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Supplemental Table S2. Primers used for qPCR.

S5. Bufexamac treatment impairs the viability of triple-negative WHIM2 PDX cells.

Immunohistochemistry of CypA+/+ and CypA-/- wk 14 virgins

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.

Supplementary Figures 1-4 from Prolyl Isomerase Cyclophilin A Regulation of Janus-Activated Kinase 2 and the Progression of Human Breast Cancer

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.

Epidemiological, cellular, and genetic analyses indicate the hormone prolactin (PRL) and its cognate receptor in humans (hPRLr) are significantly involved in breast cancer pathogenesis. Recent evidence demonstrated that a truncated mouse PRLr (mPRLrT) is oncogenic when expressed alongside its canonical long form counterpart (mPRLrL). mPRLrT shares significant sequence homology with a naturally-occurring and widely-expressed hPRLr splice variant, the intermediate hPRLr (hPRLrI). To confirm the oncogenic potential of hPRLrI, isoform-specific hPRLrI knock-down (KD) was performed in breast cancer cell line MCF7. hPRLrI KD resulted in a significant decrease in proliferation, migration, and anchorage-independent growth. Next, given the similarity between mPRLrT and hPRLrI, we hypothesized hPRLrI may also be sufficient to induce transformation, when expressed alongside wild-type long hPRLr (hPRLrL). Like the mPRLrT, hPRLrI co-expression with hPRLrL in the immortalized but not transformed human breast cell line MCF10A resulted in a significant increase in proliferation, migration, and anchorage-independent growth. These results were not observed following overexpression of either isoform alone, demonstrating that hPRLrL/I co-expression is necessary to induce transformation of normal mammary epithelia. To test our hypothesis in vivo, we established MCF10A xenografts using female NOD scid gamma (NSG) mice. Following intraductal injection, we observed rapid tumor growth in the hPRLrL/I co-expression cohort, significantly over that of the cohorts harboring either isoform alone. To determine mechanisms of transformation, we examined both differential protein stability and altered signaling events. In analyzing receptor degradation, a cycloheximide assay revealed hPRLrL stability is increased when heterodimerized with hPRLrI. hPRLrL turnover has been reported to be impaired in human breast cancer, indicating this phenomenon may be involved in the observed hPRLrI-mediated transformation. In regards to differential signaling, we examined the Jak2/Stat5a pathway. Jak2 is a promiscuous kinase whose significant oncogenic actions are well-characterized, while Stat5a is a transcription factor whose activities are critical in attenuating the actions of Jak2. Following PRL stimulation, it was observed that hPRLrL/I co-expression induced approximately two-fold greater Jak2-Y1007/1008 phosphorylation (pJak2) compared to that induced by hPRLrL expression alone. Further, it was observed that hPRLrL/I co-expression induced ten-fold less Stat5a-Y694 phosphorylation (pY-Stat5a) than hPRLrL expression alone. These data indicate unchecked pJak2 activity may also be a contributing mechanism in the observed transformation. Overall, these results demonstrate that hPRLrI, alongside hPRLrL, is sufficient for transformation of normal breast tissue. Citation Format: Jacqueline M Grible, Shannon E Hedrick, Charles V Clevenger. The human intermediate prolactin receptor is a breast cancer oncogene [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-08-02.

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.

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.

The neuroendocrine hormone prolactin (PRL) and its cognate receptor (PRLr) have been implicated in the pathogenesis of breast cancer. PRL signaling relies on activating kinases such as the tyrosine kinase Jak2 and serine/threonine kinases ERK1/2, Nek3, PI3K, and AKT. In the canonical pathway of PRL signaling, Jak2 phosphorylates the transcription factor Stat5a at tyrosine residue 694 (pY694-Stat5a), preceding Stat5a nuclear translocation and transcriptional activity. However, Stat5a exists with functional duality as a transcription factor, having both pro-differentiative and pro-proliferative target genes. Other Stat family members (Stats 1, 3, and 6) have been shown to have transcriptional activity in the un-phosphorylated (upY) state, distinct from that of pY-Stat activity. This distinction (upY vs. pY) may underlie the duality of Stat5a, coupled with additional regulatory non-canonical post-translational modifications. Within this notion, Stat5a contains two serine residues, S726 and S780, whose phosphorylation are necessary for hematopoietic transformation. However, their functions in PRL-mediated breast cancer pathogenesis have not been examined. We hypothesize that Stat5a serine phosphorylation regulates Stat5a nuclear activity in a non-canonical fashion, contributing to its role in mammary oncogenesis. As shown in a tissue microarray (TMA), human breast cancer tissues express both pS726- and pS780-Stat5a. Nuclear Allred score for pS726-Stat5a increases two-fold with increasing tumor grade, with no difference in staining associated with estrogen or progesterone receptor (ER, PR) status, nor other clinical characteristics. Likewise, patient derived xenograft (PDX) tumors of various molecular subtypes express pS726- and pS780-Stat5a. Phosphorylation of S726-Stat5a is PRL-responsive in vitro. Pharmacologic inhibition of ERK1/2 prevents this phosphorylation, uncovering a novel pathway in which ERK1/2 mediates Stat5a activity in response to PRL in breast cancer. To examine the functional significance of Stat5a serine phosphorylation in vitro, we have performed Stat5a knockdown (KD) in the breast cancer cell line MCF7. Following Stat5a KD, cells were rescued with phospho-site specific Stat5a mutant constructs. Characteristics of breast cancer examined in these mutation-carrying cells, including anchorage-independent growth and proliferation, show distinct phenotypes compared to controls. PRL-induced expression of the CISH gene is significantly decreased up to 65% in the mutation-carrying cells compared to wild type Stat5a. Mechanistic studies will examine the ability of these Stat5a mutants to undergo nuclear translocation and interact with other transcription factors. Collectively, these studies have the potential to provide novel insights into the role of the non-canonical pathway of Stat5a activation in breast cancer pathogenesis.

