Evaluation of the NRF1-proteasome axis as a therapeutic target in breast cancer.

Proteasomes are multi-subunit complexes that specialize in protein degradation. Cancer cells exhibit a heightened dependence on proteasome activity, presumably to support their enhanced proliferation and other cancer-related characteristics. Here, a systematic analysis of TCGA breast cancer datasets revealed that proteasome subunit transcript levels are elevated in all intrinsic subtypes (luminal, HER2-enriched, and basal-like/triple-negative) when compared to normal breast tissue. Although these observations suggest a pan-breast cancer utility for proteasome inhibitors, our further experiments with breast cancer cell lines and patient-derived xenografts (PDX) pointed to triple-negative breast cancer (TNBC) as the most sensitive subtype to proteasome inhibition. Finally, using TNBC cells, we extended our studies to in vivo xenograft experiments. Our previous work has firmly established a cytoprotective role for the transcription factor NRF1 via its ability to upregulate proteasome genes in response to proteasome inhibition. In further support of this notion, we show here that NRF1 depletion significantly reduced tumor burden in an MDA-MB-231 TNBC xenograft mouse model treated with carfilzomib. Taken together, our results point to TNBC as a particularly vulnerable breast cancer subtype to proteasome inhibition and provide a proof-of-principle for targeting NRF1 as a viable means to increase the efficacy of proteasome inhibitors in TNBC tumors.

Prolactin levels and breast cancer risk by tumor expression of prolactin-related markers.

BACKGROUND: Higher circulating prolactin has been associated with increased breast cancer risk. Prolactin binding to the prolactin receptor (PRLR) can activate the transcription factor STAT5, thus, we examined the association between plasma prolactin and breast cancer risk by tumor expression of PRLR, STAT5, and the upstream kinase JAK2. METHODS: Using data from 745 cases and 2454 matched controls in the Nurses' Health Study, we conducted polytomous logistic regression to examine the association between prolactin (> 11 ng/mL vs. ≤ 11 ng/mL) measured within 10 years of diagnosis and breast cancer risk by PRLR (nuclear [N], cytoplasmic [C]), phosphorylated STAT5 (pSTAT5; N, C), and phosphorylated JAK2 (pJAK2; C) tumor expression. Analyses were conducted separately in premenopausal (n = 168 cases, 765 controls) and postmenopausal women (n = 577 cases, 1689 controls). RESULTS: In premenopausal women, prolactin levels > 11 ng/mL were positively associated with risk of tumors positive for pSTAT5-N (OR 2.30, 95% CI 1.02-5.22) and pSTAT5-C (OR 1.64, 95% CI 1.01-2.65), but not tumors that were negative for these markers (OR 0.98, 95% CI 0.65-1.46 and OR 0.73, 95% CI 0.43-1.25; p-heterogeneity = 0.06 and 0.02, respectively). This was stronger when tumors were positive for both pSTAT5-N and pSTAT5-C (OR 2.88, 95% CI 1.14-7.25). No association was observed for PRLR or pJAK2 (positive or negative) and breast cancer risk among premenopausal women. Among postmenopausal women, plasma prolactin levels were positively associated with breast cancer risk irrespective of PRLR, pSTAT5, or pJAK2 expression (all p-heterogeneity ≥ 0.21). CONCLUSION: We did not observe clear differences in the association between plasma prolactin and breast cancer risk by tumor expression of PRLR or pJAK2, although associations for premenopausal women were observed for pSTAT5 positive tumors only. While additional studies are needed, this suggests that prolactin may act on human breast tumor development through alternative pathways.

Breast Cancer and Prolactin - New Mechanisms and Models.

The pathogenesis of breast cancer is driven by multiple hormones and growth factors. One of these, prolactin (PRL), contributes to both mammary differentiation and oncogenesis, and yet the basis for these disparate effects has remained unclear. The focus of this review is to examine and place into context 2 recent studies that have provided insight into the roles of PRL receptors and PRL in tumorigenesis and tumor progression. One study provides novel evidence for opposing actions of PRL in the breast being mediated in part by differential PRL receptor (PRLr) isoform utilization. Briefly, homomeric complexes of the long isoform of the PRLr (PRLrL-PRLrL) promotes mammary differentiation, while heteromeric complexes of the intermediate and long PRLr (PRLrI-PRLrL) isoforms trigger mammary oncogenesis. Another study describes an immunodeficient, prolactin-humanized mouse model, NSG-Pro, that facilitates growth of PRL receptor-expressing patient-derived breast cancer xenografts. Evidence obtained with this model supports the interactions of physiological levels of PRL with estrogen and ERBB2 gene networks, the modulatory effects of PRL on drug responsiveness, and the pro-metastatic effects of PRL on breast cancer. This recent progress provides novel concepts, mechanisms and experimental models expected to renew interest in harnessing/exploiting PRLr signaling for therapeutic effects in breast cancer.

