Breast cancer prevention with pharmacologic agents requires that the breast be exposed to an effective drug; systemic exposure is unnecessary, and its harms lead many eligible women to decline ...preventive therapy. Local transdermal therapy (LTT) to the breast involves the application of active drugs to the breast skin, resulting in high concentrations in the breast but low systemic exposure. It is non-invasive, self-delivered, and not dependent on hepatic metabolism. Existing data on LTT include investigations demonstrating relief of mastalgia with topical 4-hydroxytamoxifen (4-OHT, an active tamoxifen metabolite). Two presurgical window trials in women with invasive breast cancer, and ductal carcinoma in situ (DCIS) demonstrate that LTT decreases proliferation of invasive and non-invasive cancer cells to a similar degree as oral tamoxifen, with low systemic levels, and no effect on coagulation proteins. These data are promising regarding the use of LTT for the primary prevention of breast cancer, and for therapy of DCIS, since systemic exposure is not required for either of these purposes. They also suggest that an LTT approach could be developed for any small, lipophilic molecule with good dermal permeation, thus greatly expanding the menu of drugs that could be tested for breast cancer prevention.
Aggressive estrogen receptor (ER) positive breast cancer is frequently tamoxifen-resistant; alternative endocrine approaches exist for therapy, but not for prevention, particularly in premenopausal ...women. We examined the efficacy of the selective ER modulator (Z-endoxifen) as monotherapy and in combination with the selective progesterone receptor modulators (onapristone and ulipristal acetate) in the tamoxifen-insensitive C3(1)/SV40TAg mouse mammary tumorigenesis model. Unlike tamoxifen at human equivalent dose (HED) 101 mg/day, endoxifen at HED 24 mg/day significantly increased latency and reduced tumor growth relative to untreated controls. Ulipristal acetate (UPA) at HED 81 mg/day also significantly increased latency however failed to inhibit tumor growth, while onapristone (HED 98 mg/day) had no tumor prevention efficacy in this model. Addition of UPA to endoxifen did not enhance preventive efficacy over endoxifen alone. The expression of genes associated with cell cycle, cell proliferation and extracellular matrix remodeling was similarly repressed by endoxifen and UPA however only endoxifen significantly downregulated prominent genes associated with poor prognosis (Col11a1, Il17b, Pdgfa, Tnfrsf11a). Our results indicate that endoxifen can prevent breast cancers, even when tamoxifen-resistant, through its role in favorable tissue remodeling and immunomodulation.
•SERMs and SPRMs displayed variable cancer preventative efficacy in C3(1)/SV40TAg mice.•Tamoxifen does not prevent mammary cancer in C3(1)/SV40TAg mice.•Z-endoxifen can prevent mammary cancers, even when tamoxifen-resistant.•Combination therapy of Z-endoxifen with SPRMs at the selected doses had no additive effects.•Z- endoxifen inhibited cell proliferation and induced a tumor suppressive microenvironment.
The ovarian hormones estrogen and progesterone (EP) are implicated in breast cancer causation. A specific consequence of progesterone exposure is the expansion of the mammary stem cell (MSC) and ...luminal progenitor (LP) compartments. We hypothesized that this effect, and its molecular facilitators, could be abrogated by progesterone receptor (PR) antagonists administered in a mouse model.
Ovariectomized FVB mice were randomized to 14 days of treatment: sham, EP, EP + telapristone (EP + TPA), EP + mifepristone (EP + MFP). Mice were then sacrificed, mammary glands harvested, and mammary epithelial cell lineages separated by flow cytometry using cell surface markers. RNA from each lineage was sequenced and differential gene expression was analyzed using DESeq. Quantitative PCR was performed to confirm the candidate genes discovered in RNA seq. ANOVA with Tukey post hoc analysis was performed to compare relative expression. Alternative splicing events were examined using the rMATs multivariate analysis tool.
Significant increases in the MSC and luminal mature (LM) cell fractions were observed following EP treatment compared to control (p < 0.01 and p < 0.05, respectively), whereas the LP fraction was significantly reduced (p < 0.05). These hormone-induced effects were reversed upon exposure to TPA and MFP (p < 0.01 for both). Gene Ontology analysis of RNA-sequencing data showed EP-induced enrichment of several pathways, with the largest effect on Wnt signaling in MSC, significantly repressed by PR inhibitors. In LP cells, significant induction of Wnt4 and Rankl, and Wnt pathway intermediates Lrp2 and Axin2 (confirmed by qRTPCR) were reversed by TPA and MFP (p < 0.0001). Downstream signaling intermediates of these pathways (Lrp5, Mmp7) showed similar effects. Expression of markers of epithelial-mesenchymal transition (Cdh1, Cdh3) and the induction of EMT regulators (Zeb1, Zeb2, Gli3, Snai1, and Ptch2) were significantly responsive to progesterone. EP treatment was associated with large-scale alternative splicing events, with an enrichment of motifs associated with Srsf, Esrp, and Rbfox families. Exon skipping was observed in Cdh1, Enah, and Brd4.
