The synthesis of specific, potent progesterone antagonists adds potential agents to the breast cancer prevention and treatment armamentarium. The identification of individuals who will benefit from ...these agents will be a critical factor for their clinical success.
We utilized telapristone acetate (TPA; CDB-4124) to understand the effects of progesterone receptor (PR) blockade on proliferation, apoptosis, promoter binding, cell cycle progression, and gene expression. We then identified a set of genes that overlap with human breast luteal-phase expressed genes and signify progesterone activity in both normal breast cells and breast cancer cell lines.
TPA administration to T47D cells results in a 30 % decrease in cell number at 24 h, which is maintained over 72 h only in the presence of estradiol. Blockade of progesterone signaling by TPA for 24 h results in fewer cells in G2/M, attributable to decreased expression of genes that facilitate the G2/M transition. Gene expression data suggest that TPA affects several mechanisms that progesterone utilizes to control gene expression, including specific post-translational modifications, and nucleosomal organization and higher order chromatin structure, which regulate access of PR to its DNA binding sites.
By comparing genes induced by the progestin R5020 in T47D cells with those increased in the luteal-phase normal breast, we have identified a set of genes that predict functional progesterone signaling in tissue. These data will facilitate an understanding of the ways in which drugs such as TPA may be utilized for the prevention, and possibly the therapy, of human breast cancer.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Context: Clinical study of breast cancer patients in Chicago, IL, USA.
Objective: Ascertain the utility of measurements of single-strand breaks (SSB) in DNA for assessment of breast cancer risk.
...Methods: Fine-needle aspirates of the breast, SSB by nick translation, percent breast density (PBD), Gail model risk, cumulative methylation index (CMI), enzymes of DNA repair and tissue antioxidants.
Results: DNA repair enzymes and 4-hydroxyestradiol were negatively associated with SSB; CMI and PBD were positively associated.
Conclusions: Quantitative measurement of SSBs by this procedure indicates the relative number of SSBs and is related to promoter methylation, antioxidant availability and percent breast density.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Abstract
Background: CDB4124 a progesterone receptor modulator suppressed the development of precancerous lesions and carcinogen-induced ER+ mammary tumors in rats, and may have implications for ...prevention and treatment of human breast cancer; however liver toxicity in human is the major problem of systemic delivery, and transdermal local delivery to the breast may be an excellent solution. We tested transdermal CDB4124 for in vitro human skin permeation and in vivo tissue distribution in rats compared to s.c delivery.
Methods: We tested CDB4124 (Repros Therapeutics, Inc.) alone and with ethanol and 0.5 v/v % oleic acid (OA) using split-thickness skin from mastectomy specimens, mounted in Franz diffusion cells (PermeGear, PA, USA), 0.5mg of CDB4124 in 60 % alcoholic solution +/- OA and applied to the donor chamber. Aliquots of the receiver solution were collected at pre-determined time points for 24 hr. Next, we tested the in vivo permeation of gel formulation of CDB4124 + 0.5% OA in nude rats comparing daily transdermal delivery (0.3mg/day) with a s.c pellet (30mg) for 6 weeks. Plasma and mammary fat pads(MFPs) were collected. CDB4124 and its major metabolite CDB4453 were quantified by LC-MS/MS.
Results: The permeation through human breast skin was 3.1± 0.9% for CDB4124 alone and enhanced 4-fold to 11.6±1.5% with the addition of 0.5% OA at 24 hr. The rate of CDB4124 permeation was 5-fold faster than the drug alone within 12 hr. Plasma levels of CDB4124 were 14±5 and 11±5 ng/mL for the pellet and gel groups, respectively (p= 0.16); plasma level of CDB4453 was higher in the pellet group than in the gel group, 4.6±2 vs. 1.5±0.6 ng/mL (p=0.000). CDB4124 levels in the upper MFP (the site of gel application) were 34-fold higher in the gel group than in the pellet group (1485ng/g for gel group; 44ng/g for pellet group, p=0.000); CDB4124 levels in the lower MFP were 194 ng/g for gel group; 66 ng/g for pellet group. CDB4124 and CDB4453 levels in upper MFP were significantly higher than in lower MFP (about 8-fold for CDB4124 and 7-fold for CDB4453; p=0.000, p=0.000, respectively). In contrast, the pellet group shows a similar concentration of CDB4124 and CDB4453 in the upper and lower MFPs.