Serine residues 726 and 780 have nonredundant roles regulating STAT5a activity in luminal breast cancer.

In breast cancer, prolactin-induced activation of the transcription factor STAT5a results from the phosphorylation of STAT5a tyrosine residue 694. However, its role in mammary oncogenesis remains an unsettled debate as STAT5a exhibits functional dichotomy with both pro-differentiative and pro-proliferative target genes. Phosphorylation of STAT5a serine residues, S726 and S780, may regulate STAT5a in such a way to underlie this duality. Given hematopoiesis studies showing phospho-serine STAT5a as necessary for transformation, we hypothesized that serine phosphorylation regulates STAT5a activity to contribute to its role in mammary oncogenesis, specifically in luminal breast cancer. Here, phosphorylation of S726-, S780-, and Y694-STAT5a in response to prolactin in MCF7 luminal breast cancer cells was investigated with STAT5a knockdown and rescue with Y694F-, S726A-, or S780A-STAT5a, where the phospho-sites were mutated. RNA-sequencing and subsequent Ingenuity Pathway Analysis predicted that loss of each phospho-site differentially affected both prolactin-induced gene expression as well as functional pathways of breast cancer (e.g. cell survival, proliferation, and colony formation). In vitro studies of anchorage-independent growth and proliferation confirmed distinct phenotypes: whereas S780A-STAT5a decreased clonogenicity, S726A-STAT5a decreased proliferation in response to prolactin compared to wild type STAT5a. Collectively, these studies provide novel insights into STAT5a activation in breast cancer pathogenesis.

The human intermediate prolactin receptor is a mammary proto-oncogene.

The hormone prolactin (PRL) and its receptor (hPRLr) are significantly involved in breast cancer pathogenesis. The intermediate hPRLr (hPRLrI) is an alternatively-spliced isoform, capable of stimulating cellular viability and proliferation. An analogous truncated mouse PRLr (mPRLr) was recently found to be oncogenic when co-expressed with wild-type mPRLr. The goal of this study was to determine if a similar transforming event occurs with the hPRLr in human breast epithelial cells and to better understand the mechanism behind such transformation. hPRLrL+I co-expression in MCF10AT cells resulted in robust in vivo and in vitro transformation, while hPRLrI knock-down in MCF7 cells significantly decreased in vitro malignant potential. hPRLrL+I heterodimers displayed greater stability than hPRLrL homodimers, and while being capable of activating Jak2, Ras, and MAPK, they were unable to induce Stat5a tyrosine phosphorylation. Both immunohistochemical breast cancer tissue microarray data and RNA sequencing analyses using The Cancer Genome Atlas (TCGA) identified that higher hPRLrI expression associates with triple-negative breast cancer. These studies indicate the hPRLrI, when expressed alongside hPRLrL, participates in mammary transformation, and represents a novel oncogenic mechanism.

Breast cancer liver metastasis: current and future treatment approaches.

Nearly all fatalities arising from breast tumors are attributable to distant metastases. Breast cancer liver metastasis (BCLM) is associated with poor prognoses, with the median survival time being 2 to 3 years. Tumor intrinsic subtype directs preferential metastasis to specific organs, with HER2-enriched tumors demonstrating the highest rates of metastasis to the liver, though all subtypes can grow in the liver. There is no singular established standard-of-care for BCLM; therapeutic selection is driven by histologic and molecular hallmarks of the primary tumor or biopsied metastasis samples. Given the poor prognosis of patients with hepatic spread, pre-clinical studies are necessary to identify and evaluate promising new treatment strategies. It is critical that these laboratory studies accurately recapitulate the BCLM disease process, standard progression, and histological attributes. In this review, we summarize the histologic and molecular characteristics of BCLM, evaluate the efficacy of existing surgical and medical treatment strategies, and discuss future approaches to preclinical study of BCLM.

Prolactin Drives a Dynamic STAT5A/HDAC6/HMGN2 Cis-Regulatory Landscape Exploitable in ER+ Breast Cancer.

