Yuri Yamaguchi

Identification of a GPER/GPR30 antagonist with improved estrogen receptor counterselectivity

GPER/GPR30 is a seven-transmembrane G protein-coupled estrogen receptor that regulates many aspects of mammalian biology and physiology. We have previously described both a GPER-selective agonist G-1 and antagonist G15 based on a tetrahydro-3H-cyclopenta[c]quinoline scaffold. The antagonist lacks an ethanone moiety that likely forms important hydrogen bonds involved in receptor activation. Computational docking studies suggested that the lack of the ethanone substituent in G15 could minimize key steric conflicts, present in G-1, that limit binding within the ERα ligand binding pocket. In this report, we identify low-affinity cross-reactivity of the GPER antagonist G15 to the classical estrogen receptor ERα. To generate an antagonist with enhanced selectivity, we therefore synthesized an isosteric G-1 derivative, G36, containing an isopropyl moiety in place of the ethanone moiety. We demonstrate that G36 shows decreased binding and activation of ERα, while maintaining its antagonist profile towards GPER. G36 selectively inhibits estrogen-mediated activation of PI3K by GPER but not ERα. It also inhibits estrogen- and G-1-mediated calcium mobilization as well as ERK1/2 activation, with no effect on EGF-mediated ERK1/2 activation. Similar to G15, G36 inhibits estrogen- and G-1-stimulated proliferation of uterine epithelial cells in vivo. The identification of G36 as a GPER antagonist with improved ER counterselectivity represents a significant step towards the development of new highly selective therapeutics for cancer and other diseases.

The ubiquitin ligase CHIP acts as an upstream regulator of oncogenic pathways.

CHIP is a U-box-type ubiquitin ligase that induces ubiquitylation and degradation of its substrates, which include several oncogenic proteins. The relationship between CHIP and tumour progression, however, has not been elucidated. Here, we show that CHIP suppresses tumour progression in human breast cancer by inhibiting oncogenic pathways. CHIP levels were negatively correlated with the malignancy of human breast tumour tissues. In a nude mouse xenograft model, tumour growth and metastasis were significantly inhibited by CHIP expression. In contrast, knockdown of CHIP (shCHIP) in breast cancer cells resulted in rapid tumour growth and metastastic phenotypes in mice. In cell-based experiments, anchorage-independent growth and invasiveness of shCHIP cells was significantly elevated due to increased expression of Bcl2, Akt1, Smad and Twist. Proteomic analysis identified the transcriptional co-activator SRC-3 (refs 13, 14, 15, 16, 17, 18, 19) as a direct target for ubiquitylation and degradation by CHIP. Knocking down SRC-3 in shCHIP cells reduced the expression of Smad and Twist, and suppressed tumour metastasis in vivo. Conversely, SRC-3 co-expression prevented CHIP-induced suppression of metastasis formation. These observations demonstrate that CHIP inhibits anchorage-independent cell growth and metastatic potential by degrading oncogenic proteins including SRC-3.

5α-Reductase type 1 and aromatase in breast carcinoma as regulators of in situ androgen production

Previous in vitro studies demonstrated that bioactive androgen 5α‐dihydrotestosterone (DHT) exerted antiproliferative effects through an interaction with androgen receptor (AR) in breast carcinoma cells. However, AR status has not been examined in association with DHT concentration in breast carcinoma tissues, and significance of androgenic actions remains unclear in breast carcinomas. Therefore, in our study, we first examined intratumoral DHT concentrations in 38 breast carcinoma tissues using liquid chromatography/electrospray tandem mass spectrometry. Intratumoral DHT concentration was positively associated with 5α‐reductase type 1 (5αRed1), and negatively correlated with aromatase. We then examined clinical significance of AR and 5αRed1 status in 115 breast carcinoma tissues by immunohistochemistry. Breast carcinomas positive for both AR and 5αRed1 were inversely associated with tumor size or Ki‐67. These patients showed significant associations with a decreased risk of recurrence and improved prognosis for overall survival, and the AR / 5αRed1 status was demonstrated an independent prognostic factor. Moreover, we examined possible regulation of DHT production by aromatase in in vitro studies. DHT synthesis from androstenedione in MCF‐7 cells was significantly inhibited by coculture with aromatase‐positive stromal cells, which was significantly reversed by addition of aromatase inhibitor exemestane. These results suggest that intratumoral DHT concentration is mainly determined by 5αRed1 and aromatase in breast carcinoma tissues, and antiproliferative effect of DHT may primarily occur in the cases positive for both AR and 5αRed1. Aromatase inhibitors may be more effective in these patients, possibly due to increasing local DHT concentration with estrogen deprivation.

Estrogen signaling pathway and hormonal therapy.