PR inhibition reverses known tumorigenic pathways in the mammary gland and suppresses a previously unknown effect of progesterone on RNA splicing events. In total, our results strengthen the case for reconsideration of PR inhibitors for breast cancer prevention.
Cancer Letters: Highlights for Review • Blockade of the progestogen-PR is a untested strategy for breast cancer prevention • TPA opposed the pro-hyperplastic effects of MPA in mouse mammary glands • ...TPA decreased tumor incidence, pHH3, angiogenesis, and ER/PR expression in rats • Physiological estradiol plus progesterone increased the growth of T47D spheroids • TPA, and UPA are superior to mifepristone in growth suppression of T47D spheroids
Breast cancer prevention only requires local exposure of the breast to active drug. However, oral preventive agents entail systemic exposure, causing adverse effects that limit acceptance by ...high-risk women. Drug-delivery through the breast skin is an attractive option, but requires demonstration of dermal safety and drug distribution throughout the breast. We formulated the tamoxifen metabolite (E/Z)-endoxifen for transdermal delivery and tested it in a placebo-controlled, double-blinded Phase I trial with dose escalation from 10 to 20 mg daily. The primary endpoint was dermal toxicity. Thirty-two women planning mastectomy were randomized (2:1) to endoxifen-gel or placebo-gel applied to both breasts for 3–5 weeks. Both doses of endoxifen-gel incurred no dermal or systemic toxicity compared to placebo. All endoxifen-treated breasts contained the drug at each of five sampling locations; the median per-person tissue concentration in the treated participants was 0.6 ng/g (IQR 0.4–1.6), significantly higher (p < 0.001) than the median plasma concentration (0.2 ng/mL, IQR 0.2–0.2). The median ratio of the more potent (Z)-isomer to (E)-isomer at each breast location was 1.50 (IQR 0.96–2.54, p < 0.05). No discernible effects of breast size or adiposity on tissue concentrations were observed. At the endoxifen doses and duration used, and the tissue concentration achieved, we observed a non-significant overall reduction of tumor proliferation (Ki67 LI) and significant downregulation of gene signatures known to promote cancer invasion (FN1, SERPINH1, PLOD2, PDGFA, ITGAV) (p = 0.03). Transdermal endoxifen is an important potential breast cancer prevention agent but formulations with better dermal penetration are needed.
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•Transdermal oleic acid-hydroalcoholic gel of (E/Z)-endoxifen gel at 10 and 20 mg/day was safe and tolerable over one month of treatment.•Transdermal oleic acid-hydroalcoholic gel of (E/Z)-endoxifen gel uniformly distributed endoxifen in breast, including tumors but minimized systemic exposure.•The gene panel related to cancer invasion (FN1, SERPINH1, PLOD2, PDGFA, ITGAV) was significantly downregulated at the breast concentration achieved by transdermal endoxifen gel.
Local transdermal therapy to the breast may achieve effective target-organ drug delivery, while diminishing systemic effects. We conducted a randomized, double-blind, placebo-controlled phase II ...trial comparing transdermal 4-hydroxytamoxifen gel (4-OHT) to oral tamoxifen (oral-T) in women with ductal carcinoma in situ (DCIS).
Twenty-seven pre- and postmenopausal women were randomized to 4-OHT (4 mg/day) or oral-T (20 mg/day) for 6 to 10 weeks before surgery. Plasma, nipple aspirate fluid, and breast adipose tissue concentrations of tamoxifen and its major metabolites were determined by liquid chromatography/tandem mass spectrometry. The primary endpoint was Ki67 labeling in DCIS lesions, measured by immunohistochemistry. In plasma, insulin-like growth factor-1 (IGFI), sex hormone-binding globulin (SHBG), and coagulation protein concentrations were determined.
Posttherapy Ki67 decreased by 3.4% in the 4-OHT and 5.1% in the oral-T group (P ≤ 0.03 in both, between-group P = 0. 99). Mean plasma 4-OHT was 0.2 and 1.1 ng/mL in 4-OHT and oral groups, respectively (P = 0.0003), whereas mean breast adipose tissue concentrations of 4-OHT were 5.8 ng/g in the 4-OHT group and 5.4 ng/g in the oral group (P = 0.88). There were significant increases in plasma SHBG, factor VIII, and von Willebrand factor and a significant decrease in plasma IGFI with oral-T, but not with 4-OHT. The incidence of hot flashes was similar in both groups.