Conclusions: This is an encouraging result because skin permeation of CDB4124 is similar to the level of estradiol, an efficacious transdermal agent, and indicating more extensive metabolism of CDB4124 when administered systemically. The high CDB4124 tissue level in the gel group indicate the possibility that drug smeared on the skin area other than the upper MFP, but the higher level of the metabolite CDB4453 supports the conclusion that a significant portion of the drug measured in the fat pad was in fact delivered to the tissue. Local transdermal delivery of CDB4124 seems plausible method for breast cancer prevention.
Citation Format: Oukseub Lee, David Ivancic, Ali Shidfar, Ronald Wiehle, Seema A. Khan. In vitro and in vivo transdermal delivery of CDB4124, a progesterone receptor modulator: a potential method for breast cancer prevention . abstract. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5670. doi:10.1158/1538-7445.AM2013-5670
Abstract
Background: Several lines of evidence suggest that progesterone signaling is important in the breast cancer development, particularly in young women. Therefore, we sought to establish the ...effects of the antiprogestin CDB4124 (telapristone), and to identify PR related signature genes in hormone receptor positive (ER+ PR+) breast cancer cells.
Methods: The PR expressing breast cancer cell line T47D was used to evaluate responses to PR ligands (P4, MPA and R5020, 10nM) alone or in combination with estradiol (E2, 1nM). The effects of the antiprogestin CDB4124 (0.1 or 1μM) were tested using varying hormonal conditions. The effect on (a) PRE promoter activity by dual luciferase assay; (b) cell proliferation using the MTT assay; (c) cell cycle by flow cytometry; (d) determination of gene expression signatures related to active PR responses. For the gene array experiment, cells were treated with either vehicle or R5020 (10nM) or R5020 (10nM) plus CDB4124 (1μM) in triplicate for 24hr. Total RNA was isolated and converted to cDNA and human Illumina chip microarray was performed. Data obtained from the microarray was further analyzed by Metacore Gene GO and Ingenuity Pathway Analysis. Real time PCR was performed in triplicate to confirm the expression of those genes related to the cell cycle and proliferation. ANOVA analysis and post-hoc Sidak test were used to determined the statistical significance of the data.
Results: The PRE reporter activity resulting from P4, MPA and R5020 stimulation was inhibited by 80-90% in the presence of CDB4124 at 10 to 1000nM (p< 0.001). Cell proliferation was increased by PR ligands (P4, MPA and R5020) in the presence of E2; the addition of CDB4124 caused 50% inhibition of proliferation (p< 0.01) at 72 hours. Cell cycle analysis of T47D cells treated with P4, MPA and R5020 alone or in combination with E2 showed significant increases in S and G2/M phase and decreases in G0/G1. These were blocked by CDB4124 at 0.1 or 1.0μM (p<0.05 for all). Gene GO metacore analysis of genes identified in the microarray revealed significant enrichment of cell cycle pathways (FDR, p< 1.0X10-11 ) upon treatment with R5020. The addition of CDB4124 to R5020 treated samples showed inhibition of the same cell cycle pathways (FDR,p<1.0X10-14). A 16-gene panel related to G2/M phase of cell cycle was selected based on >1.5 fold upregulation (p<0.001) during treatment with R5020,10nM and blockade by CD4124. Real time PCR confirmed upregulation of this 16 gene panel ≥2.0 (p<0.05) in the presence of PR ligands alone or in combination with E2 which were significantly blocked by the addition of CDB4124.
Conclusion: These data demonstrate that PR mediated cell proliferation occurs upon treatment with three different ligands of PR (P4, MPA and R5020); that PR actively engages key genes involved in the G2/M phase of the cell cycle to drive proliferation of ER+ and PR+ cells; and that the antiprogestin, CBD4124 is a potent transcriptional inhibitor for blockade of PR mediated cell proliferation in hormone receptor positive breast cancer cells.
Citation Format: Akash Gupa, Mi Ran Choi, Susan E Clare, Jun Wang, Oukseub Lee, David Z Ivancic, J Julie Kim, Seema A Khan. Progesterone receptor (PR) blockade by antiprogestin, CDB4124 in hormone receptor positive breast cancer cells leads to significant inhibition of G2/M cell cycle genes abstract. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-04-10.
