RESUMO
The acquisition of estrogen-deprivation resistance and estrogen receptor (ER) signal-independence in ER-positive breast cancer is one of the crucial steps in advancing the aggressiveness of breast cancer; however, this has not yet been elucidated in detail. To address this issue, we established several estrogen-deprivation-resistant (EDR) breast cancer cell lines from our unique MCF-7 cells, which had been stably transfected with an ERE-GFP reporter plasmid. Three cell lines with high ER activity and another 3 cell lines with no ER activity were established from cell cloning by monitoring GFP expression in living cells. The former three ERE-GFP-positive EDR cell lines showed the overexpression of ER and high expression of several ER-target genes. Further analysis of intracellular signaling factors revealed a marked change in the phosphorylation status of ERα on Ser167 and Akt on Thr308 by similar mechanisms reported previously; however, we could not find any changes in MAP-kinase factors. Comprehensive phospho-proteomic analysis also indicated the possible contribution of the Akt pathway to the phosphorylation of ERα. On the other hand, constitutive activation of c-Jun N-terminal kinase (JNK) was observed in ERE-GFP-negative EDR cells, and the growth of these cells was inhibited by a JNK inhibitor. An IGF1R-specific inhibitor diminished the phosphorylation of JNK, which suggested that a novel signaling pathway, IGF1R-JNK, may be important for the proliferation of ER-independent MCF-7 cells. These results indicate that ER-positive breast cancer cells can acquire resistance by more than two mechanisms at a time, which suggests that multiple mechanisms may occur simultaneously. This finding also implies that breast cancers with different resistance mechanisms can concomitantly occur and mingle in an individual patient, and may be a cause of the recurrence of cancer.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Elementos de Resposta , Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células , Moduladores de Receptor Estrogênico/farmacologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Estrogênios/fisiologia , Feminino , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Células MCF-7 , Fosforilação , Processamento de Proteína Pós-Traducional , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais , Fator de Transcrição AP-1/metabolismoRESUMO
Aromatase inhibitors (AIs) have been reported to exert their antiproliferative effects in postmenopausal women with hormone receptor-positive breast cancer not only by reducing estrogen production but also by unmasking the inhibitory effects of androgens such as testosterone (TS) and dihydrotestosterone (DHT). However, the role of androgens in AI-resistance mechanisms is not sufficiently understood. 5α-Androstane-3ß,17ß-diol (3ß-diol) generated from DHT by 3ß-hydroxysteroid dehydrogenase type 1 (HSD3B1) shows androgenic and substantial estrogenic activities, representing a potential mechanism of AI resistance. Estrogen response element (ERE)-green fluorescent protein (GFP)-transfected MCF-7 breast cancer cells (E10 cells) were cultured for 3 months under steroid-depleted, TS-supplemented conditions. Among the surviving cells, two stable variants showing androgen metabolite-dependent ER activity were selected by monitoring GFP expression. We investigated the process of adaptation to androgen-abundant conditions and the role of androgens in AI-resistance mechanisms in these variant cell lines. The variant cell lines showed increased growth and induction of estrogen-responsive genes rather than androgen-responsive genes after stimulation with androgens or 3ß-diol. Further analysis suggested that increased expression of HSD3B1 and reduced expression of androgen receptor (AR) promoted adaptation to androgen-abundant conditions, as indicated by the increased conversion of DHT into 3ß-diol by HSD3B1 and AR signal reduction. Furthermore, in parental E10 cells, ectopic expression of HSD3B1 or inhibition of AR resulted in adaptation to androgen-abundant conditions. Coculture with stromal cells to mimic local estrogen production from androgens reduced cell sensitivity to AIs compared with parental E10 cells. These results suggest that increased expression of HSD3B1 and reduced expression of AR might reduce the sensitivity to AIs as demonstrated by enhanced androgen metabolite-induced ER activation and growth mechanisms. Androgen metabolite-dependent growth of breast cancer cells may therefore play a role in AI-resistance.