4436s Vol. 7, 4436s-4442s, December 2001 (Suppl.) Clinical Cancer Research Inhibition of erbB Receptor (HER) Tyrosine Kinases as a Strategy to Abrogate Antiestrogen Resistance in Human Breast Cancer 1 Hirokazu Kurokawa and Carlos L. Arteaga 2 duced growth of established MCF-7/HER2 xenografts in Departments of Medicine and Cancer Biology and Vanderbilt-Ingram athymic nude mice. Similar results have been obtained with Cancer Center, Vanderbilt University School of Medicine, Nashville, ZD1839 ("Iressa"), an epidermal growth factor receptor Tennessee 37232 (HER1) tyrosine kinase inhibitor. Taken together, these data suggest that exogenous inhibitors of the HER-signaling net- Abstract work and other mitogenic pathways can abrogate or delay the emergence of antiestrogen resistance, thus providing an It has been proposed that binding of ligand to the estrogen receptor (ER) releases its association with tran- evaluable therapeutic strategy in human breast carcinoma. scriptional corepressors, allowing the ER to recruit coacti- vators, which possess histone acetylase activity, and induce Introduction transcription of gene promoters containing estrogen re- The classical ER 3 oL belongs to the superfamily of nuclear sponse elements. It has also been proposed that the anties- steroid receptors that function as transcription factors on binding trogen tamoxifen recruits transcriptional corepressors to the by their respective ligands (1). The ER contains 595 amino acids AF-2 region of the hormone-binding domain of the ER, thus blocking ER-mediated transcription. The ER cross-talks with a central DNA-binding domain and a COOH-terminal, with a number of mitogenic signaling pathways and second hormone-binding domain. More recently, a smaller 530-amino messengers, like the epidermal growth factor receptor, the acid ER~3 was cloned (2). Binding of estradiol to ER induces insulin-like growth factor-I receptor, mitogen-activated pro- conformational changes in the receptor and ER dimerization tein (MAP) kinase, phosphatidylinositol-3 kinase/Akt, do- with subsequent high-affinity binding to DNA through their pamine, and cyclic AMP. Some of these molecules may: (a) DNA-binding domain at specific EREs present in the promoter support ligand-independent ER transcription; (b) increase of estrogen-responsive genes. ER-induced gene transcription is the association of ER with coactivators of transcription; mediated by two distinct transactivation domains: the hormone- and/or (c) reduce the antiestrogen-induced association of ER independent AF-1 region in the NH 2 terminus and the hormone- with corepressors. These events either alone or in combina- dependent AF-2 region in the COOH-terminal region of the tion may result in hormone independence and/or antiestro- receptor (3, 4). Studies with ER mutants have shown that, gen resistance. We have examined whether signaling by although AF-1 and AF-2 are required for maximal ER transcrip- HER2/neu (erbB-2) receptor tyrosine kinase, which can in- tional activity in most cells, certain gene promoters, sometimes duce antiestrogen resistance, can also disrupt the tamoxifen- as a function of their cellular context, can be independently induced interaction of ER with transcriptional corepressors. transactivated by AF-1 or AF-2 alone (5). This promoter and/or Notably, tamoxifen-induced association of ER with the tran- cell dependence of ER-mediated transcription appears to be scriptional corepressors N-CoR or SMRT was reduced in modulated by the recruitment of coactivator and corepressor HER2-overexpressing breast tumor cells but not in cells proteins, which serve as signaling intermediates between the with low HER2 levels. Small molecule inhibitors of the DNA-bound receptor dimer and the basal transcriptional ma- HER2 kinase or MAP extracellular signal-regulated kinase chinery (Refs. 1, 6, and references therein). These coactivators 1/2 or dominant-negative MAP extracellular signal-regu- and corepressors of transcription possess intrinsic histone acety- lated kinase 1/2 constructs restored the inhibitory effect of lase or histone deacetylase activities, respectively. Coactivators tamoxifen on both ER-mediated transcription and tumor can acetylate histones, loosen their interaction with DNA and, cell proliferation. Treatment with both tamoxifen and the therefore, expose motifs required for gene transcription. On the small molecule HER1/2 kinase inhibitor AG1478 reduced other hand, corepressor molecules can silence steroid-respon- mitogen-activated protein kinase activity and markedly re- sive genes by binding to the unliganded receptor, deacetylating histones, and potentially keeping the DNA in a conformation that occludes coactivator binding sites. Presented at the First International Conference