Shiuan Chen

The role of microRNA-128a in regulating TGFbeta signaling in letrozole-resistant breast cancer cells

Resistance to endocrine therapy agents has presented a clinical obstacle in the treatment of hormone-dependent breast cancer. Our laboratory has initiated a study of microRNA regulation of signaling pathways that may result in breast cancer progression on aromatase inhibitors (AI). Microarray analysis of hormone refractory cell lines identified 115 differentially regulated microRNAs, of which 49 microRNAs were believed to be hormone-responsive. A group of microRNAs were inversely expressed in the AI-resistant lines versus LTEDaro and tamoxifen-resistant. We focused our work on hsa-miR-128a which was hormone-responsive and selectively up-regulated in the letrozole-resistant cell lines. Human miR-128a was predicted to target the TGFβ signaling pathway and indeed sensitivity to TGFβ was compromised in the letrozole-resistant cells, as compared to parental MCF-7aro. Human miR-128a was shown to negatively target TGFβRI protein expression by binding to the 3′UTR region of the gene. Inhibition of endogenous miR-128a resulted in resensitization of the letrozole-resistant lines to TGFβ growth inhibitory effects. These data suggest that the hormone-responsive miR-128a can modulate TGFβ signaling and survival of the letrozole-resistant cell lines. To our knowledge, this is the first study to address the role of microRNA regulation as well as TGFβ signaling in AI-resistant breast cancer cell lines. We believe that in addition to estrogen-modulation of gene expression, hormone-regulated microRNAs may provide an additional level of post-transcriptional regulation of signaling pathways critically involved in breast cancer progression and AI-resistance.

ERRγ Suppresses Cell Proliferation and Tumor Growth of Androgen-Sensitive and Androgen-Insensitive Prostate Cancer Cells and Its Implication as a Therapeutic Target for Prostate Cancer

Estrogen receptor-related receptors (ERR) are orphan nuclear receptors, which are constitutively activated without estrogen binding. Recent evidence indicates that the ligand-independent ERRs may be involved in similar ER-mediated regulatory pathways and modulate estrogen responsiveness in certain target cells. We recently showed that an ERR subtype, ERRgamma, is coexpressed with ERbeta in normal human prostatic epithelial cells and exhibits reduced expression in many prostate cancer cell lines and clinical neoplastic prostate tissues. Based on this, we hypothesize that ERRgamma may have growth regulatory roles in prostate and prostate cancer. We showed in this study that ERRgamma was expressed in epithelial cell nuclei in fetal and pubertal human prostates, whereas its nuclear expression became reduced in advanced prostate cancer lesions. Stable ERRgamma expression by retroviral transduction suppressed significantly both in vitro cell growth and in vivo tumorigenicity of two prostate cancer cell lines, LNCaP and DU145, as evidenced by a cell-cycle arrest at G(1)-S transition and also induction of two cyclin-dependent kinase inhibitors p21(WAF1/CIP1) and p27(KIP1). We further showed by reporter assay that induction of p21 and p27 by ERRgamma was mediated through direct transactivation of their gene promoters. Moreover, we also showed that a selective ERRgamma-agonist, DY131, could potentiate the ERRgamma-induced growth inhibition in LNCaP-ERRgamma and DU145-ERRgamma cells in a dose-dependent manner compared with respective parental cells. Taken together, our results show that ERRgamma may perform an antiproliferative or tumor-suppressing function in prostate cancer cells. More importantly, our results suggest that ERRgamma could be a novel therapeutic target for prostate cancer treatment.

The red clover ( Trifolium pratense ) isoflavone biochanin A inhibits aromatase activity and expression

Biochanin A is an isoflavone isolated from red clover (Trifolium pratense), and is a commercially available nutraceutical for women suffering from postmenopausal symptoms. Isoflavones resemble the structure of oestrogen, and display agonistic and antagonistic interactions with the oestrogen receptor. Overexposure of oestrogen is a major contributing factor in the development of breast cancer, and cytochrome P450 (CYP) 19 enzyme, or aromatase, catalyses the reaction converting androgen to oestrogen. In the present study the effect of biochanin A on the gene regulation and enzyme activity of aromatase was investigated. By assaying MCF-7 cells stably transfected with CYP19, biochanin A inhibited aromatase activity and hampered cell growth attributing to the enzyme activity. In addition, 25 microm-biochanin A significantly reduced CYP19 mRNA abundance in the oestrogen receptor-negative breast cancer cells SK-BR-3. The transcriptional control of the CYP19 gene is exon-specific, and promoter regions I.3 and II have been shown to be responsible for CYP19 expression in SK-BR-3 cells. Luciferase reporter gene assays also revealed that biochanin A could repress the transcriptional control dictated by the promoter regulation. Interestingly, genistein did not inhibit aromatase but it might down regulate promoter I.3 and II transactivation. Since genistein is a major metabolite of biochanin A, it might contribute to biochanin As suppressive effect on CYP19 expression. The present study illustrated that biochanin A inhibited CYP19 activity and gene expression.

