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Endocrinology, doi:10.1210/en.2007-1320
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Endocrinology Vol. 149, No. 5 2219-2229
Copyright © 2008 by The Endocrine Society

Transcriptional Activation of Signal Transducer and Activator of Transcription (STAT) 3 and STAT5B Partially Mediate Homeobox A1-Stimulated Oncogenic Transformation of the Immortalized Human Mammary Epithelial Cell

Kumarasamypet M. Mohankumar, Jo K. Perry, Nagarajan Kannan, Kimitoshi Kohno, Peter D. Gluckman, B. Starling Emerald and Peter E. Lobie

The Liggins Institute and National Research Centre for Growth and Development (K.M.M., J.K.P., N.K., P.D.G., B.S.E., P.E.L.), and Department of Molecular Medicine and Pathology, Faculty of Medical Health Sciences (P.E.L.) University of Auckland, Auckland 1142, New Zealand; and Departments of Molecular Biology and Surgery (K.K.), University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan

Address all correspondence and requests for reprints to: Peter E. Lobie, The Liggins Institute, University of Auckland, 2–6 Park Avenue, Private Bag 92019, Auckland 1142, New Zealand. E-mail: p.lobie{at}auckland.ac.nz.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously demonstrated that the p44/42 MAPK pathway is one pathway involved in homeobox (HOX) A1-stimulated oncogenesis. However, inhibition of MAPK kinase 1 does not completely prevent HOXA1-stimulated oncogenic transformation, suggesting the involvement of additional signal transduction pathways. Here, we report that forced expression of HOXA1 in immortalized human mammary epithelial cells significantly increased levels of signal transducer and activator of transcription (STAT) 3, 5A, and 5B mRNA by transcriptional up-regulation. The protein levels of STAT3 and 5B, but not STAT5A, and protein phosphorylation levels of STAT3 and 5B were significantly increased by forced expression of HOXA1. Forced expression of STAT3 or STAT5B was sufficient to transform oncogenically an immortalized human mammary epithelial cell line. Accordingly, inhibition of STAT3 or STAT5B activity with dominant negative STAT3 or STAT5B abrogated the ability of HOXA1 to stimulate cell proliferation, survival, oncogenic transformation, and generation of large disorganized multiacinar structures in three-dimensional culture. These results suggest that HOXA1 partially mediates oncogenic transformation of the immortalized human mammary epithelial cell through modulation of the STAT3 and STAT5B pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HOMEOBOX (HOX)-containing genes are a family of genes encoding transcription factors that possess pivotal roles in development. Transcription factors encoded by the HOX genes are essential for maintaining the positional identity of cells along the major body axis (1). Comparative analysis of HOX gene expression between normal and neoplastic tissues has demonstrated altered expression of particular HOX genes in certain types of carcinomas (2). Accordingly, HOXA1 is expressed in the normal human mammary gland, and increased expression is observed in mammary ductal carcinoma (Perou breast study, Oncomine database: www.oncomine.org). Recently, we have demonstrated that forced expression of HOXA1 in the immortalized human mammary epithelial cell line, MCF-10A, concomitantly enhances proliferation and cell survival resulting in abnormal mammary acinar morphogenesis, oncogenic transformation, and tumor formation in vivo (3). HOXA1 has been demonstrated to stimulate oncogenicity through modulation of the p44/42 MAPK pathway by increasing growth factor receptor-bound protein 2 (GRB2) and MAPK kinase (MEK) 1 mRNA, and protein expression (4). However, treatment with a MEK1 inhibitor does not completely prevent HOXA1-stimulated oncogenic transformation, which suggests the involvement of other contributing mechanisms in HOXA1-mediated oncogenic transformation of human mammary epithelial cells.

HOXA1 has been previously defined as an autocrine human GH (hGH) regulated gene required for autocrine hGH-mediated oncogenic transformation of immortalized human mammary epithelial cells (5). One of the signal transduction pathways activated by autocrine hGH is the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway (6). In particular, STAT1, 3, 5A, and 5B have been demonstrated to mediate hGH signal transduction resulting in the regulation of a number of downstream gene targets. This raises the possibility that, in addition to the involvement of the p44/42 MAPK pathway (4), HOXA1-mediated oncogenic transformation of human mammary epithelial cells could occur through activation of STAT1, 3, 5A, or 5B.

Recent studies have demonstrated a potential role of STATs in oncogenesis (7). Seven STAT genes have been identified (STAT-1, -2, -3, -4, -5A, -5B, and -6) with demonstrated roles in cell differentiation, proliferation, survival, and angiogenesis through stimulation by cytokine, growth factor, and hormone-mediated signal transduction. Among the STAT family members, STAT1, 3, and 5 have played an important role in embryogenesis and in the development of the mammary gland (7). STAT1, 3, and 5 are also involved in regulating cell-cycle progression and apoptosis, and, thus, may contribute to oncogenesis (8). Constitutive activation of STATs 1, 3, 5A, and 5B has been demonstrated in a number of human cancer cell lines and primary tumors, including mammary carcinoma (9, 10, 11, 12). STAT3, 5A, and 5B are involved in the development and progression of cancer, whereas a tumor suppressor function has been demonstrated for STAT1 (13).

Here, we demonstrate that HOXA1 transcriptionally up-regulates STAT3, 5A, and 5B, resulting in increased activation of the STAT3 and 5B signaling pathways in immortalized human mammary epithelial cells. Thus, increased activation of STAT3 and 5B mediates HOXA1-stimulated oncogenic transformation of immortalized human mammary epithelial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and transfections
Stable cell lines
MCF10A-VECTOR and MCF10A-HOXA1 were established using methods as described (4). MCF-10A cells and derivatives were cultured using conditions as described (3). For all transient transfection experiments, 2 µg plasmids per well was transfected into six-well plates using FuGENE 6 Transfection Reagent (Roche Diagnostics Corp., Indianapolis, IN), and analysis was performed after 48 h in culture.

