Global profiling of prolactin-modulated transcripts in breast cancer in vivo

  • Takahiro Sato1,

    Affiliated with

    • Thai H Tran1,

      Affiliated with

      • Amy R Peck1,

        Affiliated with

        • Chengbao Liu1,

          Affiliated with

          • Adam Ertel1,

            Affiliated with

            • Justin Lin1,

              Affiliated with

              • Lynn M Neilson1 and

                Affiliated with

                • Hallgeir Rui1Email author

                  Affiliated with

                  Molecular Cancer201312:59

                  DOI: 10.1186/1476-4598-12-59

                  Received: 21 February 2013

                  Accepted: 15 May 2013

                  Published: 12 June 2013

                  Abstract

                  Background

                  Prolactin (PRL) is essential for normal mammary gland development. PRL promotes mammary tumor formation in rodents and elevated serum prolactin is associated with increased risk of estrogen-receptor positive breast cancer in women. On the other hand, PRL may also exert pro-differentiation effects and act to suppress invasive features of established breast cancer. Previously published limited global transcript profiling analyses of prolactin-regulated gene expression in human breast cancer cells have exclusively been performed in vitro. The present study aimed to shed new light on how PRL modulates estrogen receptor (ER)-positive breast cancer through global transcript profiling of a human breast cancer xenograft model in vivo.

                  Methods

                  The prolactin-responsive human T47D breast cancer cell line was xenotransplanted into nude mice and global transcript profiling was carried out following treatment with or without human PRL for 48 h. A subset of PRL-modulated transcripts was further validated using qRT-PCR and immunohistochemistry.

                  Results

                  The in vivo analyses identified 130 PRL-modulated transcripts, 75 upregulated and 55 downregulated, based on fold change >1.6 and P-value <0.05. From this initial panel of transcripts, a subset of 18 transcripts with established breast cancer-relevance were selected and validated by qRT-PCR. Some but not all of the transcripts were also PRL-modulated in vitro. The selected PRL-modulated transcripts were tested for dependence on Stat5, Jak1 or Jak2 activation, and for co-regulation by 17β-estradiol (E2). The protein encoded by one of the PRL-regulated transcripts, PTHrP, was examined in a panel of 92 human breast cancers and found by in situ quantitative immunofluorescence analysis to be highly positively correlated with nuclear localized and tyrosine phosphorylated Stat5. Gene Ontology analysis revealed that PRL-upregulated genes were enriched in pathways involved in differentiation. Finally, a gene signature based on PRL-upregulated genes was associated with prolonged relapse-free and metastasis-free survival in breast cancer patients.

                  Conclusions

                  This global analysis identified and validated a panel of PRL-modulated transcripts in an ER-positive human breast cancer xenotransplant model, which may have value as markers of relapse-free and metastasis-free survival. Gene products identified in the present study may facilitate ongoing deciphering of the pleiotropic effects of PRL on human breast cancer.

                  Keywords

                  Breast cancer Prolactin Stat5

                  Background

                  Prolactin (PRL) is a pituitary hormone that is critical for normal mammary gland development by promoting proliferative expansion of the secretory alveolar cell compartment during pregnancy and for terminal differentiation of these milk-producing cells during lactation. Prolactin is also strongly implicated in breast cancer. On one hand, accumulating evidence suggests that PRL promotes breast cancer initiation and growth. In vivo, PRL over-expressing transgenic mice have an increased incidence of mammary tumors [1], while PRL knock-out mice have a reduced incidence of mammary tumors [2]. In women, elevated PRL is associated with increased risk of ER-positive breast cancer [3, 4]. Furthermore, 70-95% of human breast cancers express PRL receptor (PRLR) [5, 6], and many breast cancer cell lines express high levels of PRLR with evidence of proliferative or survival responses to PRL in vitro[7, 8]. PRL was also able to enhance 17β-Estradiol (E2) dependent proliferation of breast cancer cells [911]. On the other hand, evidence suggests that PRL acts to preserve cellular differentiation of breast cancer. Stat5 transcription factors, principal mediators of PRL effects [12, 13], are frequently inactivated during metastatic progression in clinical breast cancer specimens, and loss of Stat5 signaling is associated with unfavorable prognosis and increased risk of anti-estrogen therapy failure [1417]. In experimental breast cancer models, activation of Stat5 increased cell surface E-cadherin expression, induced homotypic cell clustering, and reduced invasion through Matrigel [18, 19]. Restoration of PRL-Stat5 signaling in the mesenchymal-like MDA-MB-231 cells reverted their invasive phenotype, while blocking autocrine PRL signaling in the epithelial T47D cell line was associated with EMT and enhanced invasive properties [20]. In T47D cells, PRL also blocked progestin-induction of a tumor-initiating CK5-positive cell population through a mechanism that involved PRL-suppression of progestin-induced BCL6 [21].

                  Due to the importance of PRL in breast cancer growth and differentiation, identifying genes regulated by the PRL-Stat5 pathway may provide new insights into the pleiotropic effects of PRL in breast cancer. Several studies have identified genes regulated by PRL in the normal mouse mammary gland [2226], but only a limited number of studies have been carried out in human breast cancer cells. More importantly, global profiling for PRL-modulated gene expression in human breast cancer in vivo has not been reported. One in vitro study identified genes regulated by PRL, E2, and PRL + E2 in cultured ER-positive T47D cells using genome-wide transcript profiling [9], while a second in vitro study used subtractive hybridization to identify PRL-regulated genes in the ER-negative, Her2-overexpressing SKBR3 cell line [27]. However, neither study confirmed whether the identified transcripts were regulated by Stat5 or remained PRL-modulated in vivo. Work from our laboratory used selective overexpression of either Stat5a or Stat5b in the ER-positive MCF-7 human breast cancer cell line in vitro followed by PRL exposure to explore differences in Stat5a and Stat5b regulated transcripts on the Affymetrix platform, but this study also failed to confirm any of the modulated transcripts by qRT-PCR or validate the data in vivo[17]. An additional very recent study also examined the MCF-7 model and reported PRL regulated genes, as well as genes uniquely modulated through activation of the PRLR transactivation domain [28], but the investigators did not examine whether the identified transcripts were regulated by Stat5 or remained PRL-modulated in vivo.

                  Based on observed differences in hormone-modulated transcriptional programs in human cancer cells in vitro and in vivo[29], PRL-modified transcripts identified in an in vivo environment are expected to be more clinically relevant than transcripts modulated in cells cultured on plastic. The present study reports a panel of 130 PRL regulated transcripts in the human T47D breast cancer xenotransplant model in estrogenized nude mice. T47D xenografts were established in nude mice and mice were exposed to human PRL or saline for 48 h before RNA isolation from tumor extracts. Of the 130 transcripts, 75 were up-regulated and 55 were down-regulated. Modulated transcripts were identified based on fold change (>1.6) and P-value (<0.05). From this initial transcript set, 18 transcripts were selected based on known breast cancer relevance for validation by qRT-PCR. Many but not all of the in vivo validated transcripts were PRL-modulated in vitro. We also determined the dependence of PRL-modulated transcripts on Stat5, Jak1 or Jak2, and whether individual modulated genes are co-regulated by PRL and E2. One of the PRL-modulated genes identified, parathyroid hormone-related peptide (PTHrP), was found by fluorescence-based quantitative immunohistochemistry to positively correlate with levels of nuclear localized, tyrosine phosphorylated Stat5 (Nuc-pYStat5) at the protein level in clinical human breast cancer specimens. Gene Ontology (GO) analysis of PRL-upregulated genes demonstrated enrichment in differentiation pathways. Finally, a gene signature based on PRL-upregulated genes was associated with prolonged relapse-free and metastasis-free survival in human breast cancer patients. Studies are ongoing to determine how modulation of these genes, including PTHrP, may mediate PRL effects in breast cancer.

                  Results

                  Global transcript profiling reveals novel PRL-modulated genes in human T47D breast cancer xenotransplants

                  Global gene expression analysis was performed using the hormone receptor positive T47D xenotransplant model. Tumor-bearing nude mice received either human prolactin (N = 10) or vehicle (N = 10) subcutaneously every 12 h for 48 h. Representative images of T47D xenograft tissues display robust tyrosine phosphorylated Stat5 (pY-Stat5) staining in response to PRL (Figure 1A). For the microarray analyses, RNA isolated from individual tumors was pooled into 3 groups from PRL injected mice and 3 groups from control mice, with each group containing RNA from tumors of 3 or 4 mice. Global profiling on the Affymetrix U133 platform identified 75 upregulated transcripts (Table 1) and 55 down-regulated transcripts (Table 2) based on P-values (<0.05) and fold difference (>1.6). From this panel, 18 transcripts were selected for further analysis based on established breast cancer relevance (Table 3). CISH was induced by PRL and included in subsequent analyses as a positive control, since CISH is an established STAT5 target gene [30]. Based on the microarray data of this panel of 18 transcripts, 15 were upregulated and three were down-regulated 1.6-fold.
                  http://static-content.springer.com/image/art%3A10.1186%2F1476-4598-12-59/MediaObjects/12943_2013_Article_1126_Fig1_HTML.jpg
                  Figure 1

                  qRT-PCR validation of 18 candidate prolactin modulated transcripts. (A) Representative images of T47D xenografts treated with or without PRL for 48 h and stained with tyrosine phosphorylated Stat5 (pY-Stat5)(red) or DAPI (blue) using immunofluorescence. (B) RNA isolated from T47D xenografts were tested for 18 breast cancer relevant genes using qRT-PCR analysis. Bars represent an average of fold change from 3 independent xenograft RNA samples. (C) Immunoblot of pY-Stat5 and Stat5 in T47D cells grown in vitro and placed in serum starvation media for 24 h before treatment with vehicle or 10 nM PRL for 24 h. (D) RNA isolated from T47D cells grown in vitro were tested for the same 18 breast cancer relevant genes using qRT-PCR analysis. Bars represent an average of fold change from 3 independent experiments.