Epidemiological, cellular, and genetic analyses indicate the hormone prolactin (PRL) and its cognate receptor in humans (hPRLr) are significantly involved in breast cancer pathogenesis. Recent evidence demonstrated that a truncated mouse PRLr (mPRLrT) is oncogenic when expressed with canonical long mPRLr (mPRLrL). mPRLrT shares significant sequence homology with a naturally-occurring and widely-expressed hPRLr isoform, the intermediate hPRLr (hPRLrI). As determined by tissue microarray (TMA), hPRLrI is expressed in >85% of breast cancer, with expression increasing as a function of both tumor grade and Ki67 status. To confirm the oncogenic potential of hPRLrI, isoform-specific hPRLrI knock-down (KD) was performed in breast cancer cell line MCF7. hPRLrI KD resulted in a significant decrease in proliferation, migration, and anchorage-independent growth. Given the homology between mPRLrT and hPRLrI, we hypothesized hPRLrI may similarly induce transformation, when expressed alongside wild-type long hPRLr (hPRLrL). hPRLrL/I co-expression in the immortalized but not transformed human breast cell line MCF10A resulted in a significant increase in proliferation, migration, and anchorage-independent growth. These results were not observed following overexpression of either isoform alone, demonstrating that hPRLrL/I co-expression is necessary to induce transformation of normal mammary epithelia. To test our hypothesis in vivo, we established MCF10A xenografts using female NSG mice. Following intraductal injection, we observed rapid tumor growth in the hPRLrL/I cohort, significantly over that of expressing either isoform alone. To determine mechanisms of transformation, we examined both differential protein stability and altered signaling events. In analyzing receptor degradation, a cycloheximide assay revealed hPRLrL stability is increased when heterodimerized with hPRLrI. hPRLrL turnover is impaired in breast cancer, indicating this phenomenon may be involved in the observed hPRLrI-mediated transformation. Regarding differential signaling, we examined the Jak2/Stat5a pathway. Jak2 is a promiscuous kinase whose oncogenic actions are well-characterized, while Stat5a is a transcription factor whose activities are critical in attenuating the oncogenicity of Jak2. Following PRL stimulation, it was observed that hPRLrL/I co-expression induced approximately two-fold greater Jak2-Y1007/1008 phosphorylation (pJak2) compared to that induced by hPRLrL expression alone. Further, it was observed that hPRLrL/I co-expression induced ten-fold less Stat5a-Y694 phosphorylation (pY-Stat5a) than hPRLrL expression alone. These data indicate unchecked pJak2 activity may also be a contributing mechanism in the observed transformation. Overall, these results demonstrate that hPRLrI, alongside hPRLrL, is sufficient for transformation of normal breast tissue.

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, multi-condition STAT5A, HDAC6, and HMGN2 ChIP-seq 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. Citation Format: Charles V Clevenger, Tia Turner, Chuck Harrell, Justin Craig. Prolactin drives a dynamic STAT5A/HDAC6/HMGN2 cis-regulatory landscape exploitable in ER+ breast cancer [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 P5-04-03.

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.

Stat5 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 target Stat5, despite ample evidence that it contributes to breast cancer pathogenesis. Previous research in our lab has shown that the high mobility group nucleosome binding domain 2 (HGMN2) protein serves as a Stat5 co-activator. The activity of HMGN2 has been previously shown to be regulated by acetylation. Here, we show that deacetylation of HMGN2 on lysine residue K2 by histone deacetylase 6 (HDAC6) promotes Stat5-mediated transcription and breast cancer growth. Conversely, in vitro HDAC6 inhibition by pharmacologic and knock-down approaches enhanced HMGN2 acetylation with a concomitant reduction of in vitro Stat5-mediated signaling and global gene expression, and breast cancer growth. In vitro and in vivo treatment of traditional and patient-derived xenograft models with HDAC6 inhibitors resulted in a highly significant reduction of tumor growth. Translationally, it was also found that high levels of acetylated K2 were present in normal human breast tissue, which was lost in primary breast cancers and lymph node metastases. This suggests that blockade of HMGN2 deacetylation as described above is a novel treatment for breast cancer, given that existing HDAC6 inhibitors have a favorable toxicity profile in Phase I trials of other tumor types. Altogether, these results reveal a novel mechanism through which HDAC6 regulates the transcription of Stat5 target genes and demonstrates the utility of HDAC6 inhibition as a potential breast cancer therapeutic. Citation Format: Clevenger CV, Craig JM, Fiorillo AA, Feeney YB, Harrell JC, Medler TR. HDAC6 deacetylates HMGN2 to regulate Stat5a activity and breast cancer growth [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-06-08.

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.

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.

