CG&D
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cell Growth & Differentiation

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Humphreys, R. C.
Right arrow Articles by Hennighausen, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Humphreys, R. C.
Right arrow Articles by Hennighausen, L.
Cell Growth & Differentiation Vol. 10, 685-694, October 1999
© 1999 American Association for Cancer Research


Articles

Signal Transducer and Activator of Transcription 5a Influences Mammary Epithelial Cell Survival and Tumorigenesis1

Robin C. Humphreys2 and Lothar Hennighausen

Laboratory of Genetics and Physiology, National Institute of Digestive, Diabetes and Kidney Disease, NIH, Bethesda, Maryland 20892

Abstract

The mammary gland undergoes extensive tissue remodeling and cell death at the end of lactation in a process known as involution. We present evidence that the prolactin-activated transcription factor signal transducer and activator of transcription 5a (Stat5a) has a crucial role in the regulation of cell death during mammary gland involution. In a transforming growth factor-{alpha} transgenic mouse model that exhibited delayed mammary gland involution, the absence of Stat5a facilitated involution-associated changes in morphology of the gland and the extent and timing of programmed cell death. These Stat5a-dependent changes also affected epidermal growth factor receptor-initiated mammary gland tumorigenesis. Overexpression of the transforming growth factor {alpha} transgene in the mammary epithelium reproducibly generated mammary hyperplasia and tumors. In the presence of the activated epidermal growth factor receptor, deletion of Stat5a delayed initial hyperplasia and mammary tumor development by 6 weeks. These observations demonstrate that Stat5a is a survival factor, and its presence is required for the epithelium of the mammary gland to resist regression and involution-mediated apoptosis. We also suggest that Stat5a is one of the antecedent, locally acting molecules that initiate the process of epithelial regression and reorganization during involution.

Introduction

Involution is the phase of mammary gland development that remodels the molecular and morphological characteristics of the gland after lactation. The systemic levels of prolactin, one of the dominant hormones in the stimulation and maintenance of lactational competence of the gland, decreases at the onset of involution (1) . The characteristics of the biological activity and regulation of prolactin argue for prominent involvement of this systemic hormone in the induction of mammary gland involution. Prolactin secretion is tightly regulated and is stimulated by suckling. The half-life of prolactin is very short in the blood, and there is evidence that exogenous treatment with prolactin can delay involution at certain stages of development (2 , 3) . Despite the clear role of this systemic hormone in the initiation of involution, the role of its subservient signal transduction pathways in the regulation of the morphological and transcriptional changes of involution is unknown. The prolactin-activated transcription factor Stat5a3 is essential for the development and differentiation of the mammary gland (4) . Stat5a phosphorylation sharply declines within 12 h after weaning, suggesting a role in the onset of involution. In fact, Stat5a has been implicated in the regulation of apoptotic processes in other tissues (5 , 6) .

Mammary gland involution can be separated into two molecularly and morphologically distinct phases. The first phase is defined by changes in the expression patterns of genes involved in milk synthesis (WAP and ornithine decarbox-ylase), cell survival and death (Bax, Bcl-2, Bcl-xs, SGP-2, interleukin-converting enzyme 1, and p53), and proteins that act to regulate potential survival factors (insulin-like growth factor-1, insulin-like growth factor-binding protein 5, and TGF-ß) and modulate interactions with the extracellular matrix (tissue inhibitor of metalloproteinase 1; Refs. 3 and 7, 8, 9, 10, 11) . Importantly, dephosphorylation of the prolactin-dependent transcription factors Stat5a and Stat5b can be detected within 12 h of the initiation of involution (12) . Despite the high levels of apoptosis early in involution, the integrity of the alveolar basement membrane is not disrupted. The second phase of involution is characterized by increases in the expression of proteins and genes involved in reorganizing the extracellular matrix (gelatinase 1, stromelysin 1, and urokinase plasminogen activator) and abrogation of milk gene expression. These molecular changes precede the dramatic degradation of the lobuloalveolar acini and the absorption of apoptotic cells. It is presumed that changes in the concentrations of the systemic hormones prolactin, oxytocin, and progesterone, in concert with changes in the activity of locally acting factors, initiate and stimulate the process of involution.

The importance of the Stat proteins in the growth and differentiation of many cell types is exemplified by their ubiquitous expression and widespread utilization in the signal transduction pathways of multiple cytokines (13) . Stats are capable of transmitting both differentiative and proliferative signals, depending on the cellular and receptor context (14 , 15) . In the mammary gland, Stat5a activation by prolactin is required for functional differentiation of the epithelium during pregnancy and lactation (4 , 16) . Singular tyrosine phosphorylation by the prolactin receptor-associated intracellular kinase Jak-2 is obligatory to maintain Stat5a transcriptional activity (17) . However, novel alternative mechanisms of regulating Stat5a activity have been identified. Prolactin-activated signal transduction can tyrosine- and serine-phosphorylate multiple isoforms of the Stat5a molecules (18) . Furthermore, truncated forms of Stat5a have transcriptional dominant negative activity (19 , 20) and have been linked to the regulation of apoptosis (5) . In the liver, EGF can stimulate phosphorylation and nuclear translocation of the Stat5a isoform, Stat5b (21) . Importantly, serine phosphorylation of Stat5a by the prolactin-independent, MAPK pathway has been identified, (22) including a direct association between Stat5a and the serine kinase ERK-1/2(MAPK) (23) . Serine phosphorylation by the MAPK pathway is required for transcriptional activity of Stat1 and Stat3, but serine phosphorylation is not obligatory for prolactin-dependent transcriptional activity of Stat5 proteins (24) . Taken together, these data imply that Stat5a activity can be regulated by the prolactin and EGF signaling pathways and possesses diverse functional roles within a single cell that are dependent on its phosphorylation state and expressed isoforms.

Initiation and progression of breast cancer rely on the inappropriate temporal and qualitative activity of normal cellular signaling pathways (25) . These alterations in signaling activity often disrupt the normal mechanisms of mammary gland development. In one mouse model (WAP-TGF-{alpha}), overexpression of TGF-{alpha}, which binds to and activates the EGFR, delays mammary involution and transforms the mammary epithelium (26) . The prolactin signaling pathway has been implicated in loss of normal epithelial differentiation (4 , 16) and transformation (27 , 28) . The role of Stat5a in tumorigenesis is implicated by the appearance of Stat5a overexpression in leukemic (29 , 30) and oncogenic (31) transformed cells. Additionally, constitutive activation of the Jak/Stat pathway occurs in multiple leukemia-, v-abl-, and human T-cell lymphotrophic virus-transformed T cells (32 , 33) , and specific inhibition of constitutive Jak-2 activity in acute lymphoblastic leukemia can block cell growth and induce cell death (34) . We used the WAP-TGF-{alpha} transgenic and Stat5a-null mouse models to explore the in vivo role of Stat5a in mammary gland involution and tumorigenesis.

Results

Delayed Mammary Involution in the TGF{alpha}TG Mice Is Curtailed in the Absence of Stat5a.
The contribution of Stat5a in regulating involution and the establishment of mammary tumors was investigated in mice that carried the TGF-{alpha} transgene (TGF{alpha}TG mice) or were null for the Stat5a gene (Stat5aKO mice) and in Stat5a-null mice that expressed the transgene (Stat5aKOTGF{alpha} mice). H&E-stained mammary glands from TGF{alpha}TG, Stat5aKO, Stat5aKOTGF{alpha}, and WT nontransgenic control mice at day 18 of pregnancy and days 1, 3, and 7 of involution were examined by light microscopy (Fig. 1)Citation . Morphological differences were observed between the TGF{alpha}TG and Stat5aKOTGF{alpha} mammary glands within the first pregnancy and involution. TGF{alpha}TG mammary glands contained hyperproliferative alveoli and ducts. Alveolar lumina contained heterogenous eosin-positive staining material. A significant increase in the number of stromal cells was evident, in addition to increased deposition of stromal collagen (Fig. 1l)Citation . Involution was delayed in the TGF{alpha}TG mice, as reported previously (26) . Some cell loss and condensation of lobuloalveolar structures was observed during involution, but the gland lacked the dramatic epithelial regression of the WT gland (Fig. 1Citation , n versus p). Mammary tissue from pregnant Stat5aKOTGF{alpha} mice was histologically similar to pregnant tissue from TGF{alpha}TG mice but contained more epithelium than observed in the glands of Stat5aKO mice (Fig. 1, a–d)Citation . Secretory structures appeared to be more uniform and lacked the observed heterogeneous precipitations in the alveolar lumens. During involution, the Stat5aKOTGF{alpha} gland remodeled more rapidly and extensively than the TGF{alpha}TG gland (Fig. 1Citation , o versus p). This effect was enhanced after subsequent rounds of pregnancy and involution (data not shown). The primary and secondary ducts of the Stat5aKOTGF{alpha} mice lacked concentrations of hyperplastic cells like those found in the TGF{alpha}TG mice. On rare occasions, hyperplastic regions were evident after three or more rounds of pregnancy in Stat5aKOTGF{alpha} mice (data not shown).