                  Table 1

                  Upregulated genes from microarray with fold change >1.6 and p < 0.05

                   

                  Unigene

                  Symbol

                  Gene descriptor

                  Fold change

                  1

                  Hs.89626

                  PTHrP

                  parathyroid hormone-like hormone

                  11.9

                  2

                  Hs.473539

                  BACH1

                  BTB and CNC homology 1, basic leucine zipper transcription factor 1

                  5.2

                  3

                  Hs.46468

                  CCR6

                  chemokine (C-C motif) receptor 6

                  4.9

                  4

                  Hs.314676

                  ITCH

                  itchy homolog E3 ubiquitin protein ligase

                  4.2

                  5

                  Hs.150744

                  INVS

                  inversin

                  3.8

                  6

                  Hs.387222

                  NEK6

                  NIMA (never in mitosis gene a)-related kinase 6

                  3.8

                  7

                  Hs.76095

                  IER3

                  immediate early response 3

                  3.4

                  8

                  Hs.121520

                  AMIGO2

                  amphoterin induced gene 2

                  3.3

                  9

                  Hs.115263

                  EREG

                  epiregulin

                  3.2

                  10

                  Hs.436186

                  ERAP1

                  type 1 tumor necrosis factor receptor shedding aminopeptidase regulator

                  3.0

                  11

                  Hs.99037

                  CTEN

                  C-terminal tensin-like

                  3.0

                  12

                  Hs.252855

                  MFI2

                  antigen p97 (melanoma associated)

                  2.9

                  13

                  Hs.439658

                  MGC4796

                  Ser/Thr-like kinase

                  2.8

                  14

                  Hs.417962

                  DUSP4

                  dual specificity phosphatase 4

                  2.7

                  15

                  Hs.8257

                  CISH

                  cytokine inducible SH2-containing protein

                  2.6

                  16

                  Hs.660427

                  PAR5

                  Prader-Willi/Angelman syndrome-5

                  2.6

                  17

                  Hs.279887

                  AIPL1

                  aryl hydrocarbon receptor interacting protein-like 1

                  2.6

                  18

                  Hs.145807

                  TMC5

                  transmembrane channel-like 5

                  2.5

                  19

                  Hs.512708

                  TGM2

                  transglutaminase 2

                  2.4

                  20

                  Hs.270833

                  AREG

                  amphiregulin (schwannoma-derived growth factor)

                  2.4

                  21

                  Hs.170623

                  FGD6

                  FYVE, RhoGEF and PH domain containing 6

                  2.3

                  22

                  Hs.418138

                  FN1

                  fibronectin 1

                  2.3

                  23

                  Hs.102541

                  NTN4

                  netrin 4

                  2.2

                  24

                  Hs.354906

                  RAB39

                  RAB39, member RAS oncogene family

                  2.2

                  25

                  Hs.443906

                  EGLN3

                  egl nine homolog 3 (C. elegans)

                  2.2

                  26

                  Hs.149156

                  GLDC

                  glycine dehydrogenase

                  2.2

                  27

                  Hs.1145

                  WT1

                  Wilms tumor 1

                  2.1

                  28

                  Hs.25220

                  LARGE

                  like-glycosyltransferase

                  2.1

                  29

                  Hs.282557

                  CP

                  ceruloplasmin (ferroxidase)

                  2.0

                  30

                  Hs.78909

                  ZFP36L2

                  zinc finger protein 36, C3H type-like 2

                  2.0

                  31

                  Hs.413297

                  RGS16

                  regulator of G-protein signalling 16

                  2.0

                  32

                  Hs.182454

                  NYREN18

                  NEDD8 ultimate buster-1

                  2.0

                  33

                  Hs.96125

                  RCP

                  Rab coupling protein

                  2.0

                  34

                  Hs.308028

                  TMEM17

                  transmembrane protein 17

                  2.0

                  35

                  Hs.21894

                  PPM1H

                  protein phosphatase 1H (PP2C domain containing)

                  2.0

                  36

                  Hs.240395

                  KCNK6

                  potassium channel, subfamily K, member 6

                  1.9

                  37

                  Hs.36563

                  B7-H4

                  immune costimulatory protein B7-H4

                  1.9

                  38

                  Hs.27345

                  RNGTT

                  RNA guanylyltransferase and 5’-phosphatase

                  1.9

                  39

                  Hs.144287

                  HEY2

                  hairy/enhancer-of-split related with YRPW motif 2

                  1.8

                  40

                  Hs.269857

                  HRB2

                  HIV-1 rev binding protein 2

                  1.8

                  41

                  Hs.387871

                  TNFSF10

                  tumor necrosis factor (ligand) superfamily, member 10

                  1.8

                  42

                  Hs.80409

                  GADD45A

                  growth arrest and DNA-damage-inducible, alpha

                  1.8

                  43

                  Hs.134742

                  FAM20C

                  family with sequence similarity 20, member C

                  1.8

                  44

                  Hs.274701

                  TK2

                  thymidine kinase 2, mitochondrial

                  1.8

                  45

                  Hs.55610

                  SLC30A1

                  solute carrier family 30 (zinc transporter), member 1

                  1.8

                  46

                  Hs.82173

                  TIEG

                  TGFB inducible early growth response

                  1.8

                  47

                  Hs.404081

                  FGFR2

                  fibroblast growth factor receptor 2

                  1.8

                  48

                  Hs.31218

                  SCAMP1

                  secretory carrier membrane protein 1

                  1.8

                  49

                  Hs.350470

                  TFF1

                  trefoil factor 1

                  1.8

                  50

                  Hs.95655

                  SECTM1

                  secreted and transmembrane 1

                  1.8

                  51

                  Hs.902

                  NF2

                  neurofibromin 2

                  1.7

                  52

                  Hs.310640

                  T2BP

                  TRAF2 binding protein

                  1.7

                  53

                  Hs.252550

                  TNIK

                  TRAF2 and NCK interacting kinase

                  1.7

                  54

                  Hs.6838

                  ARHE

                  ras homolog gene family, member E

                  1.7

                  55

                  Hs.418062

                  B3GALT3

                  betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 3

                  1.7

                  56

                  Hs.270411

                  PLEKHC1

                  pleckstrin homology domain containing, family C member 1

                  1.7

                  57

                  Hs.647388

                  ARHGDIG

                  Rho GDP dissociation inhibitor (GDI) gamma

                  1.7

                  58

                  Hs.202453

                  MYC

                  v-myc myelocytomatosis viral oncogene homolog (avian)

                  1.6

                  59

                  Hs.110488

                  CHSY1

                  carbohydrate (chondroitin) synthase 1

                  1.6

                  60

                  Hs.9795

                  ACOX2

                  acyl-Coenzyme A oxidase 2, branched chain

                  1.6

                  61

                  Hs.158357

                  UNC5CL

                  unc-5 homolog C (C. elegans)-like

                  1.6

                  62

                  Hs.441972

                  IFNT1

                  interferon tau-1

                  1.6

                  63

                  Hs.221889

                  CSDA

                  cold shock domain protein A

                  1.6

                  64

                  Hs.333503

                  RNF38

                  ring finger protein 38

                  1.6

                  65

                  Hs.203581

                  DDX54

                  DEAD (Asp-Glu-Ala-Asp) box polypeptide 54

                  1.6

                  66

                  Hs.345226

                  ZNF563

                  zinc finger protein 563

                  1.6

                  67

                  Hs.30991

                  ANKRD6

                  ankyrin repeat domain 6

                  1.6

                  68

                  Hs.4113

                  AHCYL1

                  S-adenosylhomocysteine hydrolase-like 1

                  1.6

                  69

                  Hs.416077

                  SEMA4B

                  sema domain

                  1.6

                  70

                  Hs.7378

                  PHLDB2

                  pleckstrin homology-like domain, family B, member 2

                  1.6

                  71

                  Hs.369063

                  ZIC2

                  Zic family member 2 (odd-paired homolog, Drosophila)

                  1.6

                  72

                  Hs.515284

                  ZNF505

                  zinc finger protein 505

                  1.6

                  73

                  Hs.426511

                  MIPOL1

                  mirror-image polydactyly 1

                  1.6

                  74

                  Hs.108966

                  PIP5K2A

                  phosphatidylinositol-4-phosphate 5-kinase, type II, alpha

                  1.6

                  75

                  Hs.432607

                  PSMB2

                  proteasome (prosome, macropain) subunit, beta type, 2

                  1.6

                  Table 2

                  Downregulated genes from microarray with fold change > −1.6 and p < 0.05

                   

                  Unigene

                  Symbol

                  Gene descriptor

                  Fold Change

                  1

                  Hs.275464

                  KLK10

                  kallikrein 10

                  −3.3

                  2

                  Hs.307030

                  KRTAP1-5

                  keratin associated protein 1-5

                  −3.3

                  3

                  Hs.282233

                  MLLT6

                  myeloid/lymphoid leukemia translocated to, 6

                  −3.2

                  4

                  Hs.78518

                  NPR2

                  natriuretic peptide receptor B/guanylate cyclase B

                  −2.8

                  5

                  Hs.155024

                  BCL6

                  B-cell CLL/lymphoma 6

                  −2.8

                  6

                  Hs.90800

                  MMP16

                  matrix metalloproteinase 16 (membrane-inserted)

                  −2.5

                  7

                  Hs.87539

                  ALDH3B2

                  aldehyde dehydrogenase 3 family, member B2

                  −2.4

                  8

                  Hs.144906

                  METAP2

                  methionyl aminopeptidase 2

                  −2.3

                  9

                  Hs.440455

                  ALAS2

                  aminolevulinate, delta-, synthase 2

                  −2.2

                  10

                  Hs.266175

                  PAG

                  phosphoprotein associated with glycosphingolipid-enriched microdomains

                  −2.2

                  11

                  Hs.435947

                  RBM15

                  RNA binding motif protein 15

                  −2.2

                  12

                  Hs.233325

                  HFE

                  hemochromatosis

                  −2.1

                  13

                  Hs.443012

                  SEMA6A

                  sema domain,transmembrane domain(TM),and cytoplasmic domain,(semaphorin)6A

                  −2.0

                  14

                  Hs.357901

                  SOX4

                  SRY (sex determining region Y)-box 4

                  −2.0

                  15

                  Hs.415048

                  FLT4

                  fms-related tyrosine kinase 4

                  −2.0

                  16

                  Hs.380833

                  IGFBP5

                  insulin-like growth factor binding protein 5

                  −1.9

                  17

                  Hs.444881

                  CRAMP1L

                  Crm, cramped-like (Drosophila)

                  −1.9

                  18

                  Hs.398124

                  DNAH5

                  dynein, axonemal, heavy polypeptide 5

                  −1.9

                  19

                  Hs.79025

                  SNRK

                  SNF-1 related kinase

                  −1.9

                  20

                  Hs.432121

                  PRDX2

                  peroxiredoxin 2

                  −1.9

                  21

                  Hs.22370

                  NEXN

                  nexilin (F actin binding protein)

