Estrogen Receptors And Erbb Proteins In Breast Cancer

Estrogens and the estrogen receptors are key regulators of growth in the normal breast, together with a number of other hormones acting through nuclear or membrane receptors. Furthermore, in an incompletely understood fashion, they synergize with growth factors of the EGF family that activate receptor tyrosine kinases of the ERBB family to instruct proliferation and differentiation of the ductal and alveolar epithelia (Figure 18.10). Expression of the growth factors in stromal and epithelial cells and perhaps of the receptors as well is influenced by estrogens. Conversely, active ERBB receptors stimulate the MAPK cascade (^4.4) which leads to phosphorylation of the ERa and synthesis and phosphorylation of AP1 transcription factors with which the receptor interacts (—>18.1). In breast cancer, these same factors remain relevant, although in a distorted way.

At the time of presentation, in >70% of breast cancers the ERa can be detected by immunohistochemical staining or by biochemical assays such as ELISA. About half of these cancers also express the progesterone receptor. Since the PR gene is induced by the estrogen receptor, its expression is an indication that ERa is not only present, but also active. Only <5% of breast cancers express only the PR. The ER0 is usually down-regulated in breast cancer; quite often the ESR2 gene is silenced by promoter hypermethylation (^-8.3). This underlines its function as a negative growth regulator that limits proliferative responses to estrogens.

Epithelial Cell

Estrogens

Figure 18.10 Presumed interaction of estrogens and EGF-like factors in normal breast tissue It is thought that estrogen-independent breast cancers lose estrogen receptor a, increase production of EGF-like peptides and their responsiveness to them by increased expression of receptors and specifically the co-receptor ERBB2. See text for more details.

Estrogens

Mesenchymal Cell

Figure 18.10 Presumed interaction of estrogens and EGF-like factors in normal breast tissue It is thought that estrogen-independent breast cancers lose estrogen receptor a, increase production of EGF-like peptides and their responsiveness to them by increased expression of receptors and specifically the co-receptor ERBB2. See text for more details.

The presence or absence of ERa/PR provides the basis for one type of classification of breast cancers. 'ER+' breast cancers are on average better differentiated, grow more slowly, are not as strongly aneuploid, and have a slightly better prognosis than ER- breast cancers. Moreover, as a rule, their growth remains dependent on estrogens. Thus, compounds that block estrogen action or diminish the level of endogenous estrogens are often efficacious against ER+ cancers, but not at all against ER- cancers. Several strategies to specifically treat ER+ cancers are in use or are being explored (cf. 22.3). Compounds such as tamoxifen and raloxifene are partial agonists/antagonists (SERMs) that interfere with estrogen binding and with some interactions of the receptor with co-activators. They are used for chemoprevention, neoadjuvant treatment, adjuvant treatment, or treatment of systemic disease with metastases. Newer full antagonists block the ERa more efficiently and induce its degradation. They are expected to become used mostly in actual tumor treatment rather than in prevention, because of adverse effects on other tissues, such as bones and heart.

In women before menopause, when endogenous estrogens are still produced at high levels, this production must be diminished for efficient treatment. This can be achieved by surgical removal of the ovaries ('oophorectomy') or by treatment with analogues of gonadotropin-releasing hormone (GnRH or LHRH). GnRH is a hypothalamic peptide that stimulates the release of luteinizing hormone (LH) in the hypophysis. It is secreted in a cyclic fashion and regulated by neuronal inputs and by steroid feedback inhibition. Most drugs used in therapy are GnRH receptor agonistic and induce an initial burst of LH. However, since their level during treatment remains steadily high, the GnRH receptors in the pituitary become down-regulated. As a consequence, the production of LH hormone ceases, the ovaries are no longer stimulated by LH and stop to produce estrogens (Figure 18.11). Since in postmenopausal women estrogen synthesis is no longer significantly controlled by LH, a different strategy is required. Usually, inhibitors of the estrogen biosynthesis enzyme aromatase are employed.

