Aromatase In Breast Cancer Research Articles

Initial Studies with 11C-Vorozole PET Detect Overexpression of Intratumoral Aromatase in Breast Cancer.

Aromatase inhibitors are the mainstay of hormonal therapy in estrogen receptor-positive breast cancer, although the response rate is just over 50% and in vitro studies suggest that only two thirds of postmenopausal breast tumors overexpress aromatase. The goal of the present study was to validate and optimize PET with 11C-vorozole for measuring aromatase expression in postmenopausal breast cancer in vivo. Methods: Ten newly diagnosed postmenopausal women with biopsy-confirmed breast cancer were administered 11C-vorozole intravenously, and PET emission data were collected between 40 and 90 min after injection. Tracer injection and scanning were repeated 2 h after ingestion of 2.5 mg of letrozole. Mean and maximal SUVs and ratios to nontumor tissue in the contralateral breast were determined at baseline and after letrozole. Biopsy specimens from the same tumors were stained for aromatase using immunohistochemistry and evaluated for stain intensity and the percentage of immune-positive cells. Results: Seven of the 10 women (70%) demonstrated increased mean focal uptake of tracer (SUV ratio > 1.1) coinciding with the mammographic location of the lesion, whereas the other 3 women (30%) did not (SUV ratio ≤ 1.0). All patients with an SUV ratio above 1.1 had mean SUVs above 2.4, and there was no overlap (SUV ratio ≤ 1; SUVmean, 0.8-1.8). The SUV ratio relative to breast around tumor was indistinguishable from the ratio to contralateral breast. Pretreatment with letrozole reduced tracer uptake in most subjects, although the percentage of blocking varied across and within tumors. Tumors with a high SUV in vivo also showed a high immunohistochemical staining intensity. Conclusion: PET with 11C-vorozole is a useful technique for measuring aromatase expression in individual breast lesions, enabling noninvasive quantitative measurement of baseline and posttreatment aromatase availability in primary tumors and metastatic lesions.

Abstract 1689: Comparative whole genome analyses of breast cancer aromatase inhibitor resistance models

Abstract One third of breast cancer patients treated with Aromatase Inhibitors (AI) or Tamoxifen acquire resistance leading to disease recurrence and death. The clinically observed AI resistance is mimicked by in vitro models MCF-7:5C and MCF-7:2A that are susceptible to apoptosis upon E2-treatment. These models have been instrumental to the current understanding of underlying mechanisms such as the reconfiguration of survival signaling, alterations of stress-related pathways (Ariazi et al. 2011; Fan et al. 2013, 2015), and changes in miRNA expression profiles (Hoppe et al. 2016). How the model phenotypes relate to a specific genomic background is poorly understood. In order to identify somatic variants indicative of AI resistance and potentially useful as novel therapeutic targets we performed NGS analyses of the 5C and 2A clones compared to the non-resistant WS8 and MCF-7 (ATCC). Whole genome/exome libraries were sequenced on Illumina HiSeq/NovaSeq platforms, respectively. Previously we reported first results from a preliminary analysis of gains and losses of variants in both resistant derivatives (Hoppe at al. AACR 2018). Here we report an updated analysis of raw reads trimmed and aligned to GRCh38 reference using BWAkit v 0.7.15. Duplicates were removed by Picard MarkDuplicates. Base quality scores were recalibrated using GATK4 BQSR. WGS and WES alignments were merged and tested for somatic variants using GATK4 Mutect2 (Tumor-only mode) with population variation data from gnomAD as a germline source. Called and filtered variants were uploaded to IVA for functional and clinical annotation. Copy number segments were modeled using both read- and allele-counts from WGS alignments by Gaussian-kernel binary-segmentation algorithm applied in GATK4 ModelSegments tool. To interpret copy number events causative for LOH we recovered germline variants and annotated CNVs that led to loss of the corresponding wild-type allele. In line with the COSMIC database we confirmed 461 mutated loci of which 228 were lost in both resistant derivatives. Examples of 5C-specific coding mutations classified as pathogenic include splice site alterations (i.e. SEMA3A), frameshifts (i.e. ARID1A), and stop gains (i.e. CACNA1S, CDH2, NOTCH4). 2A-specific mutations include for example a stop gain affecting ADGRV1. Copy number variation analyses revealed that both resistance models share prominent genetic defects such as amplified regions at Chr6q25.1 and Chr21q22.2. A 5C-specific aberration is a large Chr18 deletion affecting most of the q-arm, and a 2A-specific aberration is the amplification of most parts of Chr6q23.3-q24.3. We will present the status of this currently ongoing comparative genome analysis of AI resistance models and discuss the putative effects of homozygous mutations at loci with copy number aberrations within the context of AI resistance. Citation Format: Reiner Hoppe, Siarhei Kandabarau, Ping Fan, V. Craig Jordan, Heather Cunliffe, Hiltrud B. Brauch. Comparative whole genome analyses of breast cancer aromatase inhibitor resistance models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1689.