3576 Background: Studies of immune checkpoint blockade therapy (ICT) outcomes have been largely performed in melanoma and lung cancer patients, both of which are enriched for White patients. For example, a National Cancer Database study found that 97% of first-line ICT treatments in melanoma have been administered to White recipients (Patel, ASCO-SITC 2020). Given expanding indication in tumor types affecting more diverse populations, we sought to study whether minority populations might be projected to have differing checkpoint blockade response rates. Methods: Ethnicity information and RNAseq expression profiles and primary site information were obtained for 7087 patients from TCGA. Ethnicity was tested for association with RNA expression of targetable checkpoint genes ( PD1, PDL1, PDL2, CTLA4, IDO1, LAG3, TIM3, TIGIT, OX40, VISTA, and GITR) in 5 tumor histology types by Wilcoxon methods with Benjamini-Hochberg correction for multiple hypothesis testing. A dataset of > 2700 cases was obtained from NantHealth, with paired whole exome/RNAseq data. Ethnicity for 579 patients was assigned using allele-fraction from ~250 single nucleotide polymorphic sites found exclusively in 6 populations within the 1000 Genomes project. Ethnicity/checkpoint associations found in TCGA were tested within this dataset. Results: Within the TCGA cohort, ethnicity was not a factor in differential expression of checkpoint molecules in lung cancer. Within melanomas, in Asian patients PDL1, CTLA4, and IDO1 were expressed at lower levels than in White patients (each p = 0.04). These associations did not remain significant after correction for multiple hypothesis testing. Breast cancers in Black patients had significantly higher PD1, CTLA4, IDO1, LAG3, GITR, and OX40 expression compared to White patients, all remaining significant after correction (adj. p 3.7e-5 to 6.4e-3). Among White patients, colon cancers showed higher expression of PDL1/2, IDO1, LAG3, TIM3, and GITR (p 0.04 to 0.0017). IDO1 was significantly higher in White patients even after correction (adj. p = 0.03), and lower in Black patients (adj. p = 0.03). Conclusions: Ethnicity may represent a significant factor for efficacy checkpoint blockade therapies. White breast cancer patients might be anticipated to exhibit reduced sensitivity to PD1/CTLA4 blockade, while Black colon cancer patients may exhibit reduced sensitivity to IDO1 therapies such as epacadostat. A biomarker-driven approach to patient selection may ameliorate ethnic disparities in ICT outcomes.

AIMS/HYPOTHESIS: Prolactin, a multifunctional hormone, is involved in regulating insulin sensitivity and glucose homeostasis in experimental studies. However, whether circulating concentrations of prolactin are associated with risk of type 2 diabetes remains uncertain. METHODS: We analysed the prospective relationship between circulating prolactin concentrations and type 2 diabetes risk in the Nurses’ Health Study (NHS) and NHSII with up to 22 years of follow-up. Total plasma prolactin was measured using immunoassay in 8615 women free of type 2 diabetes and cardiovascular disease at baseline blood collection (NHS 1989–1990; NHSII 1996–1999) and a subset of 998 NHS women providing a second blood sample during 2000–2002. Baseline bioactive prolactin was measured in a subset of 2478 women using the Nb2 bioassay. HRs were estimated using Cox regression. RESULTS: A total of 699 incident type 2 diabetes cases were documented during 156,140 person- years of follow-up. Total plasma prolactin levels were inversely associated with type 2 diabetes risk; the multivariable HR comparing the highest with the lowest quartile was 0.73 (95% CI 0.55, 0.95; p(trend)=0.02). The associations were similar by menopausal status and other risk factors (p(interaction)>0.70). Additional adjustment for sex and growth hormones, adiponectin, and inflammatory and insulin markers did not significantly alter the results. The association of plasma bioactive prolactin with type 2 diabetes risk was non-significantly stronger than that of total prolactin (HR comparing extreme quartiles, 0.53 vs 0.81 among the subset of 2478 women, p(difference)=0.11). The inverse association of total prolactin with type 2 diabetes was significant during the first 9 years after blood draw but waned linearly with time, whereas for bioactive prolactin, the inverse relationship persisted for a longer follow-up time after blood draw. CONCLUSIONS/INTERPRETATION: A normally high circulating total prolactin concentration was associated with a lower type 2 diabetes risk within 9–10 years of follow-up since blood draw in US women. Our findings are consistent with experimental evidence, suggesting that among healthy women, prolactin within the biologically normal range may play a protective role in the pathogenesis of type 2 diabetes.

Background: Prolactin, a multifunctional hormone, is involved in regulating insulin sensitivity and glucose homeostasis in experimental and cross-sectional human studies. However, whether circulating levels of prolactin are associated with risk of type 2 diabetes (T2D) remains unclear. Methods: We analyzed the prospective relationship between circulating prolactin levels and T2D risk in in the Nurses’ Health Study (NHS) and Nurses’ Health Study II (NHSII) with up to 22 years of follow-up. Total plasma prolactin was measured using immunoassay in 8,637 women free of T2D and cardiovascular disease at baseline blood collection (NHSI: 1989-1990; NHSII: 1996-1999) and a subset of 1,017 NHS women who provided a second blood sample in 2000-2002. In addition, baseline bioactive prolactin levels were measured in a subset of 2,658 women using the Nb2 lymphoma cell bioassay. We calculated hazard ratios (HRs) using Cox regressions. Results: A total of 701 incident T2D cases were documented during 159,517 person-years of follow-up. Circulating prolactin levels were inversely associated with T2D risk; the multivariable HR comparing the highest with the lowest quartile of prolactin levels was 0.71 (95% confidence interval, 0.54-0.93; P trend =0.02). The associations were similar by menopausal status and risk factors ( P >0.70 for interaction). Additional adjustment for sex hormones, growth factors, adipokine, and inflammatory markers did not alter the results. The association of bioactive prolactin with T2D risk was suggestively stronger than that of total prolactin (comparable HR=0.56 vs. 0.82 among the 2,658 women). The inverse association of total plasma prolactin with incident T2D waned linearly with time, with a significant association observed during the first 9 years after blood draw. Conclusion: High circulating prolactin level was independently associated with lower risk of T2D in women. Our findings are consistent with experimental evidence, supporting a possible protective role for prolactin in the pathogenesis of T2D.