Abstract
Background: Oral medication for breast cancer prevention is associated with systemic exposure and resulting adverse effects; there is a need for alternative drug delivery approaches. ...Transdermal drug delivery is such an option, and 4-Hydroxy-N-desmethytamoxifen (endoxifen, ENX) is an excellent candidate for development for this route. ENX is an active metabolite of tamoxifen with major therapeutic effect related to potent estrogen antagonism and ability to cause proteosomic degradation of ERα. We hypothesize that skin permeation of ENX with oleic acid (OA) will be equivalent or superior to that of estradiol (E2), which is a well-established transdermal agent.
Methods: Split-thickness human skin samples (354 ± 25 micron) were prepared from de-identified fresh mastectomy specimens under an IRB approved protocol using a surgical blade and placed in Franz diffusion cells (PermeGear, Inc.). ENX was prepared in 60 % (v/v) ethanol/phosphate buffer (PB) +/- OA (0.1to 1% v/v). E2 was used as a positive control drug, prepared in 60 % ethanol/PB. The receiver fluid was PBS, pH 7.4 with 4% (w/v) polyoxyethylene 20 oleyl ether (Sigma-Aldrich). 100 µL of the drug solution (30 µg + 0.1µCi of 3H-E2 or 3H-ENX) was loaded in the donor chamber. Receiver fluid was stirred at 37° C. A sample of receiver fluid was collected for 3H counting from 4 to 24 hr point with 2 hr interval. Each permeation experiment was performed in duplicate for each treatment condition using the breast skin of one individual. The permeation experiments were repeated three times with the skin from different individuals.
Results: Results were expressed as the mean and standard deviation (SD) of the % of the applied dose. Absorption is defined as the amount of drug passing into the receiver fluid. We found that the absorption of E2 was 0.2 ± 0.17% (at 12hr) and 3.16 ± 2.79 % (at 24hr) of the applied dose while the absorption of ENX was 0.07± 0.08 % (at 12hr) and 1.45 ± 1.07 % (at 24hr). In the presence of OA, the permeation of ENX was enhanced by 2 to 12- fold at 12 hr and by 1.2 to 3.8- fold at 24 hr. At the 12 hr point, the absorption of ENX alone is one third of that of E2 alone (p = 0.09) however in the presence of 0.25, 0.5, and 1% (v/v) of OA, the absorption of ENX was by 4.4-fold (p = 0.02), 6.4-fold (p = 0.01), and 3-fold (p = 0.02) higher than E2, respectively. At 24 hr, there was no further significant enhancement in the absorption of ENX by 0.25 and 0.5% of OA compared to that of E2. The addition of 0.1 and 1% OA made the absorption of ENX retarded than that of E2 at 24 hr.
Conclusion: The skin permeation of ENX was significantly enhanced in the presence of 0.25-1 % of oleic acid and was superior to that of E2 alone at 12 hr. Our results imply that the topical delivery of endoxifen is a potential option for breast cancer prevention with no or minimal systemic toxicity/side effects. Efficacy remains to be demonstrated, but is highly likely given the data on the parent compound (tamoxifen).
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5566. doi:10.1158/1538-7445.AM2011-5566
Abstract
Background: Adjuvant oral tamoxifen (TAM) benefits women with DCIS, but toxicity concerns have limited its acceptance. Transdermal therapy with 4-hydroxy tamoxifen (4-OHT) gel applied to the ...breast skin is a possible solution. Previous pilot data suggest equivalent anti-proliferative efficacy of TAM and 4-OHT gel, but minimal systemic exposure with transdermal therapy. We report a prospective double blinded randomized phase 2 trial comparing TAM to 4-OHT gel in women with DCIS. Methods: 107 women with estrogen receptor positive (≥10%) DCIS were randomized to TAM (20 mg/day + placebo gel) or 4-OHT gel (2mg 4-OHT gel/breast, bilaterally + oral placebo), for 4-10 weeks prior to surgery. The primary endpoint was reduction in DCIS Ki67 labeling index (LI). Secondary endpoints included the 12-gene DCIS Score assay (Exact Sciences), breast tissue and plasma concentrations of 4-OHT and endoxifen, TAM-responsive circulating proteins, and patient reported symptoms (Breast Eight Symptom Scale). We estimated that 80 evaluable participants would provide 80.5% power to establish non-inferiority of 4-OHT, defined as relative Ki67-LI decline >35% and absolute decline >2.6%, with one-sided =0.10. Non-inferiority of 4-OHT gel for Ki67-LI reduction was tested using an ANCOVA model. Statistical comparisons within- and between-arms were calculated with paired t-test and Welch Two Sample t-test, respectively. Results: 72 of 87 women adhered to the protocol, and were evaluable for the primary endpoint (39 TAM and 33 4-OHT gel). Mean treatment duration was 47 days for TAM and 44 days for 4-OHT gel (p=0.2). The median absolute decline in Ki67 labeling index was significant in the oral TAM (-3.7%, p< 0.001) but not in 4-OHT gel arm (-1.3%, p=0.2) (p=0.002). Ki67 results following menopausal stratification also favored the TAM arm: (-1.3%; p=0.06 in 37 premenopausal women and -3.7%; p=0.02 in 35 postmenopausal women). Similarly, DCIS score showed a significantly greater reduction