In vivo longitudinal imaging of RNA interference‐induced endocrine therapy resistance in breast cancer

Endocrine therapy resistance in breast cancer is a major obstacle in the treatment of patients with estrogen receptor‐positive (ER+) tumors. Herein, we demonstrate the feasibility of longitudinal, noninvasive and semiquantitative in vivo molecular imaging of resistance to three endocrine therapies by using an inducible fluorescence‐labeled short hairpin RNA (shRNA) system in orthotopic mice xenograft tumors. We employed a dual fluorescent doxycycline (Dox)‐regulated lentiviral inducer system to transfect ER+ MCF7L breast cancer cells, with green fluorescent protein (GFP) expression as a marker of transfection and red fluorescent protein (RFP) expression as a surrogate marker of Dox‐induced tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) knockdown. Xenografted MCF7L tumor‐bearing nude mice were randomized to therapies comprising estrogen deprivation, tamoxifen or an ER degrader (fulvestrant) and an estrogen‐treated control group. Longitudinal imaging was performed by a home‐built multispectral imaging system based on a cooled image intensified charge coupled device camera. The GFP signal, which corresponds to number of viable tumor cells, exhibited excellent correlation to caliper‐measured tumor size (P << .05). RFP expression was substantially higher in mice exhibiting therapy resistance and strongly and significantly (P < 1e‐7) correlated with the tumor size progression for the mice with shRNA‐induced PTEN knockdown. PTEN loss was strongly correlated with resistance to estrogen deprivation, tamoxifen and fulvestrant therapies.      

Regulation of pS2 gene expression by affinity labeling and reversibly binding estrogens and antiestrogens: comparison of effects on the native gene and on pS2-chloramphenicol acetyltransferase fusion genes transfected into MCF-7 human breast cancer cells

We have examined the effects of reversibly and irreversibly binding estrogenic and antiestrogenic ligands for the estrogen receptor on pS2 RNA accumulation in MCF-7 human breast cancer cells and on pS2-chloramphenicol acetyl transferase (CAT) fusion gene expression in transfected MCF-7 cells. In MCF-7 cells grown in the absence of estrogens, the reversibly binding estrogen, estradiol, and the affinity labeling estrogen, ketononestrol aziridine, KNA, evoked a 13-fold increase in pS2 RNA level. The reversibly binding antiestrogen trans-hydroxytamoxifen and the affinity labeling antiestrogens tamoxifen aziridine or desmethylnafoxidine aziridine behaved as partial agonists/antagonists. In thymidine kinase-chloramphenicol acetyltransferase (tk-CAT) fusion genes containing a 1000 base pair fragment of the pS2 5'-flanking region encompassing the estrogen responsive element of the gene [pS2 (-1100/-90) tk-CAT], estradiol and ketononestrol aziridine evoked a marked stimulation of CAT activity and, in transfected cells grown in both the presence or absence of the weak estrogen phenol red, the antiestrogens behaved as partial agonists/antagonists. This pS2 5'-flanking region displayed both estrogen-dependent and estrogen-independent enhancer activity as monitored by stimulation of CAT activity. Hormonal regulation of the transfected pS2 fusion gene was similar to that observed in the native pS2 gene of MCF-7 cells; however, antiestrogens, while still partial agonists-antagonists, were relatively more agonistic on the transfected fusion gene than on the native gene. One antiestrogen (ICI 164,384) that behaved as a pure estrogen antagonist on the native gene was a partial agonist-antagonist of pS2 gene expression in the plasmid. This study illustrates that the hormonal regulation of the pS2 gene, as characterized by the agonist-antagonist balance of estrogens and antiestrogens, is influenced by the DNA context of the pS2 estrogen responsive element. Also, the fact that estrogens and antiestrogens that form covalent bonds with the estrogen receptor modulate activity of the native pS2 gene and the pS2-tk-CAT fusion gene in a manner similar to that of their reversibly binding counterparts suggests that it may be possible to use these irreversibly binding ligands to follow the interaction of hormone-receptor complexes with regions regulating estrogenic stimulation of the pS2 gene.     

p53 promoter-based reporter gene in vitro assays for quick assessment of agents with genotoxic potential

The p53 promoter-based green fluorescent protein(GFP)and luciferase reporter gene assayshave been established for detecting DNA damage induced by genotoxic agents.To evaluate the system,NIH3T3 cells transfected with either pHP53-GFP or pMP53-GFP construct were treated with mitomycin or5-fluorouracil.Expression of the GFP reporter gene was significantly and specifically induced in the cellsexposed to mitomycin or 5-fluorouracil.Then we treated NIH3T3 cells harboring pHP53-Luc or pMP53-Luc vector with mitomycin,5-fluorouracil or cisplatin at various concentrations.Similarly,exposure of thecells to these agents with genotoxic potentials resulted in a dose-dependent induction in luciferase reportergene expression.Thus,these in vitro reporter gene assays could provide an ideal system for quick assess-ment or screening of agents with genotoxic potential.     

