates with PI-39K activity. However, upon stimulation with NDF, a delayed monophasic association is observed [44]. Therefore, a number of mechanisms may mediate the ability of the EGFR family to display signal specificity such as the preferred association of cytoplasmic molecules to a specific receptor, heterodimerization partners, activation of the heterodimer by specific ligands, and temporal differences in substrate association to the receptor and subsequent activity. TRANSGENIC MOUSE MODELS FOR EGFR RECEPTOR FAMILY-MEDIATED MAMMARY TUMORIGENESIS

The EGF Receptor Tyrosine Kinase The v-erbB oncogene with its cellular homolog, the 170-kDa EGFR tyrosine kinase, has been implicated as playing a major role in mammary tumorigenesis. Six specific ligands have been identified to mediate the activation of the EGF receptor: EGF, TGFa, amphiregulin, heparin-binding EGF-like growth factor, betacellulin, and epiregulin [45]. In many tumors derived from EGF receptor overexpression a concomitant increase in one or more of the ligands has also been observed [46]. Direct evidence for the importance of these EGFR ligands in mammary tumorigenesis stems from the derivation of a number of transgenic models expressing these ligands in the mammary epithelium. Mammary epithelial specific expression of TGFa in transgenic mice has been achieved by coupling the TGFa cDNA to either the whey acidic protein (WAP) or the mouse mammary tumor virus (MMTV) promoter/ enhancer [47–51]. Mammary epithelial specific expression of TGFa initially results in the induction of a range of morphological abnormalities including lobular and cystic hyperplasias. However, these transgenic

80

KIM AND MULLER

mice eventually develop focal mammary tumors that arise after a long latency period [47–51]. Given the long latency period and focal nature of these mammary tumors, it is likely that secondary genetic events are involved in the induction of these TGFa tumors. One potential event that may be involved in cooperating with TGFa to transform the mammary epithelial cell is the upregulation of ErbB-2. Indeed, overexpression of Neu is found to be associated with high levels of endogenous EGFR [46]. To address this possibility, separate transgenic strains expressing ErbB-2 and TGFa under the transcriptional control of the MMTV promoter were crossed to create dual transgene carriers that coexpress both TGFa and ErbB-2 in the mammary epithelium [52]. The results revealed that coexpression of TGFa and ErbB-2 resulted in the dramatic acceleration of tumorigenesis. Tumor progression in these strains was further associated with the tyrosine phosphorylation of both the EGFR and the ErbB-2 RTK [52]. In a related set of experiments coexpression of TGFa and c-Myc in the mammary epithelium has also been demonstrated to result in the dramatic acceleration of tumorigenesis [53]. These observations suggest that the elevated expression of either c-Myc or ErbB-2 can cooperate with TGFa to induce mammary tumors. In this regard, it is interesting to note that the elevated expression of c-Myc or ErbB-2 is frequently observed in a large proportion of human breast cancers [2, 3, 54]. In addition to the important role the EGFR plays in the induction of mammary tumors, there is considerable evidence to suggest that activation of the EGFR is critical for normal mammary gland development. Indeed, germline inactivation of several EGFR family ligands is known to result in the dramatic impairment of mammary gland development [55]. Further evidence supporting a role for the EGFR in mammary gland development stems from observations made with a naturally occurring mouse mutant that harbors a mutation that results in a catalytically impaired EGFR (Waved-2) [56, 57]. Morphological analyses of Waved-2 strains revealed that they possess a lactation defect due to aberrant mammary gland development [56, 57]. Taken together, these observations suggest that activation of the EGFR plays an instrumental role in mammary epithelial development and tumorigenesis. The ErbB-2/Neu Receptor Tyrosine Kinase The frequent amplification and overexpression of ErbB-2/Neu in human breast cancer suggest strongly that the ErbB-2/Neu RTK plays a significant role in the development of mammary tumors. Direct evidence supporting a role for ErbB-2 in mammary tumorigenesis has been obtained through the generation of transgenic mice expressing the erbB-2/neu oncogene in the

mammary epithelium. To accomplish this, several independent strains of transgenic mice carrying the erbB-2/neu oncogene under the transcriptional control of the MMTV long terminal repeat (LTR) were derived [58 – 61]. Mammary epithelial specific expression of an activated version of the erbB-2/neu oncogene in several of these transgenic strains results in the rapid induction of multifocal mammary tumors in 100% of the female transgene carriers [58 – 60]. In contrast, in another set of transgenic strains the mammary epithelial specific expression of activated erbB-2/neu resulted in the induction of focal mammary tumors that occur only after a long latency period [61]. The differences in the phenotypes of these transgenic strains may be related to the fact that these strains were derived in distinct genetic backgrounds. In fact, it has recently been reported that genetic background can have a profound effect on tumorigenesis in these transgenic strains [62, 63]. Further evidence supporting the importance of activation of the erbB-2/neu oncogene in mammary tumorigenesis stems from the introduction of retroviral vectors expressing the erbB-2/neu oncogene in the mammary epithelium of rats [64]. The results revealed that like many transgenic strains expressing activated erbB-2/neu under the transcriptional control of the MMTV promoter, expression of the activated erbB-2/ neu oncogene resulted in the rapid induction of mammary carcinomas [64]. However, examination of the penetrance of tumor induction mediated by the retroviral expression of activated erbB-2/neu indicates that although potent, mammary epithelial expression of activated erbB-2/neu is likely not sufficient to transform the mammary epithelial cell on its own [65, 66]. Indeed, examination of transgenic mammary tumors induced by the expression of activated erbB-2/neu revealed the frequent occurrence of loss of heterozygosity (LOH) at a number of chromosomal loci [67]. Taken together, these observations argue that while mammary epithelial expression of activated erbB-2/neu is capable of rapidly inducing mammary tumors, other genetic events are likely required to induce mammary tumors. Although mammary epithelial specific expression of activated erbB-2/neu is capable of rapidly inducing mammary tumors, examination of human breast cancers for comparable activating mutations in erbB-2/neu has thus far been negative [68]. Given the importance of the elevated expression of the erbB-2/neu proto-oncogene in the induction of human breast cancer, transgenic mice expressing the wild-type neu/erbB-2 protooncogene under the transcriptional control of the MMTV promoter were derived [69]. In contrast to rapid tumor progression observed in the activated erbB-2/ neu transgenic strains, mammary epithelial specific

