Richard Heinz

Multiple Molecular Subtypes of Triple-Negative Breast Cancer Critically Rely on Androgen Receptor and Respond to Enzalutamide In Vivo

Triple-negative breast cancer (TNBC) has the lowest 5-year survival rate of invasive breast carcinomas, and currently there are no approved targeted therapies for this aggressive form of the disease. The androgen receptor (AR) is expressed in up to one third of TNBC and we find that all AR+ TNBC primary tumors tested display nuclear localization of AR, indicative of transcriptionally active receptors. While AR is most abundant in the “luminal AR (LAR)” molecular subtype of TNBC, here, for the first time, we use both the new-generation anti-androgen enzalutamide and AR knockdown to demonstrate that the other non-LAR molecular subtypes of TNBC are critically dependent on AR protein. Indeed, AR inhibition significantly reduces baseline proliferation, anchorage-independent growth, migration, and invasion and increases apoptosis in four TNBC lines (SUM159PT, HCC1806, BT549, and MDA-MB-231), representing three non-LAR TNBC molecular subtypes (mesenchymal-like, mesenchymal stem–like, and basal-like 2). In vivo, enzalutamide significantly decreases viability of SUM159PT and HCC1806 xenografts. Furthermore, mechanistic analysis reveals that AR activation upregulates secretion of the EGFR ligand amphiregulin (AREG), an effect abrogated by enzalutamide in vitro and in vivo. Exogenous AREG partially rescues the effects of AR knockdown on proliferation, migration, and invasion, demonstrating that upregulation of AREG is one mechanism by which AR influences tumorigenicity. Together, our findings indicate that non-LAR subtypes of TNBC are AR dependent and, moreover, that enzalutamide is a promising targeted therapy for multiple molecular subtypes of AR+ TNBC. Mol Cancer Ther; 14(3); 769–78. ©2015 AACR.

Patterns of Sex Steroid Hormones in Nipple Aspirate Fluid during the Menstrual Cycle and after Menopause in Relation to Serum Concentrations

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

Constitutive expression of microRNA-150 in mammary epithelium suppresses secretory activation and impairs de novo lipogenesis

Profiling of RNA from mouse mammary epithelial cells (MECs) isolated on pregnancy day (P)14 and lactation day (L)2 revealed that the majority of differentially expressed microRNA declined precipitously between late pregnancy and lactation. The decline in miR-150, which exhibited the greatest fold-decrease, was verified quantitatively and qualitatively. To test the hypothesis that the decline in miR-150 is crucial for lactation, MEC-specific constitutive miR-150 was achieved by crossing ROSA26-lox-STOP-lox-miR-150 mice with WAP-driven Cre recombinase mice. Both biological and foster pups nursed by bitransgenic dams exhibited a dramatic decrease in survival compared with offspring nursed by littermate control dams. Protein products of predicted miR-150 targets Fasn, Olah, Acaca, and Stat5B were significantly suppressed in MECs of bitransgenic mice with constitutive miR-150 expression as compared with control mice at L2. Lipid profiling revealed a significant reduction in fatty acids synthesized by the de novo pathway in L2 MECs of bitransgenic versus control mice. Collectively, these data support the hypothesis that a synchronized decrease in miRNAs, such as miR-150, at late pregnancy serves to allow translation of targets crucial for lactation. Summary: A decline in miR-150 is critical for lactation, as pups nursed by dams in which this decline is prevented exhibit a dramatic decrease in survival compared with littermate controls.