                  −1.9

                  22

                  Hs.144914

                  GNMT

                  glycine N-methyltransferase

                  −1.9

                  23

                  Hs.21446

                  CENTB5

                  centaurin, beta 5

                  −1.9

                  24

                  Hs.58103

                  AKAP9

                  A kinase (PRKA) anchor protein (yotiao) 9

                  −1.8

                  25

                  Hs.387385

                  SMURF2

                  E3 ubiquitin ligase SMURF2

                  −1.8

                  26

                  Hs.324470

                  ADD3

                  adducin 3 (gamma)

                  −1.8

                  27

                  Hs.348387

                  GSTM4

                  glutathione S-transferase M4

                  −1.8

                  28

                  Hs.58419

                  TARSH

                  target of Nesh-SH3

                  −1.7

                  29

                  Hs.174051

                  SNRP70

                  small nuclear ribonucleoprotein 70 kDa polypeptide (RNP antigen)

                  −1.7

                  30

                  Hs.211601

                  MAP3K12

                  mitogen-activated protein kinase kinase kinase 12

                  −1.7

                  31

                  Hs.23964

                  SAP18

                  sin3-associated polypeptide, 18 kDa

                  −1.7

                  32

                  Hs.380929

                  LDHD

                  lactate dehydrogenase D

                  −1.7

                  33

                  Hs.390568

                  ZNF585A

                  zinc finger protein 585A

                  −1.7

                  34

                  Hs.241305

                  TRIM16

                  tripartite motif-containing 16

                  −1.7

                  35

                  Hs.403933

                  FBXO32

                  F-box only protein 32

                  −1.7

                  36

                  Hs.434756

                  AP2E

                  adaptor-related protein complex 2, epsilon subunit

                  −1.7

                  37

                  Hs.173894

                  CSF1

                  colony stimulating factor 1 (macrophage)

                  −1.6

                  38

                  Hs.104555

                  NPFF

                  neuropeptide FF-amide peptide precursor

                  −1.6

                  39

                  Hs.307015

                  KRTAP4-14

                  keratin associated protein 4-14

                  −1.6

                  40

                  Hs.301961

                  GSTM1

                  glutathione S-transferase M1

                  −1.6

                  41

                  Hs.307915

                  ABCC4

                  ATP-binding cassette, sub-family C (CFTR/MRP), member 4

                  −1.6

                  42

                  Hs.512000

                  GP1BB

                  glycoprotein Ib (platelet), beta polypeptide

                  −1.6

                  43

                  Hs.446297

                  ZNF498

                  zinc finger protein 498

                  −1.6

                  44

                  Hs.120396

                  FRMD4

                  FERM domain containing 4

                  −1.6

                  45

                  Hs.222901

                  GRIK4

                  glutamate receptor, ionotropic, kainate 4

                  −1.6

                  46

                  Hs.464896

                  ZNF397

                  zinc finger protein 397

                  −1.6

                  47

                  Hs.16232

                  CNKSR1

                  connector enhancer of kinase suppressor of Ras 1

                  −1.6

                  48

                  Hs.255526

                  DTNA

                  dystrobrevin, alpha

                  −1.6

                  49

                  Hs.107203

                  PLAC2

                  placenta-specific 2

                  −1.6

                  50

                  Hs.9029

                  KRT23

                  keratin 23 (histone deacetylase inducible)

                  −1.6

                  51

                  Hs.91753

                  SMPD3

                  sphingomyelin phosphodiesterase 3, neutral membrane (neutral sphingomyelinase II)

                  −1.6

                  52

                  Hs.331555

                  SPINK5

                  serine protease inhibitor, Kazal type 5

                  −1.6

                  53

                  Hs.391858

                  TIA1

                  TIA1 cytotoxic granule-associated RNA binding protein

                  −1.6

                  54

                  Hs.109122

                  MPP5

                  membrane protein, palmitoylated 5 (MAGUK p55 subfamily member 5)

                  −1.6

                  55

                  Hs.445072

                  ARGBP2

                  Arg/Abl-interacting protein ArgBP2

                  −1.6

                  Table 3

                  18 genes chosen for further study based on breast cancer relevance

                   

                  Unigene

                  Symbol

                  Gene descriptor

                  Fold Change

                  1

                  Hs.89626

                  PTHrP

                  parathyroid hormone-like hormone

                  11.9

                  2

                  Hs.46468

                  CCR6

                  chemokine (C-C motif) receptor 6

                  4.9

                  3

                  Hs.76095

                  IER3

                  immediate early response 3

                  3.4

                  4

                  Hs.121520

                  AMIGO2

                  amphoterin induced gene 2

                  3.3

                  5

                  Hs.115263

                  EREG

                  epiregulin

                  3.2

                  6

                  Hs.436186

                  ERAP1

                  type 1 tumor necrosis factor receptor shedding aminopeptidase regulator

                  3.0

                  7

                  Hs.417962

                  DUSP4

                  dual specificity phosphatase 4

                  2.7

                  8

                  Hs.8257

                  CISH

                  cytokine inducible SH2-containing protein

                  2.6

                  9

                  Hs.145807

                  TMC5

                  transmembrane channel-like 5

                  2.5

                  10

                  Hs.270833

                  AREG

                  amphiregulin (schwannoma-derived growth factor)

                  2.4

                  11

                  Hs.418138

                  FN1

                  fibronectin 1

                  2.3

                  12

                  Hs.102541

                  NTN4

                  netrin 4

                  2.2

                  13

                  Hs.1145

                  WT1

                  Wilms tumor 1

                  2.1

                  14

                  Hs.80409

                  GADD45A

                  growth arrest and DNA-damage-inducible, alpha

                  1.8

                  15

                  Hs.404081

                  FGFR2

                  fibroblast growth factor receptor 2

                  1.8

                  16

                  Hs.155024

                  BCL6

                  B-cell CLL/lymphoma 6

                  −2.8

                  17

                  Hs.357901

                  SOX4

                  SRY (sex determining region Y)-box 4

                  −2.0

                  18

                  Hs.415048

                  FLT4

                  fms-related tyrosine kinase 4

                  −2.0

                  qRT-PCR validation of 18 candidate prolactin modulated genes

                  To validate the data from the global microarray analysis, qRT-PCR analysis was carried out on the 18 selected genes using the same xenograft RNA samples that were used for the microarray-based profiling. Of the upregulated transcripts, 12 out of 15 transcripts were confirmed to be upregulated by qRT-PCR >1.6-fold, whereas upregulation of GADD45A, ERAP1, FGFR2 was not confirmed by qRT-PCR. Among the selected three down-regulated transcripts, BCL6 was confirmed down-regulated >1.6 fold by qRT-PCR, whereas FLT4 and SOX4 were down-regulated 1.3-fold and 1.6-fold, respectively, by qRT-PCR analysis (Figure 1B).

                  We then identified PRL-modulated transcripts in T47D xenotransplants that were also modulated by PRL T47D cells in vitro. Immunoblot analysis of T47D cells cultured in vitro displayed robust pY-Stat5 in response to 10 nM human PRL for 24 h (Figure 1C). qRT-PCR analysis of mRNA extracted from T47D cells treated with or without PRL for 24 h established that 9 of the 12 qRT-PCR validated upregulated transcripts also were upregulated over 1.6-fold in vitro, while three transcripts, CCR6, AMIGO2, and DUSP4 were not modulated by PRL in vitro (Figure 1D). Transcripts for GADD45A, ERAP1, FGFR2, which were not confirmed by qRT-PCR analysis of transcripts in vivo, remained unmodulated in vitro (Figure 1D). Out of the three selected down-regulated transcripts, BCL6 maintained PRL induced down-regulation of >1.6-fold in vitro, whereas FLT4 and SOX4 again were reproducibly down-regulated but only 1.5-fold and 1.4-fold respectively (p < 0.05) (Figure 1D). Since these values were only marginally lower than 1.6, we elected to keep FLT4 and SOX4 in the subsequent analyses. In subsequent experiments we focused on the set of 12 transcripts out of original selected panel of 18 transcripts that were PRL-responsive both in vivo and in vitro.

                  Stat5 regulates novel PRL-modulated transcripts

                  PRL activates multiple signaling pathways in breast cancer cells [31], with Stat5 constituting a principal mediator of PRL actions during development and differentiation of the mammary gland [32]. To determine whether the observed PRL-modulated transcripts were regulated through the Stat5 pathway, we overexpressed Stat5a, Stat5b, or a dominant-negative Stat5a/b (DNStat5) in T47D cells using adenoviral gene delivery, and treated cells with or without PRL for 24 h. Immunoblot analysis of total cell lysates verified over-expression of the correct Stat5 variants and robust PRL-induction of pY-Stat5 in cells following adenoviral gene delivery (Figure 2A). The overexpression was sufficiently high that basal levels of Stat5 were not detectable without further exposure of the blots (not shown). qRT-PCR analysis revealed that DNStat5 blocked PRL induction of each of the 9 upregulated transcripts. PRL induction of most of these 9 transcripts were further enhanced by overexpression of Stat5a or Stat5b, either basal or PRL-induced (Figure 2B). Although levels of PRL-induced CISH and WT1 transcripts did not rise further by Stat5 overexpression, PRL-induction of both transcripts was effectively abrogated by DNStat5, thus supporting Stat5-mediated regulation of these genes. Stat5 overexpression also enhanced down-regulation of both SOX4 and FLT4. The ERAP1 transcript was included as a negative control since it was not regulated by PRL as shown in Figure 1. Stat5a, Stat5b, or DN-Stat5 overexpression had no effect on ERAP1 transcript levels, indicating that enhancement of PRL-induction detected for the other transcripts is specific (Figure 2). BCL6 was omitted from this analysis since we have published this separately [33].
                  http://static-content.springer.com/image/art%3A10.1186%2F1476-4598-12-59/MediaObjects/12943_2013_Article_1126_Fig2_HTML.jpg
                  Figure 2

                  Stat5 regulates novel prolactin modulated transcripts. (A) Immunoblot of pY-Stat5 and Stat5 in cells treated with adenovirus containing LacZ (Control), Stat5a, Stat5b, or Dominant-Negative Stat5 (DNStat5) for 24 h, then serum starved and treated with vehicle or 10 nM PRL for 24 h. (B) RNA isolated from T47D cells treated with adenovirus was tested for 12 PRL modulated transcripts using qRT-PCR analysis.