ROLE OF EGFR FAMILY IN MAMMARY TUMORIGENESIS AND METASTASIS

expression of the wild-type erbB-2/neu proto-oncogene resulted in the induction of focal mammary tumors that arose only after a long latency period. Tumorigenesis in these strains was further associated with activation of the intrinsic tyrosine kinase activity of ErbB2/Neu [69]. Another interesting facet of these erbB-2/ neu transgenic strains is that they frequently develop metastatic lesions to the lung. Therefore, as with ErbB-2/neu-expressing human breast cancers [5], expression of the erbB-2/neu proto-oncogene in this transgenic model is associated with a poor prognostic outcome. Genetic and biochemical analyses of tumor induction in these strains have revealed that activation of ErbB2/Neu tyrosine kinase activity in these strains is associated with the frequent occurrence of somatic mutations in the transgene [70]. Indeed, at least 70% of the mammary tumors derived from these transgenic mice exhibit evidence of in-frame deletions, insertions, or point mutations in a region of the ErbB-2/Neu extracellular domain (ECD) located just outside the transmembrane domain of the protein. Importantly, all of the deletions altered the cysteine-rich region found in the juxtamembrane region of the ECD [70]. Significantly, introduction of these alterations into an otherwise wild-type erbB-2/neu cDNA resulted in its oncogenic conversion [70]. Further genetic and biochemical analyses of these sporadic deletions in the erbB-2/neu transgene revealed that oncogenic activation of these altered erbB-2/neu receptors was due to their ability to form cysteine disulfide bonds between receptor monomers resulting in constitutive receptor dimerization [71]. Taken together, these observations suggested that one of the rate-limiting steps in tumor induction in these transgenic strains was oncogenic activation of the erbB-2/neu transgene. To directly assess the in vivo transforming activity of these activated erbB-2/neu alleles, transgenic mice carrying several activated erbB-2/neu alleles under the transcriptional control of the MMTV LTR have recently been derived [24]. Mammary epithelial specific expression of the activated erbB-2/neu alleles resulted in the rapid induction of multifocal mammary tumors suggesting that the occurrence of these somatic mutations was an important rate-limiting step in mammary tumor progression [24]. Because other EGFR family members are known to heterodimerize with ErbB-2/ Neu [1], the levels of the other EGFR family members were also measured in these activated erbB-2/neu strains. The results of these analyses revealed that among the four known EGFR family members, ErbB2/Neu-induced mammary tumors consistently express elevated levels of ErbB-3. In fact, quantitative measurement of the levels of ErbB-3 protein revealed a 10to 20-fold increase in ErbB-3 levels in tumors compared to adjacent normal tissue. Interestingly, the ob-

81

served increase in ErbB-3 protein was not due to an increase in the levels of erbB-3 transcript since comparable levels of erbB-3 transcript were detected in both normal and tumor tissues [24]. Consistent with these transgenic observations, examination of a cohort of erbB-2/neu-expressing breast tumors revealed that the majority expressed elevated levels of erbB-3 transcript [24]. Taken together, these observations argue that coexpression of erbB-2/neu and erbB-3 is a frequent event in the induction of mammary tumors. Although the significance of erbB-2/neu and erbB-3 coexpression is unclear, one potential explanation for requirement for the concerted expression of these EGFR family members is that they recruit complementary signaling pathways that cooperate in tumorigenesis. Indeed, activated ErbB-2/neu is known to recruit a number of adapter proteins that funnel through the Ras signaling pathway [72], whereas ErbB-3 is believed to be involved in the recruitment of the PI-39K signaling pathway [23, 32]. Given the importance of the PI-39K signaling pathway in preventing apoptotic cell death to a number stimuli [73], it is conceivable that elevated ErbB-3 protein levels provide important cell survival signals in erbB-2-induced tumorigenesis. Indeed, elevated ErbB-3 levels are observed in mammary tumors induced by a mutant Polyomavirus middle T (PyV mT) oncogene decoupled from the PI-39K pathway [74]. Moreover, mammary tumors obtained from MMTV/NDF transgenic mice express elevated levels of tyrosine-phosphorylated ErbB-2/Neu and ErbB-3 [75]. Taken together, these observations argue that elevated expression of ErbB-3 may be a frequent event in the induction of mammary tumors. One of the intriguing observations concerning the induction of mammary tumors in transgenic mice expressing the erbB-2/neu proto-oncogene is the strong biological selection for the occurrence of activating mutations in the transgene. Although comparable somatic mutations in erbB-2/neu have not been detected in human breast cancer, several studies have recently reported the expression of an alternatively spliced erbB-2 transcript that carries an in-frame 16-aminoacid deletion in primary breast cancers [24, 76]. Remarkably, this alternatively spliced form closely resembles the sporadic erbB-2 mutations observed in the wild-type erbB-2/neu transgenic strains. Indeed, as with the sporadic erbB-2/neu mutants, this alternatively spliced erbB-2/neu variant is transforming due to its capacity to undergo constitutive disulfide-bonded dimers [24, 76]. Because expression of the erbB-2/neu oncogene in these transgenic mice is driven by erbB-2/ neu cDNA [69], the selection for sporadic mutations in this model may reflect the requirement to mimic this alternative spliced form. Given the importance of erbB2/neu in the induction of breast cancer, future studies