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.

The prolactin (PRL) receptor is over-expressed in breast cancer, and pre-clinical data indicate that it contributes to breast oncogenesis. Cabergoline is a potent dopamine receptor agonist of D2 receptors and has a direct inhibitory effect on pituitary PRL secretion. A phase II study of cabergoline in patients with metastatic breast cancer was conducted. The primary end point of the study was to determine the clinical benefit rate (CBR) at 2 months. Eligible patients had tumors of any receptor status with no limit of prior lines of therapy. Measurable and unmeasurable diseases were allowed. Cabergoline 1 mg orally, twice weekly (1 cycle = 4 weeks) was given until disease progression or unacceptable toxicity. PRL receptor immunohistochemical staining was performed on available baseline tumor tissue; serial serum PRL levels were assessed. Twenty women were enrolled; 18 were evaluable for CBR. Tumor receptor status was distributed as follows: HR−any/HER2+ 2(10%), HR+/HER2− 18 (90%). The CBR was 33% (6/18), median progression free survival was 1.8 months, and median overall survival was 10.4 months. Two patients experienced disease control for over 12 months. Most common treatment-related adverse events were nausea (30%), fatigue (25%), and elevation in alkaline phosphatase (15%). Nine patients had baseline tissue for analysis; there was no association between baseline tumor PRL receptor expression and clinical benefit (p = 0.24). Change in serum PRL level and response were not correlated after 2 months of treatment (p = 0.64). Cabergoline was well tolerated, and while the ORR was low, a small subset of patients experienced extended disease control.

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.

Prolactin (PRL) and its receptor (PRLr) have been implicated in the development and progression of human breast cancer. PRL activates its receptor and induces activation of proximal Janus kinase 2 (Jak2). Jak2 associates with PRLr, phosphorylates c-terminus of the PRLr, which leads to Stat5 recruitment and activation. Cyclophilin A (CypA) is a peptidyl-prolyl isomerase (PPI), which is constitutively bound to the PRLr that catalyze the cis-trans interconversion of proline imide bonds. Treatment with Cyclosporine (CsA) inhibited CypA binding to the PRLr and blocked PRLr-driven activation of Jak2/Stat5. Recently, Waters et al., utilizing Fluorescence Resonance Energy Transfer (FRET) with transfectants expressing CFP and YFP-tagged forms of the growth hormone receptor (GHR) showed that GHR activation induced a rotational movement in C-terminus of the GHR, resulting in a loss of baseline FRET signal. Like the GHR, PRL stimulation of transfectants expressing CFP/YFP-tagged PRLr constructs resulted in a loss of FRET efficiency. In contrast, treatment with NIM811 (a non-immunosuppressive form of CsA) or siRNA knockdown of CypA resulted in a return of FRET signal in the presence of PRL. These studies reveal that ligand stimulation of the PRLr results in a conformational change as measured by FRET signal of the receptor that is reversed by CypA inhibition or knockdown, implicating CypA as the mediator of this conformational change and ligand-induced signaling. To further assess the consequences of CypA inhibition or knockdown on the PRLr/Jak2 mediated signaling/functions, analyses of phospho-tyrosine residues that are believed to be important for interactions/signaling were investigated in breast cancer cells. It was found that NIM811 inhibition or CypA shRNA knockdown significantly reduced prolactin-stimulated phosphorylation of PRLr/Jak2 intermediates in ER+/PR+ T47D cells in a time dependent manner. A microarray analysis revealed that NIM811 inhibited approximately 66% of the top 50 PRL induced genes. NIM811 inhibited ER-/+, and HER2+ breast cancer cell proliferation, survival, migration and anchorage-independent growth. Subsequent pre-clinical testing of NIM811 in relevant mouse mammary cancer models has found that NIM811 treatment of a TNBC xenograft inhibited primary tumor growth, outgrowth of macro-metastasis and induced central tumor necrosis. Furthermore, loss of CypA (by constitutive genetic deletion) in the MMTV-PyMT mouse model demonstrated inhibition of tumorigenesis with significant reduction in lung and lymph node metastasis. Overall, CypA modulates conformational change in the C-terminus of the PRLr through its PPI activity, and alters PRLr/Jak2 complex signaling/functions in breast cancer and mammary epithelium, identifying this isomerase as a novel target for therapeutic intervention as a chemo-preventive and as an inhibitor of metastasis. Citation Format: Shawn Hakim, Shannon E. Hedrick, Justin M. Craig, Charles V. Clevenger. Cyclophilin A as a molecular switch regulating PRLr-Jak2 complex-mediated signaling, mammary tumorigenesis, and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4508.