                  Jak2 and Jak1 are critical for PRL gene regulation in T47D breast cancer cells

                  Conditional gene knock-out in mice demonstrated that Jak2 is the key Stat5 tyrosine kinase in breast epithelial cells during and outside of pregnancy and lactation [34]. However, we have reported that in human breast cancer cell lines Jak1 is also recruited in a Jak2-dependent manner for maximal PRL-activation of Stat5 and other signaling mediators [31]. To determine whether PRL-recruitment of Jak1 was required for maximal modulation of PRL-regulated transcripts, T47D cells were infected with lentivirus carrying shRNAs targeting either Jak1 or Jak2 followed by treatment with or without PRL for 24 h. Jak1 shRNA was effective and selective, as judged by marked down-regulation of Jak1 mRNA but not Jak2 mRNA (Figure 3). Conversely, Jak2 shRNA effectively suppressed Jak2 mRNA but not Jak1 mRNA (Figure 3). All nine PRL-upregulated transcripts showed complete dependence on Jak2 (Figure 3). Importantly, all nine PRL-induced transcripts were also partially suppressed by Jak1 knockdown, consistent with a significant role for Jak1 recruitment by PRL in breast cancer cells to maximize downstream signals. Among the three down-regulated transcripts, BCL6 down-regulation by PRL was dependent on Jak2 but not on Jak1, whereas down-regulation of FLT4 and SOX4 by PRL was not significant under these experimental conditions, possibly due to cell stress during lentiviral infection (Figure 3). Collectively, these data are consistent with a general model in which Jak2 is critical for PRL receptor signaling and Jak1 recruitment is needed for maximal signal.
                  http://static-content.springer.com/image/art%3A10.1186%2F1476-4598-12-59/MediaObjects/12943_2013_Article_1126_Fig3_HTML.jpg
                  Figure 3

                  Jak2 and Jak1 are critical for PRL transcript regulation in T47D breast cancer cells. T47D cells were treated with lentivirus containing NTC, Jak1 shRNA, or Jak2 shRNA overnight. Then, cells were placed in serum starvation media for 24 h and then treated with vehicle or 10 nM PRL for another 24 h before RNA isolation and qRT-PCR analysis of the 12 PRL regulated transcripts.

                  Synergistic enhancement by E2 of PRL-induced proliferation and select PRL-modulated genes

                  Previous studies have indicated that PRL may enhance E2-induced proliferation as well as additively or synergistically increase transcription of certain PRL or E2 target genes [911]. To verify that the PRL-E2 interaction occurred in T47D cells under our culture conditions, we treated T47D cells with varying doses of PRL (0, 1, 10, 20, 37, and 100 nM) while keeping E2 constant (1 nM), and determined cell number after 72 h. PRL concentrations as low as 10 nM in the presence of E2 were associated with an increase in cell number compared to E2 alone, and this effect was maintained at higher PRL concentrations (Figure 4A). Next, we maintained constant PRL concentration (20 nM) while varying the E2 dose (0.001, 0.01, 0.1, 1, 10 nM), and counted cell numbers after 72 h. At every concentration of E2, we observed a PRL-induced increase in cell number (Figure 4B). We also determined the effect of PRL on E2-driven growth in soft agar and measured colony size after 2 weeks. While PRL alone had limited effect on colony size, E2 increased colony size dramatically, and PRL further increased E2-driven colony size (Figure 4C). Representative images of the colonies in soft agar are displayed in Figure 4D. These data established under our culture conditions that PRL positively interacts with E2 to induce proliferation of luminal T47D breast cancer cells.
                  http://static-content.springer.com/image/art%3A10.1186%2F1476-4598-12-59/MediaObjects/12943_2013_Article_1126_Fig4_HTML.jpg
                  Figure 4

                  Enhancement by E2 of PRL-induced proliferation and select PRL modulated transcripts. (A) T47D cells were treated with varying doses of PRL (0, 1, 10, 20, 37, and 100 nM) in the presence of constant E2 (1 nM) for 72 h. Bars represent triplicates of each condition that were counted and averaged. (B) T47D cells were treated with varying doses of E2 (0.001, 0.01, 0.1, 1, 10 nM) in the presence of constant PRL (20 nM) for 72 h. Bars represent triplicates of each condition that were counted and averaged. (C) T47D cells were treated with vehicle, 20 nM PRL, 1 nM E2, or 20 nM PRL + 1 nM E2 for 2 weeks. Images from four independent wells were analyzed for colony size through ImageJ. (D) Representative images of T47D cells grown on soft agar and treated with vehicle, PRL, E2, or PRL + E2 for 2 weeks. (E) T47D cells were treated with PRL, E2, or PRL + E2 for 24 h. RNA was isolated and qRT-PCR analysis was performed on the 12 PRL modulated transcripts.

                  To determine whether the PRL-modulated transcript panel was affected by co-treatment with E2, we treated T47D cells with vehicle, PRL, E2, or PRL + E2 for 24 h and performed qRT-PCR analysis of the 12 transcripts. Seven out of the nine upregulated transcripts displayed further induction with E2 present (NTN4 and TMC5 had no further induction) (Figure 4E). None of the three PRL-downregulated genes displayed further downregulation in the presence of E2 (Figure 4E). This data indicates that E2 is selectively modulating some but not all PRL-Stat5 regulated transcripts, and these specific transcripts may be playing a role in PRL’s ability to increase E2-driven breast cancer cell proliferation.

                  PTHrP protein levels correlate with levels of pYStat5 in human breast cancer tissues

                  To begin to assess the clinical relevance of the newly identified PRL-modulated transcripts, we selected the gene product most strongly upregulated by PRL in the in vivo xenotransplant tumors, PTHrP, for protein expression analysis in clinical breast cancer specimens. We hypothesized that cellular PTHrP protein levels would be positively correlated with levels of nuclear localized and tyrosine phosphorylated Stat5 (Nuc-pYStat5). Nuc-pYStat5 is an indirect measure of PRL receptor activation in breast epithelia, and we documented evidence of Stat5-dependence of PRL-upregulation of PTHrP in T47D cells. Levels of cellular PTHrP and Nuc-pYStat5 were analyzed using fluorescence-based quantitative immunohistochemistry on a breast cancer progression array containing 40 normal and 140 malignant breast tissues. Representative images of PTHrP and pY-Stat5 staining are shown in Figure 5A, where Case 1 has high PTHrP and Nuc-pYStat5 levels, while Case 2 has low PTHrP and Nuc-pYStat5 levels. Evaluable levels of cellular PTHrP and Nuc-pYStat5 were obtained in 92 breast cancer specimens and in support of our hypothesis, a statistically significant positive correlation was detected (r = 0.51, P < 0.001) (Figure 5B).
                  http://static-content.springer.com/image/art%3A10.1186%2F1476-4598-12-59/MediaObjects/12943_2013_Article_1126_Fig5_HTML.jpg
                  Figure 5

                  PTHrP protein levels correlate with levels of nuc-pYStat5 in human breast cancer tissues. (A) Representative immunofluroescent images of two cases of breast cancer stained for pY-Stat5 (Red), PTHrP (Red), cytokeratin (Green), and DNA (Blue) using immunohistochemistry. (B) Scatter plot and Pearson correlation analysis between nuc-pYStat5 and cellular PTHrP protein levels on 92 breast cancer specimens.

                  Gene ontology (GO) terms are enriched in differentiation markers

                  Analysis of GO biological process terms using the 75 PRL-upregulated transcripts identified in this study revealed 24 GO terms that had a false discovery rate (FDR) below 25% (Table 4). Many of the pathways that were identified were homeostasis-related, correlating with the ability of the PRL-Stat5 pathway to maintain cellular differentiation, consistent with established pro-differentiation effects of PRL on normal and malignant luminal breast epithelial cells. In addition, proliferation-regulation and negative regulation of apoptosis were also identified, which is consistent with the reported role of PRL contributing to breast cancer initiation and growth.
                  Table 4

                  Gene Ontology (GO) analysis of PRL upregulated genes with false discovery rate (FDR) < 25

                  GO Term

                  Description

                  P value

                  FDR

                  GO:0030005

                  cellular di-, tri-valent inorganic cation homeostasis

                  0.002

                  2.452

                  GO:0042592

                  homeostatic process

                  0.002

                  2.848

                  GO:0055066

                  di-, tri-valent inorganic cation homeostasis

                  0.002

                  3.079

                  GO:0030003

                  cellular cation homeostasis

                  0.003

                  4.015

                  GO:0055080

                  cation homeostasis

                  0.004

                  6.550

                  GO:0006879

                  cellular iron ion homeostasis

                  0.006

                  8.210

                  GO:0043123

                  positive regulation of I-kappaB kinase/NF-kappaB cascade

                  0.006

                  9.136

                  GO:0060249

                  anatomical structure homeostasis

                  0.008

                  10.970

                  GO:0055072

                  iron ion homeostasis

                  0.008

                  11.045

                  GO:0019725

                  cellular homeostasis

                  0.008

                  11.791

                  GO:0043122

                  regulation of I-kappaB kinase/NF-kappaB cascade

                  0.008

                  11.809

                  GO:0048514

                  blood vessel morphogenesis

                  0.009

                  12.950

                  GO:0051052

                  regulation of DNA metabolic process

                  0.010

                  13.580

                  GO:0042127

                  regulation of cell proliferation

                  0.010

                  13.992

                  GO:0043066

                  negative regulation of apoptosis

                  0.011

                  15.532

                  GO:0043069

                  negative regulation of programmed cell death

                  0.012

                  16.371

                  GO:0060548

                  negative regulation of cell death

                  0.012

                  16.542

                  GO:0048878

                  chemical homeostasis

                  0.013

                  17.642

                  GO:0006873

                  cellular ion homeostasis

                  0.014

                  18.854

                  GO:0055082

                  cellular chemical homeostasis

                  0.014

                  19.975

                  GO:0001568

                  blood vessel development

                  0.015

                  20.556

                  GO:0010627

                  regulation of protein kinase cascade

                  0.015

                  20.807

                  GO:0001944

                  vasculature development

                  0.016

                  22.079

                  Prolactin-upregulated gene signature is associated with prolonged time to relapse and metastasis

                  We then determined whether the PRL-upregulated gene signature was associated with clinical outcome, using an available 49 of 75 PRL-upregulated genes (Table 5) in a cohort of 936 primary invasive breast cancer patients. We divided the patients into three groups based on their degree of expression of the PRL-gene signature (upper quartile, interquartile range, and lower quartile). The patients in the upper quartile had significantly prolonged time to metastasis compared to patients associated with the lower quartile (Figure 6A). In addition, patients that were in the upper quartile also had significantly prolonged disease-free survival than patients in the lower quartile (Figure 6B).
                  Table 5