The hormone prolactin has been implicated in breast cancer pathogenesis and regulates chromatin engagement by the transcription factor, STAT5A. STAT5A is known to inducibly bind promoters and cis-regulatory elements genome-wide, though the mechanisms by which it exerts specificity and regulation of target gene expression remain enigmatic. We previously identified HDAC6 and HMGN2 as cofactors that facilitate prolactin-induced, STAT5A-mediated gene expression. Here, multicondition STAT5A, HDAC6, and HMGN2 chromatin immunoprecipitation and sequencing with parallel condition RNA-seq are utilized to reveal the cis-regulatory landscape and cofactor dynamics underlying prolactin-stimulated gene expression in breast cancer. We find that prolactin-regulated genes are significantly enriched for cis-regulatory elements bound by HDAC6 and HMGN2, and that inducible STAT5A binding at enhancers, rather than promoters, conveys specificity for prolactin-regulated genes. The selective HDAC6 inhibitor, ACY-241, blocks prolactin-induced STAT5A chromatin engagement at cis-regulatory elements as well as a significant proportion of prolactin-stimulated gene expression. We identify functional pathways known to contribute to the development and/or progression of breast cancer that are activated by prolactin and inhibited by ACY-241. Additionally, we find that the DNA sequences underlying shared STAT5A and HDAC6 binding sites at enhancers are differentially enriched for estrogen response elements (ESR1 and ESR2 motifs) relative to enhancers bound by STAT5A alone. Gene set enrichment analysis identifies significant overlap of ERα-regulated genes with genes regulated by prolactin, particularly prolactin-regulated genes with promoters or enhancers co-occupied by both STAT5A and HDAC6. Lastly, the therapeutic efficacy of ACY-241 is demonstrated in in vitro and in vivo breast cancer models, where we identify synergistic ACY-241 drug combinations and observe differential sensitivity of ER+ models relative to ER- models.

Inhibition of the Activity of Cyclophilin A Impedes Prolactin Receptor-Mediated Signaling, Mammary Tumorigenesis, and Metastases.

Prolactin (PRL) and its receptor (PRLr) play important roles in the pathogenesis of breast cancer. Cyclophilin A (CypA) is a cis-trans peptidyl-prolyl isomerase (PPI) that is constitutively associated with the PRLr and facilitates the activation of the tyrosine kinase Jak2. Treatment with the non-immunosuppressive prolyl isomerase inhibitor NIM811 or CypA short hairpin RNA inhibited PRL-stimulated signaling, breast cancer cell growth, and migration. Transcriptomic analysis revealed that NIM811 inhibited two-thirds of the top 50 PRL-induced genes and a reduction in gene pathways associated with cancer cell signaling. In vivo treatment of NIM811 in a TNBC xenograft lessened primary tumor growth and induced central tumor necrosis. Deletion of CypA in the MMTV-PyMT mouse model demonstrated inhibition of tumorigenesis with significant reduction in lung and lymph node metastasis. The regulation of PRLr/Jak2-mediated biology by NIM811 demonstrates that a non-immunosuppressive prolyl isomerase inhibitor can function as a potential breast cancer therapeutic.

Cyclophilin A Function in Mammary Epithelium Impacts Jak2/Stat5 Signaling, Morphogenesis, Differentiation, and Tumorigenesis in the Mammary Gland.

The prolyl isomerase cyclophilin A (CypA) regulates the Jak2/Stat5 pathway, which is necessary for mammary differentiation and the pathogenesis of breast cancer. In this study, we assessed the role of this isomerase during mammary gland development and erbB2-driven tumorigenesis. Genetic deletion of CypA resulted in delayed mammary gland morphogenesis and differentiation with corresponding decrease in Jak2/Stat5 activation; mammary gland cross-transplantation confirmed this defect was epithelial in nature. Analysis of mammary stem and progenitor populations revealed significant disruption of epithelial maturation. Loss of CypA in the erbB2 transgenic mouse model revealed a marked increase in mammary tumor latency that correlated with decreased Stat5 activation, associated gene expression, and reduced epithelial cell proliferation. These results demonstrate an important role for CypA in the regulation of Jak2/Stat5-mediated biology in mammary epithelium, identifying this isomerase as a novel target for therapeutic intervention.Significance: These findings reveal cyclophilin A functions in normal mammary epithelial development and ErbB2-driven mammary tumorigenesis and suggest therapies targeting cyclophilin A may be efficacious for breast cancer treatment.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/14/3877/F1.large.jpg Cancer Res; 78(14); 3877-87. ©2018 AACR.