82

KIM AND MULLER

designed to assess the biological significance of the splice variant in human breast cancer are warranted. Requirement for Other Signaling Pathways in ErbB2/Neu-Induced Mammary Tumorigenesis Whereas activation of erbB-2/neu in these models is a critical rate-limiting step, genetic and biochemical analyses of tumorigenesis in these strains suggest that activation of other signaling pathways also plays a critical role in ErbB-2/Neu-induced mammary tumorigenesis. One important signaling pathway that is believed to play a critical role in mammary tumorigenesis in these strains is activation of the Src family tyrosine kinases [40 – 42, 77, 78]. Indeed, examination of primary mammary tumors from these erbB-2/neu transgenic strains has revealed that the specific activities of c-Src and c-Yes are substantially increased which further correlates with the direct and specific binding of these Src family kinases to the ErbB-2/Neu RTK [41, 42, 78]. Although the significance of activation of these Src family kinases in ErbB-2/Neu-induced breast tumors is unclear, inactivation of c-src can result in a dramatic reduction in mammary tumor formation in transgenic mice expressing the PyV mT oncogene [79]. Moreover, examination of tyrosine-phosphorylated proteins associated with c-Src in PyV mT and erbB-2/ neu mammary tumors has revealed common tyrosinephosphorylated substrates suggesting the activation of related signaling pathways [42]. Future crosses with the erbB-2/neu strains with c-src null mice should allow the significance of c-Src in ErbB-2/Neu-induced tumorigenesis to be addressed. Genetic analyses of tumors derived from the various erbB-2/neu transgenic strains have revealed that tumorigenesis is frequently associated with the LOH of specific genomic loci [67, 80]. One important LOH in human breast cancer involves the loss of the p53 tumor suppressor gene [81– 83]. Mutations within the p53 gene and its translated product have also been noted to be one of the most frequent events found in tumors. One of the most common p53 mutations includes an Arg to His conversion at amino acid 172 (p53-172H) converting it to a dominant negative allele. Consistent with the importance of p53 mutations in mammary tumorigenesis, 37% of the mammary tumors derived from the erbB-2/neu strains harbor missense mutations that involve p53 [84]. To explore the significance of the p53 mutation in the induction of erbB-2/neu mammary tumors, separate transgenic strains carrying either MMTVerbB-2/neu or WAP/mutant p53 were interbred [84]. Coexpression of a dominant negative allele of p53 and erbB-2/neu resulted in a dramatic acceleration of mammary tumorigenesis [84]. Interestingly, unlike the parental MMTVerbB-2/neu strains, tumors derived from these strains failed to display

evidence of activating mutations in the transgene suggesting that the presence of two alterations in independent genes involved in tumor suppression and oncogenesis is sufficient to drive forward transformation [84]. Another example of cooperative interaction of signaling pathways in erbB-2/neu-induced tumorigenesis involves the matrix metalloproteinase matrilysin [85]. Coexpression of matrilysin with erbB-2/neu results in a significant acceleration of mammary tumors. Again, the mammary tumors derived from these bitransgenic strains fail to display evidence of activating mutations in the transgene [85]. It is conceivable that inactivation of p53 or activation of matrilysin function may have induced the expression of EGFR ligands, thus obviating the selection for activating mutations. Future studies with these crosses should allow these issues to be addressed. FUTURE DIRECTIONS

The ability to address the significance of a given gene in pathogenesis has been aided by the ability to generate transgenic mouse models that express high levels of a specific gene in target tissues. The MMTV transgenic mouse model has provided a wealth of knowledge on basic molecular mechanisms such as receptor–receptor interactions, receptor activation, downstream signaling pathways, and synergistic effects of specific proteins involved in mammary tumorigenesis. With the large body of evidence that implicates the EGFR family in mammary tumorigenesis, this transgenic system is ideal for addressing the basic questions surrounding the role of this receptor family in mammary tumor progression. An alternative method to address the significance of genes in development and tumorigenesis has been via the ablation of specific genes via homologous recombination. Indeed, this method has been utilized on tumor suppressor genes such as p53 where gene ablation results in an increase in the susceptibility of tumor formation [86]. The generation of mice homozygous for the EGFR family has revealed its importance in the proper development of the mouse. Indeed, targeted disruption of the EGFR results in either embryonic lethality or death postpartum due to general epithelial cell defects [87, 88]. Similarly, ErbB-2 and ErbB-3 homozygotes are embryonic lethal and display severe cardiac and neurological defects, respectively [89, 90]. The fact that the ablation of these family members results in embryonic lethality, the role of this receptor family in mammary gland development and tumorigenesis, obviously cannot be addressed. The advent of conditional gene targeting utilizing Lox P1 flanked (floxed) genes and the expression of a mammary-specific Cre recombinase has created a mechanism in which specific genes can now be disrupted within the mammary

ROLE OF EGFR FAMILY IN MAMMARY TUMORIGENESIS AND METASTASIS

gland [91–93]. For example, the ablation of the BRCA1 gene in the mammary gland results in tumor formation with a long latency period associated with genetic instability [94]. With the importance of heterodimerization in EGFR family signaling, the Cre–Lox system in conjunction with transgenic mice that express specific EGFR family members as well as their ligands can address the biological significance of each receptor partner in mammary tumor progression. Although transgenic mice have been very successful in addressing the role of specific genes in mammary tumorigenesis, the fact remains that there are limitations to this system. For example, the overexpression of a specific gene is via an exogenous viral promotor, thus raising the question of whether overexpression of the gene is of biological significance. In addition, it is well known that hormonal factors play a critical role in normal mammary gland development and possibly in epithelial cell transformation [95]. Indeed, in the great majority of clinical cases the strong inverse relationship between patient prognosis and ErbB-2 receptor expression is also coupled with a low probability of a positive response from therapies that involve estrogen antagonisis. Many of the primary human breast cancer cases that display high levels of expression of the ErbB-2 receptor also display a loss of estrogen receptor function [96 –99] suggesting an intimate connection between the EGFR family and hormonal regulation during mammary tumor formation. Based on the ability to disrupt genes via homologous recombination, specific genes that have been altered can now be introduced into an endogenous loci or “knocked-in.” Thus, the possibility exists where one can investigate the significance of an activated allele such as ErbB-2 in the context of the endogenous promotor, capable of responding to physiological hormone levels typically found throughout development. This type of analysis will prove very important in the role of hormonal regulation and EGFR family activation in mammary tumor progression. The ability to generate conditional alleles can lead to the analysis of associated genes involved in tumor progression. For example, a central issue in cancer management is the ability to rapidly identify primary lesions and promptly remove them before tissue invasion and metastasis can occur to a secondary organ site [100]. A number of genes have been implicated in promoting tumor cell invasion leading to metastasis. For example, matrix metalloproteinases such as stromelysin [101, 102] and growth factors such as vascular endothelial growth factor (VEGF) and its cognate receptor Flk-1/KDR [103–105] have been implicated in angiogenesis and subsequent metastasis. By generating floxed genes implicated in angiogenesis and metastasis and exposing them to the mammary-specific expression of Cre recombinase, one can address the