Abstract 3432: Evolving genomes of breast cancer aromatase inhibitor resistance models

Abstract Acquired endocrine resistance is a severe barrier in the anti-hormonal treatment of breast cancer and is responsible for its failure in about half of the treated patients. In vitro models of long-term estrogen deprived breast cancer cells serve as laboratory tools to explore new therapeutic strategies. The models MCF-7:5C and MCF-7:2A mimic clinical AI resistance but regress with E2 due to the reconfiguration of survival signaling known as E2-inducible apoptosis (5C rapid, 2A delayed reaction). Alterations of stress related pathways including the accumulation of endoplasmic reticulum stress, oxidative stress, and inflammatory stress that occur prior to E2-induced apoptosis (Ariazi et al. 2011; Fan et al. 2013, 2015; Sweeney et al. 2014) and specific miRNA expression profiles (Hoppe et al Oncotarget 2016) have been linked with these resistance and E2-inducible apoptosis phenotypes. Little is known on their underlying genetic variability. Here we compare the mutational profiles of 5C and 2A clones with those of the estradiol stimulated growth sensitive MCF-7:WS8 and standard MCF-7 (ATCC) cells. Whole genome libraries were sequenced on the Illumina HiSeq platform (2x150bp reads). Alignments were manually inspected (bcftools mpileup) to avoid false negative variant calling in poorly (<20x) covered regions. Loci with at least 10x coverage and absence of any variant reads were classified as wildtype. The coding genome positions defined as mutant or wildtype in all four cell types served as a basis to select candidate mutations for the verification of MCF-7 variants via comparison to the COSMIC database (http://cancer.sanger.ac.uk/cosmic) and identification of novel mutations by variant calling (GATK HaplotypeCaller). Putative protein effects of variants were predicted by the SnpEff tool. We verified 421 point mutations in MCF-7 (COSMIC) of which 203 mutations were lost in our models, with additional specific losses of 4, 4, and 18 mutations in WS8, 2A and 5C, respectively. In 5C cells new mutations affected 261 genes with 31 high impact mutations based on splice site alterations in 7 genes (i.e. CERS2, SEMA3A, TDRD7, ATP5H) and gain of stop codons in 24 genes (i.e. CACNA1S, NOTCH4, SUN1, JPH3, SERPINB2). Similarly, in 2A cells there were 12 high impact mutations among 122 affected genes (e.g. ARHGEF5, ARHGAP35, ARAP2). We will report on the integrative genomic analysis including larger structural variants (short indels and CNVs) towards the evolution of these models for a comprehensive understanding of the biology of E2-inducible apoptosis. Citation Format: Reiner Hoppe, Siarhei Kandabarau, Ping Fan, V Craig Jordan, Hiltrud B. Brauch. Evolving genomes of breast cancer aromatase inhibitor resistance models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3432.

Aryl hydrocarbon receptor induced intratumoral aromatase in breast cancer.

