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.
The mammary gland consists of cells with gene expression patterns reflecting their cellular origins, function, and spatiotemporal context. However, knowledge of developmental kinetics and mechanisms ...of lineage specification is lacking. We address this significant knowledge gap by generating a single-cell transcriptome atlas encompassing embryonic, postnatal, and adult mouse mammary development. From these data, we map the chronology of transcriptionally and epigenetically distinct cell states and distinguish fetal mammary stem cells (fMaSCs) from their precursors and progeny. fMaSCs show balanced co-expression of factors associated with discrete adult lineages and a metabolic gene signature that subsides during maturation but reemerges in some human breast cancers and metastases. These data provide a useful resource for illuminating mammary cell heterogeneity, the kinetics of differentiation, and developmental correlates of tumorigenesis.
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•Establishment of a developmental mammary transcriptome atlas•Chronology of mammary epithelial lineage bifurcation events•fMaSCs exhibit mixed lineage chromatin accessibility and gene expression patterns•fMaSC metabolic programs lost in differentiation are resurrected in breast cancer
Single-cell RNA sequencing of developing mouse mammary epithelia reveals the timing of lineage specification. Giraddi et al. find that fetal mammary stem cells co-express factors that define distinct lineages in their progeny and bear functionally relevant metabolic program signatures that change with differentiation and are resurrected in human breast cancers and metastases.
Fractionation of steroids allows for multiple assays to be run on a single low volume liquid biopsy, whereas performing the same number of assays without fractionation would require increasing the ...sample volume by dilution, rendering the concentration of steroids below the level of detection for most, if not all, downstream assays. Briefly, steroids are extracted from a biofluid sample using solvent phase extraction to separate the aqueous (conjugated) steroids from the non-aqueous (non-conjugated) steroids in the organic phase. The latter is further separated by high-performance liquid chromatography (HPLC) and collected in an automated fraction collector based on the UV detection of internal standards. Commercially available immunoassays are then used to quantify the < ng/ml concentrations of steroids in each fraction. This protocol was designed for small samples of nipple aspirate fluid (minimum 2 µL), but it can be modified to fractionate steroids from homogenized solid tissue samples or other liquid biopsies. Included in this protocol are precautions to help ensure reproducibility and minimize matrix effects and other errors of measurement, given that samples requiring fractionation are fundamentally precious and, like other quantitative procedures of small samples, can be prone to contamination by solvent residues and other factors.•The method permits quantitative analysis of multiple steroids from very small volumes of biofluid.•Fractionation by HPLC provides a highly purified sample for quantification.•The immunoassay end point provides specificity without expensive equipment.
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•A SNP of HSD17B12 increased breast fluid but not serum estradiol by >2-fold.•A SNP of CYP1B1 decreased breast fluid but not serum estradiol by 49%.•A SNP of SRD5A1 increased breast ...fluid and serum androstenedione similarly by 32%.•Two SNPs of CYP19A1 had no significant effect on estradiol in breast or serum.
Although alterations of concentrations in circulating steroids have been linked to single nucleotide polymorphisms (SNPs) of steroidogenic enzymes, we hypothesized that SNPs of such enzymes located within the breast affect local steroid concentrations more than products of such SNPs absorbed from the circulation.
Steroids (estradiol, estrone, testosterone, androstenedione, DHEA, DHEA sulfate, progesterone) in nipple aspirate fluid (NAF) were purified by HPLC and they along with serum steroids were quantified by immunoassays. Polymorphisms of the transporter SLCO2B1 and enzymes HSD3B1, CYP19A1, HSD17B12, AKR1C3, CYP1B1, and SRD5A1 were measured in white blood cell DNA.
Steroid concentrations in NAF of subjects with homozygous minor genotypes differed from those with heterozygotes, i.e., SLCO2B1 (rs2851069) decreased DHEAS (p = 0.04), HSD17B12 (rs11555762) increased estradiol (p < 0.004), and CYP1B1 (rs1056836) decreased estradiol (p = 0.017) and increased progesterone (p = 0.05). Also, in serum, CYP19A1 (rs10046 and rs700518) both decreased testosterone (p = 0.02) and SRD5A1 increased androstenedione (p = 0.006). Steroids in subjects with major homozygotes did not differ from those with heterozygotes indicating recessive characteristics.