                  PRL-upregulated genes used to generate PRL gene signature

                  Gene Symbol

                  Correlation

                  Gene symbol

                  Correlation

                  with signature average

                  with signature average

                  AREG

                  0.6

                  TNIK

                  0.25

                  DUSP4

                  0.51

                  PPM1H

                  0.17

                  PTHLH

                  0.46

                  PAR5

                  0.15

                  EREG

                  0.45

                  INVS

                  0.14

                  SCAMP1

                  0.43

                  CSDA

                  0.13

                  TMC5

                  0.42

                  MYC

                  0.12

                  GADD45A

                  0.42

                  WT1

                  0.09

                  CHSY1

                  0.41

                  CISH

                  0.08

                  TFF1

                  0.4

                  CP

                  0.07

                  FGD6

                  0.39

                  RGS16

                  0.07

                  BACH1

                  0.38

                  NF2

                  0.07

                  AHCYL1

                  0.37

                  FGFR2

                  0.04

                  EGLN3

                  0.34

                  RNGTT

                  0.03

                  AMIGO2

                  0.33

                  FN1

                  0.02

                  TNFSF10

                  0.33

                  ITCH

                  −0.01

                  IER3

                  0.33

                  LARGE

                  −0.11

                  ANKRD6

                  0.32

                  PSMB2

                  −0.12

                  ACOX2

                  0.31

                  SECTM1

                  −0.15

                  SLC30A1

                  0.31

                  GLDC

                  −0.2

                  ERAP1

                  0.31

                  AIPL1

                  −0.21

                  RNF38

                  0.3

                  TGM2

                  −0.22

                  ZFP36L2

                  0.3

                  DDX54

                  −0.23

                  CCR6

                  0.28

                  MFI2

                  −0.27

                  HEY2

                  0.25

                  ARHGDIG

                  −0.27

                  TK2

                  0.25

                    
                  http://static-content.springer.com/image/art%3A10.1186%2F1476-4598-12-59/MediaObjects/12943_2013_Article_1126_Fig6_HTML.jpg
                  Figure 6

                  PRL upregulated gene signature is associated with metastasis-free and relapse-free survival. (A) Patients with high levels of a genes signature of PRL-upregulated had reduced risk of developing metastasis compared to patients with low levels of the PRL-upregulated gene signature. (B) Patients with high levels of the PRL-induced gene signature had prolonged relapse-free survival compared to patients with low levels of the PRL gene signature.

                  Discussion

                  The present study reports a novel panel of PRL-modulated transcripts based on analysis of human breast cancer xenograft tumors in vivo. We identified 75 up-regulated and 55 down-regulated transcripts that were significantly modulated based on at least a 1.6-fold change with a P-value less than 0.05. From this panel of 130 PRL-modulated transcripts, a subset of 18 transcripts with established breast cancer-relevance was selected for further analysis and validation. Validation by qRT-PCR documented significant modulation of 12 of 18 transcripts in vitro. We further documented that the majority of in vitro-modulated transcripts were Stat5- and Jak2-dependent, and showed that Jak1 was required for maximal PRL-modulation. Consistent with PRL-enhancement of E2-driven proliferation of breast cancer cells, select PRL-modulated transcripts displayed positive co-regulation by E2, including the growth factors EREG and AREG. These molecular results are summarized in Table 6. Furthermore, quantitative immunofluorescence analyses of clinical breast cancer specimens from a cohort of 92 patients documented a significant positive correlation between tumor levels of PTHrP protein, one of the PRL-stimulated transcripts, and nuclear localized and tyrosine phosphorylated Stat5, a marker of PRL signaling. Gene ontology analysis revealed that prolactin-upregulated genes were associated most frequently with terms involved in homeostasis and differentiation. Finally, a gene signature generated with PRL-upregulated genes was associated with prolonged relapse-free survival as well as metastasis-free survival in a cohort of breast cancer patients. Collectively, the validation studies support the value of the transcript data and are expected to facilitate better understanding of PRL action in breast cancer.
                  Table 6

                  Summary of transcript regulation

                  Gene

                  Fold Change (microarray)

                  in vivo Regulation

                  in vitro Regulation

                  Stat5 Regulation

                  Jak2 Regulation

                  Jak1 Regulation

                  E2 Regulation

                  PTHrP

                  11.9

                  Y

                  Y

                  Y

                  Y

                  Y

                  Y

                  CCR6

                  4.9

                  Y

                  N

                  Y

                  Y

                  Y

                  Y

                  IER3

                  3.4

                  Y

                  Y

                  Y

                  Y

                  Y

                  Y

                  AMIGO2

                  3.3

                  Y

                  N

                  NA

                  NA

                  NA

                  NA

                  EREG

                  3.2

                  Y

                  Y

                  Y

                  Y

                  Y

                  Y

                  ERAP1

                  3.0

                  N

                  N

                  NA

                  NA

                  NA

                  NA

                  DUSP4

                  2.7

                  Y

                  N

                  NA

                  NA

                  NA

                  NA

                  CISH

                  2.6

                  Y

                  Y

                  Y

                  Y

                  Y

                  Y

                  TMC5

                  2.5

                  Y

                  Y

                  Y

                  Y

                  Y

                  N

                  AREG

                  2.4

                  Y

                  Y

                  Y

                  Y

                  Y

                  Y

                  FN1

                  2.3

                  Y

                  Y

                  Y

                  Y

                  Y

                  Y

                  NTN4

                  2.2

                  Y

                  Y

                  Y

                  Y

                  Y

                  N

                  WT1

                  2.1

                  Y

                  Y

                  Y

                  Y

                  Y

                  Y

                  GADD45A

                  1.8

                  N

                  N

                  NA

                  NA

                  NA

                  NA

                  FGFR2

                  1.8

                  N

                  N

                  NA

                  NA

                  NA

                  NA

                  BCL6

                  −2.8

                  Y

                  Y

                  Y

                  Y

                  N

                  N

                  SOX4

                  −2.0

                  Y

                  Y

                  Y

                  NA

                  NA

                  N

                  FLT4

                  −2.0

                  Y

                  Y

                  Y

                  NA

                  NA

                  N

                  PRL activates both Stat5a and Stat5b, which have 92% amino acid similarity [35], but are encoded by different genes and may mediate overlapping and distinct effects in breast cancer cells [17, 33, 3638]. In the present study, which focused on PRL-modulated transcripts in the T47D breast cancer model, experimental overexpression of Stat5a or Stat5b enhanced to a comparable extent PRL-modulation of most transcripts tested. However, TMC5, NTN4, and AREG displayed greater degree of enhanced PRL-modulation when Stat5a was overexpressed rather than Stat5b, supporting the notion that certain genes are more responsive to Stat5a than Stat5b. PRL-modulation of all examined transcripts with the exception of PRL-induced down-regulation of FLT4 mRNA was disrupted by overexpression of a C-terminally truncated Stat5 variant that acts as a dominant-negative molecule for both Stat5a and Stat5b. This exception is consistent with previous reports that repression of certain Stat5 target genes is unaffected by alterations in the Stat5 transactivation domain [33, 39]. We have recently reported that Stat5a but not Stat5b expression was lost during progression of human breast cancer, and in cultured MCF-7 cells there was only a limited overlap in transcripts modulated by the two PRL-activated transcription factors [17]. It is possible that in T47D cells the higher basal levels of Stat5a and especially Stat5b make it more difficult to ascertain differences between the two transcription factors in overexpression studies. Future transcript analyses will focus on further characterizing the differences in gene regulation between Stat5a and Stat5b in human breast cancer.

                  Jak1 was reported to be activated by PRL signaling in human breast cancer lines and cooperate with Jak2 to enhance signaling pathways downstream of PRL receptors, including Stat5, Stat3 and Erk activation [31]. The present study validates the notion that Jak1-coactivation enhances PRL-Jak2 signaling by demonstrating that maximal PRL-modulation of target transcripts required PRL-induced co-activation of Jak1. Future studies to identify the mechanism of activation of Jak1 by PRL receptors in breast cancer and the effect of Jak1-activation on PRL-modulated biology of breast cancer are now warranted.

                  Furthermore, whereas PRL alone exerted limited proliferative effect on T47D breast cancer cells in vitro, PRL enhanced E2-driven cell proliferation both on plastic and soft agar. PRL positively interacted with E2 to further elevate several transcripts encoding growth and progression factors for breast cancer, including AREG, EREG, PTHrP and WT1. Considering the established role for AREG as a paracrine mediator of E2-induced proliferation of luminal breast epithelial cells during pubertal growth [40], AREG may be directly involved in PRL stimulation of E2-driven growth of human breast cancer. A recent study has implicated PRL receptors in the maintenance of ER expression and responsiveness of breast cancer cells to estrogen [28], which is consistent with our findings which demonstrate significant crosstalk between the two pathways. Since Stat5 has been associated with response to anti-estrogen therapy in clinical breast cancer specimens [16, 17], this synergistic stimulation of proliferation with estrogen may be a mechanism behind increased responsiveness to anti-estrogen treatments. In addition, PRL biological action may vary depending on the hormonal environment, especially given our recent observation that PRL effectively counteracts progestin-induction of the tumor-initiating CK5-positive cell population [21].

                  We identified PRL-suppression of BCL6 transcript and protein based on this global transcript analysis of T47D xenotransplants tumors, and we reported a negative correlation between levels of BCL6 protein and Nuc-pYStat5 in clinical breast cancer specimens [33]. The fact that the protein products of two of the PRL-modulated genes identified in this global transcript profiling, PTHrP and BCL6, both correlated with Nuc-pYStat5 in human clinical breast cancer specimens indicates that transcripts identified in the present study are relevant in clinical specimens and may become useful human breast tumor markers of PRL activation.

                  Supporting the validity of the identified panel of PRL-modulated transcripts in T47D cells in vivo, 12 out of the 57 transcripts identified as PRL-modulated transcripts in a recent in vitro transcript profiling study of T47D cells overlapped with our panel (AREG, WT1, PTHrP, IER3, TMC5, CISH, BCL6, DUSP4, TNIK, EGLN3, FBXO32, and AKAP9) [9]. Only WT1 and IER3 transcripts were tested and confirmed by qRT-PCR in the previous report. In addition, select transcripts identified in the previous study such as WT1 and IER3 demonstrated co-regulation by E2 [9], consistent with the findings of the present study. In vitro transcript profiling for PRL-modulated transcripts in another ER-positive human breast cancer cell line, MCF7, using Stat5 overexpression to enhance PRL effects, yielded 300 candidate PRL-modulated transcripts among which 12 overlapped with the panel identified in the present study of T47D cells (CISH, EGLN3, KCNK6, PTHrP, FN1, CHSY1, BCL6, DUSP4, IGFBP5, TNIK, ABCC4, and MYC) [17]. The limited overlap between our current study of T47D cells and two recent studies of MCF-7 cells in vitro may be due to lower expression levels of Stat5 in MCF-7 cells, making it necessary to overexpress Stat5 [17] or PRL receptor [28]. While previous studies were performed in vitro and did not broadly validate identified candidate PRL-modulated transcripts by qRT-PCR, the present study also provides novel data by demonstrating PRL-regulation of transcripts in vivo and through Jak-Stat5 dependent mechanisms.