Aryl hydrocarbon receptor (AhR) inhibits estrogen receptor (ER) pathway, which may suppress estrogen-dependent cell proliferation. However, the correlation between AhR stimulation and intratumoral estrogen synthesis, especially through aromatase, has not been reported to date. In the present study, we examined this correlation in breast cancer cells. We examined AhR and aromatase immunoreactivity in 29 patients with invasive ductal carcinoma. We performed in vitro studies using three breast carcinoma cell lines, MCF-7, T47D, and MDA-MB-231. AhR stimulation induced the mRNA expression of the aromatase gene in vitro in three breast carcinoma cell lines, and increased estrogen synthesis in MCF-7 cell line. Results of microarray analysis showed that AhR-induced aromatase expression was associated with BRCA1 induction. Analysis of patients with breast cancer showed a significant positive correlation between intratumoral AhR and aromatase status. We also compared the effects of AhR stimulation on the induction of intratumoral estrogen synthesis and inhibition of the ER signaling pathway, because AhR exerts contradictory effects on estrogen action in breast carcinoma cells. AhR-induced aromatase expression persisted for a significantly longer duration than AhR-induced ER pathway inhibition. Moreover, breast carcinoma cells treated with an AhR agonist tended to show earlier cell proliferation after removing the agonist than cells not treated with the AhR agonist. The results of the present study suggest that AhR stimulates estrogen-dependent progression of breast carcinoma by inducing aromatase expression under some conditions. These results provide new insights on the possible roles of environmental toxins in breast cancer development.

Semiemperical Investigation of the Postmenopausal Breast Cancer Treatment Potential of Xanthone Derivatives

Aromatase, a catalyst in the aromatization reaction of androgens to estrogen, is a member of the cytochrome p450 superfamily, known as monooxygenases. The synthesized estrogen by Aromatase in breast cancer nourishes the cancer cells and assesses the hormonally growing of cancer cells. Therefore, Aromatase is considered as a potential target in treatment of breast cancer. Nowadays, major considerations in drug's selection in the treatment of cancers are shifted to natural sources due to their low toxicity profiles and better therapeutic functionality. In the present study, we have identified the binding modes of various xanthones, dietary supplements obtained from different plant sources, by using of efficient Biocomputational tools. Through docking studies, it is clear that 10 out of 13 ligands showing hydrogen bonds with amino acids like THR 310, PRO 249, ARG 113, GLY 439 and CYS 347. Sameathxanthone A showed highest dock score of -7.96 with the binding energy -38.46 kcal/mol and two hydrogen bonds with LEU 477 and VAL 373. Subsequently, the ADMET properties of compounds have been predicted computationally.

Promestriene Affects <i>GREB</i>1 Expression in Estrogen Sensitive Breast Cancer Cells

Promestriene (3-propyl ethyl, 17B-methyl estradiol) is a synthetic estrogen analogue with reported minimal systemic absorption which has been suggested for topical treatment of vaginal atrophy. Promestriene’s ability to stimulate proliferation and estrogen responsive gene expression was analyzed in estrogen receptor (ER+) positive breast cancer cell lines MCF-7, T-47D, and BT-474 using CFSE flow cytometric analysis, and quantitative RT-PCR analysis of GREB1 RNA expression, an estrogen responsive gene involved in estrogen receptor alpha expression. In estrogen replete conditions, Promestriene did not stimulate proliferation even at high concentrations (100,000 pg/ml). However, anti-estradiol depletion allowed low dose Promestriene (2 - 10 pg/ml) to stimulate GREB1 expression in all three cell lines at levels equal to that induced by estradiol (BT-474) or significantly higher than estradiol (MCF7 and T-47D). These findings suggest that Promestriene has the potential to support estrogen like cell signaling, a possible contraindication for use in treatment of vaginal atrophy associated with breast cancer aromatase inhibitor therapy.

Treatment-associated musculoskeletal and vasomotor symptoms and relapse-free survival in the NCIC CTG MA.27 adjuvant breast cancer aromatase inhibitor trial.