Constructs
Human STAT5A and STAT5B promoter luciferase constructs (pA1Luc, pA2Luc, pA3Luc, pB4Luc, and pB5Luc) were generous gifts from Dr. Matilde Valeria Ursini (Naples, Italy). The wild-type (wt) human STAT5B-pCI and dominant negative (DN) STAT5B-pCI constructs were generously provided by Warren Leonard (Bethesda, MD). The human STAT3 promoter-luciferase constructs were obtained from Dr. Kimitoshi Kohno (Fukuoka, Japan). The human STAT3 expression constructs [pcDNA3.1-V5/His-STAT3-wt, constitutively active mutant (CA), or DN] were the kind gift from Dr. Jaharul Haque (Cleveland, OH). The Spi-GLE1-Luc plasmid was a generous gift from Dr. Haldosen (Karolinska, Sweden), and the {alpha}-2 macroglobulin luciferase reporter was obtained from Dr. Xinmin Cao (Institute for Molecular and Cell Biology, Proteos, Singapore).

The human STAT3 expression constructs (wt, CA) were generated by subcloning either the wt or CA cDNAs from either pcDNA3.1-V5/His-STAT3-wt or pcDNA3.1-V5/His-STAT3-CA vectors into the expression vector pcDNA3.1(+) by HindIII/XhoI digestion. The new vectors were designated pcDNA3.1-STAT3 wt and pcDNA3.1-STAT3 CA. Similarly, human STAT5B expression constructs (wt, CA) were generated by subcloning a KpnI/NotI fragment from STAT5B-pCI or pCI-STAT5B CA vectors into pcDNA3.1(+) to generate pcDNA3.1-STAT5B wt and pcDNA3.1-STAT5B CA, respectively.

DN human STAT3 or STAT5B constructs were generated by subcloning a HindIII/XhoI or KpnI/NotI from pcDNA3.1-V5/His-STAT3 DN or DN STAT5B-pCI vectors, respectively, into pcDNA3.1/Hygro(+) to generate pcDNA3.1-DN STAT3 or pcDNA3.1-DN STAT5B. All plasmids were prepared with the plasmid maxiprep kit from QIAGEN (Hilden, Germany).

Site-directed mutagenesis
A CA STAT5B mutation was generated by PCR mutagenesis using the Quikchange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. The primer pairs used were: 5'-AAT GTT TTG GCA TCT GAT GCC-3' and 5'-GGC ATC AGA TGC CAA AAC ATT-3'. The resulting plasmid was termed CA STAT5B.

RT-PCR
Extraction of total RNA and the RT-PCR assay were performed as described previously (14). The sequences of the primers used are listed as supplemental data 1, which is published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org.

Western blot analysis
Cells were treated as described previously, and Western blot analysis was performed as described (14). The anti-HOXA1, anti-STAT3, anti-STAT5A, and anti-STAT5B antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); antibodies against STAT1, STAT2, and phospho STAT5A/B (Y694/Y699) were purchased from Upstate Cell Signaling (Lake Placid, NY). Anti-phospho-STAT3 (Tyr705) (3E2) mouse monoclonal antibody was purchased from Cell Signaling Technology, Inc. (Danvers, MA). Anti-STAT4 and anti-STAT6 antibodies were obtained from Zymed Laboratories, Inc. (San Francisco, CA). Immunoprecipitation was performed as previously described (15).

Luciferase reporter assay
Transient transfection was performed with the respective luciferase constructs and other expression vectors as appropriate in complete medium for 24 h. Results were normalized to the level of β-galactosidase activity and protein concentration in the samples

Cell number and oncogenicity assays
MCF10-A cells and derived cell lines were grown in complete medium until 50–60% confluent and then transient transfection was performed with the appropriate expression vectors. Twenty-four hour post-transfection cells were trypsinized with 0.5% trypsin, and 5 x 104 of MCF10-A cells and its derived cell lines were seeded into six-well plates in monolayers in complete media. Total cell number was determined as previously described (16). Mitogenesis was directly assayed by measuring the incorporation of bromodeoxyuridine (17). Apoptotic cell death was measured by fluorescent microscopical analysis of nuclear DNA staining patterns with Hoechst 33258 as previously described (17). The soft agar colony formation assay was performed as previously described (3).

Confocal laser-scanning microscopy
MCF10-A cells were grown in complete medium until 50–60% confluent. Cells were transiently transfected with a HOXA1 expression plasmid or empty vector control, and immunostaining was performed as previously described (18).

Morphogenesis assays
The three-dimensional culture of MCF-10A cells on basement membrane was performed as previously described (19). Assay medium with 2% Matrigel (BD, Franklin Lakes, NJ) was replaced every 4 d.

