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Induction of Mammary Neoplasia in Transgenic Mice
From the Cellular and Molecular Biology Program,* the Department of Comparative Biosciences, and the Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, Wisconsin
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Abstract |
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(ER)-positive and ER-negative adenocarcinomas. However, disease in vivo occurs in the context of dysregulation of multiple pathways. In this study, we investigated the ability of prolactin to modulate carcinogenesis when co-expressed with the potent oncogene transforming growth factor (TGF) in bitransgenic mice. Prolactin and TGF cooperated to reduce dramatically the latency of mammary macrocyst development, the principal lesion type induced by TGF. In combination, prolactin and TGF also increased the incidence and reduced the latency of other preneoplastic lesions and increased cellular turnover in structurally normal alveoli and ducts compared with single transgenic females. Bitransgenic glands contained higher levels of phosphorylated ERK1/2 compared with single TGF transgenic glands, suggesting that this kinase may be a point of signaling crosstalk. Furthermore, transgenic prolactin also reversed the decrease in ER induced by neu-related lipocalin-TGF. Our findings demonstrate that locally produced prolactin can strikingly potentiate the car-cinogenic actions of another oncogene and modify ovarian hormone responsiveness, suggesting that prolactin signaling may be a potential thera-peutic target.
(ER)-positive and ER-negative mammary adenocarcinomas and increased ER expression in preneoplastic lesions in virgin females.8
This model provides a system to examine the roles of both local and circulating PRL in mammary oncogenesis in a defined transgenic system.
Although these studies demonstrate that PRL alone is sufficient to promote neoplasia and modulate estrogen receptor expression, in clinical disease, it would act in the context of dysregulation of multiple other pathways. Transforming growth factor (TGF) is a prime candidate molecule to evaluate in the context of local mammary PRL expression. The epidermal growth factor (EGF) family, including TGF, plays important roles in breast cancer and like PRL has complex actions in normal mammary development.9-13
TGF is overexpressed in 50 to 70% of human breast tumors.14,15
It binds to epidermal growth factor receptor (EGFR, ErbB1) and activates both EGFR homodimers and EGFR/ErbB2 heterodimers. Elevated TGF, EGFR, and ErbB2 are associated with ER loss and antiestrogen resistance in breast tumors.15-17
TGF is also a potent, well-characterized oncogene in transgenic mice.18
Transgenic mammary TGF overexpression driven by several promoters, including whey acidic protein (WAP), MT, and mouse mammary tumor virus (MMTV), result in premature alveolar development, delayed involution after lactation, increased number of preneoplastic lesions, and eventually tumors.19-23
Interactions between PRL receptor and EGFR pathways have been examined in several mammary cell lines. However, these studies have resulted in disparate conclusions: mutual inhibition, as observed in NMuMG and HC11 cells24-27 ; positive interactions, as reported in SK-BR3 breast cancer cells28 ; or variable outcomes in primary mammary epithelial cell cultures, depending on the concentration of EGF and physiological state of the cells.29 These divergent results may reflect spurious differences in cell lines, limited sampling of pathways and end points, or alternatively, modulation of receptors and available downstream pathways by physiological context and/or accumulating neoplastic changes.
In view of the prevalence of TGF overexpression in human breast cancer and the recently recognized ability of local mammary PRL to contribute to this disease, it is critical to establish the consequences of the interaction between these pathways in mammary cancer pathogenesis in the in vivo setting. Therefore, we investigated the effects of co-expression of mammary PRL and TGF directed by the NRL promoter. We found that PRL and TGF cooperate to dramatically reduce the latency of macrocyst development in virgin FVB/N females, the principal lesion elicited by transgenic TGF in this strain. This was associated with increased phosphorylation of ERK1/2, a potential signal to mediators of dysregulated cell cycle progression. Furthermore, transgenic PRL countered the fall in ER expression observed in NRL-TGF glands. These findings demonstrate that PRL strikingly amplifies the neoplastic actions of another oncogene as well as potentially modifies ovarian hormonal responsiveness of the gland.
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Materials and Methods |
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The following antibodies were used for immunohistochemical and/or Western analyses: 5-bromo-2-deoxyuridine (BrdU) (MAS-250) from Accurate Scientific (Westbury, NY), ERK1/2 (9102) and phospho-ERK1/2 (Thr202/Tyr204; 9101) from Cell Signaling Technology (Beverly, MA), EGFR (SC-03) and ER (SC-542) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and keratin 8 (RB-9095) from Labvision (Fremont, CA). Recombinant hPRL (Lot AFP795) was obtained through the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, and Dr. A. F. Parlow (National Hormone and Peptide Program, Harbor-UCLA Medical Center). TGF was purchased from Peprotech (Rocky Hill, NJ).