Treatment-emergent symptoms with adjuvant tamoxifen and aromatase inhibitors (AIs) have been associated with superior recurrence-free survival (RFS). We hypothesized that MA.27 anastrozole- or exemestane-treated patients with new or worsening vasomotor and/or joint symptoms would have improved RFS. MA.27 randomly assigned 7,576 postmenopausal women with breast cancer to 5 years of anastrozole or exemestane. Patient-reported symptoms were collected using the Common Terminology Criteria for Adverse Events version 3.0 at protocol-specified baseline and 6- and 12-month clinical visits. Symptoms were considered present with either vasomotor and/or joint complaints. Associations between symptoms and baseline patient characteristics were examined with χ(2) and Fisher's exact tests. Subsequent effects of new or worsening symptoms on RFS were examined with landmark analyses and stratified univariable and multivariable Cox models. We examined the effects of 3-month symptoms arising from unplanned clinic visits as a result of severe toxicity. Patients were assessable if eligible for the MA.27 trial, received some trial therapy, and had no disease recurrence at the end of a symptom assessment period; 96% of patients (n = 7,306 patients) were included at 6 months, and 96% (n = 7,246) were included at 12 months. Thirty-four percent of patients had baseline symptoms. For patients without baseline symptoms, 25% and 52% had new symptoms by 6 and 12 months, respectively. Neither treatment-emergent nor baseline symptoms significantly impacted RFS (P > .10) in patients with or without baseline symptoms. In MA.27, anastrozole or exemestane treatment-emergent symptoms were not associated with improved RFS. Women should be supported through treatment and encouraged to remain on their AI regardless of their symptoms.

Melatonin and Aromatase in Breast Cancer

Melatonin and Aromatase in Breast Cancer

Design, Synthesis, Antioxidant, and Anti‐Breast Cancer Activities of Novel Diethyl(alkyl/aryl/heteroarylamino)(4‐(pyridin‐2‐yl)phenyl)methylphosphonates

A series of new diethyl(alkyl/aryl/heteroarylamino)(4-(pyridine-2-yl)phenyl)methylphosphonates (4a-t) were synthesized via three-component Kabachnik-Field's reaction of 4-(pyridin-2-yl)benzaldehyde, diethylphosphite and various primary amines, catalyzed by cupric acetate monohydrate [Cu(OAc)(2) ∙ H(2)O] under solvent-free and microwave irradiation conditions. Their computational docking analysis supported them as good therapeutic agents to the breast cancer aromatase enzyme and ascertained 4a, 4h, 4m, 4n, and 4t as potential molecules with good binding affinities varying from -9.0 to -9.6 kcal/mol and containing the 4-(pyridine-2-yl)phenyl moiety as a pharmacophore. Their in vitro screening performed for the anti-cell proliferation activity against MBC-MCF7 cells by MTT and Trypan blue assays confirmed 4m, 4n, and 4q as promising compounds to sustain a low percentage of cell viability at 20 µg/mL concentration. These compounds were also evaluated for their antioxidant activity by the DPPH method and the results established that compounds 4m, 4n, and 4q show around 10% higher activity than the standard antioxidant ascorbic acid.

SIRT1 Positively Regulates Breast Cancer Associated Human Aromatase (CYP19A1) Expression

Breast cancer remains one of the leading causes of death in women diagnosed with cancer. In breast cancer, aberrant expression of the CYP19A1 gene, which encodes the aromatase enzyme, contributes to increased intratumoral levels of estradiol. Regardless of whether this estrogen is produced by peripheral tissues or within specific subpopulations of cells within the breast tumor, it is clear that the aromatase enzymatic activity is critical for the growth of estrogen-dependent tumors. Currently, aromatase inhibitors have proven to be highly effective in blocking the growth of estrogen-dependent forms of breast cancer. CYP19A1 transcription is tightly controlled by 10 tissue-specific promoters. In breast cancer, however, aromatase transcription is driven by multiple promoters that somehow override the tissue-specific regulation of normal tissue. Here, we explore the role that the deacetylase, sirtuin-1 (SIRT1), plays in positively regulating aromatase in breast cancer. We demonstrate that the use of cambinol and the SIRT1/2 inhibitor VII, 2 small molecule inhibitors of SIRT1 and SIRT2, as well as small molecule inhibitors and small interfering RNA specific to SIRT1, all reduce the levels of aromatase mRNA. We further demonstrate that pharmacologic inhibition causes a marked reduction in aromatase protein levels. Additionally, by chromatin immunoprecipitation, we demonstrate that SIRT1 occupies the promoter regions PI.3/PII and PI.4, and its inhibition leads to increased acetylation of estrogen-related receptorα, a transcription factor that positively regulates CYP19A1 transcription in epithelial cells. Finally, we demonstrate by immunohistochemistry that SIRT1 is significantly up-regulated in invasive ductal carcinoma relative to normal tissue adjacent to tumor, further suggesting a role of SIRT1 in breast cancer. This work uncovers a new mechanism for the regulation of aromatase and provides rationale for further investigation of how the inhibition of specific sirtuins may provide a unique strategy for inhibiting aromatase that may complement or synergize with existing therapies.