Statistics
All data are expressed as means ± SEM of triplicate determinants. All experiments were performed three times for conformation of results, and the results of a representative experiment are shown. Data were analyzed using the unpaired two-tailed t test or ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
HOXA1 up-regulates expression of STAT3 and 5B in human mammary epithelial cells
To identify potential mechanisms by which HOXA1 may result in oncogenic transformation, we stably transfected the immortalized human mammary epithelial cell line MCF10A with an expression vector containing the HOXA1 cDNA (designated MCF10A-HOXA1). A second cell line transfected with an empty expression vector was generated for control purposes (MCF10A-VECTOR). Cell lines were established by pooling five individual colonies. Characterization of these cell lines has been described previously (4). MCF10A-HOXA1 stable cells exhibited increased levels of HOXA1 mRNA and HOXA1-mediated transcriptional activity when compared with the control cell line, MCF10A-VECTOR. Previous reports have demonstrated that high levels of STAT protein activation, in particular STAT3 and 5, are associated with oncogenesis (9, 10, 11, 12). To determine whether HOXA1 modulates the STAT pathway, we first examined the mRNA levels of STAT1–6 in MCF10A-VECTOR and MCF10A-HOXA1 cells by semiquantitative RT-PCR. β-Actin was used as a control for RNA quality and loading. As observed in Fig. 1AGo, forced expression of HOXA1 increased the mRNA levels of STAT3, 5A, and 5B, whereas the mRNA levels of STAT4 were decreased. The mRNA levels of STAT1, 2 and 6 were similar between MCF10A-VECTOR and MCF10A-HOXA1 cells under the conditions studied. Thus, HOXA1 specifically increased the mRNA level of STAT3, 5A, and 5B.


Figure 1
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  		FIG. 1. HOXA1 regulates the expression of STATs in immortalized human mammary epithelial cells. A, MCF10A-VECTOR and MCF10A-HOXA1 cells were cultured in complete media, and the mRNA level of STAT1–6 was determined by semiquantitative RT-PCR. β-Actin was used as a loading control. B, The protein levels of STAT1–6 were determined by Western blotting. β-Actin was used as a loading control. Densitometric quantification of the protein level in MCF10A-VECTOR vs. MCF10A-HOXA1 cells demonstrated increased STAT3 (2.63 ± 0.21-fold increase with forced expression of HOXA1) and STAT5B (2.24 ± 0.19-fold increase with forced expression of HOXA1) protein in MCF10A-HOXA1 cells. C, MCF10A cells were transfected with 2 µg of the HOXA1 expression vector. HOXA1 (red) and STAT3 (green) or HOXA1 (red) and STAT5B (green) (D) were localized by confocal laser-scanning microscopy; in the inset, a single cell is shown in higher magnification. Magnification for C and D, x63. E, Effect of HOXA1 on the reporter activity of different STAT promoter-luciferase constructs in MCF10A cells. MCF10A-VECTOR and MCF10A-HOXA1 were transfected with a total of 900 ng of the different constructs containing the STAT3 promoter (pHST3-Luc-1), STAT5A promoters (pA1Luc, pA2Luc, and pA3Luc) or STAT5B promoters (pB4Luc and pB5Luc) plus 100 ng of a β-galactosidase expression plasmid as transfection control. Results represent the mean of triplicate determinations. Bars represent SE. *, P < 0.001.

 
We next determined whether the altered STAT mRNA levels observed in MCF10A-HOXA1 resulted in corresponding altered protein expression. Western blot analysis of STATs 1–6 demonstrated that MCF10A-HOXA1 cells exhibited higher expression of STAT3 and STAT5B proteins compared with the MCF10A-VECTOR cell line (Fig. 1BGo). Surprisingly, the protein expression level of STAT5A was not altered in MCF10A-HOXA1 when compared with the MCF10A-VECTOR cell line. This discordance in STAT5A mRNA and protein levels may be due to differential regulation of STAT5A translation (20). Consistent with the RT-PCR results, STAT1, 2, and 6 protein levels were not altered, whereas STAT4 expression was decreased by HOXA1 overexpression (Fig. 1BGo). These results were subsequently confirmed by confocal laser-scanning microscopy. After transient transfection with HOXA1 cDNA, HOXA1 protein was detected by immunofluorescence (Fig. 1Go, C and D). Cells with forced expression of HOXA1 exhibited increased STAT3 and 5B protein when compared with neighboring untransfected cells. Thus, forced expression of HOXA1 in human mammary epithelial cells resulted in increased levels of STAT3 and 5B protein.

HOXA1 transcriptionally activates the STAT3, 5A, and 5B genes
HOX family members bind to a core DNA recognition sequence (TAAT), which is located in the promoter region of a number of HOX target genes, resulting in transcriptional activation of the gene (21). To determine if the increased STAT3, 5A, and 5B mRNA observed in MCF10A-HOXA1 cells was mediated through transcriptional up-regulation of these genes by HOXA1, we examined the effect of forced expression of HOXA1 on STAT3, 5A, and 5B promoter-dependent reporter activity. The human STAT3 promoter-luciferase reporter construct contains a luciferase coding sequence downstream of the human STAT3 promoter fragment (1909 bp) (a schematic representation of the promoter constructs is given in supplemental data 2) (22). We also used three human STAT5A reporter constructs composing three regions of the STAT5A promoter fused to a luciferase reporter vector (pA1-Luc contains nucleotides –430 to +244 of the STAT5A promoter, pA2-Luc contains nucleotides –430 to +1075, and pA3-Luc from –2119 to +244) and two human STAT5B reporter constructs composing two regions of the STAT5B promoter (pB4-Luc contains nucleotides +1739 to +100 of the STAT5B promoter, and pB5-Luc contains nucleotides –1690 to +236) (supplemental data 2) (23). Forced expression of HOXA1 increased STAT3 promoter activity by 5.4-fold (Fig. 1EGo). In addition, the STAT5A-A3 promoter region (pA3-Luc) and the STAT5B-B5 promoter region (pB5-Luc) mediated luciferase activity were increased by 2.8- and 3.2-fold, respectively, in MCF10A-HOXA1 cells when compared with the MCF10A-VECTOR control cell line. No increase in luciferase gene expression was observed with the STAT5A1, A2, and STAT5-B4 promoter fragments. These results were further supported by the fact that small interfering RNA mediated depletion of HOXA1 decreased STAT3, STAT5A-A3, and STAT5B-B5 promoter-mediated luciferase activity compared with that of the vector-transfected cells (data not shown). Thus, HOXA1 specifically increases STAT3, 5A, and 5B transcription by modulation of promoter activity in immortalized human mammary epithelial cells. Because forced expression of HOXA1 increased transcriptional activation of STAT5A but not protein levels, we subsequently focused on the role of STAT3 and 5B in HOXA1-mediated oncogenic transformation.