Aberrantly high expression of the CUB and zona pellucida-like domain-containing protein 1 (CUZD1) in mammary epithelium leads to breast tumorigenesis.

The peptide hormone prolactin (PRL) and certain members of the epidermal growth factor (EGF) family play central roles in mammary gland development and physiology, and their dysregulation has been implicated in mammary tumorigenesis. Our recent studies have revealed that the CUB and zona pellucida-like domain-containing protein 1 (CUZD1) is a critical factor for PRL-mediated activation of the transcription factor STAT5 in mouse mammary epithelium. Of note, CUZD1 controls production of a specific subset of the EGF family growth factors and consequent activation of their receptors. Here, we found that consistent with this finding, CUZD1 overexpression in non-transformed mammary epithelial HC11 cells increases their proliferation and induces tumorigenic characteristics in these cells. When introduced orthotopically in mouse mammary glands, these cells formed adenocarcinomas, exhibiting elevated levels of STAT5 phosphorylation and activation of the EGF signaling pathway. Selective blockade of STAT5 phosphorylation by pimozide, a small-molecule inhibitor, markedly reduced the production of the EGF family growth factors and inhibited PRL-induced tumor cell proliferation in vitro Pimozide administration to mice also suppressed CUZD1-driven mammary tumorigenesis in vivo Analysis of human MCF7 breast cancer cells indicated that CUZD1 controls the production of the same subset of EGF family members in these cells as in the mouse. Moreover, pimozide treatment reduced the proliferation of these cancer cells. Collectively, these findings indicate that overexpression of CUZD1, a regulator of growth factor pathways controlled by PRL and STAT5, promotes mammary tumorigenesis. Blockade of the STAT5 signaling pathway downstream of CUZD1 may offer a therapeutic strategy for managing these breast tumors.

Histone H1 and Chromosomal Protein HMGN2 Regulate Prolactin-induced STAT5 Transcription Factor Recruitment and Function in Breast Cancer Cells.

The hormone prolactin (PRL) contributes to breast cancer pathogenesis through various signaling pathways, one of the most notable being the JAK2/signal transducer and activator of transcription 5 (STAT5) pathway. PRL-induced activation of the transcription factor STAT5 results in the up-regulation of numerous genes implicated in breast cancer pathogenesis. However, the molecular mechanisms that enable STAT5 to access the promoters of these genes are not well understood. Here, we show that PRL signaling induces chromatin decompaction at promoter DNA, corresponding with STAT5 binding. The chromatin-modifying protein high mobility group nucleosomal binding domain 2 (HMGN2) specifically promotes STAT5 accessibility at promoter DNA by facilitating the dissociation of the linker histone H1 in response to PRL. Knockdown of H1 rescues the decrease in PRL-induced transcription following HMGN2 knockdown, and it does so by allowing increased STAT5 recruitment. Moreover, H1 and STAT5 are shown to function antagonistically in regulating PRL-induced transcription as well as breast cancer cell biology. While reduced STAT5 activation results in decreased PRL-induced transcription and cell proliferation, knockdown of H1 rescues both of these effects. Taken together, we elucidate a novel mechanism whereby the linker histone H1 prevents STAT5 binding at promoter DNA, and the PRL-induced dissociation of H1 mediated by HMGN2 is necessary to allow full STAT5 recruitment and promote the biological effects of PRL signaling.

NSG Mice Provide a Better Spontaneous Model of Breast Cancer Metastasis than Athymic (Nude) Mice.

Metastasis is the most common cause of mortality in breast cancer patients worldwide. To identify improved mouse models for breast cancer growth and spontaneous metastasis, we examined growth and metastasis of both estrogen receptor positive (T47D) and negative (MDA-MB-231, SUM1315, and CN34BrM) human breast cancer cells in nude and NSG mice. Both primary tumor growth and spontaneous metastases were increased in NSG mice compared to nude mice. In addition, a pattern of metastasis similar to that observed in human breast cancer patients (metastases to the lungs, liver, bones, brain, and lymph nodes) was found in NSG mice. Furthermore, there was an increase in the metastatic burden in NSG compared to nude mice that were injected with MDA-MB-231 breast cancer cells in an intracardiac experimental metastasis model. This data demonstrates that NSG mice provide a better model for studying human breast cancer metastasis compared to the current nude mouse model.

Identification of NEK3 Kinase Threonine 165 as a Novel Regulatory Phosphorylation Site That Modulates Focal Adhesion Remodeling Necessary for Breast Cancer Cell Migration.