Methodological considerations in estrogen assays of breast fluid and breast tissue

Estradiol (E2) in nipple aspirate fluid (NAF), ductal lavage fluid (DLF), and random fine needle aspirates (rFNA) are compared. Quantification was by immunoassay or tandem MS. The percent of women yielding NAF varied between 24% and 48% and for DLF was 86.3%. Variation between ducts within a breast was not less than variation between breasts within women but variation between breasts and within women over time was significantly less than variation between women. Serum E2 was highly significantly different among phases of the menstrual cycle but NAF E2 was not different. The correlation between serum and breast fluid E2 concentrations in premenopausal women had coefficients of determination of less than 15%. The correlation between serum and NAF in studies of postmenopausal women varied greatly and may depend on patient selection. The difference between NAF E2 between pre- and postmenopausal women was only 22%; for rFNA it was non-significantly 44% lower in a similar group of postmenopausal women. Progesterone was 96% and 98% lower in postmenopausal NAF and rFNA samples, respectively. Measurements of E2 in breast fluid or breast tissue appears to provide similar estimates of E2 exposure. E2 levels in breast fluid do not reflect the rapid changes that occur in serum and, thus, serum availability of E2 is only one factor determining its levels in the breast. The similarity of levels between breasts and between ducts suggests that estimates of estrogen exposure does not require multiple samples, however, unavailability of fluid may require rFNA in some cases.

Hormonal Determinants of Nipple Aspirate Fluid Yield among Breast Cancer Cases and Screening Controls

Background: Nipple aspiration fluid (NAF) use as a biosample is limited by the variable yield across studies. We investigated the endocrine determinants of yield in an ongoing breast cancer case–control study. Methods: One-hundred and eighteen women yielding ≥2 μL NAF and 120 non-yielders were included; serum hormones were measured; differences in median hormones were assessed using the Wilcoxon rank-sum test. ORs and 95% confidence intervals (95% CI) for yielder status relative to hormone levels were estimated using logistic regression, adjusting for parity and lactation, and, in premenopausal women, menstrual cycle phase (MCP). Results: Prolactin concentrations were higher in yielders than non-yielders (premenopausal: 7.6 and 2.5 ng/mL, P < 0.01; postmenopausal 5.3 and 2.2 ng/mL; P < 0.01). Among premenopausal-yielders, estradiol was lower (64.3 vs. 90.5 pg/mL, MCP-adjusted P = 0.02). In separate menopausal status and parity-adjusted models, significant case–control differences persisted in prolactin: case OR 1.93 (95% CI, 1.35–2.77), control OR 1.64 (95% CI, 1.17–2.29). Premenopausal control yielders had higher progesterone (OR, 1.70; 95% CI, 1.18–2.46) and sex-hormone binding-globulin (OR, 2.09; 95% CI, 1.08–4.05) than non-yielders. Among parous women, further adjustment for lactation suggested a stronger positive association of serum prolactin with yield in cases than controls. Conclusion: NAF-yielders show higher prolactin than non-yielders, regardless of menopause and parity; implications of this and other endocrine differences on NAF biomarkers of breast cancer risk deserve further study. Impact: NAF yield is associated with a distinct endocrine environment that must be considered in studies of NAF-based breast cancer risk markers. Cancer Epidemiol Biomarkers Prev; 22(12); 2277–84. ©2013 AACR.

Breast Hormone Concentrations in Random Fine-Needle Aspirates of Healthy Women Associate with Cytological Atypia and Gene Methylation

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 r = 0.34, Padj = 0.001 and r = 0.69, Padj < 0.0006, respectively). Progesterone concentration was significantly higher in the premenopausal breast (Padj < 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 (P = 0.026 and Padj = 0.208). Breast androgens were significantly correlated with breast density (Spearman r = 0.27, Padj = 0.02 for testosterone) and CMI (Spearman r = 0.3, Padj = 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. Cancer Prev Res; 11(9); 557–68. ©2018 AACR.

Abstract 5756: Protein biomarkers for breast cancer risk are specifically correlated with local steroid hormones in nipple aspirate fluid