in the TAM (-14, p< 0.001) but not in the 4-OHT gel arm (-4, p=0.1). Tissue 4-OHT concentrations were non-significantly higher in the TAM arm and were similar between superficial and deep sampling locations (superficial 6.1 and 4.2 ng/g for TAM and 4-OHT gel, respectively, p= 0.55; deep 5.7 and 3.8 ng/g, respectively, p= 0.06), whereas plasma 4-OHT concentration was markedly lower in the gel group (2 ng/mL and 0.24 ng/mL for TAM and 4-OHT gel, respectively, P < 0.001). Endoxifen was abundant in plasma (11 ng/mL) and deep tissue (13 ng/g) of the TAM arm, but present in trace amounts in the 4-OHT gel arm (undetectable in plasma and 0.31 ng/g in tissue; p < 0.001). Circulating TAM responsive markers (insulin like growth factor 1, sex hormone binding globulin, von Willebrand factor, and protein S total) and vasomotor symptoms were significantly and unfavorably modulated by TAM, but not by 4-OHT gel therapy. Conclusions: The non-inferiority of transdermal 4-OHT gel to Tam in terms of anti-proliferative effect in DCIS lesions was not demonstrated at the doses used for this study. DCIS Score analysis gave similar results. Tissue 4-OHT concentration in 4-OHT gel and Tam-treated subjects was roughly similar. However, endoxifen exposure was higher with oral TAM therapy and may partially explain the observed differences in major endpoints. In future studies, use of higher 4-OHT gel doses, longer duration of treatment, or different formulation may overcome these.
Citation Format: Oukseub Lee, Xinlei Mi, Yanfei Xu, Luis Blanco, Azza M. Akasha, Kelly Benante, Shanshan Zhang, Carissa LaBoy, Thomas Helland, Melissa Pilewskie, Amy Degnim, Zahraa Al-Hilli, Amanda L. Amin, E Shelley Hwang, Joseph M. Guenther, Simon Steinar Hustad, Demirkan B. Gursel, Masha Kocherginsky, Gunnar Mellgren, Eileen Dimond, Marjorie Perloff, Brandy M. Heckman-Stoddard, Seema Khan. PD15-12 A pre-surgical window trial of oral tamoxifen versus transdermal 4-hydroxytamoxifen gel in women with estrogen receptor positive duct carcinoma in situ (DCIS) abstract. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD15-12.
Two recent studies with pre- and postmenopausal female cynomolgus monkeys suggest that dietary exposure alone of soy protein for periods of 12 to 36 months, as the human equivalent of 129 mg/d of ...isoflavones (containing 91 -mg genistein, 31 -mg daidzein, and 7-mg aglycone equivalents of glycitein) is not a ágnificant estrogen agonist for breast tissue. 4,5 This dietary exposure did not induce proliferation in mammary tissue; instead, mammary gland proliferation (Ki67 labeling index) induced by estradiol was antagonized by soy in postmenopausal monkeys. In humans, studies of breast tissue isoflavones concentrations following exposure to dietary soy products are also reassuring. In two small trials, women undergoing aesthetic breast reductions tested a several-day intervention of soy supplementation and/or soy milk, followed by measurement of isoflavones (genistein, daidzein, and equol) by LC-MS/MS in breast tissue compared with serum and urinary levels. 6,7 Both studies found that concentrations of total isoflavones were in the subnanomolar7 to low nanomolar 6 ranges in hydrolyzed breast tissue, whereas they were generally 100-fold higher in the corresponding serum and urine samples; thus serum concentrations may overestimate tissue exposure. Finally, the issue of proliferative response in the breast epithelium of premenopausal women given a soy isoflavone supplement needs to be addressed 18, 19; cell proliferation in normal breast tissue is a surrogate marker for adverse effects on the breast, and agents that increase it are generally considered to increase cancer risk. The evidence suggesting a pro-estrogenic effect of soy components on the breast comes not only from research linking soy with increased cell proliferation in premenopausal women,18 but also from a gene expression signal consistent with induction of estrogenic pathways, described in the same study. Additionally, in a study examining nipple fluid biomarkers following soy supplementation, levels of pS2 in nipple fluid were increased, although cell proliferation was not. 19 Other studies examining this issue have been small, the interventions have been short-term, 20 and the fraction of patients yielding samples sufficient for Ki67 evaluation has been low. 21 Thus although there is no robust evidence of harm in the form of increased cancer rates or mortality, studies that show no effect on proliferation are limited and do not fully counter the possibility of a weak pro-estrogenic effect of soy on the breast. The injunction against processed forms of soy does not pose any hardship to women who want to consume soyfoods as part of a healthy, varied, plant-based diet. It does not deprive women of the healthful effects of soy consumption on other organs. Therefore, until better evidence is available for the effects of soy on women from non-soyconsuming countries, it seems reasonable to limit consumption to soyfoods, and to avoid high-dose supplements of processed soy components.