Dysregulated metabolism and the regulation of aromatase in breast adipose stromal cells in obesity and cancer

Background The risk of breast cancer in postmenopausal women is increased two-fold with obesity, and the majority of breast tumours that arise are oestrogen-dependent. After menopause, when the ovaries cease to produce oestrogens, it is the local production of oestrogens within the breast adipose stromal cells (ASCs) which promotes and sustains tumour growth. This is largely due to the increased expression of aromatase, responsible for the conversion of androgens to oestrogens. Aromatase expression in breast cancer is known to be under the control of a proximal promoter, promoter PII, which is maximally activated by cAMP-dependent mechanisms. We have previously demonstrated that the LKB1/AMPK pathway is a key negative regulator of aromatase expression within the breast by inhibiting the nuclear translocation of the CREB co-activator CRTC2 [1]. We have also demonstrated that the tumour-derived factor prostaglandin E2 (PGE2) and the obesity-associated factor leptin stimulate aromatase expression by inhibiting LKB1 and AMPK expression and activity. Hypoxia inducible factor-1a (HIF1a) is emerging as a potent regulator of glycolysis in tumour cells and we have identified a putative hypoxia response element in aromatase promoter Pll immediately adjacent the cAMP response element, known to be bound by the CREBCRTC2 complex in ASCs in breast cancer. We therefore hypothesise that HIF1a may be involved in regulating aromatase in breast cancer. Materials and methods Primary human breast ASCs were isolated from tissue after breast reduction surgery. Real-time PCR, Western blotting, immunofluorescence and high content screening were used to assess HIF1a expression and localisation after PGE2 treatment. Chromatin immunoprecipitation (ChIP) was performed to examine the interaction of HIF1a with aromatase promoter PII and reporter assays were performed to assess the effect of HIF1a on PII activity. Double immunohistochemistry for HIF1a and aromatase was also performed on sections of formalin-fixed paraffin-embedded breast tissue from breast cancer patients and cancer-free patients.

ChemInform Abstract: Synthesis of a New Carbon‐11‐Labeled Sulfamate Derivative (I) as a Potential PET Tracer for Imaging of Breast Cancer Aromatase and Steroid Sulfatase Expression.

ChemInformVolume 42, Issue 38 Heterocyclic Compounds ChemInform Abstract: Synthesis of a New Carbon-11-Labeled Sulfamate Derivative (I) as a Potential PET Tracer for Imaging of Breast Cancer Aromatase and Steroid Sulfatase Expression. Min Wang, Min Wang Dep. Radiol., Indiana Univ. Sch. Med., Indianapolis, IN 46202, USASearch for more papers by this authorMingzhang Gao, Mingzhang Gao Dep. Radiol., Indiana Univ. Sch. Med., Indianapolis, IN 46202, USASearch for more papers by this authorKathy D. Miller, Kathy D. Miller Dep. Radiol., Indiana Univ. Sch. Med., Indianapolis, IN 46202, USASearch for more papers by this authorQi-Huang Zheng, Qi-Huang Zheng Dep. Radiol., Indiana Univ. Sch. Med., Indianapolis, IN 46202, USASearch for more papers by this author Min Wang, Min Wang Dep. Radiol., Indiana Univ. Sch. Med., Indianapolis, IN 46202, USASearch for more papers by this authorMingzhang Gao, Mingzhang Gao Dep. Radiol., Indiana Univ. Sch. Med., Indianapolis, IN 46202, USASearch for more papers by this authorKathy D. Miller, Kathy D. Miller Dep. Radiol., Indiana Univ. Sch. Med., Indianapolis, IN 46202, USASearch for more papers by this authorQi-Huang Zheng, Qi-Huang Zheng Dep. Radiol., Indiana Univ. Sch. Med., Indianapolis, IN 46202, USASearch for more papers by this author First published: 25 August 2011 https://doi.org/10.1002/chin.201138131Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume42, Issue38September 20, 2011 RelatedInformation