Hormonal therapy, such as estrogen-targeting therapy, has undergone remarkable development in recent several years, using drugs such as LH–RH agonists, new SERMs and third-generation aromatase inhibitors. Several ongoing large-scale international clinical trials for hormonal therapy are establishing the standard protocol for treatments with these drugs. On the other hand, there have been attempts to predict the individual efficacy of hormonal therapy using classical molecular biomarkers such as ER and PgR. However, approximately one-third of ERα-positive patients do not respond to endocrine therapy, while some ERα-negative patients are responsive. These discrepancies may be due to the different estrogen-related intracellular signaling pathways in breast cancer cells. Furthermore, the ineffectiveness of hormonal therapy in some individuals (due to, for example, aromatase inhibitor resistance) may be caused by these mechanisms. In this paper, we discuss the molecular mechanisms of these different responses to hormonal therapies and their implications for the estrogen signaling pathway in breast cancer cells. Furthermore, we touch upon basic studies into predicting the efficacy of hormonal therapy and new strategies in this field.

Early growth responsive gene 3 in human breast carcinoma: a regulator of estrogen-meditated invasion and a potent prognostic factor

Early growth responsive gene 3 (EGR3) is a zinc-finger transcription factor and plays important roles in cellular growth and differentiation. We recently demonstrated estrogen-mediated induction of EGR3 in breast carcinoma cells. However, EGR3 has not yet been examined in breast carcinoma tissues and its significance remains unknown. Therefore, in this study, we examined biological functions of EGR3 in the breast carcinoma by immunohistochemistry, in vitro study, and nude mouse xenograft model. EGR3 immunoreactivity was detected in carcinoma cells in 99 (52%) out of 190 breast carcinoma tissues and was associated with the mRNA level. EGR3 immunoreactivity was positively associated with lymph node status, distant metastasis into other organs, estrogen receptor alpha, or EGR3 immunoreactivity in asynchronous recurrent lesions in the same patients, and was negatively correlated with tubule formation. EGR3 immunoreactivity was significantly associated with an increased risk of recurrence and adverse clinical outcome by both uni- and multivariate analyses. Egr3-expressing transformant cell lines derived from MCF-7 Tet-Off cells (Eg-10 and Eg-11) significantly enhanced the migration and invasion properties according to the treatment of doxycyclin, but did not significantly change the cell proliferation. Moreover, Eg-11 cells injected into athymic mice irregularly invaded into the adjacent peritumoral tissues, although Clt-7, which was stably transfected with empty vector as a control, demonstrated a well-circumscribed tumor. Eg-11 cells were significantly associated with invasive components and less tubule formation in the xenograft model. These results suggest that EGR3 plays an important role in estrogen-meditated invasion and is an independent prognostic factor in breast carcinoma.

Recent trends in microRNA research into breast cancer with particular focus on the associations between microRNAs and intrinsic subtypes.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate the function of target genes at the post-transcriptional phase. miRNAs are considered to have roles in the development, progression and metastasis of cancer. Recent studies have indicated that particular miRNA signatures are correlated with tumor aggressiveness, response to drug therapy and patient outcome in breast cancer. On the other hand, in routine clinical practice, the treatment regimens for breast cancer are determined based on the intrinsic subtype of the primary tumor. Previous studies have shown that miRNA expression profiles of each intrinsic subtypes of breast cancer differ. In hormone receptor-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer, miRNA expressions are found to be correlated with endocrine therapy resistance, progesterone receptor expression and heat shock protein activity. Some miRNAs are associated with resistance to HER2-targeted therapy and HER3 expression in HER2-positive breast cancer. In triple-negative breast cancer, miRNA expressions are found to be associated with BRCA mutations, immune system, epithelial–mesenchymal transition, cancer stem cell properties and androgen receptor expression. As it has been clarified that the expression levels and functions of miRNA differ among the various subtypes of breast cancer, and it is necessary to take account of the characteristics of each breast cancer subtype during research into the roles of miRNA in breast cancer. In addition, the discovery of the roles played by miRNAs in breast cancer might provide new opportunities for the development of novel strategies for diagnosing and treating breast cancer.

Production of interleukin 6 and its relation to the macrophage differentiation of mouse myeloid leukemia cells (M1) treated with differentiation-inducing factor and 1α,25-dihydroxyvitamin D3

We have studied the production of interleukin 6 (IL-6) and its relation to the macrophage differentiation in murine myeloid leukemia cells (M1). As has been reported, differentiation-inducing factor (D-factor), 1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25(OH)2D3], and recombinant IL-6 similarly induced differentiation of M1 cells into macrophages. The three compounds also induced mRNA expression of IL-6 in M1 cells. M1 cells treated with D-factor or 1 alpha, 25(OH)2D3 produced biologically active IL-6, but the amounts of IL-6 secreted into culture media did not appear to be enough to induce differentiation of M1 cells. Furthermore, simultaneous addition of anti-IL-6 antibody did not suppress the differentiation of M1 cells induced by D-factor or 1 alpha, 25(OH)2D3. These results show that IL-6 production is an essential property associated with the macrophage differentiation of M1 cells, but it may not be responsible for the D-factor- and 1 alpha, 25(OH)2D3-induced differentiation.