Antiestrogens inhibit the mitogenic effect of growth factors on breast cancer cells in the total absence of estrogens

The antiproliferative effect of antiestrogens in breast cancer is believed to be entirely due to the inhibition of estrogen induced growth. We show here that non-steroidal antiestrogens inhibit the growth of the human breast cancer MCF7 cells in the complete absence of estrogens (phenol-red-free medium) when cell proliferation is stimulated by insulin or epidermal growth factor. This non-antiestrogenic effect of antiestrogens is, however, mediated by accessible estrogen receptor sites, as it is not observed in receptor negative hormone-independent breast cancers, and is rescued by estradiol but not by insulin. We conclude that antiestrogens inhibit cell proliferation by inhibiting growth factor action as well as estrogen action and that in both cases, accessible estrogen receptors are required.     

2,5-Diphenylfuran-based pure antiestrogens with selectivity for the estrogen receptor alpha

The estrogen receptor alpha (ERalpha) is understood to play an important role in the progression of breast cancer. Therefore, pure antiestrogens with a preference for this receptor form are of interest as new agents for the treatment of this malignancy. Several chemical structures with selective binding affinity for ERalpha have been identified and might be useful for the synthesis of ERalpha-selective pure antiestrogens. In this study we applied the 2,5-diphenylfuran system which is closely related to the triphenylfurans described by others. Various side chains with amino and/or sulfur functions were linked to C3 to convert the furans to estrogen antagonists without residual estrogenic activity. The degree of alpha-selectivity which ranges from 2.5- to 236-fold is strongly influenced by the alkyl group at C4. Antiestrogenic potency was determined in MCF-7/2a breast cancer cells stably transfected with a luciferase gene under the control of an ERE. The 2,5-bis(4-hydroxyphenyl)furan with an ethyl substituent and a 6-[N-methyl-N-(3-pentylthiopropyl)amino]hexyl side chain exerted the strongest antiestrogenic effect in this series with an IC(50) value of 50 nM in cells stimulated with 1 nM estradiol. The RBA values of this derivative were 18% (ERalpha) and 3.4% (ERbeta) of estradiol, respectively. It inhibited the growth of wild-type MCF-7 cells with an IC(50) value of 22 nM. The data show that the 2,5-diphenylfuran system is appropriate for the development of pure antiestrogens with preference for ERalpha.     

Compound profiling using a panel of steroid hormone receptor cell-based assays

A major focus in the current discovery of drugs targeting nuclear receptors (NRs) is identifying drugs with reduced side effects by improving selectivity, not only from other receptors but also by selective modulation of the NR of interest. Cellular assays not only provide valuable information on functional activity, potency, and selectivity but also are ideally suited for differentiating partial agonists and antagonists. The ability to partially activate a receptor is believed to be closely tied to the ability to selectively modulate the NR, resulting in expression of a subset of the normally regulated genes. To this end, the authors have built a complete panel of cell-based steroid hormone receptor assays for the androgen receptor, estrogen receptor alpha, estrogen receptor beta, glucocorticoid receptor, mineralocorticoid receptor, and progesterone receptor by stably engineering a Gal4 DNA-binding domain/nuclear receptor ligand-binding domain fusion protein into an upstream activation sequence beta-lactamase reporter cell line. Each assay was validated with known agonists and antagonists for correct pharmacology and high-throughput compatibility. To demonstrate the utility of these assays, the authors profiled 35 pharmacologically relevant compounds in a dose-response format against the panel in both agonist and antagonist modes. The results demonstrated that selective estrogen receptor modulators can be identified and differentiated, as well as mixed and partial agonists and antagonists easily detected in the appropriate assays. Importantly, a comparison of the chimeric assays with full-length reporter gene assay data from the literature shows a good degree of correlation in terms of selectivity and pharmacology of important ligands. Taken together, these steroid hormone receptor assays provide good selectivity, sensitivity, and appropriate pharmacology for high-throughput screening and selectivity profiling of modulators of steroid hormone receptors.     