The antiproliferative effect of 4-OHT gel applied to breast skin was similar to that of oral-T, but effects on endocrine and coagulation parameters were reduced. These findings support the further evaluation of local transdermal therapy for DCIS and breast cancer prevention.
Tamoxifen is a drug that has been in worldwide use for the treatment of estrogen receptor (ER)-positive breast cancer for over 30 years; it has been used in both the metastatic and adjuvant settings. ...Tamoxifen's approval for breast cancer risk reduction dates back to 1998, after results from the Breast Cancer Prevention Trial, co-sponsored by the National Cancer Institute and the National Surgical Adjuvant Breast and Bowel Project, showed a 49% reduction in the incidence of invasive, ER-positive breast cancer in high-risk women. Despite these positive findings, however, the public's attitude toward breast cancer chemoprevention remains ambivalent, and the toxicities associated with tamoxifen, particularly endometrial cancer and thromboembolic events, have hampered the drug's uptake by high-risk women who should benefit from its preventive effects. Among the strategies to overcome such obstacles to preventive tamoxifen, two novel and potentially safer modes of delivery of this agent are discussed in this paper. Low-dose tamoxifen, expected to confer fewer adverse events, is being investigated in both clinical biomarker-based trials and observational studies. A series of systemic biomarkers (including lipid and insulin-like growth factor levels) and tissue biomarkers (including Ki-67) are known to be favorably affected by conventional tamoxifen dosing and have been shown to be modulated in a direction consistent with a putative anti-cancer effect. These findings suggest possible beneficial clinical preventive effects by low-dose tamoxifen regimens and they are supported by observational studies. An alternative approach is topical administration of active tamoxifen metabolites directly onto the breast, the site where the cancer is to be prevented. Avoidance of systemic administration is expected to reduce the distribution of drug to tissues susceptible to tamoxifen-induced toxicity. Clinical trials of topical tamoxifen with biological endpoints are still ongoing whereas pharmacokinetic studies have already shown that appropriate formulations of drug successfully penetrate the skin to reach breast tissue, where a preventive effect is sought.
Soy isoflavone consumption may protect against breast cancer development. We conducted a phase IIB trial of soy isoflavone supplementation to examine its effect on breast epithelial proliferation and ...other biomarkers in the healthy high-risk breast. One hundred and twenty-six consented women underwent a random fine-needle aspiration (rFNA); those with 4,000 or more epithelial cells were randomized to a double-blind 6-month intervention of mixed soy isoflavones (PTIG-2535) or placebo, followed by repeat rFNA. Cells were examined for Ki-67 labeling index and atypia. Expression of 28 genes related to proliferation, apoptosis, and estrogenic effect was measured using quantitative reverse transcriptase PCR. Hormone and protein levels were measured in nipple aspirate fluid (NAF). All statistical tests were two-sided. Ninety-eight women were evaluable for Ki-67 labeling index. In 49 treated women, the median Ki-67 labeling index was 1.18 at entry and 1.12 post intervention, whereas in 49 placebo subjects, it was 0.97 and 0.92 (P for between-group change: 0.32). Menopausal stratification yielded similar results between groups, but within premenopausal soy-treated women, Ki-67 labeling index increased from 1.71 to 2.18 (P = 0.04). We saw no treatment effect on cytologic atypia or NAF parameters. There were significant increases in the expression of 14 of 28 genes within the soy, but not the control group, without significant between-group differences. Plasma genistein values showed excellent compliance. A 6-month intervention of mixed soy isoflavones in healthy, high-risk adult Western women did not reduce breast epithelial proliferation, suggesting a lack of efficacy for breast cancer prevention and a possible adverse effect in premenopausal women.
Previous studies have shown that progesterone concentrations in serum and nipple aspirate fluid (NAF) are significantly correlated
in premenopausal women, but estradiol concentrations are not. We ...therefore sought to ascertain the patterns of both steroids
in NAF throughout the menstrual cycle and in postmenopausal women. Simultaneous samples of blood and NAF were obtained from
40 premenopausal and 16 postmenopausal women. Premenopausal samples were backdated from the following menstrual period. Steroids
were purified by high-performance liquid chromatography before quantification by immunoassays. Serum steroids and NAF progesterone
followed the expected pattern across the menstrual cycle, with a midcycle peak of estradiol and a midluteal peak of progesterone.
