Tamoxifen induces a pluripotency signature in breast cancer cells and human tumors

Abstract

Tamoxifen is the treatment of choice in estrogen receptor alpha breast cancer patients that are eligible for adjuvant endocrine therapy. However, ∼50% of ERα-positive tumors exhibit intrinsic or rapidly acquire resistance to endocrine treatment. Unfortunately, prediction of de novo resistance to endocrine therapy and/or assessment of relapse likelihood remain difficult. While several mechanisms regulating the acquisition and the maintenance of endocrine resistance have been reported, there are several aspects of this phenomenon that need to be further elucidated. Altered metabolic fate of tamoxifen within patients and emergence of tamoxifen-resistant clones, driven by evolution of the disease phenotype during treatment, appear as the most compelling hypotheses so far. In addition, tamoxifen was reported to induce pluripotency in breast cancer cell lines, in vitro. In this context, we have performed a whole transcriptome analysis of an ERα-positive (T47D) and a triple-negative breast cancer cell line (MDA-MB-231), exposed to tamoxifen for a short time frame (hours), in order to identify how early pluripotency-related effects of tamoxifen may occur. Our ultimate goal was to identify whether the transcriptional actions of tamoxifen related to induction of pluripotency are mediated through specific ER-dependent or independent mechanisms. We report that even as early as 3 hours after the exposure of breast cancer cells to tamoxifen, a subset of ERα-dependent genes associated with developmental processes and pluripotency are induced and this is accompanied by specific phenotypic changes (expression of pluripotency-related proteins). Furthermore we report an association between the increased expression of pluripotency-related genes in ERα-positive breast cancer tissues samples and disease relapse after tamoxifen therapy. Finally we describe that in a small group of ERα-positive breast cancer patients, with disease relapse after surgery and tamoxifen treatment, ALDH1A1 (a marker of pluripotency in epithelial cancers which is absent in normal breast tissue) is increased in relapsing tumors, with a concurrent modification of its intra-cellular localization. Our data could be of value in the discrimination of patients susceptible to develop tamoxifen resistance and in the selection of optimized patient-tailored therapies.

Keywords: Breast cancer; Cell lines; Endocrine resistance; Patient data; Pluripotency; Tamoxifen; Transcriptomic analysis.

Figure 1

Short‐term incubation of ERα‐positive and negative breast cancer cells with tamoxifen, results in massive transcriptional changes. T47D and MDA‐MB‐231 breast cancer cells were incubated for 3 h with 10−6 M tamoxifen and transcriptional changes were identified with the Affymetrix HU133plus2 microarray. In A, the number of up‐ and down‐regulated genes in each closed area of the Venn diagram (shown in B) is depicted. In B, the strategy for the identification of ERα, other ERs and ER‐independent action of tamoxifen is presented. Panels C–E present analysis (in terms of p‐value, in Logarithmic scale) of significantly modified GO‐terms, analyzed separately for each category presented in B, with the online resource DAVID (data are presented in Supplemental Table 3). Finally, panel F presents the 173 genes found in category “Anatomical Structure Morphogenesis” (GO:0009653), identified with the blue arrow in panel C. Pluripotency‐related genes are identified in color.

Figure 2

Short‐term incubation of T47D cells with tamoxifen induces pluripotency. T47D cells were incubated for the indicated time‐periods with 10−6 M tamoxifen. In panel A qRT‐PCR time‐course expression analysis of Myc, SOX2, Oct1, KLF4 and ALDH1A1 after up to 24 h exposure of T47D cells to 10−6 M tamoxifen (data of a single experiment are presented, repeated three times in duplicates). Data are normalized as per 18s RNA expression. B. Cells were incubated for 24 or 48 h with 10−6 M tamoxifen, fixed and the expression of Myc, Nanog, Sox2 and ALDH1A1 were assayed by immunocytochemistry, as detailed in Material and Methods. Bar = 100 μm. The lower part of the Figure presents the quantitation of three different experiments (5 different fields per experiment), performed with the Image J (NIH) program. Mean ± SE are shown. C. T47D cells were incubated with tamoxifen (10−6 M) for 7 days in a low adherence substrate (see Material and Methods). Seven days after seeding, the number of primary mammospheres was measured, mammospheres were dissociated and an equal number of cells were further incubated under the same conditions, without tamoxifen, for seven additional days. The number of secondary mammospheres was counted at the end of this second incubation. Mean ± SE of three different experiments performed in triplicate.

Figure 3

Primary tumors of tamoxifen‐resistant breast cancer patients present early elements of pluripotency. A. Heat‐map of the 21 genes, being differentially expressed between tamoxifen‐responders and tamoxifen‐relapsing patients (0 and 1 in the tumor identification). Presented data were derived from the re‐analysis of the set GSE9195, as described in Material and Methods and Results sections. B. Identified transcription factors differentially modified in tamoxifen‐sensitive and relapsing tumor specimens of GSE9195 data‐set, by the use of the T‐Facts web resource (www.tfacts.org). See Material and Methods for further details. C. Network of protein–protein interactions of the identified transcription factors presented in B, through the web resource SNOW (http://babelomics.bioinfo.cipf.es/functional.html). See Results for further details.

Figure 4

Tamoxifen‐resistant and sensitive breast cancer primary tumors express elements of pluripotency. One hundred sixty three tamoxifen‐treated breast cancer cases were retrieved from the Cancer Genome Atlas collection (www.cancergenome.nih.gov). In A, gene set enrichment analysis of the “Sox‐targets” GO category is presented, while in B, GSEA of genes related to POU3F2 transcription factor are depicted. See Material and Methods and Results sections for additional details.

Figure 5

Immunohistochemistry of tumor specimens in tamoxifen‐responders and relapsing tumors present elements of pluripotency. A. Immunohistochemical determination of Myc, Nanog, Sox2 and ALDH1A1 in a case of tamoxifen relapsing (#1 in Table 1) tumor. In the first row the initial tumor is presented, while in the middle row the relapsing tumor is shown. The lower row of the Figure presents the immunohistochemical analysis of a tamoxifen‐sensitive (non‐relapsed) tumor specimen (#1 in Table 1). ×400, bar = 50 μm. B. ALDH1A1 presents a specific intracellular redistribution in the cytosol of tamoxifen resistant patients' tumors. Membrane localization of ALDH1A1 immunopositivity (evidenced mainly in left panel, before Tamoxifen treatment) is marked by black arrows. A typical case is presented (#2 in Table 1). Bar = 50 μm.