Accumulating evidence supports a role for prolactin (PRL) in the development and progression of human breast cancer. Although PRL is an established chemoattractant for breast cancer cells, the precise molecular mechanisms of how PRL regulates breast cancer cell motility and invasion are not fully understood. PRL activates the serine/threonine kinase NEK3, which was reported to enhance breast cancer cell migration, invasion, and the actin cytoskeletal reorganization necessary for these processes. However, the specific mechanisms of NEK3 activation in response to PRL signaling have not been defined. In this report, a novel PRL-inducible regulatory phosphorylation site within the activation segment of NEK3, threonine 165 (Thr-165), was identified. Phosphorylation at NEK3 Thr-165 was found to be dependent on activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway using both pharmacological inhibition and siRNA-mediated knockdown approaches. Strikingly, inhibition of phosphorylation at NEK3 Thr-165 by expression of a phospho-deficient mutant (NEK3-T165V) resulted in increased focal adhesion size, formation of zyxin-positive focal adhesions, and reorganization of the actin cytoskeleton into stress fibers. Concordantly, NEK3-T165V cells exhibited migratory defects. Together, these data support a modulatory role for phosphorylation at NEK3 Thr-165 in focal adhesion maturation and/or turnover to promote breast cancer cell migration.

HDAC6 Deacetylates HMGN2 to Regulate Stat5a Activity and Breast Cancer Growth.

Stat5a is a transcription factor utilized by several cytokine/hormone receptor signaling pathways that promotes transcription of genes associated with proliferation, differentiation, and survival of cancer cells. However, there are currently no clinically approved therapies that directly target Stat5a, despite ample evidence that it contributes to breast cancer pathogenesis. Here, deacetylation of the Stat5a coactivator and chromatin-remodeling protein HMGN2 on lysine residue K2 by HDAC6 promotes Stat5a-mediated transcription and breast cancer growth. HDAC6 inhibition both in vitro and in vivo enhances HMGN2 acetylation with a concomitant reduction in Stat5a-mediated signaling, resulting in an inhibition of breast cancer growth. Furthermore, HMGN2 is highly acetylated at K2 in normal human breast tissue, but is deacetylated in primary breast tumors and lymph node metastases, suggesting that targeting HMGN2 deacetylation is a viable treatment for breast cancer. Together, these results reveal a novel mechanism by which HDAC6 activity promotes the transcription of Stat5a target genes and demonstrate utility of HDAC6 inhibition for breast cancer therapy. IMPLICATIONS: HMGN2 deacetylation enhances Stat5a transcriptional activity, thereby regulating prolactin-induced gene transcription and breast cancer growth. Mol Cancer Res; 14(10); 994-1008. ©2016 AACR.

Immunoassay and Nb2 lymphoma bioassay prolactin levels and mammographic density in premenopausal and postmenopausal women the Nurses' Health Studies.

Higher circulating prolactin levels have been associated with higher percent mammographic density among postmenopausal women in some, but not all studies. However, few studies have examined associations with dense area and non-dense breast area breast or considered associations with prolactin Nb2 lymphoma cell bioassay levels. We conducted a cross-sectional study among 1,124 premenopausal and 890 postmenopausal women who were controls in breast cancer case-control studies nested in the Nurses' Health Study (NHS) and NHSII. Participants provided blood samples in 1989-1990 (NHS) or 1996-1999 (NHSII) and mammograms were obtained from around the time of blood draw. Multivariable linear models were used to assess the associations between prolactin levels (measured by immunoassay or bioassay) with percent density, dense area, and non-dense area. Among 1,124 premenopausal women, percent density, dense area, and non-dense area were not associated with prolactin immunoassay levels in multivariable models (p trends = 0.10, 0.18, and 0.69, respectively). Among 890 postmenopausal women, those with prolactin immunoassay levels in the highest versus lowest quartile had modestly, though significantly, higher percent density (difference = 3.01 percentage points, 95 % CI 0.22, 5.80) as well as lower non-dense area (p trend = 0.02). Among women with both immunoassay and bioassay levels, there were no consistent differences in the associations with percent density between bioassay and immunoassay levels. Postmenopausal women with prolactin immunoassay levels in the highest quartile had significantly higher percent density as well as lower non-dense area compared to those in the lowest quartile. Future studies should examine the underlying biologic mechanisms, particularly for non-dense area.

Bioactive prolactin levels and risk of breast cancer: a nested case-control study.