83

importance of specific genes involved in metastasis. Interbreeding of the current transgenic mouse models of mammary tumorigenesis with these floxed alleles may address the ability of the tumor to vascularize and potentially metastasize to secondary sites. With the knowledge of the significance the EGFR family plays in mammary tumorigenesis, logical therapies of intervention can be developed that take advantage of the key roles such receptors play. It is clear that ErbB-2/Neu is an important partner in receptor dimerization and activation. In addition, many specific cytoplasmic proteins that contain SH2 and PTB domains directly associate with the EGFR family; however, recent data derived from ErbB-2/Neu-mediated transformation experiments [72] suggest that therapies that inhibit these associated proteins may not be ideal targets for development. Molecular analyses of the signaling pathways that are relevant to ErbB-2/Neu have revealed that redundant pathways exist that can lead to transformation. Indeed, the majority of the redundant pathways present mediate the activation of the Ras pathway from ErbB-2/Neu. These pathways, when independent from the other known signaling pathways, can efficiently transform cells in culture [72]. Logical targets may therefore include inhibition of ErbB-2/Neu RTK directly. Indeed, downregulation of ErbB-2/Neu with antibodies specific to the receptor has been found to interfere with the proliferative ability of mammary carcinoma cell lines [106 –108]. Alternatively, specific Ras inhibitors may prove valuable in conjunction with therapies against ErbB-2/Neu since evidence suggests that the Ras pathway is a common pathway from ErbB-2/Neu RTK. To this end, the generation of specific farnesyltransferase inhibitors that inhibit Ras association to the lipid membrane has proven useful in inhibiting Ras transformation [109]. The molecular mechanisms that are involved in mammary tumorigenesis are slowly being elucidated with the advent of technologies such as transgenics, homologous recombination, knock-in, and tissue-specific gene ablation. These tools have been extremely valuable in the clarification of the steps involved in EGFR family signaling in mammary tumorigenesis. W.J.M. is a recipient of the Medical Research Council of Canada Scientist award. H.K. was supported by a studentship from the Medical Research Council of Canada.

REFERENCES 1.

Hynes, N. E., and Stern, D. F. (1994). The biology of erbB-2/ neu/HER-2 and its role in cancer. Biochim. Biophys. Acta 1198, 165–184.

2.

Slamon, D. J., Clark, G. M., Wong, S. G., Levin, W. J., Ullrich, A., and McGuire, W. L. (1987). Human breast cancer: Correlation of relapse and survival with the amplification of the HER2/neu oncogene. Science 235, 177–182.

84 3.

KIM AND MULLER Slamon, D. J., Godolphin, W., Jones, L. A., Holt, J. A., Wong, S. G., Keith, D. E., Levin, W. J., Stuart, S. G., Udove, J., Ullrich, A., and Press, M. F. (1989). Studies of the HER-2/neu proto-oncogene in human breast and ovaian cancer. Science 244, 707–712.

isoforms activate distinct receptor combinations. J. Biol. Chem. 271, 19029 –19032. 18.

Pinkas-Kramarski, R., Soussan, L., Waterman, H., Levkowitz, G., Alroy, I., Klapper, L., Lavi, S., Seger, R., Ratzkin, B. J., Sela, M., and Yarden, Y. (1996). Diversification of Neu differentiation factor and epidermal growth factor signaling by combinatorial receptor interactions. EMBO J. 15, 2452–2467.

4.

Ross, J. S., and Fletcher, J. A. (1999). The HER-2/neu oncogene: Prognostic factor, predictive factor and target for therapy. Semin. Cancer Biol. 9, 125–138.

19.

5.

Andrulis, I. L., Bull, S. B., Blackstein, M. E., Sutherland, D., Mak, C., Sidlofsky, S., Pritzker, K. P., Hartwick, R. W., Hanna, W., Lickley, L., Wilkinson, R., Qizilbash, A., Ambus, U., Lipa, M., Weizel, H., Katz, A., Baida, M., Mariz, S., Stoik, G., Dacamara, P., Strongitharm, D., Geddie, W., and McCready, D. (1998). neu/erbB-2 amplification identifies a poor-prognosis group of women with node-negative breast cancer. Toronto Breast Cancer Study Group. J. Clin. Oncol. 16, 1340 –1349.

Pinkas-Kramarski, R., Shelly, M., Guarino, B. C., Wang, L. M., Lyass, L., Alroy, I., Alimandi, M., Kuo, A., Moyer, J. D., Lavi, S., Eisenstein, M., Ratzkin, B. J., Seger, R., Bacus, S. S., Pierce, J. H., Andrews, G. C., Yarden, Y., and Alamandi, M. (1998). ErbB tyrosine kinases and the two neuregulin families constitute a ligand–receptor network. Mol. Cell. Biol. 18, 6090 –101. [published erratum appears in Mol. Cell. Biol. 18(12), 7602, 1998]

20.

6.

Plowman, G. D., Culouscou, J. M., Whitney, G. S., Green, J. M., Carlton, G. W., Foy, L., Neubauer, M. G., and Shoyab, M. (1993). Ligand-specific activation of HER4/p180erbB4, a fourth member of the epidermal growth factor receptor family. Proc. Natl. Acad. Sci. USA 90, 1746 –1750.

Cohen, B. D., Kiener, P. A., Green, J. M., Foy, L., Fell, H. P., and Zhang, K. (1996). The relationship between human epidermal growth-like factor receptor expression and cellular transformation in NIH3T3 cells. J. Biol. Chem. 271, 30897– 30903.

21.

7.

Lacroix, H., Iglehart, J. D., Skinner, M. A., and Kraus, M. H. (1989). Overexpression of erbB-2 or EGF receptor proteins present in early stage mammary carcinoma is detected simultaneously in matched primary tumors and regional metastases. Oncogene 4, 145–151.

Kokai, Y., Myers, J. N., Wada, T., Brown, V. I., LeVea, C. M., Davis, J. G., Dobashi, K., and Greene, M. I. (1989). Synergistic interaction of p185c-neu and the EGF receptor leads to transformation of rodent fibroblasts. Cell 58, 287–292.

22.

Alimandi, M., Romano, A., Curia, M. C., Muraro, R., Fedi, P., Aaronson, S. A., Di Fiore, P. P., and Kraus, M. H. (1995). Cooperative signaling of ErbB3 and ErbB2 in neoplastic transformation and human mammary carcinomas. Oncogene 10, 1813–1821.

23.

Prigent, S. A., and Gullick, W. J. (1994). Identification of c-erbB-3 binding sites for phosphatidylinositol 39-kinase and SHC using an EGF receptor/c-erbB-3 chimera. EMBO J. 13, 2831–2841.

24.

Siegel, P. M., Ryan, E. D., Cardiff, R. D., and Muller, W. J. (1999). Elevated expressions of activated forms of Neu/ErbB-2 and ErbB-3 are involved in the induction of mammary tumors in transgenic mice: Implications for human breast cancer. EMBO J. 18, 2149 –2164.