Epidemiological, cellular, and genetic analyses indicate the hormone prolactin (PRL) and its cognate receptor in humans (hPRLr) are significantly involved in breast cancer pathogenesis. Recent evidence demonstrated that a truncated mouse PRLr (mPRLrT) is oncogenic when expressed alongside its canonical long form counterpart (mPRLrL) [Cell Reports 17, 249-260]. The mPRLrT shares significant sequence homology with a naturally-occurring and widely-expressed hPRLr splice variant, the intermediate hPRLr (hPRLrI) isoform. Given this similarity, we hypothesized hPRLrI may also induce transformation, when expressed alongside wild-type long hPRLr (hPRLrL). Like the mPRLrT, hPRLrI co-expression with hPRLrL in the immortalized but not transformed human breast cell line MCF10A resulted in a significant increase in proliferation, migration, viability, and anchorage-independent growth. These results were not observed following overexpression of either isoform alone, demonstrating that hPRLrL and hPRLrI co-expression is necessary to induce transformation of normal mammary epithelia. To further characterize this transformation, we established MCF10A xenografts using female NOD scid gamma (NSG) mice. Following intraductal injection, we observed rapid tumor growth in the hPRLrL/I co-expression cohort, significantly over that of the cohorts harboring either isoform alone, validating our in vitro findings in vivo. To determine mechanisms of transformation, we examined both differential protein stability and altered signaling events. In analyzing receptor degradation, a cycloheximide assay revealed hPRLrL stability is increased when heterodimerized with hPRLrI. hPRLrL turnover has been reported to be impaired in human breast cancer, indicating this phenomenon may be involved in the observed hPRLrI-mediated transformation. In regards to differential signaling, we examined the Jak2/Stat5a pathway. Jak2 is a promiscuous kinase whose significant oncogenic actions are well-characterized, while Stat5a is a transcription factor whose activities are critical in attenuating the actions of Jak2. Following PRL stimulation, it was observed that hPRLrL/I co-expression induced approximately two-fold greater Jak2-Y1007/1008 phosphorylation (pJak2) compared to that induced by hPRLrL expression alone. Further, it was observed that hPRLrL/I co-expression induced ten-fold less Stat5a-Y694 phosphorylation (pY-Stat5a) than hPRLrL expression alone. These data indicate unchecked pJak2 activity may also be a contributing mechanism in the observed transformation. Overall, these results demonstrate that hPRLrI, alongside hPRLrL, is sufficient for transformation of normal breast tissue. Citation Format: Jacqueline M. Grible, Charles V. Clevenger. The role of the intermediate prolactin receptor in breast cancer oncogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 870.

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.

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.

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.

The multidrug transporter, breast cancer resistance protein, ABCG2, is up-regulated in certain chemoresistant cancer cells and in the mammary gland during lactation. We investigated the role of the lactogenic hormone prolactin (PRL) in the regulation of ABCG2. PRL dose-dependently induced ABCG2 expression in T-47D human breast cancer cells. This induction was significantly reduced by short-interfering RNA-mediated knockdown of Janus kinase 2 (JAK2). Knockdown or pharmacologic inhibition of the down-stream signal transducer and activator of transcription-5 (STAT5) also blunted the induction of ABCG2 by PRL, suggesting a role for the JAK2/STAT5 pathway in PRL-induced ABCG2 expression. Corroborating these findings, we observed PRL-stimulated STAT5 recruitment to a region containing a putative γ-interferon activation sequence (GAS) element at -434 base pairs upstream of the ABCG2 transcription start site. Introduction of a single mutation to the -434 GAS element significantly attenuated PRL-stimulated activity of a luciferase reporter driven by the ABCG2 gene promoter and 5'-flanking region containing the -434 GAS motif. In addition, this GAS element showed strong copy number dependency in its response to PRL treatment. Interestingly, inhibitors against the mitogen-activated protein kinase and phosphoinositide-3-kinase signaling pathways significantly decreased the induction of ABCG2 by PRL without altering STAT5 recruitment to the GAS element. We conclude that the JAK2/STAT5 pathway is required but not sufficient for the induction of ABCG2 by PRL.

Aromatase is essential for estrogen production and is the target of aromatase inhibitors, the most effective endocrine treatment for postmenopausal breast cancer. Peripheral tissues in women, including the breast, express aromatase via alternative promoters. Female mice lack the promoters that drive aromatase expression in peripheral tissues; thus, we generated a transgenic humanized aromatase (Aromhum) mouse line containing a single copy of the human aromatase gene to study the link between aromatase expression in mammary adipose tissue and breast pathology. Aromhum mice expressed human aromatase, driven by the proximal human promoters II and I.3 and the distal promoter I.4, in breast adipose fibroblasts and myoepithelial cells. Estrogen levels in the breast tissue of Aromhum mice were higher than in wild-type mice, whereas circulating levels were similar. Aromhum mice exhibited accelerated mammary duct elongation at puberty and an increased incidence of lobuloalveolar breast hyperplasia associated with increased signal transducer and activator of transcription-5 phosphorylation at 24 and 64 wk. Hyperplastic epithelial cells showed remarkably increased proliferative activity. Thus, we demonstrated that the human aromatase gene can be expressed via its native promoters in a wide variety of mouse tissues and in a distribution pattern nearly identical to that of humans. Locally increased tissue levels, but not circulating levels, of estrogen appeared to exert hyperplastic effects on the mammary gland. This novel mouse model will be valuable for developing tissue-specific aromatase inhibition strategies.