In the breast, SNPs were associated with decreased uptake of DHEAS (SLCO2B1), increased estradiol concentrations through increased oxidoreductase activity (HSD17B12), or decreased estradiol concentrations by presumed formation of 4-hydroxyestradiol (CYP1B1). CYP19A1 was associated with decreased testosterone concentrations in serum but had no significant effect on estrogen or androgen concentrations within the breast. The hormone differences observed in NAF were not usually evident in serum, indicating the importance of assessing the effect of these SNPs within the breast.
The objective of the study was to evaluate all available systematic reviews on the use of prone positional ventilation in adult patients with acute respiratory distress syndrome (ARDS). An umbrella ...review on the efficacy of prone positional ventilation in adult patients ventilation in adult patients with acute respiratory distress syndrome was conducted. We performed a systematic search in the database of Medline (Pubmed), Scopus, Cochrane Library, Web of Science, and Epistemonikos. The ROBIS tools and GRADE methodology were used to assess the risk of bias and certainty of evidence. We estimated the necessary number of patients to be treated to have benefit. For the synthesis of the result, we selected the review with the lowest risk of bias. Sixteen systematic reviews including 64 randomized clinical trials and evaluating the effect of prone positional ventilation, with or without other ventilation strategies were included. Aoyama 2019 observed prone positioning, without complementary ventilation strategies, leading to a reduction in the 28-day mortality only when compared to high-frequency oscillatory ventilation (RR 0.61; 95% CI 0.39–0.95) and lung-protective ventilation in the supine position (RR 0.69; 95% CI 0.48–0.98), with an ARR of 9.32% and 14.94%, an NNTB of 5.89 and 8.04, and a low and moderate certainty of evidence, respectively. Most reviews had severe methodological flaws that led to results with very low certainty of evidence. The review with the lowest risk of bias presented results in favor of prone positional ventilation compared with high-frequency oscillatory ventilation and lung-protective ventilation. There is a need to update the available reviews to obtain more accurate results.
Abstract
Background
CRIPTO is a multi-functional signaling protein that promotes stemness and oncogenesis. We previously developed a CRIPTO antagonist, ALK4
L75A
-Fc, and showed that it causes loss ...of the stem cell phenotype in normal mammary epithelia suggesting it may similarly inhibit CRIPTO-dependent plasticity in breast cancer cells.
Methods
We focused on two triple negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) to measure the effects of ALK4
L75A
-Fc on cancer cell behavior under nutrient deprivation and endoplasmic reticulum stress. We characterized the proliferation and migration of these cells in vitro using time-lapse microscopy and characterized stress-dependent changes in the levels and distribution of CRIPTO signaling mediators and cancer stem cell markers. We also assessed the effects of ALK4
L75A
-Fc on proliferation, EMT, and stem cell markers in vivo as well as on tumor growth and metastasis using inducible lentiviral delivery or systemic administration of purified ALK4
L75A
-Fc, which represents a candidate therapeutic approach.
Results
ALK4
L75A
-Fc inhibited adaptive responses of breast cancer cells under conditions of nutrient and ER stress and reduced their proliferation, migration, clonogenicity, and expression of EMT and cancer stem cell markers. ALK4
L75A
-Fc also inhibited proliferation of human breast cancer cells in stressed tumor microenvironments in xenografts and reduced both primary tumor size and metastatic burden.
Conclusions
Cancer cell adaptation to stresses such as nutrient deprivation, hypoxia, and chemotherapy can critically contribute to dormancy, metastasis, therapy resistance, and recurrence. Identifying mechanisms that govern cellular adaptation, plasticity, and the emergence of stem-like cancer cells may be key to effective anticancer therapies. Results presented here indicate that targeting CRIPTO with ALK4
L75A
-Fc may have potential as such a therapy since it inhibits breast cancer cell adaptation to microenvironmental challenges and associated stem-like and EMT phenotypes.
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.
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.
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.
.
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.
...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).
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.
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.
NAF yield is associated with a distinct endocrine environment that must be considered in studies of NAF-based breast cancer risk markers.
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