8.

9.

Kraus, M. H., Issing, W., Miki, T., Popescu, N. C., and Aaronson, S. A. (1989). Isolation and characterization of ERBB3, a third member of the ERBB/epidermal growth factor receptor family: Evidence for overexpression in a subset of human mammary tumors. Proc. Natl. Acad. Sci. USA 86, 9193–9197. Normanno, N., Ciardiello, F., Brandt, R., and Salomon, D. S. (1994). Epidermal growth factor-related peptides in the pathogenesis of human breast cancer. Breast Cancer Res. Treat. 29, 11–27.

10.

Salomon, D. S., Bianco, C., and De Santis, M. (1999). Cripto: A novel epidermal growth factor (EGF)-related peptide in mammary gland development and neoplasia. Bioessays 21, 61–70.

11.

Kenney, N. J., Smith, G. H., Maroulakou, I. G., Green, J. H., Muller, W. J., Callahan, R., Salomon, D. S., and Dickson, R. B. (1996). Detection of amphiregulin and Cripto-1 in mammary tumors from transgenic mice. Mol. Carcinog. 15, 44 –56.

25.

Graus-Porta, D., Beerli, R. R., and Hynes, N. E. (1995). Singlechain antibody-mediated intracellular retention of ErbB-2 impairs Neu differentiation factor and epidermal growth factor signaling. Mol. Cell. Biol. 15, 1182–1191.

12.

Peles, E., and Yarden, Y. (1993). Neu and its ligands: From an oncogene to neural factors. Bioessays 15, 815– 824.

26.

13.

Karunagaran, D., Tzahar, E., Beerli, R. R., Chen, X., GrausPorta, D., Ratzkin, B. J., Seger, R., Hynes, N. E., and Yarden, Y. (1996). ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: Implications for breast cancer. EMBO J. 15, 254 –264.

Graus-Porta, D., Beerli, R. R., Daly, J. M., and Hynes, N. E. (1997). ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling. EMBO J. 16, 1647–1655.

27.

Wada, T., Qian, X. L., and Greene, M. I. (1990). Intermolecular association of the p185neu protein and EGF receptor modulates EGF receptor function. Cell 61, 1339 –1347.

Qian, X., Dougall, W. C., Hellman, M. E., and Greene, M. I. (1994). Kinase-deficient neu proteins suppress epidermal growth factor receptor function and abolish cell transformation. Oncogene 9, 1507–1514.

28.

Qian, X., LeVea, C. M., Freeman, J. K., Dougall, W. C., and Greene, M. I. (1994). Heterodimerization of epidermal growth factor receptor and wild-type or kinase-deficient Neu: A mechanism of interreceptor kinase activation and transphosphorylation. Proc. Natl. Acad. Sci. USA 91, 1500 –1504.

29.

Qian, X., O’Rourke, D. M., Zhao, H., and Greene, M. I. (1996). Inhibition of p185neu kinase activity and cellular transformation by co-expression of a truncated neu protein. Oncogene 13, 2149 –2157.

30.

Spivak-Kroizman, T., Rotin, D., Pinchasi, D., Ullrich, A., Schlessinger, J., and Lax, I. (1992). Heterodimerization of c-erbB2 with different epidermal growth factor receptor mutants elicits stimulatory or inhibitory responses. J. Biol. Chem. 267, 8056 – 8063.

14.

15.

16.

17.

Tzahar, E., Waterman, H., Chen, X., Levkowitz, G., Karunagaran, D., Lavi, S., Ratzkin, B. J., and Yarden, Y. (1996). A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor. Mol. Cell. Biol. 16, 5276 –5287. Tzahar, E., Levkowitz, G., Karunagaran, D., Yi, L., Peles, E., Lavi, S., Chang, D., Liu, N., Yayon, A., Wen, D., et al. (1994). ErbB-3 and ErbB-4 function as the respective low and high affinity receptors of all Neu differentiation factor/heregulin isoforms. J. Biol. Chem. 269, 25226 –25233. Pinkas-Kramarski, R., Shelly, M., Glathe, S., Ratzkin, B. J., and Yarden, Y. (1996). Neu differentiation factor/neuregulin

ROLE OF EGFR FAMILY IN MAMMARY TUMORIGENESIS AND METASTASIS 31.

Wong, L., Deb, T. B., Thompson, S. A., Wells, A., and Johnson, G. R. (1999). A differential requirement for the COOH-terminal region of the epidermal growth factor (EGF) receptor in amphiregulin and EGF mitogenic signaling. J. Biol. Chem. 274, 8900 – 8909.

32.

Soltoff, S. P., Carraway, K. L., III, Prigent, S. A., Gullick, W. G., and Cantley, L. C. (1994). ErbB3 is involved in activation of phosphatidylinositol 3-kinase by epidermal growth factor. Mol. Cell. Biol. 14, 3550 –3558.

33.

Wilson, L. K., Luttrell, D. K., Parsons, J. T., and Parsons, S. J. (1989). pp60c-src tyrosine kinase, myristylation, and modulatory domains are required for enhanced mitogenic responsiveness to epidermal growth factor seen in cells overexpressing c-src. Mol. Cell. Biol. 9, 1536 –1544.

34.

Luttrell, D. K., Luttrell, L. M., and Parsons, S. J. (1988). Augmented mitogenic responsiveness to epidermal growth factor in murine fibroblasts that overexpress pp60c-src. Mol. Cell. Biol. 8, 497–501.

35.

Osherov, N., and Levitzki, A. (1994). Epidermal-growth-factordependent activation of the src-family kinases. Eur. J. Biochem. 225, 1047–1053.

36.

Sato, K., Sato, A., Aoto, M., and Fukami, Y. (1995). c-Src phosphorylates epidermal growth factor receptor on tyrosine 845. Biochem. Biophys. Res. Commun. 215, 1078 –1087.

37.

Maa, M. C., Leu, T. H., McCarley, D. J., Schatzman, R. C., and Parsons, S. J. (1995). Potentiation of epidermal growth factor receptor-mediated oncogenesis by c-Src: Implications for the etiology of multiple human cancers. Proc. Natl. Acad. Sci. USA 92, 6981– 6985.

38.

Tice, D. A., Biscardi, J. S., Nickles, A. L., and Parsons, S. J. (1999). Mechanism of biological synergy between cellular Src and epidermal growth factor receptor. Proc. Natl. Acad. Sci. USA 96, 1415–1420.