The molecular mechanisms that modulate the activity of the signal transducers and activators of transcription 5 (Stat5) during the progression of breast cancer remain elusive. Here, we present evidence that the calcineurin/nuclear factor of activated T cells (NFAT) pathway negatively regulates the activation of Stat5, and vice versa in breast cancer. NFAT1 interacts with Stat5 in breast cancer cells, and their physical association is mediated by the DNA binding and transactivation domains of Stat5. Ectopically expressed NFAT1 is capable of inhibiting Stat5-dependent functions, including Stat5 transactivation, Stat5-mediated transcription of the downstream target gene expression, and binding of Stat5a to the Stat5 target promoter. By contrast, overexpression of a selective NFAT inhibitor VIVIT reversed NFAT1-mediated suppression of Stat5-dependent gene expression, whereas silencing of NFAT1 through RNA interference enhanced prolactin-induced, Stat5-mediated gene transcription, and breast cancer cell proliferation. A reciprocal inhibitory effect of Stat5 activity on NFAT1 signaling was also observed, implying these two signaling cascades antagonize each other in breast cancer. Importantly, analysis of a matched breast cancer progression tissue microarray revealed a negative correlation between levels of NFAT1 and Stat5 (pY694) during the progression of breast cancer. Taken together, these studies highlight a novel negative cross talk between the NFAT1- and Stat5-signaling cascades that may affect breast tumor formation, growth, and metastasis.

The direct actions of transmembrane receptors within the nucleus remain enigmatic. In this report, we demonstrate that the prolactin receptor (PRLr) localizes to the nucleus where it functions as a coactivator through its interactions with the latent transcription factor signal transducer and activator of transcription 5a (Stat5a) and the high-mobility group N2 protein (HMGN2). We identify a novel transactivation domain within the PRLr that is activated by ligand-induced phosphorylation, an event coupled to HMGN2 binding. The association of the PRLr with HMGN2 enables Stat5a-responsive promoter binding, thus facilitating transcriptional activation and promoting anchorage-independent growth. We propose that HMGN2 serves as a critical regulatory factor in Stat5a-driven gene expression by facilitating the assembly of PRLr/Stat5a onto chromatin and that these events may serve to promote biological events that contribute to a tumorigenic phenotype. Our data imply that phosphorylation may be the molecular switch that activates a cell surface receptor transactivation domain, enabling it to tether chromatin-modifying factors, such as HMGN2, to target promoter regions in a sequence-specific manner.

Cyclophilin A (CypA) is a member of the immunophilin family of peptidyl prolyl isomerases (PPI) that catalyze the cis-trans interconversion of proline imide bonds of peptides. In this role, cyclophilins have been found to function as signaling switches, regulating the activity of receptors, kinases and transcription factors. The PPI activity of CypA is inhibited by the immunosuppressive drug cyclosporine A (CsA), and in turn the CypA-CsA complex inhibits calcineurin-mediated NFAT activation. Our laboratory has recently demonstrated that CypA is necessary for the prolactin (PRL)-induced activation of Jak2/Stat5 signaling and gene expression (Cancer Res 68:7769, 2008). In addition, we have also shown that CsA inhibits the in vitro and in vivo growth and progression of both ER+ and ER-breast cancer cell lines. Here, we show that the non-immunosuppressive CsA analog NIM811 blocked PRL-stimulated activation of the Jak2/Stat5 pathway, and the kinases AKT and MAPK in T47D cells. NIM811 also inhibited ER+, ER-, and Her2+ breast cancer cell proliferation, survival, motility and anchorage independent growth in a dose-dependent manner. NIM811 inhibited PRL induced gene expression, such as CISH and Cyclin D1. NIM811 also blocked the PRL-induced association between Stat5 and cMyb, and at higher doses, triggered PARP cleavage. In summary, these results indicate that non-immunosuppressive cyclophilin inhibitor NIM811 has significant potential as a novel for breast cancer therapeutic. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-19-04.

The hormone prolactin (PRL) contributes to the pathogenesis of breast cancer in part through its activation of Janus-activated kinase 2 (Jak2)/signal transducer and activator of transcription 5 (Stat5), a PRL receptor (PRLr)–associated pathway dependent on cross-talk signaling from integrins. It remains unclear, however, how this cross-talk is mediated. Following PRL stimulation, we show that a complex between the transmembrane glycoprotein signal regulatory protein-α (SIRPα) and the PRLr, β1 integrin, and Jak2 in estrogen receptor–positive (ER+) and ER− breast cancer cells is formed. Overexpression of SIRPα in the absence of collagen 1 significantly decreased PRL-induced gene expression, phosphorylation of PRLr-associated signaling proteins, and PRL-stimulated proliferation and soft agar colony formation. In contrast, overexpression of SIRPα in the presence of collagen 1 increased PRL-induced gene expression; phosphorylation of Jak2, Stat5, and Erk; and PRL-stimulated cell growth. Interestingly, overexpression of a tyrosine-deficient SIRPα (SIRPα-4YF) prevented the signaling and phenotypic effects mediated by wild-type SIRPα. Furthermore, overexpression of a phosphatase-defective mutant of Shp-2 or pharmacologic inhibition of Shp-2 produced effects comparable with that of SIRPα-4YF. However, the tyrosine phosphorylation of SIRPα was unaffected in the presence or absence of collagen 1. These data suggest that SIRPα modulates PRLr-associated signaling as a function of integrin occupancy predominantly through the alteration of Shp-2 activity. This PRLr-SIRPα-integrin complex may therefore provide a basis for integrin-PRLr cross-talk and contribute to the biology of breast cancer. Mol Cancer Res; 8(10); 1413–24. ©2010 AACR.