HOXA1 modulates STAT3 and 5B phosphorylation
The activation of STAT3 and 5B by various growth factors has previously been demonstrated to require tyrosine phosphorylation at positions Tyr705 in STAT3 and position Tyr699 in STAT5B (24, 25). Because forced expression of HOXA1 increased both mRNA and protein expression levels of STAT3 and 5B, it was, therefore, possible that forced expression of HOXA1 also results in increased activation of STAT3 and 5B. Western blot analysis was used to determine levels of active STAT3 and 5B in MCF10A-HOXA1 cells using an antibody that specifically recognizes phosphorylated STAT3 and 5B at the respective residues (24, 25). β-Actin was used as a loading control. The level of phosphorylated STAT3 and 5B protein was higher in cells with forced expression of HOXA1 when compared with that of the control cell line MCF10A-VECTOR (Fig. 2Go, A and B). Therefore, forced expression of HOXA1 regulates the activity of STAT3 and 5B in human mammary epithelial cells.


Figure 2
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  		FIG. 2. Activation of STAT3/5B dependent transcription by HOXA1 in MCF10A cells. A, Western blot analysis of tyrosine phosphorylation of STAT3 protein with anti-phospho-STAT3 (Tyr705) antibody. B, Lysates were immunoprecipitated with anti-STAT5B and then immunoblotted with antiphospho-STAT5A/B (Y694/Y699) antibody to demonstrate the activation of STAT3 and STAT5B in MCF10A-Vector and MCF10A-HOXA1 cells. After normalization to total STAT3/5B protein, respectively, densitometric quantification shows a 1.91 ± 0.22-fold increase in the level of phospho-STAT3 protein and 2.12 ± 0.13-fold increase in level of phospho-STAT5B protein in MCF10A-HOXA1 cells compared with that of the MCF10A-VECTOR. C, MCF10A-VECTOR and MCF10A-HOXA1 stable cell lines were grown to 60–80% confluence and were transiently transfected with the {alpha}2-macroglobulin-luciferase reporter construct in complete media. Cells were processed for luciferase assay as described in the Materials and Methods. D, The same procedure was followed for the SPI 2.1 promoter-luciferase reporter construct in complete media. Results represent the mean of triplicate determinations. Bars represent SE. *, P < 0.001, significant when compared with its respective control; **, P <0.001; significant when comparing transfected and control experiments from the same cell line.

 
Increased phosphorylation of STAT3 and 5B results in transcriptional activation of downstream effector molecules. One of the downstream targets activated by STAT3 is {alpha}2-macroglobulin (26). Therefore, we examined the effect of forced expression of HOXA1 on STAT3-mediated transcription using a {alpha}2-macroglobulin promoter-luciferase construct. {alpha}2-Macroglobulin promoter-mediated luciferase activity was increased almost 3.3-fold by forced expression of HOXA1 compared with that of the respective vector only control (Fig. 2CGo). We also used a DN human STAT3 expression construct to assess whether the HOXA1-mediated increase in {alpha}2-macroglobulin promoter transcription is STAT3 dependent. The DN STAT3 construct possesses an amino acid substitution at tyrosine residue 705 (Tyr-Phe) required for STAT3 activity, and this mutation renders STAT3 inactive (27). As shown in Fig. 2CGo, the HOXA1-stimulated increase in {alpha}2-macroglobulin promoter activity was largely prevented by transient transfection of DN STAT3. In the absence of serum and growth factors (insulin and epidermal growth factor), {alpha}2-macroglobulin promoter-mediated luciferase activity was increased 2.3-fold by forced expression of HOXA1 compared with that of the respective vector (data not shown). Therefore, HOXA1 increased STAT3-mediated transcription in human mammary epithelial cells.

We next investigated the effect of forced expression of HOXA1 on STAT5B-mediated transcriptional activation using a serine protease inhibitor (SPI) 2.1 promoter-luciferase reporter construct. The expression of SPI 2.1 is tightly controlled by STAT5A and 5B at the transcriptional level (28). We observed a 3.6-fold increase in SPI 2.1 promoter-mediated transcription with forced expression of HOXA1 compared with the MCF10A-VECTOR control cell line (Fig. 2DGo). To determine whether the HOXA1-stimulated increase in SPI 2.1 transcription was STAT5B dependent, cells were transiently transfected with a DN STAT5B construct. The DN STAT5B construct has an amino acid substitution at tyrosine residue 699 (Tyr-Phe) of the wt STAT5B protein required for activity and, therefore, renders this protein inactive. In the presence of DN STAT5B, the HOXA1-stimulated increase in SPI 2.1 promoter activity was significantly reduced (Fig. 2DGo). Thus, STAT5B-mediated transcription is regulated by HOXA1 in human mammary epithelial cells.