39.

Biscardi, J. S., Maa, M. C., Tice, D. A., Cox, M. E., Leu, T. H., and Parsons, S. J. (1999). c-Src-mediated phosphorylation of the epidermal growth factor receptor on Tyr845 and Tyr1101 is associated with modulation of receptor function. J. Biol. Chem. 274, 8335– 8343.

40.

Muthuswamy, S. K., and Muller, W. J. (1994). Activation of the Src family of tyrosine kinases in mammary tumorigenesis. Adv. Cancer Res. 64, 111–123.

41.

Muthuswamy, S. K., and Muller, W. J. (1995). Direct and specific interaction of c-Src with Neu is involved in signaling by the epidermal growth factor receptor. Oncogene 11, 271– 279.

42.

Muthuswamy, S. K., and Muller, W. J. (1995). Activation of Src family kinases in Neu-induced mammary tumors correlates with their association with distinct sets of tyrosine phosphorylated proteins in vivo. Oncogene 11, 1801–1810.

43.

Olayioye, M. A., Beuvink, I., Horsch, K., Daly, J. M., and Hynes, N. E. (1999). ErbB receptor-induced activation of stat transcription factors is mediated by Src tyrosine kinases. J. Biol. Chem. 274, 17209 –17218.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

Olayioye, M. A., Graus-Porta, D., Beerli, R. R., Rohrer, J., Gay, B., and Hynes, N. E. (1998). ErbB-1 and ErbB-2 acquire distinct signaling properties dependent upon their dimerization partner. Mol. Cell. Biol. 18, 5042–5051.

59.

Burden, S., and Yarden, Y. (1997). Neuregulins and their receptors: A versatile signaling module in organogenesis and oncogenesis. Neuron 18, 847– 855.

60.

DiGiovanna, M. P., Lerman, M. A., Coffey, R. J., Muller, W. J., Cardiff, R. D., and Stern, D. F. (1998). Active signaling by Neu in transgenic mice. Oncogene 17, 1877–1884.

85

Arteaga, C. L., Dugger, T. C., and Hurd, S. D. (1996). The multifunctional role of transforming growth factor (TGF)-beta s on mammary epithelial cell biology. Breast Cancer Res. Treat. 38, 49 –56. Jhappan, C., Stahle, C., Harkins, R. N., Fausto, N., Smith, G. H., and Merlino, G. T. (1990). TGF alpha overexpression in transgenic mice induces liver neoplasia and abnormal development of the mammary gland and pancreas. Cell 61, 1137– 1146. Matsui, Y., Halter, S. A., Holt, J. T., Hogan, B. L., and Coffey, R. J. (1990). Development of mammary hyperplasia and neoplasia in MMTV-TGF alpha transgenic mice. Cell 61, 1147– 1155. Sandgren, E. P., Luetteke, N. C., Palmiter, R. D., Brinster, R. L., and Lee, D. C. (1990). Overexpression of TGF alpha in transgenic mice: Induction of epithelial hyperplasia, pancreatic metaplasia, and carcinoma of the breast. Cell 61, 1121– 1135. Sandgren, E. P., Schroeder, J. A., Qui, T. H., Palmiter, R. D., Brinster, R. L., and Lee, D. C. (1995). Inhibition of mammary gland involution is associated with transforming growth factor alpha but not c-myc-induced tumorigenesis in transgenic mice. Cancer Res. 55, 3915–3927. Muller, W. J., Arteaga, C. L., Muthuswamy, S. K., Siegel, P. M., Webster, M. A., Cardiff, R. D., Meise, K. S., Li, F., Halter, S. A., and Coffey, R. J. (1996). Synergistic interaction of the Neu proto-oncogene product and transforming growth factor alpha in the mammary epithelium of transgenic mice. Mol. Cell. Biol. 16, 5726 –5736. Amundadottir, L. T., Nass, S. J., Berchem, G. J., Johnson, M. D., and Dickson, R. B. (1996). Cooperation of TGF alpha and c-Myc in mouse mammary tumorigenesis: Coordinated stimulation of growth and suppression of apoptosis. Oncogene 13, 757–765. Escot, C., Theillet, C., Lidereau, R., Spyratos, F., Champeme, M. H., Gest, J., and Callahan, R. (1986). Genetic alteration of the c-myc protooncogene (MYC) in human primary breast carcinomas. Proc. Natl. Acad. Sci. USA 83, 4834 – 4838. Luetteke, N. C., Qiu, T. H., Fenton, S. E., Troyer, K. L., Riedel, R. F., Chang, A., and Lee, D. C. (1999). Targeted inactivation of the EGF and amphiregulin genes reveals distinct roles for EGF receptor ligands in mouse mammary gland development. Development 126, 2739 –2750. Luetteke, N. C., Phillips, H. K., Qiu, T. H., Copeland, N. G., Earp, H. S., Jenkins, N. A., and Lee, D. C. (1994). The mouse waved-2 phenotype results from a point mutation in the EGF receptor tyrosine kinase. Genes Dev. 8, 399 – 413. Fowler, K. J., Walker, F., Alexander, W., Hibbs, M. L., Nice, E. C., Bohmer, R. M., Mann, G. B., Thumwood, C., Maglitto, R., Danks, J. A., et al. (1995). A mutation in the epidermal growth factor receptor in waved-2 mice has a profound effect on receptor biochemistry that results in impaired lactation. Proc. Natl. Acad. Sci. USA 92, 1465–1469. Guy, C. T., Cardiff, R. D., and Muller, W. J. (1996). Activated neu induces rapid tumor progression. J. Biol. Chem. 271, 7673–7678. Muller, W. J., Sinn, E., Pattengale, P. K., Wallace, R., and Leder, P. (1988). Single-step induction of mammary adenocarcinoma in transgenic mice bearing the activated c-neu oncogene. Cell 54, 105–115. Lucchini, F., Sacco, M. G., Hu, N., Villa, A., Brown, J., Cesano, L., Mangiarini, L., Rindi, G., Kindl, S., Sessa, F., et al. (1992). Early and multifocal tumors in breast, salivary, harderian and epididymal tissues developed in MMTY-Neu transgenic mice. Cancer Lett. 64, 203–209.

86

KIM AND MULLER

61.

Bouchard, L., Lamarre, L., Tremblay, P. J., and Jolicoeur, P. (1989). Stochastic appearance of mammary tumors in transgenic mice carrying the MMTV/c-neu oncogene. Cell 57, 931– 936.