Background: Temporal variability of biomarkers should be evaluated before their use in epidemiologic studies. Methods: We evaluated the reproducibility, using intraclass correlation coefficients (ICC), of 27 plasma, 50 red blood cell, and 9 urinary biomarkers over 1 to 3 years among premenopausal (n = 40) and postmenopausal (n = 35-70) participants from the Nurses' Health Study and Nurses' Health Study II. Results: Plasma and urinary stress hormones and melatonin were measured among premenopausal women, whereas melatonin and the remaining biomarkers were measured in postmenopausal women. ICCs were good to excellent for plasma carotenoids (0.73-0.88), vitamin D analytes (0.56-0.72), bioactive somatolactogens (0.62), soluble leptin receptor (0.82), resistin (0.74), and postmenopausal melatonin (0.63). Reproducibility was lower for some of the red blood cell fatty acids (0.38-0.72), plasma matrix metalloproteinases (0.07-0.91), and premenopausal melatonin (0.44). The ICCs for plasma and urinary phytoestrogens were poor (≤0.09) except for enterolactone (plasma, 0.44; urinary, 0.52). ICCs for the stress hormones among premenopausal women ranged from 0 (plasma cortisol) to 0.45 (urinary dopamine). Conclusions: Our results indicate that for the majority of these markers, a single measurement can reliably estimate average levels over a 1- to 3-year period in epidemiologic studies. For analytes with fair to good ICCs, reproducibility data can be used for measurement error correction. Analytes with poor ICCs should only be used in settings with multiple samples per subject or in populations in which ICCs are higher. Impact: This article summarizes the feasibility of the use of >80 biomarkers in epidemiologic studies in which only one biospecimen is available to represent longer term exposure. Cancer Epidemiol Biomarkers Prev; 19(4); 938–46. ©2010 AACR.

Academic pathologists perform clinical duties, as well as valuable nonclinical activities. Nonclinical activities may consist of research, teaching, and administrative management among many other important tasks. While clinical duties have many clear metrics to measure productivity, like the relative value units of Medicare reimbursement, nonclinical performance is often difficult to measure. Despite the difficulty of evaluating nonclinical activities, nonclinical productivity is used to determine promotion, funding, and inform professional evaluations of performance. In order to better evaluate the important nonclinical performance of academic pathologists, we present an evaluation system for leadership use. This system uses a Microsoft Excel workbook to provide academic pathologist respondents and reviewing leadership a transparent, easy-to-complete system that is both flexible and scalable. This system provides real-time feedback to academic pathologist respondents and a clear executive summary that allows for focused guidance of the respondent. This system may be adapted to fit practices of varying size, measure performance differently based on years of experience, and can work with many different institutional values.

Signal transducer and activator of transcription 3 (Stat3) is the major mediator of interleukin-6 (IL-6) family cytokines. In addition, Stat3 is known to be involved in the pathophysiology of many malignancies. Here, we show that the cis-trans peptidyl-prolyl isomerase cyclophilin (Cyp) B specifically interacts with Stat3, whereas the highly related CypA does not. CypB knockdown inhibited the IL-6-induced transactivation potential but not the tyrosine phosphorylation of Stat3. Binding of CypB to Stat3 target promoters and alteration of the intranuclear localization of Stat3 on CypB depletion suggested a nuclear function of Stat3/CypB interaction. By contrast, CypA knockdown inhibited Stat3 IL-6-induced tyrosine phosphorylation and nuclear translocation. The Cyp inhibitor cyclosporine A (CsA) caused similar effects. However, Stat1 activation in response to IL-6 or interferon-gamma was not affected by Cyp silencing or CsA treatment. As a result, Cyp knockdown shifted IL-6 signaling to a Stat1-dominated pathway. Furthermore, Cyp depletion or treatment with CsA induced apoptosis in IL-6-dependent multiple myeloma cells, whereas an IL-6-independent line was not affected. Thus, Cyps support the anti-apoptotic action of Stat3. Taken together, CypA and CypB both play pivotal roles, yet at different signaling levels, for Stat3 activation and function. These data also suggest a novel mechanism of CsA action.

Cyclophilin B (CypB) is a 21-kDa protein with peptidyl-prolyl cis-trans isomerase activity that functions as a transcriptional inducer for Stat5 and as a ligand for CD147. To better understand the global function of CypB in breast cancer, T47D cells with a small interfering RNA-mediated knockdown of CypB were generated. Subsequent expression profiling analysis showed that 663 transcripts were regulated by CypB knockdown, and that many of these gene products contributed to cell proliferation, cell motility, and tumorigenesis. Real-time PCR confirmed that STMN3, S100A4, S100A6, c-Myb, estrogen receptor alpha, growth hormone receptor, and progesterone receptor were all down-regulated in si-CypB cells. A linkage analysis of these array data to protein networks resulted in the identification of 27 different protein networks that were impacted by CypB knockdown. Functional assays demonstrated that CypB knockdown also decreased cell growth, proliferation, and motility. Immunohistochemical and immunofluorescent analyses of a matched breast cancer progression tissue microarray that was labeled with an anti-CypB antibody demonstrated a highly significant increase in CypB protein levels as a function of breast cancer progression. Taken together, these results suggest that the enhanced expression of CypB in malignant breast epithelium may contribute to the pathogenesis of this disease through its regulation of the expression of hormone receptors and gene products that are involved in cell proliferation and motility.