Synthesis of a New Carbon-11–Labeled Sulfamate Derivative as a Potential PET Tracer for Imaging of Breast Cancer Aromatase and Steroid Sulfatase Expression

A carbon-11-labeled sulfamate derivative was designed and synthesized as a new potential positron-emission-tomography dual aromatase–steroid sulfatase inhibitor radiotracer for imaging of aromatase and steroid sulfatase expression in breast cancer. The target tracer 2-chloro-4-(((4-cyanophenyl)(4H-1,2,4-triazol-4-yl)amino)methyl)-6-[11C]methoxyphenyl sulfamate ([11C]7) was prepared from its corresponding precursor 2-chloro-4-(((4-cyanophenyl)(4H-1,2,4-triazol-4-yl)amino)methyl)-6-hydroxyphenyl sulfamate (18) with [11C]CH3OTf under basic conditions through the O-[11C]methylation and isolated by the reversed-phase high-performance liquid chromatography in 40–45% radiochemical yields based on [11C]CO2 and decay corrected to end of bombardment. The specific activity at end of synthesis was 111-185 GBq/μmol.

Expression of the forkhead transcription factor FOXL2 correlates with good prognosis in breast cancer patients treated with tamoxifen

Aromatase is an important enzyme in the local synthesis of oestrogens and its expression has been shown to be increased in breast cancer through the activation of multiple promoters. However, the mechanisms behind this are not yet fully understood. A novel candidate in this context is the transcription factor forkhead box L2 (FOXL2), which has been recognised to be co-expressed with aromatase and transcriptionally active promoter II in developing goat and chicken ovaries. We propose that FOXL2 could be involved in the increased expression of aromatase in breast cancer. We examined FOXL2 and its relation to aromatase in 132 post-menopausal breast cancer patients by immunohistochemistry. Using in silico analysis, we further searched for FOXL2 binding-elements in the aromatase gene promoters. The results demonstrate that FOXL2 is expressed in breast cancer and influences clinical outcome with improved recurrence-free survival in cases with nuclear expression. In a multivariate Cox model, nuclear FOXL2 was a significant prognostic factor in ER-positive patients treated with tamoxifen (HR=0.18, 95% confidence interval (CI)=0.04-0.81, P=0.03). Tumours expressing nuclear FOXL2 were also more likely positive for stromal and/or cytoplasmic aromatase (P=0.03 and P=0.008, respectively). In silico analyses revealed binding elements of FOXL2 in promoters I.3, II and I.7 of the aromatase gene of which promoter I.7 was most significant. In conclusion, this is the first study to report that FOXL2 is expressed in breast cancer and correlates with aromatase as well as with clinical outcome. The results further strengthen a possible binding of FOXL2 to aromatase promoter I.7. Nevertheless, whether FOXL2 is a direct activator of aromatase requires further investigation.