View larger version (141K):
[in this window]
[in a new window]
 
Fig. 1. Involution is enhanced in the absence of Stat5a. H&E staining of inguinal mammary glands from Stat5a-null nontransgenic (NTGKO), WT, Stat5a-null TGF-{alpha} transgenic (KOTG), and TGF-{alpha} transgenic (TG) mice at 18 days of pregnancy (p18, a–d) and days 1 (i1, e–h), 3 (i3, i–l), and 7 (i7, m–p) of involution. Samples were collected from mice during or immediately after their first pregnancy. Compare epithelial condensation at day 3 and day 7 of involution in the WT (j) versus the KOTG (k) and TG (l). Bar in a, 200 µm.

 
Apoptosis and Proliferation Levels Are Altered in the Stat5aKOTGF{alpha} and TGF{alpha}TG Mice.
High levels of apoptosis, decreased proliferation, and the collapse of lobuloalveolar structures in the mammary epithelium are hallmarks of involution in the mammary gland (7) . To establish and de-fine the characteristics of mammary involution in the Stat5aKOTGF{alpha} and TGF{alpha}TG mice, the levels of apoptosis and proliferation were analyzed by TUNEL and BrdUrd immunohistochemistry, respectively (Tables 1Citation  and 2)Citation . WT glands exhibited increased levels of apoptosis within 1 day of the initiation of weaning and exhibited maximal levels at day 3, as expected (9 , 35) . Consistent with an inhibition or delay in involution in the TGF{alpha}TG gland, the number of apoptotic cells was slightly reduced at day 1 of involution compared with WT and Stat5aKOTGF{alpha} glands (Fig. 2A)Citation . Apoptosis was reduced in the TGF{alpha}TG gland at day 7 when compared with WT gland at day 7. Whereas Stat5aKOTGF{alpha} mammary glands at day 18 of pregnancy (P = 0.02) and at day 1 of involution (P = 0.015) had significantly higher levels of apo-ptosis than TGF{alpha}TG and WT mammary glands over the same time period, the levels were similar at days 3 and 7. Interestingly, Stat5aKO mammary glands also displayed significantly high levels of apoptosis at day 18 of pregnancy and day 1 of involution (P = 0.03).


View this table:
[in this window]
[in a new window]
 
Table 1 Percentage of apoptotic cells detected with TUNEL

 

View this table:
[in this window]
[in a new window]
 
Table 2 Percentage of proliferating cells detected with BrdUrd labeling

 


View larger version (57K):
[in this window]
[in a new window]
 
Fig. 2. A, KOTG mammary glands display elevated levels of apoptotic cells during pregnancy and early involution. Apoptosis levels in Stat5a-null TGF-{alpha} transgenic (KOTG), TGF-{alpha} transgenic (TG), Stat5a-null nontransgenic (NTGKO), and WT mammary glands after analysis by the TUNEL technique. Values represent the percentage of cells (means ± SE) displaying the label in a minimum population of 1000 cells. Analysis was performed on mice during or immediately after their first pregnancy. Serial sections of tissue from the same mouse were used for the determination of proliferation and apoptosis. A minimum of three mice were analyzed from each genotype (see "Materials and Methods."). Total number of cells counted, 7.5 x 105. a, b, and c denote significant differences between the KOTG and NTGKO values when compared with apoptosis levels in WT glands at day 18 of pregnancy and day 1 of involution. Specific values for Fig. 2, A and BCitation , are shown in Tables 1Citation and 2Citation . B, proliferation is higher in TG and KOTG mammary glands through involution. Proliferation levels in Stat5a-null TGF-{alpha} transgenic (KOTG), TGF-{alpha} transgenic (TG), Stat5a-null nontransgenic (NTGKO), and WT mammary glands after analysis by BrdUrd labeling. Values represent the percentage of cells (means ± SE) displaying the label in a minimum population of 1000 cells. Analysis was performed on mice during or immediately after their first pregnancy. Serial sections of tissue from the same mouse were used for determination of proliferation and apoptosis. A minimum of three mice were analyzed from each genotype. Total number of cells counted, 6.5 x 105. a, b, c, and d significant differences between TG and KOTG samples when compared with WT at day 1, 3, and 7 of involution. C, proliferation analysis of mammary gland in Stat5a-null TGF-{alpha} transgenic mammary glands. This figure demonstrates the appearance of BrdUrd-labeled apoptotic cells in late pregnancy (p18) and days 1 and 7 of involution (i1 and i7). Mice were injected 2 h before sacrifice with 20 µg/g body weight BrdUrd, as described in "Materials and Methods." Gray arrows, BrdUrd-labeled apoptotic cells. Black arrowheads, intact BrdUrd-labeled nuclei. Bar in p18, 100 µm.

 
Levels of proliferation, as measured by BrdUrd incorporation, were equivalent in mammary tissue of pregnant Stat5aKOTGF{alpha}, TGF{alpha}TG, Stat5aKO, and WT mice (Fig. 2B)Citation . Generally, 6–10% of the cells in the pregnant gland were labeled with BrdUrd (Fig. 2B)Citation . Proliferation was low in involuting tissue from Stat5aKO and WT mice (<1%). In contrast, the TGF{alpha}TG and Stat5aKOTGF{alpha} glands had detectable, persistent proliferation levels of 4–7% throughout involution. Apoptotic cells were observed in Stat5aKOTGF{alpha} mammary glands at day 18 of pregnancy and day 1 of involution in animals labeled exclusively with BrdUrd (Fig. 2CCitation , arrows). The appearance of apoptotic, BrdUrd-labeled cells demonstrated that DNA synthesis in these cells preceded the initiation of PCD.

WAP-TGF-{alpha} Transgene Expression Is Present throughout Mammary Gland Development in Stat5aKOTGF{alpha} and TGF{alpha}TG Mice.
The WAP gene promoter has been exploited to target high-level expression of transgenes, including TGF-{alpha}, to the mammary epithelium of pregnant mice (36, 37, 38, 39, 40) . To assure that the hormonal and morphological changes associated with involution and the absence of Stat5a did not disrupt transgene expression, TGF-{alpha} expression was evaluated in mammary tissue of pregnant and involuting TGF{alpha}TG, WT, and Stat5aKOTGF{alpha} mice. Northern blot analysis of TGF-{alpha} transgene expression in TGF{alpha}TG and Stat5aKOTGF{alpha} mice revealed expression of an appropriately sized transcript at day 18 of pregnancy. Transgene expression persisted through days 1, 3, and 7 of involution in multiple animals (Fig. 3)Citation . This expression pattern deviates from the expression of the endogenous WAP gene, which declines at the initiation of involution (41) . Endogenous TGF-{alpha} expression was only detected in an overloaded lane containing wildtype tissue at day 1 of involution. TGF-{alpha} transgene expression persisted through multiple rounds of pregnancy (three to nine rounds) in TGF{alpha}TG and Stat5aKOTGF{alpha} mice (data not shown).



View larger version (40K):
[in this window]
[in a new window]
 
Fig. 3. TGF-{alpha} transgene expression persists through involution in KOTG and TG mammary glands. Northern analysis of TGF-{alpha} expression in TG, KOTG, and WT mammary glands. Total RNA (20 µg) was separated and analyzed on a single Northern blot. Each lane represents RNA from a single animal. Samples were collected from mice during or immediately after their first pregnancy. The TGF-{alpha} blot was exposed for 4 h at -70°C. EtBr, ethidium bromide staining of RNA.

 
The EGFR-activated Serine Threonine Kinase MAPK Remains Active throughout Involution.
Binding of TGF-{alpha} to the EGFR initiates receptor dimerization and inherent kinase activation, which stimulates the Ras/Raf/Mek signaling cascade. To establish that the EGFR-dependent pathway was being activated by the expression of the TGF-{alpha} transgene, Western blot analysis was performed on protein extracts from Stat5aKOTGF{alpha}, WT, Stat5aKO, and TGF{alpha}TG animals with an antibody that only recognizes the active (phosphorylated) form of MAPK. Active MAPK was detected in TGF{alpha}TG and Stat5aKOTGF{alpha} mammary glands at day 18 of pregnancy and days 1, 3, and 7 of involution (Fig. 4, A and B)Citation . Increased levels of the active form of this enzyme were detectable after multiple rounds of pregnancy in the TGF{alpha}TG and Stat5aKOTGF{alpha} mammary glands. Very low levels of active MAPK were detected in the nontransgenic Stat5aKO gland (Fig. 4C)Citation . Levels were also very low in pregnant and involuting WT mammary glands (Fig. 4D)Citation . A significant level of MAPK is present in the lactating, WT mammary gland.