Characterization of STAT3 and 5B expression and activation in human mammary epithelial cells
To determine the functional consequences of HOXA1-mediated increase in STAT3 and 5B expression and activation, we transfected human mammary epithelial cells with wt STAT3, CA STAT3, wt STAT5B, and CA STAT5B constructs. Substitution of two Cys residues for Ala662 and Asn664 located within the C-terminal loop of the SH2 domain of STAT3 produces a mutant protein termed CA-STAT3 that dimerizes spontaneously, binds to DNA, and activates transcription (29). CA-STAT5B has a single-point mutation located in the SH2 domain, which results in an amino acid substitution (N642H), and is constitutively phosphorylated and activated in the absence of cytokine stimulation (30). MCF-10A cell lines were transiently transfected with wt or CA STAT3 and wt or CA STAT5B. Forced expression of wt STAT3 or CA STAT3, in MCF10A cells compared with a vector-transfected control cell line was verified by RT-PCR analysis (Fig. 3AGo). Accordingly, MCF-10A cells transfected with wt STAT3 or CA STAT3 also demonstrated higher levels of STAT3 protein compared with vector-transfected cells (Fig. 3CGo). We also examined STAT3 protein tyrosine phosphorylation (at position 705) in MCF10A cells expressing the wt STAT3 or CA STAT3 using Western blot analysis. Forced expression of wt STAT3 or CA STAT3 concomitantly increased the tyrosine phosphorylation of STAT3 (Fig. 3CGo). Cells transfected with either wt STAT3 or CA STAT3 had equal amounts of STAT3 tyrosine phosphorylation. However, CA STAT3 generates a protein that dimerizes spontaneously, binds to DNA, and activates transcription without the requirement for tyrosine phosphorylation of STAT3 (29). Therefore, we next investigated the transactivation potential of the STAT3 variants in MCF-10A cells. MCF-10A cells were transfected with the STAT3 variants and the {alpha}2-macroglobulin promoter-luciferase construct. Transfection with wt STAT3 demonstrated a 3.7-fold induction of {alpha}2-macroglobulin promoter activity compared with that of the respective vector control (Fig. 3EGo). Transfection with the constitutively active STAT3 variant dramatically enhanced {alpha}2-macroglobulin promoter-driven luciferase gene expression by more than 13.6-fold.


Figure 3
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  		FIG. 3. Expression and activation of STAT3 or STAT5B variants in immortalized human mammary epithelial cells. MCF10A cells were transiently transfected with pcDNA3.1, wt STAT3, CA STAT3, wt STAT5B, and CA STAT5B constructs in complete media for 24 h. The level of STAT3 mRNA (A) or STAT5B mRNA (B) was determined by RT-PCR. The level of expression and tyrosine phosphorylation of STAT3 protein (C) and STAT5B protein (D) was examined with antibodies to STAT3/5B (top) or phosphorylated STAT3/5B (bottom), respectively. Transcriptional activity of the STAT3 variants on the {alpha}2-macroglobulin promoter (E) or STAT5B variants on the SPI 2.1 promoter (F) were measured by reporter assay, as indicated. β-Actin was used as a loading control where appropriate. *, P < 0.001, significant when compared with control (pCDNA3); **, P <0.001, significant when compared with wt transfection.

 
Similarly, we also verified the expression and transactivation functions of the wt STAT5B, and CA STAT5B constructs in MCF10A cells. As expected, transient transfection with wt STAT5B or CA STAT5B resulted in increased levels of STAT5B mRNA and protein (Fig. 3Go, B and D). Increased STAT5B tyrosine phosphorylation (Tyr699) was also observed in wt STAT5B and CA STAT5B transfected cells. The degree of STAT5B tyrosine phosphorylation in CA STAT5B cells was greater than in cells expressing the wt STAT5B (Fig. 3DGo). In MCF10A cells, CA STAT5B is constitutively phosphorylated and, therefore, constitutively active. Therefore, we further investigated the transactivation potential of the STAT5B variants using the SPI 2.1 promoter-luciferase reporter construct. This analysis demonstrated a 6.6-fold increase in SPI 2.1 promoter activity after transient transfection with wt STAT5B and a 28-fold increase with CA STAT5B when compared with the vector controls (Fig. 3FGo). Therefore, forced expression of wt STAT3 or wt STAT5B results in increased activation of STAT3 or STAT5B, respectively.

Forced expression and activation of STAT3 or STAT5B in MCF-10A cells result in increased cell proliferation, cell survival, and oncogenic transformation
Recent studies have demonstrated that constitutive activation of STAT3 and 5B in a variety of tumors directly contributes to an increase in cell number by inducing cell proliferation and decreasing apoptosis (8). Thus, we sought to determine the effect of STAT3 and 5B expression and activation in human mammary epithelial cells on parameters of cell growth. To investigate the role of the STAT3 and 5B pathway on total cell number, we transiently transfected MCF-10A cells with the STAT3 or STAT5B wt and CA variants, plated cells in identical numbers, and the cell number was determined after 48 h. We observed, with either wt STAT3 or wt STAT5B, an increase in cell number when compared with the vector control cell line (Fig. 4AGo). Constitutive activation of STAT3 or STAT5B dramatically increased total cell number above that of wt STAT3 and 5B over 48 h. Increased cell number may be achieved by either increased proliferation, decreased apoptotic cell death, or a combination of the two. Therefore, we proceeded to determine the relative contribution of these processes to the observed increase in cell number as a consequence of forced expression of STAT3 or STAT5B wt and CA variants. Comparison of nuclear 5-bromo-2-deoxyuridine (BrdU) incorporation between different STAT3 or STAT5B variants demonstrated that forced expression of wt STAT3 or 5B resulted in a slight increase in cell cycle progression, whereas constitutively active STAT3 or 5B expression dramatically increased cell cycle progression above that of the vector control (Fig. 4BGo). However, the proliferative effect stimulated by STAT3 was minimal compared with STAT5B. Apoptotic cell death was significantly reduced by forced expression of wt STAT3 or 5B, or CA STAT3 or 5B when compared with vector-transfected cells in serum-free conditions (Fig. 4CGo). Cells transfected with the CA STAT3 or 5B expression vectors survived better than those transfected with wt STAT3 or 5B. Thus, constitutive activation of STAT3 reduced apoptosis significantly more in mammary epithelial cells than STAT5B.