                  The established breast cancer relevance for AREG, WT1, and IER3 is discussed in a previous transcript profiling study [9]. NTN4 is a transmembrane protein whose expression levels positively correlates with better prognosis in breast cancer [41]. TMC5 is a transmembrane channel that was overexpressed in PIK3CA-mutated breast cancer [42]. FN1 is a protein present in the extracellular matrix that is a candidate serum biomarker for detecting breast cancer [43], and disrupting the interaction between FN1 and integrins in breast cancer cells led to increased apoptosis and response to radiation [44]. EREG is a ligand for EGFR and Her4, and was reported to be part of a set of four genes that promote breast cancer intravasation and metastasis to the lung [45]. PTHrP is a secreted protein critical for mammary gland development [46], and extensive research has been performed on its role in mediating breast cancer metastasis to bone [4749]. SOX4 is a transcription factor regulated by progesterone in breast cancer cells [50], and was identified as an oncogene in prostate cancer [51]. FLT4 is a member of the VEGF receptor family, and expression in vessels surrounding breast tumors was correlated to lymph node positivity and poor clinical outcome [52]. These genes clearly demonstrate the complexity of PRL effects, since PRL has the ability to suppress oncogenes (SOX4, FLT4, BCL6) and upregulate favorable prognostic markers (NTN4), while also upregulating genes involved in breast cancer growth and progression (AREG, WT1, IER3, EREG, TMC5, FN1, PTHrP). These observations are consistent with the many reported conflicting and likely context-dependent effects of PRL in breast cancer.

                  Furthermore, gene ontology (GO) analysis based on PRL-upregulated genes demonstrated a concentration in homeostasis pathways, consistent with the known pro-differentiation role of PRL in breast cancer. However, other terms such as cell proliferation and anti-apoptosis were also enriched, most likely reflecting the duality of PRL action. Consistent with the enrichment of differentiation terms in the GO analysis, a PRL gene signature generated on PRL-upregulated genes was associated with prolonged time to relapse and metastasis-free survival. These associations are consistent with the multiple reports that Stat5 is associated with favorable prognosis in breast cancer patients [1416]. In addition, we have recently reported that prolactin can suppress a therapy-resistant, tumor-initiating CK5-positive population induced by progestin [21]. Our results are consistent with a role of PRL in reducing the tumor-initiating CK5-positive cell population, which is implicated in breast cancer metastasis and relapse.

                  Conclusions

                  The present study is the first to report a panel of PRL-modulated transcripts based on global transcript profiling of human breast cancer xenotransplant tumors in vivo. Some but not all transcripts were also modulated by PRL in vitro. PRL-enhancement of E2-driven proliferation of T47D cells in vitro may be mediated by observed co-regulation by PRL and E2 of growth-promoting genes including AREG, EREG, WT1 and PTHrP. PRL-modulated transcripts reported in this study are expected to facilitate deciphering of the mechanisms underlying the pleotropic effects of PRL on breast cancer. PRL-upregulated genes were frequently associated with differentiation pathways. Finally, select transcripts or their protein complement identified in this study also may be useful as breast cancer tumor marker signatures of PRL activation, which is highly relevant considering our report that PRL gene signature is associated with relapse-free survival and metastasis-free survival, and the already documented association of Stat5 with breast cancer prognosis and hormone therapy responsiveness.

                  Materials and methods

                  Tissue culture

                  T47D cells were cultured in RPMI (Cellgro) containing 10% fetal bovine serum (FBS) and 1 mM sodium pyruvate (Cellgro). For PRL induction, confluent T47D cells were put in serum starvation media (RPMI without FBS) for 24 h, and then stimulated with either PBS vehicle or 10 nM of recombinant human prolactin (AFP795, provided by Dr. A.F. Parlow at National Hormone and Pituitary Program) for 24 h. 24 h prior to experiments involving β-Estradiol (Sigma), media was changed to RPMI containing 5% Charcoal Stripped Serum (Thermo Scientific) and 1 mM sodium pyruvate.

                  Xenotransplants

                  Nude mice (N = 20) were implanted with 17β-estradiol pellets (0.72 mg; Innovative Research of America) and injected subcutaneously with T47D cells (5 × 106) suspended in Matrigel into two dorsolateral sites. Tumors were allowed to grow for 6 weeks and subsequently were injected subcutaneously with either saline (N = 10) or 5 μg/g body mass of human prolactin (N = 10) every 12 h for 48 h total. Tumors were harvested and processed for immunohistochemistry and qRT-PCR. All research involving mice were approved by Thomas Jefferson IACUC (protocol 789C to H.R.) in accordance with international guidelines for ethical treatment of animals.

                  Microarray

                  RNA was pooled into 3 groups from PRL injected mice and 3 groups from saline injected mice, with each group containing RNA from 3–4 mice. Microarray analysis was performed for each group using the Affymetrix HG-U133 GeneChip Set (Expression Analysis). Two group comparison analyses were conducted on normalized expression values that were individually transformed using base 2 logarithms. On the log-transformed scale, the mean is calculated for every gene within each group and a two-sample, two-sided t-test is conducted to test the equality of those means. The P-value indicates the significance level of this test.

                  Quantitative reverse transcription polymerase chain reaction

                  Quantitative RT-PCR assays were performed with RNA samples isolated from T47D cells using RNeasy kit (Qiagen). cDNA was generated using Iscript (Bio-Rad) according to the manufacturer’s protocol. cDNAs were subjected to qPCR using corresponding primers (Table 7).
                  Table 7

                  Primer sequences

                  Gene

                  Forward primer

                  Reverse primer

                  PTHrP

                  GTTCCTGGTGAGCTACGCG

                  CTTGGATGGACTTCCCCTTG

                  CCR6

                  TGCTACCGCTGCCTGTGAGC

                  AAAATAATCTTCACTGGAGTCG

                  IER3

                  CGTCCTCGAGCCCTTTAATCT

                  AGGTCCAGAGCGTAGTCCGA

                  AMIGO2

                  CCGGTGTCTTTTCCACCG

                  GAGCCCACGAGGCTCC

                  EREG

                  GCTCTGCCTGGGTTTCCATC

                  CCACACGTGGATTGTCTTCTGTC

                  ERAP1

                  GCCATTCTAGCTGCAGTGGG

                  CAACTGTGTACGGGAGCCC

                  DUSP4

                  TACAAGTGCATCCCAGTGGA

                  CCCGTTTCTTCATCATCAGG

                  CISH

                  CTGCTGTGCATAGCCAAGAC

                  GTGCCTTCTGGCATCTTCTG

                  TMC5

                  TATCCTTCAGCTCAATTGCTG

                  AGAGGACGCTGGTTCCAAAC

                  AREG

                  GGTGGTGCTGTCGCTCTTG

                  TCAGCACTGTGGTCCCCAG

                  FN1

                  TTCTACTCCTGCACACAGAAG

                  CCCTCAGAAGTGCAATCAGTG

                  NTN4

                  CATGGTGGGATACTGGGGC

                  TCAGGAACTTCATGATACCAGTC

                  WT1

                  GAGAGCCAGCCCGCTATTC

                  CATGGGATCCTCATGCTTG

                  GADD45A

                  TCAGCGCACGATCACTGTC

                  CCAGCAGGCACAACACCAC

                  FGFR2

                  CTCACTCTCACAACCAATGAGG

                  AGGAAGGCATGGTTCGTAAG

                  BCL6

                  CTGCAGATGGAGCATGTTGT

                  TCTTCACGAGGAGGCTTGAT

                  SOX4

                  GTGAGCGAGATGATCTCGGG

                  CAGGTTGGAGATGCTGGACTC

                  FLT4

                  CAGGATGAAGACATTTGAGG

                  AAGAAAATGCTGACGTAT

                  GAPDH

                  AATCCATCACCATCTTCCA

                  TGGACTCCACGACGTACTCA

                  ERAP1

                  GCCATTCTAGCTGCAGTGGG

                  CAACTGTGTACGGGAGCCC

                  Immunohistochemistry

                  Immunohistochemistry and AQUA analyses were performed on a tissue array generated by cutting-edge matrix-assembly containing 100 deidentified primary invasive breast carcinoma specimens in a cohort described previously [33]. Immunohistochemistry was performed as described previously [33] using pY-Stat5 (Epitomics, 1:200), PTHrP (Santa Cruz, 1:200), and cytokeratin (DAKO, 1:100). AQUA analysis was performed using AQUA/PM2000 (HistoRx) as described previously [33].

                  Adenoviral and lentiviral production and infection

                  Lentiviral particle production was performed as described previously [33]. shRNA lentiviral vectors (Open Biosystems, Lafayette, CO, USA) were obtained for scrambled control (SC002), and Jak1 (TRCN0000003102), and Jak2 (TRCN0000003180). The cells were infected with lentivirus overnight and allowed to grow for 48 h before hormone induction for an additional 24 h. LacZ, Stat5a, Stat5b, and Dominant-negative-Stat5 (DN-Stat5) adenoviruses were prepared using double cesium chloride centrifugation [53] and used for gene delivery into T47D cells (1.5×106/well in 6 well dish; multiplicity of infection = 5). 24 h after infection, cells were incubated with hormones for another 24 h and subsequently harvested for immunoblot analysis.

                  Cell proliferation and soft agar assays

                  T47D cells were treated with vehicle, PRL, or 17β-Estradiol (E2) (Sigma) for 72 h. Triplicates of each condition were plated and counted using the Countess Automated Cell Counter (Invitrogen). For the soft agar assay, T47D cells suspended in 0.3% agarose were plated on top of 0.6% agarose. T47D cells were treated with media containing either vehicle, PRL (20 nM), E2 (10 nM), or PRL + E2 for 2 weeks, with fresh media and hormones added every 3 days. Each condition was done in quadruplicate. Images were taken from each well at 2 weeks and were analyzed for colony size using ImageJ.