View larger version (44K):
[in this window]
[in a new window]
 
Fig. 4. MAPK is active during pregnancy and involution in TG and KOTG mammary glands. Phosphorylation of MAPK was examined by Western blot analysis with an antibody specific for the active phosphorylated form of MAPK and is shown in the top panel of each section. 18p, i1, i3, and i7 represent samples collected from TG (A) KOTG (B), NTGKO (C), and WT (D) mammary glands. 4L, a control sample from a WT, 4 day lactating mammary gland. {alpha}, a sample from a KOTG mammary gland after three pregnancies. ß, a TG mammary gland after three pregnancies. Each immunoblot was stripped and reblotted with an antibody to MAPK to determine loading, and the result is shown directly under the phosphorylated MAPK immunoblot in each panel. Arrows in A indicate the two subunits of MAPK, ERK-1 (top) and ERK-2 (bottom).

 
Stat5a Proteins Are Phosphorylated during Involution in TGF{alpha}TG and Stat5aKOTGF{alpha} Mice.
Stat5a is phosphorylated on tyrosine 694 by Jak-2, which is associated with the dimerized prolactin receptor (13) . Stat5a must remain phosphorylated to maintain its transcriptional activity. One of the earliest molecular events after the onset of involution is the dephosphorylation of Stat5a and Stat5b (42) . Immunoprecipitation and Western blot analysis of protein extracts from TGF{alpha}TG mice detected high levels of tyrosine-phosphorylated Stat5a and Stat5b at days 1, 3, and 7 of involution (Fig. 5)Citation . Conversely, the phosphorylation of Stat5a and Stat5b was decreased at day 1 and remained low to day 7 of involution in WT controls, as reported previously and shown in Fig. 5BCitation (42) . Additionally, tyrosine-phosphorylated Stat5a and Stat5b were also detected by immunoprecipitation and Western blot analysis in multiple individual TGF{alpha}TG tumors (data not shown). Stat5aKOTGF{alpha} mammary glands, lacking any Stat5a protein, were assayed for the phosphorylation of Stat5b to determine whether there was a compensation for the absence of Stat5a phosphorylation. A slight increase in Stat5b phosphorylation was detected on days 1, 3, and 7 of involution relative to WT controls.



View larger version (45K):
[in this window]
[in a new window]
 
Fig. 5. A, Stat5a remains phosphorylated during involution in TG mammary glands. Stat5a and Stat5b phosphorylation was examined in KOTG and TG mice mammary glands. Protein extracts were immunoprecipitated with anti-Stat5a antibodies (Stat5a, 1:2 x 105) or anti-Stat5b antibodies (Stat5b, 1:1 x 105) overnight. Immunoprecipitates were separated by PAGE and immunoblotted with anti-phosphotyrosine (p-Tyr, 1:1 x 103) antibodies. Samples were collected from mice during or immediately after their first pregnancy. Immunoblots probed initially with p-Tyr were stripped and reblotted with anti-Stat5a antibodies in the TG samples (5a/5b; 5a/5b) or anti-Stat5b antibodies in the KOTG samples (5a/5b; 5a/5b), respectively, to determine protein loading. i1, i3, and i7, days 1, 3, and 7 of involution, respectively. B, Stat5a and Stat5b are dephosphorylated during involution. Stat5a and Stat5b phosphorylation was examined in NTGKO and WT mice mammary glands. Protein extracts were immunoprecipitated with anti-Stat5a antibodies (Stat5a, 1:2 x 105) or anti-Stat5b antibodies (Stat5b, 1:1 x 105) overnight. Immunoprecipitates were separated by PAGE and immunoblotted with anti-phosphotyrosine (p-Tyr, 1:1 x 103) antibodies. Samples were collected from mice during or immediately after their first pregnancy. Immunoblots probed initially with p-Tyr were stripped and reblotted with anti-Stat5a antibodies or anti-Stat5b antibodies to determine protein loading. p18, i1, i3, and i7, day 18 of pregnancy and days 1, 3, and 7 of involution, respectively.

 
TGF-{alpha}-initiated Mammary Tumorigenesis Is Delayed in Stat5aKOTGF{alpha} Mice.
The rat TGF-{alpha} transgene under control of the WAP gene promoter induces mammary tumors in mice bred ad lib after three pregnancies (26) . The contribution of Stat5a to the establishment of mammary hyperplasias and tumors was investigated in TGF{alpha}TG and Stat5aKOTGF{alpha} mice. Stat5aKOTGF{alpha} and TGF{alpha}TG mice were bred ad lib and examined after each round of pregnancy for palpable hyperplasias and tumors. All 48 TGF{alpha}TG mice contained palpable hyperplasia in multiple glands by the third pregnancy. TGF{alpha}TG mammary glands consistently displayed a swollen appearance by the second pregnancy, emblematic of hypertrophic glands and hyperplasias (data not shown). Gross histological examination revealed the presence of multiple hyperplastic alveolar nodules in multiple glands. TGF{alpha}TG glands also contained fluid-filled cysts and stromal proliferation. Some removed tumors were characterized by abnormal hyperproliferation of glandular structures with relatively little hyperplasia. The latency of mammary hyperplasia and tumor appearance and the histology of the hyperplasias and tumors were consistent with the results described previously (26) . Hyperplasia and hypertrophy appeared less frequently in the Stat5aKOTGF{alpha} mammary gland. There was a significant difference in the rate of initial hyperplasia and tumor formation between the TGF{alpha}TG and Stat5aKOTGF{alpha} mice, (P = 0.003; Fig. 6Citation ). The initial appearance in the Stat5aKOTGF{alpha} mice was delayed 1.5 months compared with the TGF{alpha}TG mice. In Stat5aKOTGF{alpha} mice that did develop hyperplasias or tumors, usually only a single gland was involved, as compared with multiple gland involvement in the TGF{alpha}TG mice.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 6. Absence of Stat5a delays EGFR-mediated hyperplasia and tumor formation. Kaplan-Meier plot of the percentage of hyperplasia and tumor-free KOTG and TG mice over a 9-month time period. All mice were examined at each pregnancy for palpable tumors. There is a significant difference between the two curves (P = 0.0357). TG, n = 48. KOTG, n = 34.

 
Discussion

Loss of Stat5a Activity Is an Early Event in Mouse Mammary Gland Involution.
Dephosphorylation and hence inactivation of the transcription factors Stat5a and Stat5b and de novo phosphorylation of Stat3 occur in the first phase within several hours of pup removal (42) , implicating the Stats and prolactin as early regulatory molecules in the regression of the gland. Using a combination of transgenic and gene deletion mouse models, we have now demonstrated a role for Stat5a in involution that is dependent on its phosphorylation and can be regulated by constitutive EGFR signal transduction. Involution is abrogated on continued activation of the EGFR (and Stat5a) through the expression of a TGF-{alpha} transgene. In mammary tissue of TGF{alpha}TG mice, Stat5a remained phosphorylated, as did Stat5b. However, on deletion of the Stat5a gene, involution proceeded unabated, despite expression of the TGF-{alpha} transgene. Rapid involution in the absence of Stat5a was accompanied by increased levels of apoptotic cells. Our experiments not only demonstrate that Stat5a is a survival factor in vivo but also show that its presence is mandatory to avoid the initiation of PCD in mammary epithelial cells.

Despite the lack of known genetic targets for Stat5a in the involuting gland, there is evidence to suggest that prolactin and Stat5a can control cell survival. Activation of Stat5a has been implicated in the inhibition of apoptosis in T cells through interleukin 2 (6) and interleukin 9 (43) signaling. In addition, selection of apoptosis-resistant immature T cells leads to the activation of a dominant negative form of Stat5a (5) . Prolactin can also stimulate cell survival in mammary epithelial cells in vitro (44) , and prolactin and growth hormone can promote cell survival in the involuting rat mammary gland (2 , 3) . The presence of apoptotic cells in the mammary tissue of pregnant mice that lack the Stat5a gene and express the TGF-{alpha} transgene suggests that cell dependence on the presence of phosphorylated Stat5a precedes the actual systemic hormonal switch into involution. This observation also implies that the Stat5a-dependent mechanism of apoptosis induction may be hormonally independent. We did not observe this apoptotic phenomena in the WT and TGF-{alpha} transgenic mice. This increased number of apoptotic cells during pregnancy was also detected in BrdUrd-labeled Stat5aKOTGF{alpha} mice. Analysis of these glands revealed the presence of BrdUrd-labeled apoptotic cells at day 18 of pregnancy and at days 1, 3, and 7 of involution. These cells replicated their DNA and then proceeded directly into PCD. This association between the cell’s ability to replicate its DNA and the progression into the apoptotic program has been reported previously (45) . Cells may be programmed to initiate PCD before they enter the cell cycle; consequently, BrdUrd-labeled cells undergoing apoptosis are detected. Interestingly, not all the cells in the Stat5aKOTGF{alpha} pregnant gland undergo PCD. This suggests that some cells have obviated the requirement for Stat5a, possibly through an unknown compensatory mechanism. Alternatively, these cells may have a unique differentiation state that precludes them from entering the apoptotic pathway.