62.

Rowse, G. J., Ritland, S. R., and Gendler, S. J. (1998). Genetic modulation of neu proto-oncogene-induced mammary tumorigenesis. Cancer Res. 58, 2675–2679.

63.

Thompson, J. A., Eades-Perner, A. M., Ditter, M., Muller, W. J., and Zimmermann, W. (1997). Expression of transgenic carcinoembryonic antigen (CEA) in tumor-prone mice: An animal model for CEA-directed tumor immunotherapy. Int. J. Cancer 72, 197–202.

64.

Wang, B., Kennan, W. S., Yasukawa-Barnes, J., Lindstrom, M. J., and Gould, M. N. (1991). Frequent induction of mammary carcinomas following neu oncogene transfer into in situ mammary epithelial cells of susceptible and resistant rat strains. Cancer Res. 51, 5649 –5654.

65.

Tai, Y. T., and Gould, M. N. (1997). The genetic penetrance of the activated neu oncogene for the induction of mammary cancer in vivo. Oncogene 14, 2701–2707.

77.

Amundadottir, L. T., and Leder, P. (1998). Signal transduction pathways activated and required for mammary carcinogenesis in response to specific oncogenes. Oncogene 16, 737–746.

78.

Muthuswamy, S. K., Siegel, P. M., Dankort, D. L., Webster, M. A., and Muller, W. J. (1994). Mammary tumors expressing the neu proto-oncogene possess elevated c-Src tyrosine kinase activity. Mol. Cell. Biol. 14, 735–743.

79.

Guy, C. T., Muthuswamy, S. K., Cardiff, R. D., Soriano, P., and Muller, W. J. (1994). Activation of the c-Src tyrosine kinase is required for the induction of mammary tumors in transgenic mice. Genes Dev. 8, 23–32.

80.

Ritland, S. R., Rowse, G. J., Chang, Y., and Gendler, S. J. (1997). Loss of heterozygosity analysis in primary mammary tumors and lung metastases of MMTV-MTAg and MMTV-neu transgenic mice. Cancer Res. 57, 3520 –3525.

81.

Callahan, R., Cropp, C. S., Merlo, G. R., Liscia, D. S., Cappa, A. P., and Lidereau, R. (1992). Somatic mutations and human breast cancer. A status report. Cancer 69, 1582–1588.

82.

Chen, L. C., Neubauer, A., Kurisu, W., Waldman, F. M., Ljung, B. M., Goodson, W. d., Goldman, E. S., Moore, D. d., Balazs, M., Liu, E., et al. (1991). Loss of heterozygosity on the short arm of chromosome 17 is associated with high proliferative capacity and DNA aneuploidy in primary human breast cancer. Proc. Natl. Acad. Sci. USA 88, 3847–3851.

66.

Tai, Y. T., and Gould, M. N. (1995). Hormonal modulation of neu-induced mammary carcinogenesis: Evidence against a single-step induction of mammary cancer by neu. Carcinogenesis 16, 1455–1459.

67.

Cool, M., and Jolicoeur, P. (1999). Elevated frequency of loss of heterozygosity in mammary tumors arising in mouse mammary tumor virus/neu transgenic mice. Cancer Res. 59, 2438 – 2444.

83.

Singh, S., Simon, M., Meybohm, I., Jantke, I., Jonat, W., Maass, H., and Goedde, H. W. (1993). Human breast cancer: Frequent p53 allele loss and protein overexpression. Hum. Genet. 90, 635– 640.

68.

Lemoine, N. R., Staddon, S., Dickson, C., Barnes, D. M., and Gullick, W. J. (1990). Absence of activating transmembrane mutations in the c-erbB-2 proto-oncogene in human breast cancer. Oncogene 5, 237–239.

84.

Li, B., Rosen, J. M., McMenamin-Balano, J., Muller, W. J., and Perkins, A. S. (1997). neu/ERBB2 cooperates with p53-172H during mammary tumorigenesis in transgenic mice. Mol. Cell. Biol. 17, 3155–3163.

69.

Guy, C. T., Webster, M. A., Schaller, M., Parsons, T. J., Cardiff, R. D., and Muller, W. J. (1992). Expression of the neu protooncogene in the mammary epithelium of transgenic mice induces metastatic disease. Proc. Natl. Acad. Sci. USA 89, 10578 –10582.

85.

Rudolph-Owen, L. A., Chan, R., Muller, W. J., and Matrisian, L. M. (1998). The matrix metalloproteinase matrilysin influences early-stage mammary tumorigenesis. Cancer Res. 58, 5500 –5506.

86.

70.

Siegel, P. M., Dankort, D. L., Hardy, W. R., and Muller, W. J. (1994). Novel activating mutations in the neu proto-oncogene involved in induction of mammary tumors. Mol. Cell. Biol. 14, 7068 –7077.

Donehower, L. A., Harvey, M., Slagle, B. L., McArthur, M. J., Montgomery, C. A., Jr., Butel, J. S., and Bradley, A. (1992). Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356, 215–221.

87.

71.

Siegel, P. M., and Muller, W. J. (1996). Mutations affecting conserved cysteine residues within the extracellular domain of Neu promote receptor dimerization and activation. Proc. Natl. Acad. Sci. USA 93, 8878 – 8883.

Threadgill, D. W., Dlugosz, A. A., Hansen, L. A., Tennenbaum, T., Lichti, U., Yee, D., LaMantia, C., Mourton, T., Herrup, K., Harris, R. C., et al. (1995). Targeted disruption of mouse EGF receptor: Effect of genetic background on mutant phenotype. Science 269, 230 –234.

72.

Dankort, D. L., Wang, Z., Blackmore, V., Moran, M. F., and Muller, W. J. (1997). Distinct tyrosine autophosphorylation sites negatively and positively modulate neu-mediated transformation. Mol. Cell. Biol. 17, 5410 –5425.

88.

Sibilia, M., and Wagner, E. F. (1995). Strain-dependent epithelial defects in mice lacking the EGF receptor. Science 269, 234 –238. [published erratum appears in Science 269(5226), 909, 1995]

73.

Hemmings, B. A. (1997). Akt signaling: Linking membrane events to life and death decisions. Science 275, 628 – 630.

89.

74.