However, the estradiol peak in NAF occurred about a week after the serum peak in the midluteal phase, when serum estradiol
had declined to less than half the value at midcycle. NAF estrone was also elevated at the midluteal phase. Potential estrogen
precursors androstenedione, estrone sulfate, and dehydroepiandrosterone sulfate declined in NAF from midcycle to the midluteal
phase as NAF estradiol was increasing. Progesterone concentrations were significantly lower in NAF in postmenopausal women
than in premenopausal women, but estrogen concentrations were not. This is the first description of the temporal relationships
of sex steroids in NAF and serum relative to the menstrual cycle. These results provide insights into the lack of correlation
of NAF and breast tissue estrogens with serum estrogens, and generate new hypotheses. Cancer Epidemiol Biomarkers Prev; 19(1);
275–9
Abstract
Background: Women carrying a germline BRCA1 mutation, have 60% risk of developing breast cancer during their lifetime and frequently develop triple-negative, basal-like, aggressive breast ...tumors. Humans produce around 150,000 different proteins from their 25,000-30,000 genes. This is accomplished by alternative splicing (AS). We hypothesized that AS in BRCA1 mutation carriers is different from those women with wild type BRCA1, therefore, we examined AS, expression of genes encoding splicing factors, and enrichment of binding motifs for RNA-binding proteins in BRCA1 carriers (BRCA1mut/+) and controls (BRCA1 normal). Methods: Prophylactic mastectomy (BRCA1mut/+) and reduction mammoplasty (BRCA1 normal) specimens were collected at Prentice Women’s Hospital of Northwestern Medicine under approval by Northwestern’s Institutional Review Board (NU15B07). We isolated organoids from these tissues, and cryopreserved them for future use. We selected organoids of premenopausal women, age-matched (28-46) without oral contraceptive usage within 3 years: BRCA1mut/+ (n=12) and BRCA1 normal (n=4). Organoids were maintained in complete MammoCult medium in ultra-low attachment plates at 37 °C, 5% CO2 for 72 hrs, and harvested for RNA extraction (NucleoSpin RNA Plus, TaKaRa). 100 ng of total RNA (RIN 7+) were used for RNA sequencing assay with the KAPA mRNA Hyper Prep Kit (Roche Corporate) and NovaSeq 6000 for 100b paired-end sequencing (Illumina, Inc). The preprocessed reads were aligned to the human genome (hg38) using STAR. Differential expression analysis (DESeq2, nominal p-value < 0.05) and pathway analysis (GSEA, size ≥30, NES >2, FDR q-value <0.05, and enriched genes ≥ 5) were performed to compare BRCA1mut/+ with BRCA1 normal. We used rMATS- turbo v4.1.0 to perform multivariate analysis of transcript splicing and chose an FDR < 0. 05, to identify significant differential alternative splicing events. To determine which RNA binding proteins and splicing factors were employed in BRCA1mut/+ tissue, we examined the cis-regulatory motifs adjacent to splice sites using rMAPs. Results: BRCA1mut/+ organoids displayed 155 upregulated genes compared to BRCA1 normal organoids (P <0.05). Among top 20 enriched pathways, we identified that expression of mRNA spliceosome (POLR2L, U2AF2, NUP188, SNRPF, CSTF3, HNRNPM, SRSF2), and, in addition, cell cycle, oxidative phosphorylation, HIV infection, and protein metabolism genes were significantly upregulated in BRCA1mut/+ organoids. We identified significant differential AS events (FDR <0.05) between BRCA1mut/+ and BRCA1 normal organoids: 2159 genes (skipped exon), 433 genes (mutually exclusive exon), 325 genes (retained intron), 304 genes (alternative 3’ splice sites), 168 genes (alternative 5’ splice sites). RNA splicing analysis indicated that highly expressed genes (SRSF2, ALG3, AP1B1 and PMF1) in BRCA1mut/+ organoids were also alternatively spliced. In addition, many other serine/arginine-rich splicing factors (SRSF) themselves (SRSF1, 2, 3, 5, 6, 7) were alternatively spliced (skipped exon events). An examination of binding motifs of RNA-binding proteins with significantly increased expression in BRCA1 carriers revealed multiple significantly enhanced and silenced regions flanking exons involved in AS. Conclusions: Our data suggest that AS of BRCA1mut/+ mammary tissue significantly differ from BRCA1 normal tissue, and may alter RNA spliceosome activity, and consequently play a role in malignant transformation and warrant further study.
Citation Format: Oukseub Lee, Mi-Ran Choi, Gannon Cottone, Priyam Patel, Susan E Clare, Seema A. Khan. Heterozygous BRCA1 mutation influences alternative splicing in human benign mammary organoids abstract. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-13-03.
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