Selective regulation of aromatase expression for drug discovery

Aromatase is a particularly attractive drug target in the treatment of hormone-responsive breast cancer, and aromatase activity in breast cancer patients is greater in or near the tumor tissue compared with the normal breast tissue. Complex regulation of aromatase expression in human tissues involves alternative promoter sites that provide tissue-specific control. Previous studies in our laboratories suggested a strong association between aromatase ( CYP19) gene expression and the expression of cyclooxygenase ( COX) genes. Additionally, COX selective inhibitors can suppress CYP19 gene expression and decrease aromatase activity. Our current hypothesis is that pharmacological regulation of aromatase can act locally to decrease the biosynthesis of estrogen and may provide additional therapy options for patients with hormone-dependent breast cancer. Two pharmacological approaches are being developed, one approach utilizing small molecule drug design and the second approach involving mRNA silencing technology. The small molecule drug design approach focuses on the synthesis and biological evaluation of a novel series of sulfonanilide analogs derived from COX-2 selective inhibitors. Combinatorial chemistry approaches were used to generate diversely substituted novel sulfonanilides. The compounds suppress aromatase enzyme activity in SK-BR-3 breast cancer cells in a dose and time dependent manner, and structure activity analysis does not find a correlation between aromatase suppression and COX inhibition. Real-time PCR analysis demonstrates that the sulfonanilide analogs decrease aromatase gene transcription in breast cells. Furthermore, the sulfonanilide compounds selectively decrease aromatase gene expression in several breast cancer cells, without exhibiting cytotoxic or apoptotic effects at low micromole concentrations. A ligand-based pharmacophore model for selective aromatase modulation (SAM) by the novel sulfonanilides identified an aromatic ring, two hydrogen bond acceptors, and a hydrophobic function as four key chemical features. In the second approach, short interfering RNAs (siRNA) were designed targeting human aromatase mRNA. Treatment of breast cancer cells with siRNAs targeting aromatase (siAROMs) completely masked the aromatase enzyme activity and resulted in suppression of CYP19 mRNA. Thus, these results suggest that the novel sulfonanilides and the siRNAs targeting aromatase expression may be valuable tools for selective regulation of aromatase in breast cancer.

Breast cancer aromatase expression evaluated by the novel antibody 677: Correlations to intra-tumor estrogen levels and hormone receptor status

Breast cancer tissue estrogen levels on an average exceed plasma as well as benign breast tissue levels. To evaluate the contribution of intra-tumor aromatization to individual tumor estrogen levels (estradiol, E 2; estrone, E 1; estrone sulfate, E 1S), breast cancer tissue sections obtained during mastectomy in 28 postmenopausal breast cancer patients were stained for aromatase protein expression using the aromatase antibody 677. The findings were correlated to intra-tumor estrogen levels determined with a highly sensitive HPLC-RIA. Staining with 677 alone (irrespective of the hormone receptor status) revealed no difference in tumor E 2 levels comparing 677+ versus 677− tumors, although a non-significant trend towards higher tumor E 1 and E 1S levels was observed in 677+ breast cancers. In contrast, tumor levels of E 2 were significantly higher in ER+ tumors compared to ER− tumors ( P < 0.001) and to benign breast tissue from the same breast ( P < 0.001). Analysing the additional effect of positive staining with the aromatase antibody 677 on tumor estrogen levels in the subgroup of ER+ tumors, revealed significantly higher tumor levels of E 2 (mean level of 544.7 versus 197.1 fmol/g tissue) as well as a non-significant trend concerning tumor E 1 (mean level of 296.9 versus 102.1 fmol/g tissue). The mean tumor tissue E 1S level was observed somewhat lower in ER+677+ (103.5 fmol/g) versus ER+677− tumors (190.1 fmol/g). In the subgroup of ER+PgR+ tumors, tissue levels of E 2 were also found to be significantly higher among 677+ compared to 677− tumors: 873.2 fmol/g (95% CI 395.9–1925.6) versus 217.9 fmol/g (95% CI 88.8–534.9) ( P = 0.015). In conclusion, our results indicate a moderate effect of aromatase enzyme expression evaluated by IHC using the antibody 677 on intra-tumor estrogen levels among ER+ breast cancers. A substantial interindividual variation in the ratios between the individual estrogen fractions suggests additional effects, like alterations in other enzymes to be involved in the intra-tumor estrogen homeostasis.