                  Immunoblotting

                  T47D cells were lysed as described previously [54]. Proteins were resolved by SDS-PAGE and immunoblotted with antibodies to phospho-Stat5 (AX1, Advantex), and total Stat5 (BD #610192), followed by secondary antibodies Alexa Fluor 680-conjugated goat anti-mouse IgG (Invitrogen) or IRDye 800 CW-conjugated goat anti-rabbit IgG (Licor, Lincoln, NE, USA) depending on primary antibodies. Immunoblots were scanned using the Odyssey Infrared Imaging System (Licor).

                  Gene ontology analysis and survival analyses

                  The PRL-regulated genes identified using mRNA expression microarrays were analyzed for enrichment of Gene Ontology Biological Process terms using the Database for Annotation, Visualization and Integrated Discovery (DAVID ) v6.7 [55, 56]. A previously described collection of mRNA microarray datasets compiled from public repositories [57] was used to evaluate the set of PRL-induced transcripts in the context of clinical outcome. Transcription levels for PRL-upregulated genes were averaged into an expression signature and used to divide samples into the lower quartile, interquartile range, and upper quartile. Relapse-free and metastasis-free survival differences in these groups were evaluated for significance using the log rank test.

                  Declarations

                  Acknowledgements

                  We thank Jessica Davison for editorial assistance with the manuscript. This work was supported by Komen for the Cure Promise Grant KG091116 (H.R., T.S., A.R.P., C.L.), NIH grants CA101841 (H.R.), CA118740 (H.R.), and NCI Support Grant 1P30CA56036 to the Kimmel Cancer Center. The Project is funded, in part, under a Commonwealth University Research Enhancement Program grant with the Pennsylvania Department of Health (H.R.). The Department specifically disclaims responsibility for any analyses, interpretations or conclusions.

                  Authors’ Affiliations

                  (1)
                  Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University