By and large, the growth of ER+ breast cancers appears to be promoted by overactivity of those mechanisms that stimulate normal breast epithelial cells to grow during the proliferative phase of the monthly cycle or during pregnancy. Of course, in the cancers, neither the cyclic regression phase nor the terminal differentiation of duct cells take place in an orderly fashion. In contrast, ER- tumors neither depend on nor respond to estrogens. The estrogen receptors are not expressed and the ESR1 gene as well is quite frequently silenced by hypermethylation. In many ER- breast cancers, a large fraction of the proliferative stimulus appears to be provided through ERBB proteins.

In man, the ERBB family named after their first identified member, the retroviral v-erbB oncogene (^4.1) comprises four structurally related membrane receptors, ERBB1 - ERBB4. The designations HER1 - HER4 are also in use (Table 18.3). Each receptor protein comprises an «600 amino acid extracellular ligand-binding domain, a 24 amino acid single-pass helical transmembrane domain and a >500 amino acid cytoplasmatic tyrosine kinase domain with multiple autophosphorylation sites and an autoinhibitory loop (cf. Figure 4.4).

ERBB1, also known as the EGF receptor, of course binds the epidermal growth factor, but also several further peptides that share with EGF a conserved motif with three cystine disulfide bridges, termed the EGF motif. In fact, in most physiological circumstances, the structurally related TGFa, amphiregulin, and heparin-binding EGF (HB-EGF) are probably more relevant than EGF itself. HB-EGF as well as betacellulin (BTC) and epiregulin (EPR) also bind and activate HER4. Neuregulins 1 - 4 (NRGs) are specific for ERBB3 and ERBB4, NRGs 1&2 being predominantly recognized by ERBB3 and NRGs 3&4 by ERBB4. This leaves ERBB2 with no known ligand and there may indeed be none (Table 18.3). Rather, all ERBB receptors form dimers after ligand binding and all prefer to form heterodimers with ERBB2, although homodimers are also active.

Ligand binding induces a conformation change that relieves auto-inhibition of the tyrosine kinase by the pseudosubstrate loop to allow cross-phosphorylation with subsequent docking of adaptor and substrate proteins (^4.4). Signals emanate from the ERBB receptors mainly through the MAPK, PI3K, and STAT pathways (^4.4, 6.3, 6.8).

As ERBB2 is the odd member of the family with respect to ligand binding, ERBB3 is unusual with respect to kinase activity. Crucial residues in its active center are not conserved and ERBB3 may have no kinase activity at all. Thus, its tyrosine kinase activity is provided by its ERBB2 dimerization partner.

Figure 18.11 Regulation of steroid hormone production by pituitary hormones and mode of action of GnRH receptor agonists

In normal breast epithelial cells, both proliferation and differentiation are influenced by several EGF-related peptides and each member of the ERBB family is involved, at least in a subset of the cells. Production of the peptide growth factors takes place in stromal and epithelial cells and is regulated by estrogens. Both their production and the cellular responses to the growth factors are influenced by crosstalk with other hormones and growth factors. For instance, release of HB-EGF requires proteolytic cleavage by metalloproteinases (^9.3) which are stimulated by endothelin-1 and bombesin acting through G-coupled serpentine receptors. Certain WNT factors also promote release of EGF-like factors. The relevance of WNT pathway activation (^6.10) in breast cancer may therefore have to be considered in this light. At the ERBB receptor step, cross-talk with cytokine receptors may be particularly important in breast tissue. Prolactin and growth hormone receptors induce phosphorylation of ERBB1 by JAK kinases (^6.8).

With multiple growth factors and receptors distributed between several cell types and subject to cross-talk with further pathways, the precise relationships are extremely complex. In addition, they may substantially vary between different phases of breast growth and even segments of the ducts. In summary, it appears that activation of ERBB1, but even more of the ERBB2/ERBB3 heterodimer provides the main stimulus for proliferation. At least in the case of the ERBB2/ERBB3 unit, this stimulation may predominantly be exerted through the PI3K pathway, since ERBB3 after phosphorylation by ERBB2 provides several binding sites for the PI3Ka regulatory subunit (^6.3). In contrast, the ERBB4 receptor which is

Table 18.3. The ERBB family in man

Gene

Other names

Chromosomal Localization

Ligands *

Remarks

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