Increased androgen receptor activity and cell proliferation in aromatase inhibitor-resistant breast carcinoma

Aromatase inhibitors (AI) are commonly used to treat postmenopausal estrogen-receptor (ER)-positive breast carcinoma. However, resistance to AI is sometimes acquired, and the molecular mechanisms underlying such resistance are largely unclear. Recent studies suggest that AI treatment increases androgen activity during estrogen deprivation in breast carcinoma, but the role of the androgen receptor (AR) in breast carcinoma is still a matter of controversy. The purpose of this study is to examine the potential correlation between the AR- and AI-resistant breast carcinoma. To this end, we performed immunohistochemical analysis of 21 pairs of primary breast carcinoma and corresponding AI-resistant recurrent tissue samples and established two stable variant cell lines from ER-positive T-47D breast carcinoma cell line as AI-resistance models and used them in in vitro experiments. Immunohistochemical analysis demonstrated that the expression of prostate-specific antigen (PSA) and Ki-67 were significantly higher and ER and progesterone receptor (PR) were lower in recurrent lesions compared to the corresponding primary lesions. Variant cell lines overexpressed AR and PSA and exhibited neither growth response to estrogen nor expression of ER. Androgen markedly induced the proliferation of these cell lines. In addition, the expression profile of androgen-induced genes was markedly different between variant and parental cell lines as determined by microarray analysis. These results suggest that in some cases of ER-positive breast carcinoma, tumor cells possibly change from ER-dependent to AR-dependent, rendering them resistant to AI. AR inhibitors may thus be effective in a selected group of patients.

Estrogen and growth factor signaling pathway: basic approaches for clinical application.

Estrogen and its receptor play important roles in genesis and malignant progression of estrogen-dependent cancers, together with various growth factors. Functional cross-talk between estrogen-signaling and growth factor-mediated signaling pathways has been reported. Firstly, we show an example of the cross-talk that may alter the effect of antagonist on the breast and endometrial cancer cell growth. Our observations suggest that the constitutively activated MAP kinase-signaling pathway in endometrial cancer cells might enhance the transcriptional activity of ERalpha via phosphorylation of AF-1 domain. This mechanism may cause the growth stimulative effect of tamoxifen on the endometrium. Secondly, we show our recent study for comprehensive understanding of estrogen-signaling pathway using cDNA microarray. According to the results of the expression profiling of estrogen-responsive genes in ER-positive breast cancer cells using large-scale cDNA microarray, the custom-made cDNA microarray, on which only estrogen-responsive genes were loaded, was produced. Using this microarray consisting of the narrowed gene subset, we analyzed estrogen responsiveness of various cell lines and effect of estrogen antagonists. Aim of this study is not only to address the molecular mechanisms of estrogen-dependent growth of breast cancer, but also to develop the new diagnostic tools for responsiveness to hormone therapy of primary breast cancer patients. Finally, in order to understand the local tumor biology including stroma-cancer interaction, we recently developed the new analytical system using ERE-GFP introduced into breast cancer cells. Several observations indicated that these reporter cells were useful for assessment of stimulative effects of stroma cells adjacent to breast cancer on the estrogen-signaling pathway. These studies may provide not only new clues for elucidation of the molecular mechanisms of estrogen-dependent growth of breast cancer, but also assessment of anti-estrogen responses of individual breast cancer for patient-tailored hormone therapy.

Possible role of the aromatase-independent steroid metabolism pathways in hormone responsive primary breast cancers

Aromatase inhibitors (AIs) exert antiproliferative effects by reducing local estrogen production from androgens in postmenopausal women with hormone-responsive breast cancer. Previous reports have shown that androgen metabolites generated by the aromatase-independent enzymes, 5α-androstane-3β, 17β-diol (3β-diol), androst-5-ene-3β, and 17β-diol (A-diol), also activate estrogen receptor (ER) α. Estradiol (E2) can also reportedly be generated from estrone sulfate (E1S) pooled in the plasma. Estrogenic steroid-producing aromatase-independent pathways have thus been proposed as a mechanism of AI resistance. However, it is unclear whether these pathways are functional in clinical breast cancer. To investigate this issue, we assessed the transcriptional activities of ER in 45 ER-positive human breast cancers using the adenovirus estrogen-response element-green fluorescent protein assay and mRNA expression levels of the ER target gene, progesterone receptor, as indicators of ex vivo and in vivo ER activity, respectively. We also determined mRNA expression levels of 5α-reductase type 1 (SRD5A1) and 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD type 1; HSD3B1), which produce 3β-diol from androgens, and of steroid sulfatase (STS) and 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD type 1; HSD17B1), which produce E2 or A-diol from E1S or dehydroepiandrosterone sulfate. SRD5A1 and HSD3B1 expression levels were positively correlated with ex vivo and in vivo ER activities. STS and HSD17B1 expression levels were positively correlated with in vivo ER activity alone. Elevated expression levels of these steroid-metabolizing enzymes in association with high in vivo ER activity were particularly notable in postmenopausal patients. Analysis of the expression levels of steroid-metabolizing enzymes revealed positive correlations between SRD5A1 and HSD3B1, and STS and HSD17B1. These findings suggest that the SRD5A1-HSD3B1 as well as the STS-HSD17B pathways, could contributes to ER activation, especially postmenopause. These pathways might function as an alternative estrogenic steroid-producing, aromatase-independent pathways.