Despite persistent phosphorylation of Stat5a in involuting mammary tissue, TGF{alpha}TG mice did undergo involution-dependent epithelial reorganization and activation of PCD. This morphological change was characterized by moderate condensation of alveolar structures and loss of cells through the induction of PCD. We propose that the dephosphorylation of Stat5a is not the only guiding factor for apoptosis progression in the involuting mammary gland and that other parallel signals may be required for the complete induction of both phases of involution (12) .

Transgenic TGF-{alpha} Activates the EGFR and Ensures Persistent Phosphorylation of Stat5a.
TGF-{alpha} stimulates the EGFR and activates MAPK through the Ras/Raf/Mek signaling cascade. This is exemplified by the presence of the activated form of MAPK during involution in the TGF{alpha}TG and Stat5aKOTGF{alpha} mice. Persistent activation and phosphorylation of Stat5a in the TGF{alpha}TG mice could occur through three distinct mechanisms. The EGFR can directly phosphorylate Stat1 and Stat3 in the absence of Jak activation (46, 47, 48) . Although direct phosphorylation of Stat5a by the EGFR cannot be ruled out, it was not possible to coimmunoprecipitate this receptor with anti-Stat5a antibodies in the TGF{alpha}TG mammary gland (data not shown). Secondly, stimulation of MAPK by EGF can lead to phosphorylation and nuclear translocation of the Stat5a isoform Stat5b in the liver (21) . Support for this mechanism comes from results describing that Stat5a and its shorter isoforms can be tyrosine- and serine-phosphorylated by prolactin (18) , and Stat5a and Stat5b can be tyrosine- and serine-phosphorylated by prolactin-independent, MAPK-dependent mechanisms (22 , 49) . A direct interaction was recently demonstrated between MAPK(ERK1/2) and Stat5a in growth hormone-dependent signaling (23) . Growth hormone is known to influence mammary gland involution, although this effect is not as dominant as that observed for prolactin (2) . Thirdly, EGFR could activate Stat5a through Jak-2. The EGFR is known to use Jak-1 to activate Stat1 and Stat3 (14 , 50) and can modulate Stat5 expression in mammary epithelial cells (51) . Theoretically, the constitutively active EGFR in the TGF{alpha}TG and Stat5aKOTGF{alpha} mice could stimulate Jak-2, although this was not formally proven. Alternatively, EGFR could stimulate a secondary Jak, which inappropriately uses Stat5a as a substrate that has been demonstrated in IFN-{gamma} signaling (52 , 53) . Presumably, the stimulation of the EGFR kinase in the presence of the TGF-{alpha} transgene leads to phosphorylation and activation of a downstream kinase, possibly including a Jak, that maintains Stat5a in its phosphorylated state. We suggest that one or more of these mechanisms regulate the phosphorylation of Stat5a and control apoptosis in the mouse mammary gland.

Persistent Stat5a Activity Contributes to Mouse Mammary Tumorigenesis, and Its Absence Acts to Delay Hyperplasitic Epithelial Formation.
One of the dominant pathways commonly altered in breast cancer is the EGF signal transduction pathway (25 , 54) . Gene deletion studies in mice have demonstrated that the EGFR is not dispensable for normal mammary development (55 , 56) , and that aberrant signaling through this receptor induces neoplastic growth and frank tumors (26 , 57, 58, 59) . Using an EGF-dependent transgenic mouse model, we can now show that the persistent activation of Stat5a is linked to the transformation of the mammary gland. We suggest that the transformation of the mammary epithelium occurs at the expense of a loss of cell survival regulation, which is contingent on the persistent phosphorylation of Stat5a and permits the inappropriate survival of epithelial cells. The appearance of tumors and hyperplasias in Stat5aKOTGF{alpha} mice suggests that the absence of Stat5a will not completely block mammary epithelial transformation, and that the TGF-{alpha} transgene can activate alternative (non-Stat5a) pathways to bypass PCD and involution and support tumor formation. Such pathways may include Stat5b, which, under specific circumstances, substitutes for Stat5a (60) . The involvement of a single gland in a majority of the Stat5aKOTGF{alpha} tumors suggests that significant numbers of potentially transformed cells are destroyed during involution. This loss of cells probably contributes to the observed lower rate of tumor formation and the delay in initial tumor appearance. In spite of single gland involvement, survival of the Stat5aKOTGF{alpha} mice was no greater than that observed for the TGF{alpha}TG mice after initial tumor appearance.

Our findings that Stat5a is a survival factor and that its absence reduces mammary tumorigenesis support findings from other settings. Prolactin and other type 1 cytokines do possess mitogenic activity, implicating the associated Stat molecules with contributing to the mitogenesis of these organs (13) . Constitutive Stat activity has been observed in cells transformed by oncogenic viruses (32) and oncogenes (61) and in leukemia cells (29) . Moreover, constitutive activation of Jak expression can lead to transformation (27 , 62) .

Stat5a Is a Survival Factor for the Mammary Epithelium.
The absence of Stat5a disrupts the prolactin-mediated survival of the mammary epithelium. Whereas overexpression of TGF-{alpha} in the presence of an intact Stat5a results in delayed involution, apoptosis and involution in the absence of Stat5a lead to elimination of the majority of the epithelium. The dephosphorylation or lack of de novo phosphorylation and inactivation of Stat5a that occur during involution in the WT gland are mimicked by the absence of Stat5a in the Stat5aKOTGF{alpha} mice. Therefore, we propose that the presence of the activated form of this transcription factor prolongs cell survival, and its inactivation or deletion permits PCD to occur. These experiments demonstrate that Stat5a can act as a survival factor for the epithelium of the mammary gland. In addition, they illustrate that the inhibition and/or disruption of the mechanisms that regulate this involution process are critical in mammary gland tumorigenesis. The conclusions from this study lead us to hypothesize that the inhibition or disruption of Stat5a phosphorylation can lead to protection from transformation in the mammary gland. The application of Stat and/or Jak-specific tyrosine or serine kinase inhibitors may be an approach to blunt the development of mammary tumors originating from EGFR deregulation.

Materials and Methods

Materials.
The TGF-{alpha} cDNA probe was a kind gift from Dr. David Lee (Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC). The generation of the Stat5a antibodies has been described previously (63) . Phosphorylated MAPK antibody was purchased from Promega (Madison, WI). Anti-phosphotyrosine antibody was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Goat antirabbit and rabbit antimouse secondary antibodies were purchased from Transduction Laboratories (Lexington, KY).

Generation of TGF{alpha}TG and Stat5aKOTGF{alpha} Mice.
WAP-TGF{alpha}TG mice were a gift of Dr. Eric Sandgren (University of Wisconsin, Madison, WI Ref. 26 ). The generation of the Stat5a-null mice has been described previously (4) . TGF{alpha}TG mice were interbred with Stat5a-null mice (SvEv129/C57B6) to generate F1 founders that were hemizygous for Stat5a deletion (Stat5aHT) and transgenic for TGF-{alpha} expression. Female Stat5aHTTGF{alpha} mice were bred with Stat5aHTTGF{alpha} males to generate offspring that were transgenic for TGF-{alpha} and homozygous for deletion of the Stat5a gene. These mice were backcrossed for five generations to generate a pure inbred Stat5aKOTGF{alpha} strain of mice. The same generation TGF{alpha}TG and control WT littermates was used for molecular analyses. Confirmation of the presence of the TGF-{alpha} transgene was performed by PCR analysis with TGF-{alpha} forward primer 5''-TGTCAGGCTCTGGAGAACAGC-3' and reverse primer 5'-CACAGCGAACACCCACGTACC-3'. Stat5a PCR was performed with two sets of primers to knockout (null) and WT alleles: (a) Stat5a forward (5'-CTGGATTGACGTTTCTTACCTG-3') and Stat5a reverse (5'-TGGAGTCAACTAGTCTGTCTCT-3') and (b) Neo forward (5'-AGAGGCTATTCGGCTATGACTG-3') and Neo reverse 5'-TTCGTCCAGATCATCCTGATC-3'. PCR for all primers was performed with a denaturing step of 94°C for 3 min, followed by 30 cycles of 94°C for 40 s, 56°C for 40 s, and 68°C for 40 s, followed by 10 min at 68°C. Genotype of the mice was confirmed after tissue collection by Northern and Western blot analysis for TGF-{alpha} gene expression and absence of Stat5a protein expression, respectively. All animals were housed and handled according to the approved protocol established by the Institutional Animal Care and Use Committee and NIH guidelines.