Dietary administration of the licorice flavonoid isoliquiritigenin deters the growth of MCF-7 cells overexpressing aromatase

Licorice is the sweet‐tasting rhizomes of a bean plant and is quite commonly used in Western countries for culinary purposes, while it is a medicinal herb in China. Many flavonoids have been isolated from licorice, and their pharmacological properties may be applicable in preventive medicine. Overexposure to estrogen has been implicated in the etiology of breast cancer, and cytochrome P450 (CYP) 19 enzyme, or aromatase, catalyzes the rate‐limiting reaction. Phytocompounds that are able to inhibit this enzyme may potentially suppress breast cancer development. In the present study the licorice flavonoid isoliquiritigenin (ILN) was shown to be an aromatase inhibitor in recombinant protein and MCF‐7 cells stably transfected with CYP19 (MCF‐7aro). ILN displayed a Ki value of around 3 μM, and it also blocked the MCF‐7aro cell growth pertaining to the enzyme activity in vitro. Subsequently, the compound administered in diet was given to ovariectomized athymic mice transplanted with MCF‐7aro cells. This mouse model is widely accepted for studying postmenopausal breast cancer. The phytochemical significantly deterred the xenograft growth without affecting the body weight. Subsequently, the flavonoids effect on CYP19 transcriptional control in vitro was also investigated. At the mRNA level, ILN could also suppress the expression in wild‐type MCF‐7 cells. Reporter gene assay and real‐time PCR verified that the transactivity of CYP19 driven by promoters I.3 and II was suppressed in these cells. Deactivation of C/EBP could be the underlying molecular mechanism. Our study demonstrated that ILN was an inhibitor of aromatase and a potential chemopreventive agent against breast cancer.

Inhibition of the proliferation of acquired aromatase inhibitor-resistant breast cancer cells by histone deacetylase inhibitor LBH589 (panobinostat)

Aromatase inhibitors (AIs) are important drugs for treating postmenopausal patients with hormone receptor-positive breast cancer. However, acquired resistance to AI therapies is a significant problem. Our study has revealed that the histone deacetylase inhibitor LBH589 treatment abrogated growth of AI-resistant cells in vitro and in vivo, causing cell cycle G2/M arrest and induced apoptosis. LBH589 treatment also reduced the level of NF-κB1 which is overexpressed when AI resistance develops. Analyzing paired tumor specimens from 12 patients, we found that NF-κB1 expression was increased in recurrent AI-resistant tumors as compared to the paired primary tumors before AI treatment. This finding was consistent with up-regulated NF-κB1 expression seen in a collection of well-established AI-resistant cell lines. Furthermore, knockdown of NF-κB1 expression significantly suppressed the proliferation of AI-resistant cells. Treatment of AI-resistant cell lines with LBH589 suppressed NF-κB1 mRNA and protein expression. In addition, LBH589 treatment abrogated growth of AI-resistant tumors in mice, and was associated with significantly decreased levels of NF-κB1 in tumors. In all, our findings strongly support further investigation of LBH589 as a novel therapeutic strategy for patients with AI-resistant breast cancer, in part by suppressing the NF-κB1 pathway.

The citrus flavonone hesperetin inhibits growth of aromatase-expressing MCF-7 tumor in ovariectomized athymic mice

Aromatase is responsible for the rate-determining reaction in estrogen synthesis and is a prime target for treating estrogen-receptor-positive breast cancer. Previous in vitro study has demonstrated that apigenin (APG), naringenin (NGN) and hesperetin (HSP) are three of the most potent natural aromatase inhibitors. Because the enzyme inhibition could potentially block breast cancer development, we employed an established postmenopausal breast cancer model to examine the chemopreventive effect of these flavonoids in vivo. Athymic mice were ovariectomized and transplanted with aromatase-overexpressing MCF-7 cells. Dietary administration of HSP at 1000 ppm and 5000 ppm significantly deterred the xenograft growth, while a null effect was observed in mice treated with APG or NGN. Further study illustrated that plasma estrogen in HSP-treated mice was reduced. Messenger RNA expression of the estrogen-responsive gene pS2 was also decreased in the tumors of mice treated with 1000 and 5000 ppm HSP. On the other hand, western analysis indicated that cyclin D1, CDK4 and Bcl-x(L) were reduced in the tumors. This study suggested that HSP could be a potential chemopreventive agent against breast carcinogenesis through aromatase inhibition.

