“Dwarf” Estrogen Receptor in Breast Cancer and Resistance to Tamoxifen

A major problem in breast cancer treatment and research today is why so many patients with estrogen receptor alpha (ER ) –positive tumors have de novo resistance to tamoxifen and why even more acquire resistance at a later time. An interesting study by Shi et al in this issue of Journal of Clinical Oncology suggests that one mechanism (and there are probably many) involves a truncated variant of ER , which may predict and possibly mediate resistance. In previous studies, these investigators identified a novel 36-kDa variant of ER using a homology search against a portion of the ER 66 ligand binding domain, and cloned it from human placental cDNA. Like ER 66, this ER variant is expressed from the ESR1 gene, but it is transcribed from an alternative start site. Following transcription, it also undergoes alternative splicing at the 3 end, yielding a protein lacking the AF-1 and AF-2 trans-activation domains, as well as the last 138 amino acids of ER 66, which are replaced with a unique 22–amino acid sequence. They developed an antibody specific to the unique C-terminus of ER 36, which does not recognize ER 66, and used it to show that ER 36 is expressed in a significant proportion of ER 66-positive and -negative breast cancer cell lines and human primary tumors. Interestingly, ER 36 is apparently not expressed in normal breast epithelial cells, or the MCF10A human mammary epithelial cell line. To study the function of this new variant, they stably transduced and overexpressed ER 36 in human embryonic kidney cells (HEK293), which do not express the receptor. They showed that ER 36 lacks intrinsic transcriptional activity, consistent with its lack of both trans-activation domains. In cells cotransduced with ER 66, they also demonstrated that the variant is a dominant inhibitor of ER 66 transcriptional activity. An unexpected and particularly interesting finding was that ER 36 localizes to the cytoplasm and surface membrane of cells, and that binding by both estrogen and tamoxifen activates the mitogen-activated protein kinase (MAPK) signaling pathway, stimulating cell growth. Based on these laboratory studies, they hypothesized that ER 36 expression may be associated with tamoxifen resistance in patients with ER 66-positive breast cancer. In this study, Shi et al tested this hypothesis by measuring ER 36 expression by immunohistochemistry (IHC) in archival tissue banks of several groups of patients with invasive breast cancer. The patients were treated with various combinations of tamoxifen and chemotherapy, and the associations with ER 36 expression and disease-free survival (DFS) and disease-specific survival (DSS) were evaluated. ER 36 positive was artificially defined as moderate-to-strong IHC staining in 10% or more tumor cells. The largest group of patients (n 307) had ER 66-positive tumors treated with tamoxifen with or without chemotherapy (median follow-up 7.9 years), and showed significantly decreased benefit in ER 36-positive compared with ER 36-negative tumors in both univariate and multivariate (MVA) analyses of DFS and DSS (11% and 12%, P .002, and hazard ratio [HR] 1.9 and 2.5, P .002, respectively). A separate, independently evaluated group with ER 66positive tumors treated with tamoxifen alone (n 156; median follow-up, 4.8 years) also showed significantly decreased benefit in ER 36-positive versus ER 36-negative tumors (eg, DFS HR, 5.47; P .003). In contrast, there was a hint of benefit in patients with ER 66-positive/ER 36-positive tumors (n 129) treated with chemotherapy alone (DSS HR, 0.79; P .58 in MVA), and a stronger trend towards benefit in a group with ER 66-negative/ER 36-positive tumors (n 149) treated in a similar manner (DSS HR, 0.52; P .07 in MVA). A smaller group of patients with ER 66-negative/ER 36positive tumors (n 73) treated with tamoxifen with or without chemotherapy showed a nonsignificant trend towards decreased benefit (DFS HR, 1.55; P .35 in MVA). This study has strengths and limitations. Foremost among the strengths are the tantalizing results suggesting that ER 66-positive/ ER 36-positive breast cancers are relatively resistant to adjuvant tamoxifen, which was demonstrated in two independent cohorts of patients of reasonable size and follow-up. Foremost among the limitations was the absence of an untreated group of patients for comparison, so it is difficult to determine if the apparent resistance to tamoxifen represents a truly significant negative interaction with the drug alone, or if there are additional prognostically detrimental biologic features associated with the variant receptor. Aggressive, fastgrowing breast cancers often initially respond fairly well to the types of cytotoxic chemotherapies used in this study, and there were weak trends towards benefit in ER 36-positive compared with ER 36negative tumors treated with chemotherapy alone, regardless of ER 66 status, suggesting that the variant may indeed be associated with aggressive behavior. The authors’ previous in vitro studies of ER 66-negative/ER 36-positive breast cancer cell lines showing that estrogen activates the MAPK pathway and increases proliferation also support this notion. Because of the retrospective and longitudinal study design, it is also difficult to separate de novo from acquired JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 27 NUMBER 21 JULY 2