Mammary Gland Collection.
Mammary glands were surgically removed from anesthetized and cervically dislocated mice at day 18 of pregnancy and days 1, 3, and 7 of involution. Day 1 of involution was designated as 24 h after the morning that the pups were born. Pups were immediately removed from the dam after birth and fostered onto a WT mother. The mammary lymph node was removed before homogenization of all glands. Tissues were prepared immediately for RNA and protein extraction, as described previously (42) . Whole (number 4) inguinal or (number 3) thoracic mammary glands were surgically excised from mice, spread on Omniset tissue cages (Fisher Scientific, Pittsburgh, PA), fixed for 5 h in Tellyzinckys fixative, and stored in 70% ethanol until processed by standard embedding and sectioning techniques onto Probe-On Plus slides (Fisher Scientific). Sections were stained with H&E.

Immunoprecipitations and Western Blot Analysis.
Preparation of protein extracts and immunoprecipitations have been described previously (42) . Briefly, 2 mg of fresh and frozen tissue were homogenized in 2 ml of lysis buffer with protease inhibitors; phenylmethylsulfonyl fluoride, leupeptin, and aprotinin at 50 µg/ml on ice. Protein lysates were rocked for 1 h at 4°C and then cleared by centrifugation at 14,000 x g for 15 min. The supernatants were removed, mixed with 2x loading buffer, and heated to 90°C for 3 min. Samples were spun briefly, electrophoresed under denaturing conditions on 8% precast tris-glycine gels, and transferred to polyvinylidene difluoride membranes according to manufacturer’s protocol (Novex, San Diego, CA). Western blot analysis was performed essentially as described with the following exceptions; primary antibody (Stat5a, 1:20,000 dilution; Stat5b, 1:10,000 dilution; anti-phosphotyrosine, 1:5,000 dilution; anti-MAPK, 1:5,000 dilution) was incubated overnight at 4°C with gentle rocking, and all incubations with antibodies and initial blocking were performed with 3% nonfat dried milk in 1x TBST. Detection was performed with the enhanced chemiluminescence kit according to manufacturer’s protocol (Amersham) and exposed to Kodak (Rochester, NY) MR autoradiography film. Exposure times are between 1 s and 2 min. Immunoprecipitations with anti-Stat5a antibodies were carried out as described previously (4) . Stripping was performed by incubating blots at 56°C in 6.25 mM Tris-HCl (pH 6.8), 2% SDS, and 1% ß-mercaptoethanol for 30 min. Blots were washed extensively in TBST and then blocked with 3% nonfat dried milk in TBST.

Northern Blot Analysis.
Total RNA was isolated from fresh tissue by homogenization in lysis buffer as described previously (63) . RNA was quantified by spectrophotometry and prepared for Northern blot analysis by heating in loading buffer at 65°C. Total RNA was separated by 1.5% agarose gel electrophoresis and transferred to Hybond N+ (Amersham) nylon membrane by capillary transfer with 10 x SSC. After overnight transfer, the membrane was UV-irradiated and hybridized to each cDNA probe in Quikhyb (Stratagene, La Jolla, CA). Hybridization for the TGF-{alpha} cDNA probe was performed in Quikhyb (Stratagene) at 65°C for 16 h, followed by two washes in 1 x SSC and 0.5% SDS for 30 min, followed by one wash in 0.1 x SSC and 0.5% SDS for 30 min at 56°C. Blots were hybridized for 16 h and then washed twice in 1 x SSC and 0.5% SDS for 30 min, followed by one wash in 0.1 x SSC and 0.5% SDS for 30 min. cDNA and oligo probes were random-primed and Klenow-labeled with [{alpha}-32P]dCTP. After hybridization and washing, blots were exposed from 30 min to 24 h, as specifically described in the figure legends, to Kodak MR autoradiography film at -70°C.

BrdUrd and TUNEL Assays.
Protocols for BrdUrd and TUNEL analysis have been described elsewhere (64) . Mice were injected 2 h before sacrifice with 20 µg/g body weight BrdUrd labeling reagent, as described by the manufacturer (Amersham). Each proliferation and apoptosis sample counted represents a minimum of three random fields (at x200) and a minimum of 1000 total cells/section for each mouse. A minimum of three mice per timepoint were collected and analyzed. The total number of cells counted for proliferation assay is 6.5 x 105. The total number of cells counted for the apoptosis assay is 7.5 x 105. The number of mice collected and the number of mice analyzed are listed by genotype and by timepoints (pregnancy day 18/involution day 1/involution day 3/involution day 7) for apoptosis and proliferation, respectively: KOTG, 4/4/4/3 and 4/3/3/3; TG, 3/5/3/5 and 3/3/3/5; WT, 3/4/5/3/ and 3/3/3/3; NTGKO, 3/3/2/3 and 3/3/3/3.

Hyperplasia and Tumor Analysis.
Mice were palpated at the 18th day of pregnancy for mammary hyperplasia, hypertrophy, and tumors. Animals were scored for the presence of either hyperplasia or tumor at every pregnancy. Most animals were sacrificed at the third pregnancy by anesthesia followed by cervical dislocation. In studies that required long-term breeding for tumor development, dams were rebred within 2 days after birth and removal of the pups.

Acknowledgments

We thank Dr. Eric Sandgren for the kind gift of TGF-{alpha} transgenic mice. We thank Drs. J. Shillingford and E. Rucker for critical reading of the manuscript and Drs. P. Furth and B. Gusterson for essential discussions. We thank Ashton Garett, Bill Kemp, and Sandra Price for animal husbandry and technical support and Dr. J. Shillingford for developing the Stat5a PCR assay.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Department of Defense, Medical Research and Material Command Grant DAMD17-99-1-9328 (to R. C. H.). Back

2 To whom requests for reprints should be addressed, at Laboratory of Genetics and Physiology, National Institute of Digestive, Diabetes and Kidney Disease, NIH, Building 8, Room 111, Bethesda MD 20892. Phone: (301) 435-6634; Fax: (301) 480-7312; E-mail: robinh{at}box-r.nih.gov Back

3 The abbreviations used are: Stat, signal transducer and activator of transcription; TGF, transforming growth factor; Jak, janus kinase; MAPK, mitogen-activated protein kinase; WAP, whey acidic protein; EGFR, epidermal growth factor receptor; BrdUrd, bromodeoxy uridine; WT, wild-type; PCD, programmed cell death; EGF, epidermal growth factor; TBST, Tris buffered saline Tween; TUNEL, terminal deoxynucleotidyl transferase-mediated uridine nick end labeling; ERK, extracellular signal-regulated kinase. Back

Received for publication 5/11/99. Revision received 8/ 4/99. Accepted for publication 8/10/99.