GFP-reporter for a high throughput assay to monitor estrogenic compounds

In vitro test systems using yeast cells are a useful tool for the determination of the estrogenic activity of estrogens, phyto- and xeno-estrogens and can be used for monitoring large sample numbers in a routine analysis procedure. Our conventional transactivation assay functions with an expression plasmid expressing estrogen receptor α (ERα) under the control of a copper-inducible CUP1 promoter and a reporter plasmid expressing β-galactosidase under the control of the vitellogenin estrogen response element (ERE). In the novel yeast screen system the lacZ gene in the reporter plasmid was substituted by a gene for green fluorescent protein (GFP). Incubation of yeast with various concentrations of estrogenically active substances led to expression of the reporter gene product GFP in a dose dependent manner. The yeast transactivation assay was further down-scaled to be performed in a microplate scale, which is an important step to facilitate handling of large sample numbers. The sensitivity and reproducibility of the novel test system could be confirmed by analysis of the potencies of various estrogenically active substances. Thus, the newly developed yeast estrogen screen using GFP as a reporter can substitute the assay that has been used for a period of several years.     

Development of a new xenoestrogen screening system using fission yeast Schizosaccharomyces pombe

Endocrine disrupters refer to environmental or chemical compounds, which interfere with the endocrine system of organisms. In this study, our aim was to develop a screening method to detect xenoestrogen (an endocrine disrupter that is commonly encountered in our daily life) by using fission yeast Schizosaccharomyces pombe. Although the yeast (the simplest eukaryotic cell) has no endocrine system, estrogen receptors that are created to express in the yeast cell can be activated by estrogen in a similar manner to mammalian cells. First, in order to express the human estrogen receptor (hER) in the yeast cell, we constructed a yeast expression vector that contained hER (pREP42MHN-hER). In the yeast cells that are transformed with the pREP42MHN-hER vector, estrogen receptors could recognize xenoestrogen, which allowed the determination of the presence of xenoestrogen in any given sample. Furthermore, we constructed a yeast strain that contained an estrogen responsive element (ERE) that fused with the Escherichia coli LacZ gene (pERE-LacZ) as a reporter for binding of xenoestrogen with the estrogen receptor. Since this vector system allows determination of the presence and level of xenoestrogen with simple procedures, it is expected that they can serve as an efficient assay system to detect xenoestrogen.     

Acteoside and martynoside exhibit estrogenic/antiestrogenic properties

Acteoside and martynoside are plant phenylpropanoid glycosides exhibiting anticancer, cytotoxic and antimetastatic activities. We investigated their potential to activate estrogen receptor isoforms ERalpha and ERbeta in HeLa cells transfected with an estrogen response element (ERE)-driven luciferase (Luc) reporter gene and an ERalpha or ERbeta expression vector. Their estrogenic/antiestrogenic effects were also assessed in breast cancer cells (MCF7), endometrial cancer cells (Ishikawa) and osteoblasts (KS483), by measuring IGFBP3 levels, cell viability and number of mineralized nodules, respectively, seeking for a natural selective estrogen receptor modulator (SERM). Acteoside and martynoside antagonized both ERalpha and ERbeta (p<0.001), whereas they reversed the effect of E(2) mainly via ERalpha (p<0.001). Martynoside was a potent antiestrogen in MCF-7 cells, increasing, like ICI182780, IGFBP3 levels via the ER-pathway. In osteoblasts, martynoside induced nodule mineralization, which was abolished by ICI182780, implicating an ER-mediated mechanism. Furthermore, its antiproliferative effect on endometrial cells suggests that martynoside may be an important natural SERM. Acteoside was an antiestrogen in breast cancer cells and osteoblasts, without any effect on endometrial cells. Our study suggests that the nature is rich in selective ERalpha and ERbeta ligands, the discovery of which may lead to the development of novel neutraceutical agents.     

利用非侵入性光学成像技术监测在体肿瘤的生长

利用电穿孔技术将gfp表达质粒转染于SP2／0细胞,通过G418筛选获得稳定表达GFP的小鼠骨髓瘤细胞株SP2／0—GFP细胞。将稳定的转化细胞移植于同系BALB／c小鼠后腿、耳部皮下及腹腔成瘤,每隔一天对小鼠肿瘤成像,跟踪肿瘤的生长过程。结果表明,探测到的GFP荧光强度和发光范围与肿瘤的生长或消亡相关,从而证明可以利用GFP作为肿瘤细胞的标记,结合光学成像技术,对肿瘤生长过程实时追踪。     

Visualization of agonist-induced association and trafficking of green fluorescent protein-tagged forms of both beta-arrestin-1 and the thyrotropin-releasing hormone receptor-1.