on Recent Advances and Future Directions in Endocrine Therapy for Breast Cancer, June 21-23, 2001, Cambridge, MA. Supported in part by NIH Grant R01 CA80195, Department of Defense Breast Cancer Research Program Grant DAMD 17-98-1-8262, and Vanderbilt-Ingram Cancer Center sup- 3 The abbreviations used are: ER, estrogen receptor; ERE, estrogen port Grant CA68485. response element; SERM, selective estrogen receptor modulator; EGFR, 2 To whom requests for reprints should be addressed, at Division of epidermal growth factor receptor; EGF, epidermal growth factor; Hematology-Oncology, Vanderbitt University School of Medicine, 777 MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol-3 Preston Residential Building, Nashville, TN 37232-6307. Phone: (615) kinase; MEKK, mitogen-activated protein kinase kinase kinase; MEK, 936-3524; Fax: (615) 936-1790; E-mail: carlos.arteaga@mcmail. mitogen-activated protein/extracellular signal-regulated kinase kinase; vanderbilt.edu. JNK, c-Jun-NHz-terminal kinase. Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2001 American Association for Cancer Research. Clinical Cancer Research 4437s Molecular Basis for the ER Agonist/Antagonist Role A SERM sensitive estrogen of Tamoxifen The antiestrogen tamoxifen antagonizes the transcription of genes that require the AF-2 region for ER-mediated transacti- vation. However, for gene promoters in which AF-2 function is dispensable and whose transcription is driven by the AF-1 Coactlvatom 7~s domain, tamoxifen can function as an agonist (5, 7). This ability of tamoxifen to behave as an AF-2 antagonist and AF-1 agonist NH2-I A/B I C I D I ElF I.COOH explains its antagonistic effect in breast epithelium, as well as its agonistic effect in other tissues, such as endometrium, bone, and AF-1 DBD AF-21HBD liver. In addition to blocking AF-2-dependent gene transactiva- tion, in some experimental systems, tamoxifen has been shown I transcription to repress ER-mediated transcription by recruiting corepressor proteins to the receptor (8, 9). In other experiments, overexpres- B SERM resistant sion of transcriptional coactivators, like SRC-1 or L7/SPA, results in enhancement of tamoxifen-stimulated ER reporter (~j estrogen activity (8, 10). Because of their ability to either repress or activate the transcription of estrogen target genes, mixed antag- tamoxifen / onist/agonists like tamoxifen are now more appropriately re- ferred to as SERMs. The data summarized above imply that the net cellular response to SERMs may depend on the tissue repertoire, avail- NH2-1 AJB I C IDI ElF I_COOH ability, and/or relative amounts of transcriptional coactivators and corepressors. Therefore, high levels of coactivators and/or low levels of corepressors may be permissive for the agonist AF-1 DBD AF-21HBD activity of SERMs. These results suggest that the recruitment of I coactivators or corepressors in different ratios to the ER-ERE -~ transcription complex could introduce a potential element of transcriptional Fig. 1 A molecular hypothesis to support sensitivity or resistance to control and diversity that may be more marked in some estro- SERMs. In tamoxifen-sensitive tumor cells (A), corepressors predomi- nate over coactivators in tamoxifen-occupied ERs. In this setting, the gen-responsive genes than others. Finally, these data have led to tamoxifen-mediated inhibitory effect on AF-2 is dominant, resulting in the evaluable hypothesis that de novo or acquired abundance of a blockade of hormone-induced gene transcription. With progression to coactivators and/or the reduction or loss of transcriptional core- SERM resistance (B), the ER coactivator:corepressor ratio is reversed, pressors in breast tumor cells may result in hormone independ- resulting in a loss of tamoxifen-mediated inhibition of AF-2 and/or ence and antiestrogen resistance (11). This hypothesis, illus- unmasking of its agonistic effect on AF-1 function (modified from Takimoto et al., Ref. 11). trated in Fig. 1, is supported by a recent experiment reported by Lavinsky et al. (9). On prolonged treatment with tamoxifen, ER-positive MCF-7 xenografts in athymic nude mice developed resistance to treatment and grew during continuous tamoxifen well as that induced by tamoxifen-occupied ERs (13). Neuro- therapy. Tumor levels of N-CoR were reduced in the tamoxifen- transmitters like dopamine or SKF-38393 can induce ER re- resistant xenografts, suggesting that reduced levels of the core- porter activity in the absence of added steroids (14). Exogenous pressor may enable tamoxifen to stimulate ER-mediated tran- EGF or overexpression of the EGFR-homologous HER2/neu scription through unopposed AF-1 function (9). Interestingly, (erbB2) proto-oncogene can reduce tamoxifen-induced associ- 4-hydroxytamoxifen