Webster, M. A., Hutchinson, J. N., Rauh, M. J., Muthuswamy, S. K., Anton, M., Tortorice, C. G., Cardiff, R. D., Graham, F. L., Hassell, J. A., and Muller, W. J. (1998). Requirement for both Shc and phosphatidylinositol 39 kinase signaling pathways in Polyomavirus middle T-mediated mammary tumorigenesis. Mol. Cell. Biol. 18, 2344 –2359.

Lee, K. F., Simon, H., Chen, H., Bates, B., Hung, M. C., and Hauser, C. (1995). Requirement for neuregulin receptor erbB2 in neural and cardiac development. Nature 378, 394 –398.

90.

Riethmacher, D., Sonnenberg-Riethmacher, E., Brinkmann, V., Yamaai, T., Lewin, G. R., and Birchmeier, C. (1997). Severe neuropathies in mice with targeted mutations in the ErbB3 receptor. Nature 389, 725–730.

91.

Sauer, B., and Henderson, N. (1988). Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc. Natl. Acad. Sci. USA 85, 5166 –5170.

92.

Wagner, K. U., Wall, R. J., St-Onge, L., Gruss, P., WynshawBoris, A., Garrett, L., Li, M., Furth, P. A., and Hennighausen, L. (1997). Cre-mediated gene deletion in the mammary gland. Nucleic Acids Res. 25, 4323– 4330.

75.

Krane, I. M., and Leder, P. (1996). NDF/heregulin induces persistence of terminal end buds and adenocarcinomas in the mammary glands of transgenic mice. Oncogene 12, 1781–1788.

76.

Kwong, K. Y., and Hung, M. C. (1998). A novel splice variant of HER2 with increased transformation activity. Mol. Carcinog. 23, 62– 68.

ROLE OF EGFR FAMILY IN MAMMARY TUMORIGENESIS AND METASTASIS 93.

94.

95.

96.

97.

98.

99.

100. 101.

Hamilton, D. L., and Abremski, K. (1984). Site-specific recombination by the bacteriophage P1 lox-Cre system. Cre-mediated synapsis of two lox sites. J. Mol. Biol. 178, 481– 486. Xu, X., Wagner, K. U., Larson, D., Weaver, Z., Li, C., Ried, T., Hennighausen, L., Wynshaw-Boris, A., and Deng, C. X. (1999). Conditional mutation of Brca1 in mammary epithelial cells results in blunted ductal morphogenesis and tumour formation. Nat. Genet. 22, 37– 43. Hennighausen, L., and Robinson, G. W. (1998). Think globally, act locally: The making of a mouse mammary gland. Genes Dev. 12, 449 – 455. Houston, S. J., Plunkett, T. A., Barnes, D. M., Smith, P., Rubens, R. D., and Miles, D. W. (1999). Overexpression of c-erbB2 is an independent marker of resistance to endocrine therapy in advanced breast cancer. Br. J. Cancer 79, 1220 – 1226. De Placido, S., Carlomagno, C., De Laurentiis, M., and Bianco, A. R. (1998). c-erbB2 expression predicts tamoxifen efficacy in breast cancer patients. Breast Cancer Res. Treat. 52, 55– 64. Newby, J. C., Johnston, S. R., Smith, I. E., and Dowsett, M. (1997). Expression of epidermal growth factor receptor and c-erbB2 during the development of tamoxifen resistance in human breast cancer. Clin. Cancer Res. 3, 1643–1651. Carlomagno, C., Perrone, F., Gallo, C., De Laurentiis, M., Lauria, R., Morabito, A., Pettinato, G., Panico, L., D’Antonio, A., Bianco, A. R., and De Placido, S. (1996). c-erb B2 overexpression decreases the benefit of adjuvant tamoxifen in earlystage breast cancer without axillary lymph node metastases. J. Clin. Oncol. 14, 2702–2708. Dankort, D. L., and Muller, W. J. (1996). Transgenic models of breast cancer metastasis. Cancer Treat. Res. 83, 71– 88. Sternlicht, M. D., Lochter, A., Sympson, C. J., Huey, B., Rougier, J. P., Gray, J. W., Pinkel, D., Bissell, M. J., and Werb, Z. (1999). The stromal proteinase MMP3/stromelysin-1 promotes mammary carcinogenesis. Cell 98, 137–146.

Received September 23, 1999

102.

103.

104.

105.

106.

107.

108.

109.

87

Thomasset, N., Lochter, A., Sympson, C. J., Lund, L. R., Williams, D. R., Behrendtsen, O., Werb, Z., and Bissell, M. J. (1998). Expression of autoactivated stromelysin-1 in mammary glands of transgenic mice leads to a reactive stroma during early development. Am. J. Pathol. 153, 457– 467. Millauer, B., Wizigmann-Voos, S., Schnurch, H., Martinez, R., Moller, N. P., Risau, W., and Ullrich, A. (1993). High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis. Cell 72, 835– 846. Millauer, B., Longhi, M. P., Plate, K. H., Shawver, L. K., Risau, W., Ullrich, A., and Strawn, L. M. (1996). Dominantnegative inhibition of Flk-1 suppresses the growth of many tumor types in vivo. Cancer Res. 56, 1615–1620. Fukumura, D., Xavier, R., Sugiura, T., Chen, Y., Park, E. C., Lu, N., Selig, M., Nielsen, G., Taksir, T., Jain, R. K., and Seed, B. (1998). Tumor induction of VEGF promoter activity in stromal cells. Cell 94, 715–725. Drebin, J. A., Link, V. C., Stern, D. F., Weinberg, R. A., and Greene, M. I. (1985). Down-modulation of an oncogene protein product and reversion of the transformed phenotype by monoclonal antibodies. Cell 41, 697–706. Hudziak, R. M., Lewis, G. D., Winget, M., Fendly, B. M., Shepard, H. M., and Ullrich, A. (1989). p185HER2 monoclonal antibody has antiproliferative effects in vitro and sensitizes human breast tumor cells to tumor necrosis factor. Mol. Cell. Biol. 9, 1165–1172. Klapper, L. N., Glathe, S., Vaisman, N., Hynes, N. E., Andrews, G. C., Sela, M., and Yarden, Y. (1999). The ErbB-2/ HER2 oncoprotein of human carcinomas may function solely as a shared coreceptor for multiple stroma-derived growth factors. Proc. Natl. Acad. Sci. USA 96, 4995–5000. Cox, A. D., and Der, C. J. (1997). Farnesyltransferase inhibitors and cancer treatment: Targeting simply Ras? Biochim. Biophys. Acta 1333, F51–F71.