Figure 4
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  		FIG. 4. Effect of expression of STAT3 variants or STAT5B variants in immortalized human mammary epithelial cells on cell number, cell cycle progression, apoptosis, and oncogenic transformation. MCF10A cells were transiently transfected with pcDNA3.1, wt STAT3, CA STAT3, wt STAT5B, and CA STAT5B constructs in serum-free media or in complete media as indicated. Total cell number (A), cell cycle progression (BrdU incorporation) (B), apoptotic cell death (C), and soft agar colony formation (D) were determined under the indicated conditions as detailed in Materials and Methods. Results represent the means ± SD of triplicate determinations. *, P < 0.001, significant when compared with control (pCDNA3); **, P <0.001, significant when compared with respective wt transfection.

 
To address the possible functional involvement of increased expression of STAT3 or STAT5B in the oncogenic transformation of immortalized human mammary epithelial cells, we again introduced wt STAT3 or 5B and CA STAT3 or 5B into the MCF10A cell line by transient transfection. As observed in Fig. 4DGo, expression of wt STAT3 and 5B in MCF10A cells resulted in the formation of numerous colonies in soft agar, whereas vector-transfected cells were largely ineffective in colonization of soft agar. Transfecting MCF-10A cells with CA STAT3 or 5B dramatically increased the number of colonies formed in soft agar when compared with cells transfected with wt STAT3 or 5B. Thus, increased expression and activation of STAT3 or 5B are sufficient to oncogenically transform immortalized human mammary epithelial cells.

Together, these data indicate that STAT3 and STAT5B regulate cell cycle progression, cell survival, and promote oncogenic transformation in human mammary epithelial cells.

HOXA1-stimulated cellular growth is STAT3 and 5B dependent
We have previously demonstrated that forced expression of HOXA1 in human mammary epithelial cells resulted in a significant increase in total cell number. Because STAT3 and STAT5B expression is regulated by HOXA1, and expression of STAT3 or STAT5B results in increased total cell number, proliferation, and survival, we reasoned that a proportion of the HOXA1-stimulated increase in total cell number was likely to be mediated by STAT3 or STAT5B. Therefore, we transiently transfected MCF10A-VECTOR and MCF10A-HOXA1 cells with DN STAT3 or DN STAT5B expression vectors. As observed in Fig. 5AGo, HOXA1 stimulated an increase in total mammary epithelial cell number, and this was largely prevented by transient transfection of DN STAT3 or DN STAT5B. We next determined whether HOXA1-mediated stimulation of proliferation was STAT3 and STAT5B dependent. As observed in Fig. 5BGo, the use of DN STAT3 or DN STAT5B reduced the HOXA1-stimulated increase in BrdU incorporation in MCF10A cells. Thus, the HOXA1-mediated increase in cell proliferation was demonstrated to be mediated partially by the STAT3 and STAT5B pathways.


Figure 5
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  		FIG. 5. STAT3 or STAT5B mediates HOXA1-stimulated increase in total cell number, cell proliferation, survival, and oncogenic transformation. MCF10A-Vector and MCF10A-HOXA1 cells were transiently transfected with either DN STAT3 or DN STAT5B constructs in serum-free media or in complete media as indicated. Total cell number (A), cell cycle progression (B), apoptotic cell death (C), the level of Bcl-2 mRNA (D), and soft agar colony formation (E) were determined in both cell lines as described in the Materials and Methods. Densitometric quantification determined Bcl-2 mRNA level in pCDNA3 (3.16 ± 0.23-fold difference), DN STAT3 (2.47 ± 0.18-fold difference; P < 0.05), or DN STAT5B (2.31 ± 0.32-fold difference; P < 0.05) transfected MCF10A-HOXA1 cells compared with that of MCF10A-Vector. Results represent the mean of triplicate determinations. Bars represent SE. *, P < 0.001, significant when compared with control; **, P <0.001, significant when comparing transfected and control experiments from the same cell line.

 
We also examined the dependence of HOXA1-stimulated cell survival on STAT3 or STAT5B. As shown in Fig. 5CGo, forced expression of HOXA1-protected mammary epithelial cells from apoptosis due to serum deprivation and transfection of cells with DN STAT3 or DN STAT5B constructs substantially reduced the survival effect of HOXA1. We have previously demonstrated that forced expression of HOXA1 in mammary epithelial cells resulted in decreased apoptotic cell death, and this was mediated by a specific increase in the antiapoptotic protein, B-cell leukemia/lymphoma-2 (Bcl-2) (3). Thus, HOXA1 may use the STAT3 or STAT5B pathway to mediate up-regulation of Bcl-2. Therefore, we examined the effect of inhibition of the STAT3 or STAT5B pathway on the HOXA1-stimulated increase in Bcl-2 mRNA. Concordant with previous observations, MCF-10A-HOXA1 cells exhibited higher expression of Bcl-2 mRNA in comparison with MCF-10A-vector cells in serum-free media (Fig. 5DGo). The HOXA1-stimulated increase in Bcl-2 mRNA was significantly reduced by transfection of cells with either DN STAT3 or DN STAT5B constructs. Thus, the increase in Bcl-2 mRNA levels resulting from forced expression of HOXA1 is STAT3 and STAT5B dependent.