Sex steroid hormones contribute to breast cancer development, but data on concentrations of these within breast tissue are limited. We performed simultaneous multiparameter measurement of breast sex ...steroids, breast epithelial cytology, and DNA methylation in 119 healthy women (54 pre- and 65 postmenopausal) without a history of breast cancer. Random fine-needle aspiration (rFNA) of the breast was performed simultaneously with blood collection. Breast samples were analyzed by LC/MS-MS for estrone, estradiol, progesterone, androstenedione, and testosterone. Blood samples were assayed for estradiol and progesterone by immunoassay. Cytomorphology was classified using the Masood Score, and DNA methylation of eight genes was analyzed using quantitative multiplexed methylation-specific PCR, and expressed as the cumulative methylation index (CMI). Serum and breast concentrations of estradiol and progesterone showed significant correlation (Spearman
= 0.34,
= 0.001 and
= 0.69,
< 0.0006, respectively). Progesterone concentration was significantly higher in the premenopausal breast (
< 0.0008), and showed a luteal surge. Breast estrone and estradiol concentrations did not differ significantly by menopause, but androstenedione concentration was higher in the breasts of postmenopausal women (
= 0.026 and
= 0.208). Breast androgens were significantly correlated with breast density (Spearman
= 0.27,
= 0.02 for testosterone) and CMI (Spearman
= 0.3,
= 0.038 for androstenedione). Our data indicate that future larger studies of breast steroid hormones along with other parameters are feasible. Significant associations of breast androgen concentrations with breast density and gene methylation warrant future study.
.
Surface‐modified dendron micelles (DMs) are developed on page 2442 by Seungpyo Hong and colleagues as a potential platform for topical delivery of endoxifen (EDX). The data indicates that skin ...permeation of EDX is highly dependent on the surface group of the DMs. In particular, carboxylated DMs exhibit significantly enhanced permeation of EDX through both mouse and human skin layers, offering a potential alternative administration route for chemoprevention.
Risk biomarkers that are specific to estrogen receptor (ER) subtypes of breast cancer would aid the development and implementation of distinct prevention strategies. The contralateral unaffected ...breast of women with unilateral breast cancer (cases) is a good model for defining subtype-specific risk because women with ER-negative (ER-) index primaries are at high risk for subsequent ER-negative primary cancers. We conducted random fine needle aspiration of the unaffected breasts of cases. Samples from 30 subjects 15 ER-positive (ER+) and 15 ER- cases matched for age, race and menopausal status were used for Illumina expression array analysis. Findings were confirmed using quantitative real-time PCR (qRT-PCR) in the same samples. A validation set consisting of 36 subjects (12 ER+, 12 ER- and 12 standard-risk healthy controls) was used to compare gene expression across groups. ER- case samples displayed significantly higher expression of 18 genes/transcripts, 8 of which were associated with lipid metabolism on gene ontology analysis (GO: 0006629). This pattern was confirmed by qRT-PCR in the same samples, and in the 24 cases of the validation set. When compared to the healthy controls in the validation set, significant overexpression of 4 genes (DHRS2, HMGCS2, HPGD and ACSL3) was observed in ER- cases, with significantly lower expression of UGT2B11 and APOD in ER+ cases, and decreased expression of UGT2B7 in both subtypes. These data suggest that differential expression of lipid metabolism genes may be involved in the risk for subtypes of breast cancer, and are potential biomarkers of ER-specific breast cancer risk.
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