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Breast cancer risk is related to endogenous lifetime steroid hormone exposure. The local production of steroid hormones in the breast is a stable contributor to the hormonal environment. Several risk marker proteins have been identified in nipple aspirate fluid (NAF) in unaffected contralateral breast or in high-risk subjects as indicators of changes in breast on oxidative stress (superoxide dismutase), inflammation (C-reactive protein and YKL40), protease activity (cathepsin D) and growth factors (bFGF). However, it is not clear whether the alteration of protein markers is related to the local steroid hormone synthesis and metabolism in the breast. In the study, we studied the correlation between protein markers and both systematic (serum) and local (NAF) abundance of hormones. Methods: NAF and blood samples were obtained from normal breast of 54 healthy women and from unaffected breast of 60 breast cancer patients. The 114 subjects consisted of 48 women in premenopausal, 54 in postmenopausal and 12 in perimenopausal. The abundance of five protein markers (SOD1, CRP, YKL40, CatD and bFGF) was measured using ELISA. The NAF concentration of estradiol (E2), estrone (E1), progesterone (P4), andostenedione (A4), testosterone (T), dehydroepiandrostrerone (DHEA) and DHEA sulfate (DHEAS), and the serum concentration of E2, E1, P4, A4, T, DHEA and follicular-stimulating hormone (FSH) were measured using ELISA or RIA. The correlation between protein markers and hormones were examined using Spearmans rank correlation test with Bonferroni adjusted p < 0.05 as statistical significant. Results: The correlation among the protein markers showed that SOD1 was significantly correlated with CRP (r=0.276, p=0.033) and catD (r=0.340, p=0.0036). bFGF was significantly correlated with CRP (r=0.343, p=0.0021), indicating these proteins may be affected each other in the breast. The relationship between protein markers and hormones indicated that protein markers did not have much correlation with serum hormones, except bFGF was negatively correlated with serum T level (r=−0.339, p=0.017). In contrast, protein markers showed stronger correlation with multiple estradiol precursors in NAF. Specifically, SOD1 correlated with DHEA (r=0.333, P=0.019) and DHEAS (r=0.372, p=0.0030). YKL40 correlated with NAF T level (r=0.389, p=0.0012). CatD correlated with NAF DHEAS (r=0.400, p=0.0014). bFGF negatively correlated with NAF T (r=−0.339, p=0.015). CRP did not show significant correlation with any types of measured NAF hormones. Conclusions: NAF protein markers were more strongly related to local hormone levels in the breast, rather than systematic hormones in the blood. Protein markers were specifically correlated with different components of hormone hormones, suggesting that metabolic precursors may contribute to breast cancer risk through distinct mechanisms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5756. doi:1538-7445.AM2012-5756

Abstract 4466: Local regulation of steroid hormones in the breast is related to single nucleotide polymorphism of steroid metabolism genes

Background: The pattern of steroid hormone levels in nipple aspirate fluid (NAF) significantly differs from systemic levels, but may be a better predictor of breast cancer risk than the systemic environment. We hypothesize that local production and clearance of estrogen precursors or estrogen in the breast is affected by genetic variation in the enzymes involved in steroid transport, biosynthesis, and metabolism, thereby contributing to breast cancer risk. We examined the effect of functional single nucleotide polymorphism (SNP) on the concentrations of the NAF steroid hormones, as compared to serum levels. Methods: NAF was collected from both the contralateral unaffected breast of breast cancer patients and controls (blinded study of 265 women). Selection criteria for SNPs were; 1) known/potential functional significance on activity of the resulting enzyme. 2) Expression in breast tissue. 3) Caucasian population frequency of the minor alleles. gDNA was extracted from the plasma buffy coat by a Qiagen kit, followed by TaqMan genotyping assays (Applied Biosystems). The NAF and serum level of estradiol (E2), estrone (E1), progesterone (P4), testosterone (T), androstenedione (A4), and dehydroepiandrosterone (DHEA) were measured by immunoassay. Hormone levels were compared across genotype groups using the ANCOVA in SAS (age-adjusted significance p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4466. doi:1538-7445.AM2012-4466

Abstract 4477: Progesterone (P4) and testosterone (T) concentrations in random fine needle aspirates of the breast in pre- and postmenopausal women