BACKGROUND: Prolactin is a lactogenic hormone associated with breast cancer risk in prospective studies, which used immunoassays. The immunoassay captures multiple isoforms and may not fully reflect the biologic activity of prolactin relevant to breast carcinogenesis. METHODS: We considered plasma bioactive prolactin levels measured by the Nb2 lymphoma cell bioassay, which is sensitive to the somatolactogenic activity of prolactin and growth hormone, within a nested case-control study of invasive breast cancer in the Nurses' Health Studies (NHS/NHSII). We also considered associations with breast cancer risk factors. RESULTS: We had bioassay measures on 1,329 cases and 1,329 controls. Bioassay levels were inversely associated with parity (4+ vs. 0 children = -18%, P = 0.01), body mass index (30+ vs. <22 kg/m(2) = -16%, P < 0.01), and age at menopause (53+ vs. 48 years = -18%, P = 0.03) and positively with family history of breast cancer (yes vs. no = 14%, P < 0.01). The relative risk (RR) comparing the top versus bottom quartile of bioassay levels was 1.19 [95% confidence intervals (CI), 0.94-1.51; Ptrend = 0.18]. The association was suggestively stronger for postmenopausal (RR = 1.36; 95% CI, 0.93-1.98; Ptrend = 0.12) versus premenopausal women (RR = 0.99; 95% CI, 0.71-1.37; Ptrend = 0.71). There was an association for cases diagnosed <4 years after blood draw (RR = 2.66; 95% CI, 1.45-4.89; Ptrend < 0.01), but not for cases diagnosed later. We did not observe differential associations by estrogen receptor status or other tumor characteristics. CONCLUSIONS: Our results show similar associations for prolactin levels measured by bioassay and by immunoassay with both breast cancer risk factors and risk. IMPACT: Future work examining risk prediction model of breast cancer can use the immunoassay to accurately characterize risk.

Antipsychotic treatment in breast cancer patients.

Special consideration is required when prescribing antipsychotic drugs for patients with an existing diagnosis of breast cancer. The package inserts of all approved antipsychotics contain precautions regarding their administration in this patient group. These drugs are well known to elevate serum prolactin levels to varying degrees. Overexpression of the prolactin receptor is seen in more than 95% of human breast cancers. Many genes that are activated by the prolactin receptor are associated with tumorigenesis and cancer cell proliferation. The authors discuss the pathophysiology, clinical implications, and pertinent preclinical data and make specific recommendations regarding the use of antipsychotics in patients with breast cancer.

Comment on "Progesterone/RANKL is a major regulatory axis in the human breast".

In vivo menstrual cycle data support the findings by Tanos et al. that progesterone regulates RANKL in an ex vivo microstructure model of the human breast, but dispute the suppression of estradiol on progesterone-stimulated RANKL expression. RANKL responds to progesterone in a three-dimensional organoid culture model under conditions mimicking luteal-phase hormone concentration, suggesting that the microstructure may not be crucial to demonstrate progesterone responsiveness.

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.

The prolactin receptor transactivation domain is associated with steroid hormone receptor expression and malignant progression of breast cancer.

The polypeptide hormone prolactin (PRL) stimulates breast epithelial cell growth, differentiation, and motility through its cognate receptor, PRLr. PRLr is expressed in most breast cancers; however, its exact role remains elusive. Our laboratory previously described a novel mode of PRLr signaling in which Stat5a-mediated transcription is regulated through ligand-induced phosphorylation of the PRLr transactivation domain (TAD). Herein, we used a PRLr transactivation-deficient mutant (PRLrYDmut) to identify novel TAD-specific target genes. Microarray analysis identified 120 PRL-induced genes up-regulated by wild type but not PRLrYDmut. Compared with control, PRLr expression significantly induced expression of approximately 4700 PRL-induced genes, whereas PRLrYDmut ablated induction of all but 19 of these genes. Ingenuity pathway analysis found that the PRLr TAD most profoundly affected networks involving cancer and proliferation. In support of this, PRLrYDmut expression reduced anchorage-dependent and anchorage-independent growth. In addition, pathway analysis identified a link between the PRLr TAD and the estrogen and progesterone receptors (ERα/PR). Although neither ERα nor PR was identified as a PRL target gene, a TAD mutation significantly impaired ERα/PR expression and estrogen responsiveness. TMA analysis revealed a marked increase in nuclear, but not cytoplasmic, PRLr TAD phosphorylation as a function of neoplastic progression. We propose that PRLr TAD phosphorylation contributes to breast cancer pathogenesis, in part through regulation of ERα and PR, and has potential utility as a biomarker in this disease.