References

  1. Vonderhaar B. K. Prolactin: transport, function and receptors in mammary gland development and differentiation Neville M. C. Daniel C. W. eds. . The Mammary Gland: Development, Regulation and Function, : 383-438, Plenum Publishing New York 1985.
  2. Travers M. T., Barber M. C., Tonner E., Quarrie L., Wilde C. J., Flint D. J. The role of prolactin and growth hormone in the regulation of casein gene expression and mammary cell survival: relationships to milk synthesis and secretion. Endocrinology, 137: 1530-1539, 1996.[Medline]
  3. Tonner E., Quarrie L., Travers M., Barber M., Logan A., Wilde C., Flint D. Does an IGF-binding protein (IGFBP) present in involuting rat mammary gland regulate apoptosis?. Prog. Growth Factor Res., 6: 409-414, 1995.[Medline]
  4. Liu X., Robinson G. W., Wagner K. U., Garrett L., Wynshaw-Boris A., Hennighausen L. Stat5a is mandatory for adult mammary gland development and lactogenesis. Genes Dev., 11: 179-186, 1997.[Abstract/Free Full Text]
  5. Bovolenta C., Testolin L., Benussi L., Lievens P. M., Liboi E. Positive selection of apoptosis-resistant cells correlates with activation of dominant-negative STAT5. J. Biol. Chem., 273: 20779-20784, 1998.[Abstract/Free Full Text]
  6. Zamorano J., Wang H. Y., Wang R., Shi Y., Longmore G. D., Keegan A. D. Regulation of cell growth by IL-2: role of STAT5 in protection from apoptosis but not in cell cycle progression. J. Immunol., 160: 3502-3512, 1998.[Abstract/Free Full Text]
  7. Strange R., Friis R. R., Bemis L. T., Geske F. J. Programmed cell death during mammary gland involution. Methods Cell Biol., 46: 355-368, 1995.[Medline]
  8. Marti A., Feng Z., Altermatt H. J., Jaggi R. Milk accumulation triggers apoptosis of mammary epithelial cells. Eur. J. Cell Biol., 73: 158-165, 1997.[Medline]
  9. Lund L. R., Romer J., Thomasset N., Solberg H., Pyke C., Bissell M. J., Dano K., Werb Z. Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways. Development (Camb.), 122: 181-193, 1996.[Abstract]
  10. Hadsell D. L., Greenberg N. M., Fligger J. M., Baumrucker C. R., Rosen J. M. Targeted expression of des(1–3) human insulin-like growth factor I in transgenic mice influences mammary gland development and IGF-binding protein expression. Endocrinology, 137: 321-330, 1996.[Medline]
  11. Li F., Strange R., Friis R. R., Djonov V., Altermatt H. J., Saurer S., Niemann H., Andres A. C. Expression of stromelysin-1 and TIMP-1 in the involuting mammary gland and in early invasive tumors of the mouse. Int. J. Cancer, 59: 560-568, 1994.[Medline]
  12. Li M., Liu X., Robinson G., Bar-Peled U., Wagner K. U., Young W. S., Hennighausen L., Furth P. A. Mammary-derived signals activate programmed cell death during the first stage of mammary gland involution. Proc. Natl. Acad. Sci. USA, 94: 3425-3430, 1997.[Abstract/Free Full Text]
  13. Leonard W. J., O’Shea J. J. Jaks and STATs: biological implications. Annu. Rev. Immunol., 16: 293-322, 1998.[Medline]
  14. Zhong Z., Wen Z., Darnell J. E., Jr. Stat3: a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6. Science (Washington DC), 264: 95-98, 1994.[Abstract/Free Full Text]
  15. Iwamoto Y., Chin Y. E., Peng X., Fu X. Y. Identification of a membrane-associated inhibitor(s) of epidermal growth factor-induced signal transducer and activator of transcription activation. J. Biol. Chem., 273: 18198-18204, 1998.[Abstract/Free Full Text]
  16. Teglund S., McKay C., Schuetz E., van Deursen J. M., Stravopodis D., Wang D., Brown M., Bodner S., Grosveld G., Ihle J. N. Stat5a and Stat5b proteins have essential and nonessential, or redundant, roles in cytokine responses. Cell, 93: 841-850, 1998.[Medline]
  17. Darnell J. E., Jr. STATs and gene regulation. Science (Washington DC), 277: 1630-1635, 1997.[Abstract/Free Full Text]
  18. Kirken R. A., Malabarba M. G., Xu J., Liu X., Farrar W. L., Hennighausen L., Larner A. C., Grimley P. M., Rui H. Prolactin stimulates serine/tyrosine phosphorylation and formation of heterocomplexes of multiple Stat5 isoforms in Nb2 lymphocytes. J. Biol. Chem., 272: 14098-14103, 1997.[Abstract/Free Full Text]
  19. Moriggl R., Gouilleux-Gruart V., Jahne R., Berchtold S., Gartmann C., Liu X., Hennighausen L., Sotiropoulos A., Groner B., Gouilleux F. Deletion of the carboxyl-terminal transactivation domain of MGF-Stat5 results in sustained DNA binding and a dominant negative phenotype. Mol. Cell. Biol., 16: 5691-5700, 1996.[Abstract/Free Full Text]
  20. Wang D., Stravopodis D., Teglund S., Kitazawa J., Ihle J. N. Naturally occurring dominant negative variants of Stat5. Mol. Cell. Biol., 16: 6141-6148, 1996.[Abstract/Free Full Text]
  21. Ruff-Jamison S., Chen K., Cohen S. Epidermal growth factor induces the tyrosine phosphorylation and nuclear translocation of Stat 5 in mouse liver. Proc. Natl. Acad. Sci. USA, 92: 4215-4218, 1995.[Abstract/Free Full Text]
  22. Yamashita H., Xu J., Erwin R. A., Farrar W. L., Kirken R. A., Rui H. Differential control of the phosphorylation state of proline-juxtaposed serine residues Ser725 of Stat5a and Ser730 of Stat5b in prolactin-sensitive cells. J. Biol. Chem., 273: 30218-30224, 1998.[Abstract/Free Full Text]
  23. Pircher T. J., Petersen H., Gustafsson J. A., Haldosen L. A. Extracellular signal-regulated kinase (ERK) interacts with signal transducer and activator of transcription (STAT) 5a. Mol. Endocrinol., 13: 555-565, 1999.[Medline]
  24. Wartmann M., Cella N., Hofer P., Groner B., Liu X., Hennighausen L., Hynes N. E. Lactogenic hormone activation of Stat5 and transcription of the ß-casein gene in mammary epithelial cells is independent of p42 ERK2 mitogen-activated protein kinase activity. J. Biol. Chem., 271: 31863-31868, 1996.[Abstract/Free Full Text]
  25. Dickson R. B., Lippman M. E. Growth factors in breast cancer. Endocr. Rev., 16: 559-589, 1995.[Medline]
  26. Sandgren E. P., Schroeder J. A., Qui T. H., Palmiter R. D., Brinster R. L., Lee D. C. Inhibition of mammary gland involution is associated with transforming growth factor {alpha} but not c-myc-induced tumorigenesis in transgenic mice. Cancer Res., 55: 3915-3927, 1995.[Abstract/Free Full Text]
  27. Schwaller J., Frantsve J., Aster J., Williams I. R., Tomasson M. H., Ross T. S., Peeters P., Van Rompaey L., Van Etten R. A., Ilaria R., Jr., Marynen P., Gilliland D. G. Transformation of hematopoietic cell lines to growth-factor independence and induction of a fatal myelo- and lymphoproliferative disease in mice by retrovirally transduced TEL/JAK2 fusion genes. EMBO J., 17: 5321-5333, 1998.[Abstract]
  28. Wennbo H., Gebre-Medhin M., Gritli-Linde A., Ohlsson C., Isaksson O. G., Tornell J. Activation of the prolactin receptor but not the growth hormone receptor is important for induction of mammary tumors in transgenic mice. J. Clin. Investig., 100: 2744-2751, 1997.[Medline]
  29. Zhang Q., Nowak I., Vonderheid E. C., Rook A. H., Kadin M. E., Nowell P. C., Shaw L. M., Wasik M. A. Activation of Jak/STAT proteins involved in signal transduction pathway mediated by receptor for interleukin 2 in malignant T lymphocytes derived from cutaneous anaplastic large T-cell lymphoma and Sezary syndrome. Proc. Natl. Acad. Sci. USA, 93: 9148-9153, 1996.[Abstract/Free Full Text]
  30. Hayakawa F., Towatari M., Iida H., Wakao H., Kiyoi H., Naoe T., Saito H. Differential constitutive activation between STAT-related proteins and MAP kinase in primary acute myelogenous leukaemia. Br. J. Haematol., 101: 521-528, 1998.[Medline]
  31. Yu C. L., Jove R., Burakoff S. J. Constitutive activation of the Janus kinase-STAT pathway in T lymphoma overexpressing the Lck protein tyrosine kinase. J. Immunol., 159: 5206-5210, 1997.[Abstract]
  32. Migone T. S., Lin J. X., Cereseto A., Mulloy J. C., O’Shea J. J., Franchini G., Leonard W. J. Constitutively activated Jak-STAT pathway in T cells transformed with HTLV-I. Science (Washington DC), 269: 79-81, 1995.[Abstract/Free Full Text]