Background: The availability of steroid hormones in the breast may be an important measure of the potential for proliferation of hormone-dependent breast cancer, and because retention of hormones may be influenced by serum levels, in situ formation, and the presence of binding proteins and lipids in the tissue, measurements of tissue concentrations are more informative than serum concentrations alone. Methods: Subjects were normal women, aged 36-65 yr, without breast cancer and not taking hormones. Tissue was obtained by fine needle aspiration of the breast. The cellular pellet was saved for analysis of DNA, and lipids were extracted from the supernatant including the fatty layer. Previous work had shown the cellular pellet contained a very little of the total steroids of the sample. Triglycerides were precipitated from 90% methanol at –20 °C and the remaining lipids were applied to a C18 HPLC column for purification in a gradient of methanol and acetonitrile in phosphate buffer. Fractions containing P4 and T were analyzed by immunoassays. The total DNA in the cellular pellet was isolated and quantified by UV absorbance. The steroid content was expressed as either per mg of purified lipid or per microgram of DNA. The data, when expressed as the natural log of the concentrations, followed a normal distribution. Differences in pre- and postmenopausal concentrations were analyzed by group t-tests. Results: The geometric means of the hormone concentrations are shown in the table. Comment: Despite the almost complete loss of progesterone in serum of postmenopausal women, the concentrations in the tissue are reduced but remain, on average, at more than 25% of the premenopausal level, somewhat more than the previously reported percentage in NAF of 5%. Relationships within individual women to other risk factors will be explored. Testosterone is also maintained at a relatively high level in the tissue of postmenopausal women, and may serve as a substrate for aromatase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4477. doi:1538-7445.AM2012-4477

Abstract P4-12-04: Association of single-strand breaks (SSBs) in normal breast DNA with estimates of breast cancer risk

Background: Formation of single strand breaks in DNA is a constant process, estimated to occur 10,000 times per day in each cell, either from endogenous biosynthetic errors or from interference by endogenous or exogenous agents. Base excision repair in some cases may be impaired or may be exceeded by the rate of DNA damage, leading to cancer. Nick translation of with labeled nucleotides can be used as a quantitative indicator of SSBs. Methods: Healthy women were recruited through the Love-Avon Army of Women and breast clinics of Northwestern and John Hopkins Universities. Digital or digitized mammograms were evaluated for percent density using Cumulus software. The medians (ranges) were age 51 (36 to 60); BMI 28.2 (18.7 to 51.3); life-time Gail estimate 12.5 (5.6 to 28.3); % breast density 16.5 (2.4 to 52.5); Masood score 13 (0 to 18). Breast tissue was obtained by random fine needle aspiration (rFNA). Specimens were rinsed into ice-cold phosphate buffered saline and were stored at −80°C. Thawed samples were centrifuged at 2200 g for 60 min. A kit from Norgen Inc., was used to separate DNA, RNA, and protein from the pellet. The lipids were extracted with ethyl acetate-hexane (3:2) from the supernatant fluid, and triglycerides were precipitated from cold 90% methanol, leaving a purified lipid fraction containing the steroids. Steroids were then fractionated by HPLC on a C18 column, and estradiol was analyzed by a radioimmunoassay. Blood was obtained at the same time as the rFNA samples and was frozen prior to analysis. An aliquot containing 200 ng of DNA (260/280 ratio 1.3 to 2.3) was taken for the nick translation assay. Incorporation of free nucleotides at SSBs with dCTP labeled with 3 H was catalyzed by Polymerase I from E. coli. A quality control preparation prepared from calf thymus DNA and a reagent blank without DNA was included with each set of 6 samples. Incorporation of 3 H-dCTP was linear with dose of DNA and reached a maximal at 30 min. Separation of free from incorporated 3 H-dCTP was accomplished by gel chromatography on an 80 × 15 mm column. The concordance of interassay values was 0.96. Results: Incorporation of nucleotides into DNA ranged from 0.03 to 8.59 pmol/μg, median 0.63 pmol/μg DNA. The association with other factors associated with breast cancer is shown in the Table. A significant correlation, was found with % breast density, life-time risk by the Gail model, but not serum estradiol concentrations. Conclusions: Assessment of SSBs by the nick translation procedure may be a useful indicator of breast cancer risk. Future studies will relate this method with actual risk as assessed by analysis of pre-diagnosis specimens with subsequent occurrence of breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-12-04.