Bone marrow-derived mesenchymal stromal cells (MSCs) localize to solid tumors. Defining the signaling mechanisms that regulate this process is important in understanding the role of MSCs in tumor growth. Using a combination of chromatography and electrospray tandem mass spectrometry we have identified novel soluble signaling molecules that induce MSC chemotaxis present in conditioned medium of the breast carcinoma cell line MDA-MB231. Previous work has employed survey strategies using ELISA assay to identify known chemokines that promote MSC chemotaxis. While these studies provide valuable insights into the intercellular signals that impact MSC behavior, many less well-described, but potentially important soluble signaling molecules could be overlooked using these methods. Through the less directed method of column chromatography we have identified novel candidate MSC chemotactic peptides. Two proteins, cyclophilin B and hepatoma-derived growth factor were then further characterized and shown to promote MSC chemotaxis.

Prolactin receptor signaling can modulate proliferation, survival, motility, angiogenesis, and differentiation in breast cancer. Increased serum prolactin is associated with a significantly increased risk of breast cancer in post-menopausal women. The purpose of this study was to examine the expression of prolactin receptor-associated signaling proteins in breast cancer vs benign breast tissue. Breast tissue microarrays representing 40 cases of benign and malignant pathologies were obtained from the Cooperative Human Tissue Network. Standard immunohistochemistry for prolactin and prolactin receptor-associated proteins was performed. Both positive regulators (c-Myb, Nek3, Vav2) and negative regulators (PIAS3, SIRP) of prolactin receptor signaling were examined. Virtual slides were created from the stained breast tissue microarrays. Labels were scored by region of interest and labeling indices incorporating percent target labeled and label intensity were created. Quantitative determinations of labels were made using the Clarient image system. The unpaired t-test was used to compare labels from benign and malignant tissues. Visual scoring data showed upregulation of Nek3 (P=0.000377), PIAS3 (P=0.000257), and prolactin (P=0.002576) in breast cancer vs normal/hyperplastic epithelium. c-Myb showed a trend toward upregulation, but this did not achieve statistical significance (P=0.107374). SIRP (P=0.002060) was downregulated. Vav2 showed a trend toward downregulation (P=0.107456), but this did not achieve statistical significance. Clarient analysis corroborated upregulation in cancer of Nek3 (P=0.000013), PIAS3 (P=0.000067), and prolactin (P=0.017569). In conclusion, regulators of prolactin receptor signaling show heterogeneity in their expression in benign vs malignant breast tissue. Since these species are known to regulate prolactin-mediated actions, these results suggest multiple targets for modulating prolactin receptor-mediated growth and differentiation in breast cancer.

Prolactin (PRL) activates its receptor to initiate signal transduction pathways (including activation of Janus kinases, Jak) but also stimulates downregulation of this receptor to limit the magnitude and duration of signaling. Degradation of the long form of PRL receptor (PRLr) depends on its phosphorylation on Ser349 that is required to facilitate PRLr ubiquitination. Signaling events that mediate PRL-induced degradation of PRLr remain to be elucidated. Here, we investigated the role of Jak2 activity in ligand-triggered increase of PRLr phosphorylation on Ser349, PRLr ubiquitination, endocytosis, and degradation. Using Jak2 reconstitution in Jak2-null cells as well as pharmacologic approaches, we found that treatment with PRL (but not with PRLr antagonist) promotes phosphorylation of PRLr on Ser349 and accelerates endocytosis of PRLr. Furthermore, PRL-stimulated PRLr phosphorylation, endocytosis, and degradation in Jak2-null cells reconstituted with wild type but not with catalytically inactive Jak2. We discuss how Jak2-mediated signaling might be transduced into Ser349 phosphorylation of PRLr as well as its ubiquitination and endocytosis.

Prolactin (PRL) stimulates the cytoskeletal re-organization and motility of breast cancer cells. During PRL receptor signaling, Vav2 becomes phosphorylated and activated, an event regulated by the serine/threonine kinase Nek3. Given the regulatory role of Vav2, the function of Nek3 in PRL-mediated motility and invasion was examined. Overexpression of Nek3 in Chinese hamster ovary transfectants potentiated cytoskeletal re-organization in response to PRL. In contrast, downregulation of Nek3 expression by small-interfering RNA (siRNA) attenuated PRL-mediated cytoskeletal reorganization, activation of GTPase Rac1, cell migration and invasion of T47D cells. In addition, PRL stimulation induced an interaction between Nek3 and paxillin and significantly increased paxillin serine phosphorylation, whereas Nek3 siRNA-transfected cells showed a marked reduction in paxillin phosphorylation. Analysis of breast tissue microarrays also demonstrated a significant up-regulation of Nek3 expression in malignant versus normal specimens. These data suggest that Nek3 contributes to PRL-mediated breast cancer motility through mechanisms involving Rac1 activation and paxillin phosphorylation.