  33. Danial N. N., Pernis A., Rothman P. B. Jak-STAT signaling induced by the v-ab1 oncogene. Science (Washington DC), 269: 1875-1877, 1995.[Abstract/Free Full Text]
  34. Meydan N., Grunberger T., Dadi H., Shahar M., Arpaia E., Lapidot Z., Leeder J. S., Freedman M., Cohen A., Gazit A., Levitzki A., Roifman C. M. Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor. Nature (Lond.), 379: 645-648, 1996.[Medline]
  35. Quarrie L. H., Addey C. V., Wilde C. J. Apoptosis in lactating and involuting mouse mammary tissue demonstrated by nick-end DNA labelling. Cell Tissue Res., 281: 413-419, 1995.[Medline]
  36. Wagner K. U., Young W. S., III, Liu X., Ginns E. I., Li M., Furth P. A., Hennighausen L. Oxytocin and milk removal are required for post-partum mammary gland development. Genes Funct., 1: 233-244, 1997.[Medline]
  37. Tzeng Y. J., Zimmermann C., Guhl E., Berg B., Avantaggiati M. L., Graessmann A. SV40 T/t-antigen induces premature mammary gland involution by apoptosis and selects for p53 missense mutation in mammary tumors. Oncogene, 16: 2103-2114, 1998.[Medline]
  38. Li B., Murphy K. L., Laucirica R., Kittrell F., Medina D., Rosen J. M. A transgenic mouse model for mammary carcinogenesis. Oncogene, 16: 997-1007, 1998.[Medline]
  39. Jhappan C., Geiser A. G., Kordon E. C., Bagheri D., Hennighausen L., Roberts A. B., Smith G. H., Merlino G. Targeting expression of a transforming growth factor ß 1 transgene to the pregnant mammary gland inhibits alveolar development and lactation. EMBO J., 12: 1835-1845, 1993.[Medline]
  40. Jager R., Herzer U., Schenkel J., Weiher H. Overexpression of Bcl-2 inhibits alveolar cell apoptosis during involution and accelerates c-myc-induced tumorigenesis of the mammary gland in transgenic mice. Oncogene, 15: 1787-1795, 1997.[Medline]
  41. Pittius C. W., Sankaran L., Topper Y. J., Hennighausen L. Comparison of the regulation of the whey acidic protein gene with that of a hybrid gene containing the whey acidic protein gene promoter in transgenic mice. Mol. Endocrinol., 2: 1027-1032, 1988.[Medline]
  42. Liu X., Robinson G. W., Hennighausen L. Activation of Stat5a and Stat5b by tyrosine phosphorylation is tightly linked to mammary gland differentiation. Mol. Endocrinol., 10: 1496-1506, 1996.[Medline]
  43. Bauer J. H., Liu K. D., You Y., Lai S. Y., Goldsmith M. A. Heteromerization of the {gamma}c chain with the interleukin-9 receptor {alpha} subunit leads to STAT activation and prevention of apoptosis. J. Biol. Chem., 273: 9255-9260, 1998.[Abstract/Free Full Text]
  44. Leff M. A., Buckley D. J., Krumenacker J. S., Reed J. C., Miyashita T., Buckley A. R. Rapid modulation of the apoptosis regulatory genes bcl-2 and bax by prolactin in rat Nb2 lymphoma cells. Endocrinology, 137: 5456-5462, 1996.[Medline]
  45. Evan G. I., Brown L., Whyte M., Harrington E. Apoptosis and the cell cycle. Curr. Opin. Cell Biol., 7: 825-834, 1995.[Medline]
  46. Sartor C. I., Dziubinski M. L., Yu C. L., Jove R., Ethier S. P. Role of epidermal growth factor receptor and STAT-3 activation in autonomous proliferation of SUM-102PT human breast cancer cells. Cancer Res., 57: 978-987, 1997.[Abstract/Free Full Text]
  47. David M., Wong L., Flavell R., Thompson S. A., Wells A., Larner A. C., Johnson G. R. STAT activation by epidermal growth factor (EGF) and amphiregulin. Requirement for the EGF receptor kinase but not for tyrosine phosphorylation sites or JAK1. J. Biol. Chem., 271: 9185-9188, 1996.[Abstract/Free Full Text]
  48. Leaman D. W., Pisharody S., Flickinger T. W., Commane M. A., Schlessinger J., Kerr I. M., Levy D. E., Stark G. R. Roles of JAKs in activation of STATs and stimulation of c-fos gene expression by epidermal growth factor. Mol. Cell. Biol., 16: 369-375, 1996.[Abstract/Free Full Text]
  49. Kirken R. A., Malabarba M. G., Xu J., DaSilva L., Erwin R. A., Liu X., Hennighausen L., Rui H., Farrar W. L. Two discrete regions of interleukin-2 (IL2) receptor ß independently mediate IL2 activation of a PD98059/rapamycin/wortmannin-insensitive Stat5a/b serine kinase. J. Biol. Chem., 272: 15459-15465, 1997.[Abstract/Free Full Text]
  50. Shual K., Ziemiecki A., Wilks A. F., Harpur A. G., Sadowski H. B., Gilman M. Z., Darnell J. E. Polypeptide signalling to the nucleus through tyrosine phosphorylation of Jak and Stat proteins. Nature (Lond.), 366: 580-583, 1993.[Medline]
  51. Petersen H., Haldosen L. A. EGF modulates expression of STAT5 in mammary epithelial cells. Exp. Cell Res., 243: 347-358, 1998.[Medline]
  52. Kohlhuber F., Rogers N. C., Watling D., Feng J., Guschin D., Briscoe J., Witthuhn B. A., Kotenko S. V., Pestka S., Stark G. R., Ihle J. N., Kerr I. M. A JAK1/JAK2 chimera can sustain {alpha} and {gamma} interferon responses. Mol. Cell. Biol., 17: 695-706, 1997.[Abstract/Free Full Text]
  53. Kotenko S. V., Izotova L. S., Pollack B. P., Muthukumaran G., Paukku K., Silvennoinen O., Ihle J. N., Pestka S. Other kinases can substitute for Jak2 in signal transduction by interferon-{gamma}. J. Biol. Chem., 271: 17174-17182, 1996.[Abstract/Free Full Text]
  54. Lupu R., Cardillo M., Cho C., Harris L., Hijazi M., Perez C., Rosenberg K., Yang D., Tang C. The significance of heregulin in breast cancer tumor progression and drug resistance. Breast Cancer Res. Treat., 38: 57-66, 1996.[Medline]
  55. Sebastian J., Richards R. G., Walker M. P., Wiesen J. F., Werb Z., Derynck R., Hom Y. K., Cunha G. R., DiAugustine R. P. Activation and function of the epidermal growth factor receptor and erbB-2 during mammary gland morphogenesis. Cell Growth Differ., 9: 777-785, 1998.[Abstract]
  56. Wiesen J. F., Young P., Werb Z., Cunha G. R. Signaling through the stromal epidermal growth factor receptor is necessary for mammary ductal development. Development (Camb.), 126: 335-344, 1998.[Abstract]
  57. Muller W. J., Arteaga C. L., Muthuswamy S. K., Siegel P. M., Webster M. A., Cardiff R. D., Meise K. S., Li F., Halter S. A., Coffey R. J. Synergistic interaction of the Neu proto-oncogene product and transforming growth factor {alpha} in the mammary epithelium of transgenic mice. Mol. Cell. Biol., 16: 5726-5736, 1996.[Abstract/Free Full Text]
  58. Kenney N. J., Smith G. H., Maroulakou I. G., Green J. H., Muller W. J., Callahan R., Salomon D. S., Dickson R. B. Detection of amphiregulin and Cripto-1 in mammary tumors from transgenic mice. Mol. Carcinog., 15: 44-56, 1996.[Medline]
  59. Huang S., Trujillo J. M., Chakrabarty S. Proliferation of human colon cancer cells: role of epidermal growth factor and transforming growth factor {alpha}. Int. J. Cancer, 52: 978-986, 1992.[Medline]
  60. Liu X., Gallego M. I., Smith G. H., Robinson G. W., Hennighausen L. Functional release of Stat5a-null mammary tissue through the activation of compensating signals including Stat5b. Cell Growth Differ., 9: 795-803, 1998.[Abstract]
  61. Danial N. N., Losman J. A., Lu T., Yip N., Krishnan K., Krolewski J., Goff S. P., Wang J. Y., Rothman P. B. Direct interaction of Jak1 and v-Ab1 is required for v-Ab1-induced activation of STATs and proliferation. Mol. Cell. Biol., 18: 6795-6804, 1998.[Abstract/Free Full Text]
  62. Frank D. A., Varticovski L. BCR/ab1 leads to the constitutive activation of Stat proteins and shares an epitope with tyrosine phosphorylated Stats. Leukemia (Baltimore), 10: 1724-1730, 1996.[Medline]
  63. Liu X., Robinson G. W., 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. USA, 92: 8831-8835, 1995.[Abstract/Free Full Text]
  64. Humphreys R. C., Krajewska M., Krnacik S., Jaeger R., Weiher H., Krajewski S., Reed J. C., Rosen J. M. Apoptosis in the terminal endbud of the murine mammary gland: a mechanism of ductal morphogenesis. Development (Camb.), 122: 4013-4022, 1996.[Abstract]