The molecular basis of the interaction between the proline-rich SH3-binding motif of PNRC and estrogen receptor alpha

PNRC and PNRC2 are members of a new family of nuclear receptor coactivators. We systematically determined the molecular basis and the structure/function relationship for the PNRC–ERα interaction. PNRC was found to interact with ERα mainly through its C-terminus region, amino acids 270–327, and an SH3-binding motif within this region was shown to be essential for PNRC to interact with and function as coactivator of ERα. The importance of the flanking sequences of SH3-binding motif in the interaction between PNRC and ERα was also investigated. The PNRC-interacting domain(s) on ERα was also mapped. PNRC was found to interact with both AF1 and LBD of ERα, and to function as a coactivator for both AF1 and AF2 transactivation functions. The interaction of ERα mutants, I358R, K362A, V376R, L539R and E542K, with PNRC/PNRC2 was further investigated. ERα/HBD/V376R could bind to PNRC or PNRC2, with similar affinity as wild-type ERα/HBD, and the transactivation activity of ERα/V376R was enhanced 5-fold by PNRC. Since GRIP1, a well-characterized coactivator, was found not to be able to enhance the transactivation function of this mutant, our results indicate that the PNRC–ERα interaction interface is not exactly identical to that of GRIP1–ERα interaction.

Characterization of the weak estrogen receptor α agonistic activity of exemestane

Third generation aromatase inhibitors (AI) have shown good clinical efficacy in comparison to the anti-estrogen tamoxifen. The steroidal AI, exemestane (EXE) has previously been shown to act as an androgen, but this report demonstrates the estrogen-like activity of EXE. Based on genome-wide microarray analysis, high correlation was seen between EXE-Only (EXE O, hormone-free) and hormone-containing AI-resistant lines. In addition, the top regulated genes in the EXE O lines were mostly estrogen-responsive genes. This estrogen-like activity of EXE was further validated using estrogen receptor (ER) activity assays, where in comparison to 17β-estradiol (E2), EXE was able to induce ER activity, though at a higher concentration. Also, this EXE-mediated ER activity was blocked by the ER antagonist ICI as well as the ERα-specific antagonist methyl-piperidino-pyrazole (MPP). Similarly, EXE was able to induce proliferation of breast cancer cell lines, MCF-7 and MCF-7aro, as well as activate transcription of known estrogen-responsive genes, i.e., PGR, pS2 and AREG. These results suggest that EXE does have weak estrogen-like activity.

The citrus flavonone hesperetin prevents letrozole-induced bone loss in a mouse model of breast cancer.

Aromatase is a key enzyme in estrogen synthesis, and aromatase inhibitors (AIs) have been developed for treating estrogen-responsive breast cancer. Because of its nondiscriminatory inhibition of estrogen synthesis, patients treated with AIs also contract diseases typically associated with estrogen deficiency, such as bone deterioration. Our laboratory found that the citrus flavonone hesperetin could inhibit aromatase, and the selective estrogen receptor modulator nature of flavonoid might counteract the undesirable effect of AIs. In the present study, we employed an established postmenopausal model for breast carcinogenesis to examine the drug interaction between hesperetin and letrozole, one of the AIs. Athymic mice were ovariectomized and transplanted with aromatase-overexpressing MCF-7 cells (MCF-7aro). Hesperetin was administered in the diet at 5000 ppm, and letrozole was injected sc at different doses. Results showed that either hesperetin or letrozole could reduce plasma estrogen level and inhibit tumor growth. Most importantly, the letrozole-induced bone loss measured as bone volume fraction was reversed by hesperetin without compromising on the deterrence of MCF-7aro tumor growth. Taken together, the present study suggested that hesperetin could be a potential cotherapeutic agent to AI.

Nuclear Receptor Coactivator PNRC2 Regulates Energy Expenditure and Adiposity

PNRC2 was identified in our laboratory as a general cofactor for nuclear receptors. To better characterize the physiological function of PNRC2, we used gene-targeting technology to generate PNRC2-null mice (PNRC2-/- mice). These PNRC2-/- mice are viable and fertile. PNRC2-null mice, especially male mice, are lean and are resistant to high fat diet-induced obesity but without the induction of insulin resistance. Male mice devoid of PNRC2 protein have a higher metabolic rate than wild-type mice. They consume more oxygen and produce more heat. Consistent with reduced adipose mass, the levels of leptin are lower in PNRC2-/- mice. This study provides evidence that PNRC2 plays one or more important roles in controlling the energy balance between energy storage and energy expenditure. PNRC2 may be a new target in the treatment of obesity and related metabolic diseases.