DN STAT3 and 5B suppress HOXA1-mediated oncogenic transformation of immortalized human mammary epithelial cells
Recently, we have demonstrated that forced expression of HOXA1 results in de novo oncogenic transformation of immortalized human mammary epithelial cells (3). Here, we have observed that expression and activation of STAT3 and STAT5B increased proliferation, survival, and anchorage independent growth. Therefore, it seems plausible that increased activation of STAT3 or STAT5B by HOXA1 may also mediate HOXA1-stimulated oncogenic transformation of immortalized human mammary epithelial cells. Therefore, we examined the ability of DN STAT3 and 5B to prevent soft agar colony formation in MCF-10A cells consequent to forced expression of HOXA1. As expected, forced expression of HOXA1 in MCF-10A cells produced colonies in soft agar, whereas the control vector-transfected cells exhibited a small number of spontaneously formed colonies (Fig. 5EGo). The use of either DN STAT3 or DN STAT5B substantially reduced HOXA1-stimulated soft agar colony formation by MCF-10A cells, indicative that the STAT3 and STAT5B pathway mediates HOXA1-induced oncogenic transformation of human mammary epithelial cells.

We also examined whether the effect of forced expression of HOXA1 on the architecture of acinar structures formed by immortalized human mammary epithelial cells in Matrigel is STAT3 and 5B pathway dependent. Three-dimensional acinar structures were generated by plating MCF10A-VECTOR and MCF10A-HOXA1 cells as single cells in Matrigel. After 14 d in culture, MCF10A-VECTOR cells formed small acinar like structures with a hollow lumen, reminiscent of ductal formation in the mammary gland (31, 32). Forced expression of HOXA1 in immortalized human mammary epithelial cells resulted in the generation of large disorganized multi-acinar structures (Fig. 6AGo). Of the formed acinar structures produced by MCF10A-HOXA1 cells, 61% exhibited large disorganized multi-acinar structures. After transfection with DN STAT3, only 18% of MCF10A-HOXA1 cells formed multi-acinar structures, and the majority of spheroids possessed normal acinar architecture (Fig. 6BGo). Similarly, 24% of MCF10A-HOXA1 cells formed multi-acinar spheroids after transfection with DN STAT5B (Fig. 6AGo). Thus, STAT3 and STAT5B are necessary for HOXA1-mediated disruption of acinar structure accompanied with luminal filling.


Figure 6
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  		FIG. 6. Forced expression of HOXA1 mediates disruption of acinar morphology and luminal filling in a STAT3 or STAT5B pathway dependent manner. MCF10A-VECTOR and MCF10A- HOXA1 cells were MCF10A-VECTOR and MCF10A-HOXA1 cells transiently transfected with pcDNA3 vector or DN STAT3 or DN STAT5B constructs and cultured in Matrigel for 16 d. Representative phase contrast images of MCF10A-VECTOR and MCF10A-HOXA1 cells transfected with Vector (A), DN STAT3 or DN STAT5B constructs (B). The percentage of acinar structures with filled lumen was quantified. The results are presented as the mean of triplicate determinations. Bars represent SE. *, P < 0.001, significant when compared with control; **, P <0.001, significant when comparing transfected and control experiments from the same cell line.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously demonstrated that HOXA1 increases oncogenicity of mammary carcinoma cells and stimulates oncogenic transformation of immortalized human mammary epithelial cells through the p44/42 MAPK pathway (4). However, inhibition of the p44/42 MAPK pathway with a MEK1 inhibitor did not completely abrogate HOXA1-mediated oncogenic effects, which suggested the involvement of one or more additional signal transduction pathways. In the present study, we observed that HOXA1 stimulates STAT3, 5A, and 5B gene expression in immortalized human mammary epithelial cells. Increased STAT3 and 5B activation, by virtue of increased STAT3 or 5B expression, was used by HOXA1 to execute a program of cell proliferation, survival, and anchorage independent growth, resulting in the generation of large disorganized multi-acinar structures with filled lumina. Thus, we have demonstrated that in addition to the p44/42 MAPK pathway, STAT3 and 5B-mediated signaling is another pivotal component in HOXA1-mediated oncogenic transformation of immortalized human mammary epithelial cells.

STAT proteins regulate cell growth, differentiation, and death in a number of different cell types (33). STAT1, STAT3, STAT5A, and STAT5B are thought to be responsible for cell survival and growth, whereas STAT2, STAT4, and STAT6 are preferentially involved in differentiation (8). STAT protein activity can be regulated by numerous cytokines, hormones, and growth factors, and regulation is tightly coordinated in the mammary gland. Distinct and largely reciprocal regulation of individual STAT family members suggests specific developmental roles. Concordantly the phenotypes of STAT5A null and STAT3 tissue-specific knockouts have impaired differentiation/lactation and delayed involution, respectively (34, 35, 36). In addition to playing an important role in normal growth and development, STAT3 and 5B have also been implicated in the development and progression of breast cancer (9, 10, 11, 12). Constitutive activation of STAT3 has been shown to be responsible for malignant tumor progression in a broad spectrum of tumor types and various cancer cell lines. STAT3 is activated by a number of different oncoproteins (13) such as v-Src, v-Ros, v-Eyk, as well as constitutively activated IGF-I receptor tyrosine kinase (37, 38, 39, 40). Inhibition of STAT3 function by STAT3β reduces anchorage independent growth and tumorigenesis (40). STAT5B has also been implicated in human cancer (40, 41). Recent reports illustrated that STAT5B, and not STAT5A, has a pro-proliferative effect in several cancers, including breast cancer (41, 42, 43). In addition, STAT5B is the predominantly expressed STAT family member in breast cancer cell lines (42, 44). Upon treatment with prolactin, both STAT5A and 5B are translocated to the nucleus of COS-1 cells, whereas STAT5B, but not STAT5A, translocates to the nucleus in the presence of constitutive active c-Src (45). In addition, inhibition of STAT5B but not STAT5A repressed tumor growth in xenograft models of head and neck carcinomas (41, 43). Activation of STAT3 and 5B is frequently found in breast cancer (9, 10, 11, 12). STAT3 and 5B have also been classified as protooncogenes because an activated form of STAT3 and 5B can mediate oncogenic transformation in immortalized fibroblasts and tumor formation in nude mice (29, 41). STAT3 as well as STAT5 induces progression through the cell cycle, prevents apoptosis, and up-regulates oncogenes, such as c-myc, Bcl-2, and Bcl-XL (46).