                  References

                  1. Rose-Hellekant TA, Arendt LM, Schroeder MD, Gilchrist K, Sandgren EP, Schuler LA: Prolactin induces ERalpha-positive and ERalpha-negative mammary cancer in transgenic mice. Oncogene. 2003, 22: 4664-4674. 10.1038/sj.onc.1206619PubMed CentralView ArticlePubMed
                  2. Vomachka AJ, Pratt SL, Lockefeer JA, Horseman ND: Prolactin gene-disruption arrests mammary gland development and retards T-antigen-induced tumor growth. Oncogene. 2000, 19: 1077-1084. 10.1038/sj.onc.1203348View ArticlePubMed
                  3. Tworoger SS, Eliassen AH, Sluss P, Hankinson SE: A prospective study of plasma prolactin concentrations and risk of premenopausal and postmenopausal breast cancer. J Clin Oncol. 2007, 25: 1482-1488. 10.1200/JCO.2006.07.6356View ArticlePubMed
                  4. Tworoger SS, Sluss P, Hankinson SE: Association between plasma prolactin concentrations and risk of breast cancer among predominately premenopausal women. Cancer Res. 2006, 66: 2476-2482. 10.1158/0008-5472.CAN-05-3369View ArticlePubMed
                  5. Reynolds C, Montone KT, Powell CM, Tomaszewski JE, Clevenger CV: Expression of prolactin and its receptor in human breast carcinoma. Endocrinology. 1997, 138: 5555-5560. 10.1210/en.138.12.5555PubMed
                  6. Gill S, Peston D, Vonderhaar BK, Shousha S: Expression of prolactin receptors in normal, benign, and malignant breast tissue: an immunohistological study. J Clin Pathol. 2001, 54: 956-960. 10.1136/jcp.54.12.956PubMed CentralView ArticlePubMed
                  7. Shiu RP: Prolactin receptors in human breast cancer cells in long-term tissue culture. Cancer Res. 1979, 39: 4381-4386.PubMed
                  8. Schroeder MD, Symowicz J, Schuler LA: PRL modulates cell cycle regulators in mammary tumor epithelial cells. Mol Endocrinol. 2002, 16: 45-57. 10.1210/me.16.1.45View ArticlePubMed
                  9. Rasmussen LM, Frederiksen KS, Din N, Galsgaard E, Christensen L, Berchtold MW, Panina S: Prolactin and oestrogen synergistically regulate gene expression and proliferation of breast cancer cells. Endocr Relat Cancer. 2010, 17: 809-822. 10.1677/ERC-09-0326View ArticlePubMed
                  10. Chen Y, Huang K, Chen KE, Walker AM: Prolactin and estradiol utilize distinct mechanisms to increase serine-118 phosphorylation and decrease levels of estrogen receptor alpha in T47D breast cancer cells. Breast Cancer Res Treat. 2010, 120: 369-377. 10.1007/s10549-009-0400-7View ArticlePubMed
                  11. Gutzman JH, Nikolai SE, Rugowski DE, Watters JJ, Schuler LA: Prolactin and estrogen enhance the activity of activating protein 1 in breast cancer cells: role of extracellularly regulated kinase 1/2-mediated signals to c-fos. Mol Endocrinol. 2005, 19: 1765-1778. 10.1210/me.2004-0339PubMed CentralView ArticlePubMed
                  12. Gouilleux F, Wakao H, Mundt M, Groner B: Prolactin induces phosphorylation of Tyr694 of Stat5 (MGF), a prerequisite for DNA binding and induction of transcription. EMBO J. 1994, 13: 4361-4369.PubMed CentralPubMed
                  13. Liu X, Robinson GW, Gouilleux F, Groner B, Hennighausen L: Cloning and expression of Stat5 and an additional homologue (Stat5b) involved in prolactin signal transduction in mouse mammary tissue. Proc Natl Acad Sci U S A. 1995, 92: 8831-8835. 10.1073/pnas.92.19.8831PubMed CentralView ArticlePubMed
                  14. Nevalainen MT, Xie J, Torhorst J, Bubendorf L, Haas P, Kononen J, Sauter G, Rui H: Signal transducer and activator of transcription-5 activation and breast cancer prognosis. J Clin Oncol. 2004, 22: 2053-2060. 10.1200/JCO.2004.11.046View ArticlePubMed
                  15. Yamashita H, Nishio M, Ando Y, Zhang Z, Hamaguchi M, Mita K, Kobayashi S, Fujii Y, Iwase H: Stat5 expression predicts response to endocrine therapy and improves survival in estrogen receptor-positive breast cancer. Endocr Relat Cancer. 2006, 13: 885-893. 10.1677/erc.1.01095View ArticlePubMed
                  16. Peck AR, Witkiewicz AK, Liu C, Stringer GA, Klimowicz AC, Pequignot E, Freydin B, Tran TH, Yang N, Rosenberg AL: Loss of nuclear localized and tyrosine phosphorylated Stat5 in breast cancer predicts poor clinical outcome and increased risk of antiestrogen therapy failure. J Clin Oncol. 2011, 29: 2448-2458. 10.1200/JCO.2010.30.3552PubMed CentralView ArticlePubMed
                  17. Peck AR, Witkiewicz AK, Liu C, Klimowicz AC, Stringer GA, Pequignot E, Freydin B, Yang N, Ertel A, Tran TH: Low levels of Stat5a protein in breast cancer are associated with tumor progression and unfavorable clinical outcomes. Breast Cancer Res. 2012, 14: R130- 10.1186/bcr3328PubMed CentralView ArticlePubMed
                  18. Sultan AS, Xie J, LeBaron MJ, Ealley EL, Nevalainen MT, Rui H: Stat5 promotes homotypic adhesion and inhibits invasive characteristics of human breast cancer cells. Oncogene. 2005, 24: 746-760. 10.1038/sj.onc.1208203View ArticlePubMed
                  19. Sultan AS, Brim H, Sherif ZA: Co-overexpression of Janus kinase 2 and signal transducer and activator of transcription 5a promotes differentiation of mammary cancer cells through reversal of epithelial-mesenchymal transition. Cancer Sci. 2008, 99: 272-279. 10.1111/j.1349-7006.2007.00685.xView ArticlePubMed
                  20. Nouhi Z, Chughtai N, Hartley S, Cocolakis E, Lebrun JJ, Ali S: Defining the role of prolactin as an invasion suppressor hormone in breast cancer cells. Cancer Res. 2006, 66: 1824-1832. 10.1158/0008-5472.CAN-05-2292View ArticlePubMed
                  21. Sato T, Tran TH, Peck AR, Girondo MA, Liu C, Goodman CR, Neilson LM, Freydin B, Chervoneva I, Hyslop T, Kovatich AJ, Hooke JA, Shriver CD, Fuchs SY, Rui H: Prolactin suppresses a progestin-induced CK5-positive cell population in luminal breast cancer through inhibition of progestin-driven BCL6 expression. Oncogene. 2013, In Press.
                  22. Gass S, Harris J, Ormandy C, Brisken C: Using gene expression arrays to elucidate transcriptional profiles underlying prolactin function. J Mammary Gland Biol Neoplasia. 2003, 8: 269-285.View ArticlePubMed
                  23. Harris J, Stanford PM, Sutherland K, Oakes SR, Naylor MJ, Robertson FG, Blazek KD, Kazlauskas M, Hilton HN, Wittlin S: Socs2 and elf5 mediate prolactin-induced mammary gland development. Mol Endocrinol. 2006, 20: 1177-1187. 10.1210/me.2005-0473View ArticlePubMed
                  24. Hou Z, Bailey JP, Vomachka AJ, Matsuda M, Lockefeer JA, Horseman ND: Glycosylation-dependent cell adhesion molecule 1 (GlyCAM 1) is induced by prolactin and suppressed by progesterone in mammary epithelium. Endocrinology. 2000, 141: 4278-4283. 10.1210/en.141.11.4278PubMed
                  25. Naylor MJ, Oakes SR, Gardiner-Garden M, Harris J, Blazek K, Ho TW, Li FC, Wynick D, Walker AM, Ormandy CJ: Transcriptional changes underlying the secretory activation phase of mammary gland development. Mol Endocrinol. 2005, 19: 1868-1883. 10.1210/me.2004-0254View ArticlePubMed
                  26. Ormandy CJ, Naylor M, Harris J, Robertson F, Horseman ND, Lindeman GJ, Visvader J, Kelly PA: Investigation of the transcriptional changes underlying functional defects in the mammary glands of prolactin receptor knockout mice. Recent Prog Horm Res. 2003, 58: 297-323. 10.1210/rp.58.1.297View ArticlePubMed
                  27. Perotti C, Liu R, Parusel CT, Bocher N, Schultz J, Bork P, Pfitzner E, Groner B, Shemanko CS: Heat shock protein-90-alpha, a prolactin-STAT5 target gene identified in breast cancer cells, is involved in apoptosis regulation. Breast Cancer Res. 2008, 10: R94- 10.1186/bcr2193PubMed CentralView ArticlePubMed
                  28. Fiorillo AA, Medler TR, Feeney YB, Wetz SM, Tommerdahl KL, Clevenger CV: The prolactin receptor transactivation domain is associated with steroid hormone receptor expression and malignant progression of breast cancer. Am J Pathol. 2013, 182: 217-233. 10.1016/j.ajpath.2012.09.021View ArticlePubMed
                  29. Sharma NL, Massie CE, Ramos-Montoya A, Zecchini V, Scott HE, Lamb AD, Macarthur S, Stark R, Warren AY, Mills IG, Neal DE: The androgen receptor induces a distinct transcriptional program in castration-resistant prostate cancer in man. Cancer Cell. 2013, 23: 35-47. 10.1016/j.ccr.2012.11.010View ArticlePubMed
                  30. Yoshimura A, Ohkubo T, Kiguchi T, Jenkins NA, Gilbert DJ, Copeland NG, Hara T, Miyajima A: A novel cytokine-inducible gene CIS encodes an SH2-containing protein that binds to tyrosine-phosphorylated interleukin 3 and erythropoietin receptors. EMBO J. 1995, 14: 2816-2826.PubMed CentralPubMed
                  31. Neilson LM, Zhu J, Xie J, Malabarba MG, Sakamoto K, Wagner KU, Kirken RA, Rui H: Coactivation of Jak1 positively modulates prolactin-Jak2 signaling in breast cancer: recruitment of ERK and Stat3 and enhancement of Akt and Stat5a/b pathways. Mol Endocrinol. 2007, 21: 2218-2232. 10.1210/me.2007-0173View ArticlePubMed
                  32. Liu X, Robinson GW, Wagner KU, Garrett L, Wynshaw-Boris A, Hennighausen L: Stat5a is mandatory for adult mammary gland development and lactogenesis. Genes Dev. 1997, 11: 179-186. 10.1101/gad.11.2.179View ArticlePubMed
                  33. Tran TH, Utama FE, Lin J, Yang N, Sjolund AB, Ryder A, Johnson KJ, Neilson LM, Liu C, Brill KL: Prolactin inhibits BCL6 expression in breast cancer through a Stat5a-dependent mechanism. Cancer Res. 2010, 70: 1711-1721. 10.1158/0008-5472.CAN-09-2314PubMed CentralView ArticlePubMed
                  34. Wagner KU, Krempler A, Triplett AA, Qi Y, George NM, Zhu J, Rui H: Impaired alveologenesis and maintenance of secretory mammary epithelial cells in Jak2 conditional knockout mice. Mol Cell Biol. 2004, 24: 5510-5520. 10.1128/MCB.24.12.5510-5520.2004PubMed CentralView ArticlePubMed
                  35. Grimley PM, Dong F, Rui H: Stat5a and Stat5b: fraternal twins of signal transduction and transcriptional activation. Cytokine Growth Factor Rev. 1999, 10: 131-157. 10.1016/S1359-6101(99)00011-8View ArticlePubMed
                  36. Bernaciak TM, Zareno J, Parsons JT, Silva CM: A novel role for signal transducer and activator of transcription 5b (STAT5b) in beta1-integrin-mediated human breast cancer cell migration. Breast Cancer Res. 2009, 11: R52- 10.1186/bcr2341PubMed CentralView ArticlePubMed
                  37. Fox EM, Bernaciak TM, Wen J, Weaver AM, Shupnik MA, Silva CM: Signal transducer and activator of transcription 5b, c-Src, and epidermal growth factor receptor signaling play integral roles in estrogen-stimulated proliferation of estrogen receptor-positive breast cancer cells. Mol Endocrinol. 2008, 22: 1781-1796. 10.1210/me.2007-0419PubMed CentralView ArticlePubMed
                  38. Tang JZ, Zuo ZH, Kong XJ, Steiner M, Yin Z, Perry JK, Zhu T, Liu DX, Lobie PE: Signal transducer and activator of transcription (STAT)-5A and STAT5B differentially regulate human mammary carcinoma cell behavior. Endocrinology. 2010, 151: 43-55. 10.1210/en.2009-0651View ArticlePubMed
                  39. Nakajima H, Brindle PK, Handa M, Ihle JN: Functional interaction of STAT5 and nuclear receptor co-repressor SMRT: implications in negative regulation of STAT5-dependent transcription. EMBO J. 2001, 20: 6836-6844. 10.1093/emboj/20.23.6836PubMed CentralView ArticlePubMed
                  40. Ciarloni L, Mallepell S, Brisken C: Amphiregulin is an essential mediator of estrogen receptor alpha function in mammary gland development. Proc Natl Acad Sci U S A. 2007, 104: 5455-5460. 10.1073/pnas.0611647104PubMed CentralView ArticlePubMed
                  41. Esseghir S, Kennedy A, Seedhar P, Nerurkar A, Poulsom R, Reis-Filho JS, Isacke CM: Identification of NTN4, TRA1, and STC2 as prognostic markers in breast cancer in a screen for signal sequence encoding proteins. Clin Cancer Res. 2007, 13: 3164-3173. 10.1158/1078-0432.CCR-07-0224View ArticlePubMed
                  42. Cizkova M, Cizeron-Clairac G, Vacher S, Susini A, Andrieu C, Lidereau R, Bieche I: Gene expression profiling reveals new aspects of PIK3CA mutation in ERalpha-positive breast cancer: major implication of the Wnt signaling pathway. PLoS One. 2010, 5: e15647- 10.1371/journal.pone.0015647PubMed CentralView ArticlePubMed
                  43. Ruiz-Garcia E, Scott V, Machavoine C, Bidart JM, Lacroix L, Delaloge S, Andre F: Gene expression profiling identifies Fibronectin 1 and CXCL9 as candidate biomarkers for breast cancer screening. Br J Cancer. 2010, 102: 462-468. 10.1038/sj.bjc.6605511PubMed CentralView ArticlePubMed
                  44. Nam JM, Onodera Y, Bissell MJ, Park CC: Breast cancer cells in three-dimensional culture display an enhanced radioresponse after coordinate targeting of integrin alpha5beta1 and fibronectin. Cancer Res. 2010, 70: 5238-5248. 10.1158/0008-5472.CAN-09-2319PubMed CentralView ArticlePubMed
                  45. Gupta GP, Nguyen DX, Chiang AC, Bos PD, Kim JY, Nadal C, Gomis RR, Manova-Todorova K, Massague J: Mediators of vascular remodelling co-opted for sequential steps in lung metastasis. Nature. 2007, 446: 765-770. 10.1038/nature05760View ArticlePubMed
                  46. Wysolmerski JJ, Philbrick WM, Dunbar ME, Lanske B, Kronenberg H, Broadus AE: Rescue of the parathyroid hormone-related protein knockout mouse demonstrates that parathyroid hormone-related protein is essential for mammary gland development. Development. 1998, 125: 1285-1294.PubMed
                  47. Li J, Karaplis AC, Huang DC, Siegel PM, Camirand A, Yang XF, Muller WJ, Kremer R: PTHrP drives breast tumor initiation, progression, and metastasis in mice and is a potential therapy target. J Clin Invest. 2011, 121: 4655-4669. 10.1172/JCI46134PubMed CentralView ArticlePubMed
                  48. Kohno N, Kitazawa S, Fukase M, Sakoda Y, Kanbara Y, Furuya Y, Ohashi O, Ishikawa Y, Saitoh Y: The expression of parathyroid hormone-related protein in human breast cancer with skeletal metastases. Surg Today. 1994, 24: 215-220. 10.1007/BF02032890View ArticlePubMed
                  49. Guise TA, Yin JJ, Thomas RJ, Dallas M, Cui Y, Gillespie MT: Parathyroid hormone-related protein (PTHrP)-(1–139) isoform is efficiently secreted in vitro and enhances breast cancer metastasis to bone in vivo. Bone. 2002, 30: 670-676. 10.1016/S8756-3282(02)00685-3View ArticlePubMed
                  50. Graham JD, Hunt SM, Tran N, Clarke CL: Regulation of the expression and activity by progestins of a member of the SOX gene family of transcriptional modulators. J Mol Endocrinol. 1999, 22: 295-304. 10.1677/jme.0.0220295View ArticlePubMed
                  51. Liu P, Ramachandran S, Ali Seyed M, Scharer CD, Laycock N, Dalton WB, Williams H, Karanam S, Datta MW, Jaye DL, Moreno CS: Sex-determining region Y box 4 is a transforming oncogene in human prostate cancer cells. Cancer Res. 2006, 66: 4011-4019. 10.1158/0008-5472.CAN-05-3055View ArticlePubMed
                  52. Nakamura Y, Yasuoka H, Tsujimoto M, Yang Q, Imabun S, Nakahara M, Nakao K, Nakamura M, Mori I, Kakudo K: Flt-4-positive vessel density correlates with vascular endothelial growth factor-d expression, nodal status, and prognosis in breast cancer. Clin Cancer Res. 2003, 9: 5313-5317.PubMed
                  53. Dagvadorj A, Kirken RA, Leiby B, Karras J, Nevalainen MT: Transcription factor signal transducer and activator of transcription 5 promotes growth of human prostate cancer cells in vivo. Clin Cancer Res. 2008, 14: 1317-1324. 10.1158/1078-0432.CCR-07-2024View ArticlePubMed
                  54. Johnson KJ, Peck AR, Liu C, Tran TH, Utama FE, Sjolund AB, Schaber JD, Witkiewicz AK, Rui H: PTP1B suppresses prolactin activation of Stat5 in breast cancer cells. Am J Pathol. 2010, 177: 2971-2983. 10.2353/ajpath.2010.090399PubMed CentralView ArticlePubMed
                  55. da Huang W, Sherman BT, Lempicki RA: Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009, 4: 44-57.View ArticlePubMed
                  56. da Huang W, Sherman BT, Lempicki RA: Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009, 37: 1-13. 10.1093/nar/gkn923View ArticlePubMed
                  57. Ertel A, Dean JL, Rui H, Liu C, Witkiewicz AK, Knudsen KE, Knudsen ES: RB-pathway disruption in breast cancer: differential association with disease subtypes, disease-specific prognosis and therapeutic response. Cell Cycle. 2010, 9: 4153-4163. 10.4161/cc.9.20.13454PubMed CentralView ArticlePubMed

                  Copyright

                  © Sato et al.; licensee BioMed Central Ltd. 2013

                  This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                  Advertisement