A fusion protein (beta-arrestin-1-green fluorescent protein (GFP)) was constructed between beta-arrestin-1 and a modified form of the green fluorescent protein from Aequorea victoria. Expression in HEK293 cells allowed immunological detection of an 82-kDa cytosolic polypeptide with antisera to both beta-arrestin-1 and GFP. Transient expression of this construct in HEK293 cells stably transfected to express the rat thyrotropin-releasing hormone receptor-1 (TRHR-1) followed by confocal microscopy allowed its visualization evenly distributed throughout the cytoplasm. Addition of thyrotropin-releasing hormone (TRH) caused a profound and rapid redistribution of beta-arrestin-1-GFP to the plasma membrane followed by internalization of beta-arrestin-1-GFP into distinct, punctate, intracellular vesicles. TRH did not alter the cellular distribution of GFP transiently transfected into these cells nor the distribution of beta-arrestin-1-GFP following expression in HEK293 cells lacking the receptor. To detect potential co-localization of the receptor and beta-arrestin-1 in response to agonist treatment, beta-arrestin-1-GFP was expressed stably in HEK293 cells. A vesicular stomatitis virus (VSV)-tagged TRHR-1 was then introduced transiently. Initially, the two proteins were fully resolved. Short term exposure to TRH resulted in their plasma membrane co-localization, and sustained exposure to TRH resulted in their co-localization in punctate, intracellular vesicles. In contrast, beta-arrestin-1-GFP did not relocate or adopt a punctate appearance in cells that did not express VSV-TRHR-1. Reciprocal experiments were performed, with equivalent results, following transient expression of beta-arrestin-1 into cells stably expressing VSVTRHR-1-GFP. These results demonstrate the capacity of beta-arrestin-1-GFP to interact with the rat TRHR-1 and directly visualizes their recruitment from cytoplasm and plasma membrane respectively into overlapping, intracellular vesicles in an agonist-dependent manner.     

Development of a flow cytometric assay to study glucocorticoid receptor-mediated gene activation in living cells

A flow cytometry-based reporter gene assay was developed and utilized to measure glucocorticoid receptor (GR)-mediated gene activation at the single cell level in living cells. A reporter gene was generated that contains two copies of the glucocorticoid response element and an E1b TATA box upstream of a destabilized enhanced green fluorescent protein. Glucocorticoid activation of the reporter gene in Cos-1 and HTC cell lines was measured in vivo by flow cytometry and was shown to be dose dependent, leading to an increase in total fluorescence of the cell population. Flow cytometric analysis indicated this increase in total fluorescence per sample resulted from an increase in the number of cells expressing the activated green fluorescent protein (GFP) reporter as well as an overall increase in the mean GFP fluorescence within cells. Activation of reporter gene activity was time dependent occurring as early as 1-2h after dexamethasone addition. Activation of the reporter gene was specific as it exhibited different sensitivities to a range of glucocorticoids and activation could be blocked with glucocorticoid receptor antagonists. Coexpression of the coactivator SRC-1a or P65 subunit of NF-kappa B with GR led to enhancement or repression, respectively. Taken together, these data suggest the reporter-based flow cytometry assay is an effective method for analyzing glucocorticoid receptor-mediated gene expression at the single cell level in living cells.     

Response of estrogen receptor-positive breast cancer tumorspheres to antiestrogen treatments

Estrogen signaling plays a critical role in the pathogenesis of breast cancer. Because the majority of breast carcinomas express the estrogen receptor ERα, endocrine therapy that impedes estrogen-ER signaling reduces breast cancer mortality and has become a mainstay of breast cancer treatment. However, patients remain at continued risk of relapse for many years after endocrine treatment. It has been proposed that cancer recurrence may be attributed to cancer stem cells (CSCs)/tumor-initiating cells (TICs). Previous studies in breast cancer have shown that such cells can be enriched and propagated in vitro by culturing the cells in suspension as mammospheres/tumorspheres. Here we established tumorspheres from ERα-positive human breast cancer cell line MCF7 and investigated their response to antiestrogens Tamoxifen and Fulvestrant. The tumorsphere cells express lower levels of ERα and are more tumorigenic in xenograft assays than the parental cells. Both 4-hydroxytamoxifen (4-OHT) and Fulvestrant attenuate tumorsphere cell proliferation, but only 4-OHT at high concentrations interferes with sphere formation. However, treated tumorsphere cells retain the self-renewal capacity. Upon withdrawal of antiestrogens, the treated cells resume tumorsphere formation and their tumorigenic potential remains undamaged. Depletion of ERα shows that ERα is dispensable for tumorsphere formation and xenograft tumor growth in mice. Surprisingly, ERα-depleted tumorspheres display heightened sensitivity to 4-OHT and their sphere-forming capacity is diminished after the drug is removed. These results imply that 4-OHT may inhibit cellular targets besides ERα that are essential for tumorsphere growth, and provide a potential strategy to sensitize tumorspheres to endocrine treatment.