Here, we have demonstrated that in addition to the p44/42 MAPK pathway (4), the STAT3 and 5B signaling pathway is also required for HOXA1-mediated oncogenic transformation of human mammary epithelial cells. Our results have demonstrated that HOXA1 expression results in increased levels of STAT3 and 5B mRNA expression, whereas STAT4 expression was decreased. STAT3 and 5B transcription has been activated through several mechanisms, including through epigenetic regulation of STAT5B promoter activity (23). In addition, the STAT3 promoter has been transcriptionally up-regulated by cisplatin and through STAT3 itself (22). Interestingly, analysis of the promoter regions of the human STAT3, 5A, and 5B genes identified the presence of multiple potential HOXA1 core-binding motifs in the promoter sequences of STAT3 (23), STAT5A-A3 (five), and STAT5B-B5 (six). Therefore, STAT3, 5A, and 5B transcriptional up-regulation may be mediated by direct binding of HOXA1 to HOXA1 core-binding motifs in the promoter sequences of these genes, however, this remains to be elucidated.

Autocrine hGH is a human mammary epithelial oncogene dependent on HOXA1 for oncogenic transformation (5, 47). Several signal transduction pathways, common to a number of growth factors, are activated by hGH binding to the hGH receptor. Among these are the JAK2/STAT pathway and the p44/42 MAPK pathway (Fig. 7Go) (6, 48). In addition to the direct activation of the p44/42 MAPK pathway, we have recently demonstrated that hGH also indirectly modulates this pathway through up-regulation of HOXA1 mRNA and protein expression in human mammary carcinoma cells (3, 5). Forced expression of HOXA1 was demonstrated to regulate components of the p44/42 MAPK pathway, specifically up-regulating GRB2 and MEK1 mRNA and protein expression, and activity (4). Furthermore, we have demonstrated here that HOXA1 up-regulates STAT3 and STAT5b mRNA and protein expression. In addition, we have also observed on the array analysis (4) that expression of JAK2, one of the kinases responsible for phosphorylation of STAT3/5B, is also increased by HOXA1. Thus, the oncogenic effects of autocrine hGH are partially mediated through direct activation of the JAK2/STAT and the p44/42 MAPK pathways. HOXA1, controlling the expression of components of these pathways and, therefore, the extent of activation, would act to synergize the activation of these pathways by autocrine hGH. This concept is summarized in Fig. 7Go.


Figure 7
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  		FIG. 7. Mechanism of autocrine hGH-stimulated oncogenicity. Autocrine hGH stimulates the expression of HOXA1 in a JAK2-dependent manner (3 ). Increased HOXA1 expression results in increased expression of components of the p44/42 MAPK pathway (GRB2 and MEK1) (4 ) and the JAK-STAT pathway (STAT3 and 5B). Autocrine hGH also stimulates the activation of these pathways (54 55 ), resulting in increased cell proliferation, cell survival, and oncogenicity.

 
Previous studies have demonstrated that activation of STAT3 and 5 signaling regulates the expression of numerous genes involved in te control of cell growth and survival (49, 50). Consistent with this previous observation, we have shown that the antiapoptotic protein Bcl-2 is a downstream target of STAT3 and 5B (51, 52). The HOXA1-mediated effect on cell survival and protection from anoikis requires the transcriptional up-regulation of Bcl-2 (3). Consequently, inhibition of Bcl-2 function prevents HOXA1-mediated survival and oncogenic transformation of an immortalized human mammary epithelial cell line. We have previously demonstrated that HOXA1-stimulated up-regulation of Bcl-2 is partially mediated by the p44/42 MAPK pathway (4). Here, we demonstrate that abrogation of STAT3 and 5B activities with DN STAT3 or 5B suppresses HOXA1-induced expression of Bcl-2 and induced cell death. Thus, HOXA1 may activate Bcl-2 expression through both p44/42 MAPK, and STAT3 and 5B signal transduction pathways (51, 52, 53).

In summary, we have identified HOXA1 as a novel upstream regulator of STAT3 and 5B expression and activation in immortalized human mammary epithelial cells. Increased activation of the STAT3 and 5B pathway is required for HOXA1-stimulated oncogenic transformation.


   Footnotes
 
This work was supported by grant support from the Marsden Fund, Royal Society of New Zealand; The Breast Cancer Research Trust, New Zealand; The National Research Centre for Growth and Development, New Zealand; and The Foundation for Research, Science and Technology, New Zealand.

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 14, 2008

Abbreviations: Bcl-2, B-cell leukemia/lymphoma-2; BrdU, 5-bromo-2-deoxyuridine; CA, constitutively active mutant; DN, dominant negative; GRB2, growth factor receptor-bound protein 2; HOX, homeobox; JAK, Janus kinase; MEK, MAPK kinase; SPI, serine protease inhibitor; STAT, signal transducer and activator of transcription; wt, wild type.

Received September 25, 2007.

Accepted for publication February 6, 2008.


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 Introduction
 Materials and Methods
 Results
 Discussion
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