Aromatase and COX in breast cancer: Enzyme inhibitors and beyond

Aromatase expression and enzyme activity in breast cancer patients is greater in or near the tumor tissue compared with the normal breast tissue. Complex regulation of aromatase expression in human tissues involves alternative promoter sites that provide tissue-specific control. Previous studies in our laboratories suggested a strong association between aromatase ( CYP19) gene expression and the expression of cyclooxygenase ( COX) genes. Additionally, nonsteroidal anti-inflammatory drugs (NSAIDs) and COX selective inhibitors can suppress CYP19 gene expression and decrease aromatase activity. Our current hypothesis is that pharmacological regulation of aromatase and/or cyclooxygenases can act locally to decrease the biosynthesis of estrogen and may provide additional therapy options for patients with hormone-dependent breast cancer. Two pharmacological approaches are being developed, one involving mRNA silencing by selective short interfering RNAs (siRNA) molecules and the second utilizing small molecule drug design. In the first approach, short interfering RNAs were designed against either human aromatase mRNA or human COX-2 mRNA. Treatment of breast cancer cells with siAROMs completely masked the aromatase enzyme activity. Treatment with COX-2 siRNAs decreased the expression of COX-2 mRNA; furthermore, the siCOX-2-mediated decrease also resulted in suppression of CYP19 mRNA. The small molecule drug design approach focuses on the synthesis and biological evaluation of a novel series of sulfonanilide analogs derived from the COX-2 selective inhibitors. The compounds suppress aromatase enzyme activity in SK-BR-3 breast cancer cells in a dose and time-dependent manner, and structure activity analysis does not find a correlation between aromatase suppression and COX inhibition. Real-time PCR analysis demonstrates that the sulfonanilide analogs decrease aromatase gene transcription in breast cells. Thus, these results suggest that the siRNAs and novel sulfonanilides targeting aromatase expression may be valuable tools for selective regulation of aromatase in breast cancer.

Synthesis of carbon-11 labeled sulfonanilide analogues as new potential PET agents for imaging of aromatase in breast cancer

Aromatase is a particularly good target in the treatment of estrogen receptor positive breast cancer. Novel carbon-11 labeled sulfonanilide analogues, N-[ 11C]methyl- N-(2-alkyloxy-4-nitrophenyl)-methanesulfonamides ([ 11C] 3a– f, alkyl = propyl, isopropyl, 1-ethyl-propyl, cyclopentyl, cyclohexyl, and cyclohexylethyl), were designed and synthesized as potential PET agents for imaging of aromatase in breast cancer.

Interactions between Prostaglandin E2, Liver Receptor Homologue-1, and Aromatase in Breast Cancer

Abstract Local synthesis of estrogens within breast adipose tissue by cytochrome P450 aromatase contributes to the growth of postmenopausal breast cancers. One of the major stimulators of aromatase expression in breast is prostaglandin E2 (PGE2) derived from tumorous epithelium and/or infiltrating macrophages. Recently, the orphan nuclear receptor, liver receptor homologue-1 (LRH-1), has also been shown to regulate aromatase expression in breast adipose tissue. We therefore examined the expression of, and correlations between, aromatase and LRH-1 mRNA in a panel of breast carcinoma tissues and adjacent adipose tissue. LRH-1 mRNA expression was low in normal breast tissue but markedly elevated in both breast carcinoma tissue and adipose tissue surrounding the tumor invasion (thereby paralleling aromatase expression). Laser capture microdissection localized the site of LRH-1 expression to tumor epithelial cells but not to intratumoral stromal cells. A strong correlation between LRH-1 and aromatase mRNA levels was observed in tumor-containing adipose tissue but not in tumor tissue. Ectopic expression of LRH-1 in primary human adipose stromal cells strongly activated endogenous aromatase mRNA expression and enzyme activity. Finally, treatment of adipose stromal cells with PGE2 induced expression of both LRH-1 and aromatase. We suggest that PGE2 derived from breast tumor tissue may increase aromatase expression in the surrounding adipose stroma in part by inducing LRH-1 in these cells. The roles of LRH-1 in breast cancer proliferation merit further study.

エストロゲンレセプター陽性乳癌における腫瘍組織内アロマターゼ発現の検討

エストロゲンレセプター陽性乳癌における腫瘍組織内アロマターゼ発現の検討