This article has been cited by other articles:


Home page
Mol Cancer ResHome page
T. R. Medler, J. M. Craig, A. A. Fiorillo, Y. B. Feeney, J. C. Harrell, and C. V. Clevenger
HDAC6 Deacetylates HMGN2 to Regulate Stat5a Activity and Breast Cancer Growth
Mol. Cancer Res., October 1, 2016; 14(10): 994 - 1008.
[Abstract] [Full Text] [PDF]


Home page
JCOHome page
A. R. Peck, A. K. Witkiewicz, C. Liu, G. A. Stringer, A. C. Klimowicz, E. Pequignot, B. Freydin, T. H. Tran, N. Yang, A. L. Rosenberg, et 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., June 20, 2011; 29(18): 2448 - 2458.
[Abstract] [Full Text] [PDF]


Home page
Am. J. Physiol. Endocrinol. Metab.Home page
M. C. Rudolph, T. D. Russell, P. Webb, M. C. Neville, and S. M. Anderson
Prolactin-mediated regulation of lipid biosynthesis genes in vivo in the lactating mammary epithelial cell
Am J Physiol Endocrinol Metab, June 1, 2011; 300(6): E1059 - E1068.
[Abstract] [Full Text] [PDF]


Home page
Mol. Cell. Biol.Home page
B. A. Creamer, K. Sakamoto, J. W. Schmidt, A. A. Triplett, R. Moriggl, and K. U. Wagner
Stat5 Promotes Survival of Mammary Epithelial Cells through Transcriptional Activation of a Distinct Promoter in Akt1
Mol. Cell. Biol., June 15, 2010; 30(12): 2957 - 2970.
[Abstract] [Full Text] [PDF]


Home page
Biochem. J.Home page
R. Zaragoza, A. Bosch, C. Garcia, J. Sandoval, E. Serna, L. Torres, E. R. Garcia-Trevijano, and J. R. Vina
Nitric oxide triggers mammary gland involution after weaning: remodelling is delayed but not impaired in mice lacking inducible nitric oxide synthase
Biochem. J., June 15, 2010; 428(3): 451 - 462.
[Abstract] [Full Text] [PDF]


Home page
Cancer Res.Home page
J. Nilsson, K. Helou, A. Kovacs, P.-O. Bendahl, G. Bjursell, M. Ferno, P. Carlsson, and M. Kannius-Janson
Nuclear Janus-Activated Kinase 2/Nuclear Factor 1-C2 Suppresses Tumorigenesis and Epithelial-to-Mesenchymal Transition by Repressing Forkhead Box F1
Cancer Res., March 1, 2010; 70(5): 2020 - 2029.
[Abstract] [Full Text] [PDF]


Home page
Cancer Res.Home page
T. H. Tran, F. E. Utama, J. Lin, N. Yang, A. B. Sjolund, A. Ryder, K. J. Johnson, L. M. Neilson, C. Liu, K. L. Brill, et al.
Prolactin Inhibits BCL6 Expression in Breast Cancer through a Stat5a-Dependent Mechanism
Cancer Res., February 15, 2010; 70(4): 1711 - 1721.
[Abstract] [Full Text] [PDF]


Home page
Cancer Res.Home page
K. Sakamoto, W.-c. Lin, A. A. Triplett, and K.-U. Wagner
Targeting Janus Kinase 2 in Her2/neu-Expressing Mammary Cancer: Implications for Cancer Prevention and Therapy
Cancer Res., August 15, 2009; 69(16): 6642 - 6650.
[Abstract] [Full Text] [PDF]


Home page
Clin. Cancer Res.Home page
P. Koppikar, V. W. Y. Lui, D. Man, S. Xi, R. L. Chai, E. Nelson, A. B.J. Tobey, and J. R. Grandis
Constitutive Activation of Signal Transducer and Activator of Transcription 5 Contributes to Tumor Growth, Epithelial-Mesenchymal Transition, and Resistance to Epidermal Growth Factor Receptor Targeting
Clin. Cancer Res., December 1, 2008; 14(23): 7682 - 7690.
[Abstract] [Full Text] [PDF]


Home page
Genes Dev.Home page
L. Hennighausen and G. W. Robinson
Interpretation of cytokine signaling through the transcription factors STAT5A and STAT5B
Genes & Dev., March 15, 2008; 22(6): 711 - 721.
[Abstract] [Full Text] [PDF]


Home page
J EndocrinolHome page
A. J Brennan, J. A Sharp, E. Khalil, M. R Digby, S. L Mailer, C. M Lefevre, and K. R Nicholas
A population of mammary epithelial cells do not require hormones or growth factors to survive
J. Endocrinol., March 1, 2008; 196(3): 483 - 496.
[Abstract] [Full Text] [PDF]


Home page
Cancer Res.Home page
F. Sotgia, H. Rui, G. Bonuccelli, I. Mercier, R. G. Pestell, and M. P. Lisanti
Caveolin-1, Mammary Stem Cells, and Estrogen-Dependent Breast Cancers.
Cancer Res., November 15, 2006; 66(22): 10647 - 10651.
[Abstract] [Full Text] [PDF]


Home page
DevelopmentHome page
C. M. Blakely, L. Sintasath, C. M. D'Cruz, K. T. Hahn, K. D. Dugan, G. K. Belka, and L. A. Chodosh
Developmental stage determines the effects of MYC in the mammary epithelium
Development, March 1, 2005; 132(5): 1147 - 1160.
[Abstract] [Full Text] [PDF]


Home page
JCOHome page
M. T. Nevalainen, J. Xie, J. Torhorst, L. Bubendorf, P. Haas, J. Kononen, G. Sauter, and H. Rui
Signal Transducer and Activator of Transcription-5 Activation and Breast Cancer Prognosis
J. Clin. Oncol., June 1, 2004; 22(11): 2053 - 2060.
[Abstract] [Full Text] [PDF]


Home page
Physiol. GenomicsHome page
S. P. Suchyta, S. Sipkovsky, R. G. Halgren, R. Kruska, M. Elftman, M. Weber-Nielsen, M. J. Vandehaar, L. Xiao, R. J. Tempelman, and P. M. Coussens
Bovine mammary gene expression profiling using a cDNA microarray enhanced for mammary-specific transcripts
Physiol Genomics, December 1, 2003; 16(1): 8 - 18.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
M. T. Kloth, K. K. Laughlin, J. S. Biscardi, J. L. Boerner, S. J. Parsons, and C. M. Silva
STAT5b, a Mediator of Synergism between c-Src and the Epidermal Growth Factor Receptor
J. Biol. Chem., January 17, 2003; 278(3): 1671 - 1679.
[Abstract] [Full Text] [PDF]


Home page
Mol Cancer ResHome page
E. Iavnilovitch, B. Groner, and I. Barash
Overexpression and Forced Activation of Stat5 in Mammary Gland of Transgenic Mice Promotes Cellular Proliferation, Enhances Differentiation, and Delays Postlactational Apoptosis
Mol. Cancer Res., November 1, 2002; 1(1): 32 - 47.
[Abstract] [Full Text] [PDF]


Home page
ScienceHome page
B. S. Wiseman and Z. Werb
Stromal Effects on Mammary Gland Development and Breast Cancer
Science, May 10, 2002; 296(5570): 1046 - 1049.
[Abstract] [Full Text] [PDF]


Home page
Biol. Reprod.Home page
A. V. Capuco, M. Li, E. Long, S. Ren, K. S. Hruska, K. Schorr, and P. A. Furth
Concurrent Pregnancy Retards Mammary Involution: Effects on Apoptosis and Proliferation of the Mammary Epithelium after Forced Weaning of Mice
Biol Reprod, May 1, 2002; 66(5): 1471 - 1476.
[Abstract] [Full Text]


Home page
Cell Growth Differ.Home page
L. Shan, S. A. Rouhani, H. A. J. Schut, and E. G. Snyderwine
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) Modulates Lactogenic Hormone-mediated Differentiation and Gene Expression in HC11 Mouse Mammary Epithelial Cells
Cell Growth Differ., December 1, 2001; 12(12): 649 - 656.
[Abstract] [Full Text] [PDF]


Home page
JCBHome page
K. Miyoshi, J. M. Shillingford, G. H. Smith, S. L. Grimm, K.-U. Wagner, T. Oka, J. M. Rosen, G. W. Robinson, and L. Hennighausen
Signal transducer and activator of transcription (Stat) 5 controls the proliferation and differentiation of mammary alveolar epithelium
J. Cell Biol., November 12, 2001; 155(4): 531 - 542.
[Abstract] [Full Text] [PDF]


Home page
Cell Growth Differ.Home page
A. V. Kazansky and J. M. Rosen
Signal Transducers and Activators of Transcription 5B Potentiates v-Src-mediated Transformation of NIH-3T3 Cells
Cell Growth Differ., January 1, 2001; 12(1): 1 - 7.
[Abstract] [Full Text]


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Humphreys, R. C.
Right arrow Articles by Hennighausen, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Humphreys, R. C.
Right arrow Articles by Hennighausen, L.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cell Growth & Differentiation