Mice
NRL-PRL mice (line 1647-13, TgN(Nrl-Prl)23EPS; line 1655-8, TgN(Nrl-Prl)24EPS)8
and NRL-TGF mice (line 1385-7, TgN(Nrl-TGF)25EPS; T.A. Rose-Hellekant, unpublished data) were generated as described. The NRL promoter directs expression to mammary epithelial cells and is not altered by PRL or estrogen8
(T.A. Rose-Hellekant, unpublished data). All lines were maintained in the FVB/N strain background, and studies were conducted in virgin females. Tail biopsies were collected at weaning, and offspring were screened for the PRL transgene as described previously8
and for the TGF transgene using the following polymerase chain reaction primers: forward, 5'-AAGGAAAGGTGTCTCAGGACAA-3', and reverse, 5'-CTGCTCCCTTCCCTGTCCTTC-3'. Female mice of the NRL-PRL 1655-8 but not the 1647-13 lineage have elevated circulating PRL.8
The studies described herein were performed in both NRL-PRL lines; for clarity, data only from line 1647-13 are included in the tables and Figures 3 to 7
. Mice were housed and handled in accordance with the Guide for Care and Use of Laboratory Animals in Association for Assessment and Accreditation of Laboratory Animal Care-accredited facilities. All procedures were approved by the University of Wisconsin-Madison Animal Care and Use Committee.
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Fourth inguinal mammary glands were pressed between two slides, fixed in 10% neutral buffered formalin overnight, and stored in 70% ethanol. Whole mounts were treated with 1:3 glacial acetic acid:ethanol for 1 hour, hydrated by graded ethanol, stained for 1 hour with hematoxylin, dehydrated with graded ethanol, and stored in glycerol until analysis. The whole mounts were analyzed for growth by measuring the percentage of the fat pad filled by ductal outgrowth from the nipple. Ductal growth was analyzed by dividing the area from the nipple to the leading edge of the ducts by the total area of the mammary gland.
Histological Examination of Mammary Tissue
Mammary glands were fixed in 10% neutral buffered formalin overnight, embedded in paraffin, and cut into 6-µm sections. Morphological analysis was performed on hematoxylin and eosin-stained slides. Mice were injected with 200 mg/kg body weight BrdU (Sigma Chemical Co., St. Louis, MO) 1 hour before sacrifice to label cells undergoing DNA synthesis. Proliferation, apoptosis, and ER expression were evaluated as described previously.8
BrdU, apoptotic, and ER indices were evaluated separately for morphologically normal epithelial structures and lesions as described previously.8
Stromal density was assessed by counting fibroblast nuclei under x400 magnification. The counts from four fields of view were averaged for each mouse; 10 mice from each genotype were examined.
MCF-7 Cell Culture
PRL-deficient cells derived from the human mammary adenocarcinoma cell line MCF-7 were grown in RPMI-1640 containing 10% horse serum and 50 µmol/L gangciclovir as reported previously.30
For experiments, 106 cells/60-mm plate were incubated in serum-free media for 24 hours before treatment with vehicle, 4 nmol/L PRL, and/or 30 ng/ml TGF for times as stated in the legends. Cell lysates were harvested and analyzed by immunoblotting as described previously.30
Western Analysis
Western analysis was performed as described previously.30 In brief, 30 µg protein of cellular lysate, or 60 µg of mammary gland homogenate, was fractionated by standard Laemmli SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidine fluoride membranes, and then probed with appropriate antibodies (ERK1/2, 1:1000; phospho-ERK1/2, 1:5000; keratin 8, 1:1000; and EGFR, 1:400). Signals were visualized by enhanced chemiluminescence, followed by autoradiography. For some experiments, signals were quantified by densitometry (ImageQuant software, v.4.2a; Molecular Dynamics, Inc., Sunnyvale, CA).
Statistical Analyses
Latency data were subjected to Kaplan-Meier analysis and plotted as a function of the probability of a mouse remaining tumor free versus its age in months. Differences were detected using the Mantel-Haenszel test, which compares median survival among groups. Counted indices from immunohistochemistry sections were analyzed using Kruskal-Wallis test for nonparametric data, followed by the Mann-Whitney post test. Protein levels determined by immunoblotting were analyzed by one-way analysis of variance. Statistical analyses were performed using Prism v.3.02 (GraphPad Software, Inc., San Diego, CA). Differences were considered significant at P < 0.05.
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Results |
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Females
NRL-TGF/PRL virgin females developed complex mammary macrocysts or neoplasms with 100% incidence (Figure 1
; Table 1
). Although these end-stage lesions were similar to those that developed in the mammary glands of NRL-TGF virgin females, the macrocysts formed with greatly decreased latency in the bitransgenic mice (Figure 1
; Table 1
). The latency to development of macrocysts was not significantly different between the bitransgenic lines, despite the presence of elevated circulating PRL in only one of the NRL-PRL lines, consistent with the importance of locally produced PRL.
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under control of the NRL promoter (T.A. Rose-Hellekant, M.D. Schroeder, J.L. Brockman, M.N. Gould, L.A. Schuler, and E.P. Sandgren, unpublished data), as well as the WAP31
and MT promoters21
in the FVB/N train background. Microscopically, the multiloculated macrocysts consisted of a single layer of simple or papillary epithelium surrounding proteinaceous fluid (Figure 2, A and B)
. The cells were uniform in size with an increased nuclear-to-cytoplasmic ratio, one to two nucleoli in each nucleus, and highly vacuolated cytoplasm, suggesting high secretory activity. Focal areas within the cysts of NRL-PRL/TGF bitransgenic mice displayed a more complex histotype, with combinations of both papillary and simple epithelium present around the same lumen and foci with multiple cell layers in the cyst lining and occasionally solid centers (Figure 2, D and E)
. This complex histotype was found more often in bitransgenic females than NRL-TGF females (Table 1)
. For clarity, only the analysis of mice derived from NRL-PRL line 1647-13 (normal circulating PRL) is shown; glands derived from NRL-PRL line 1655-8 were similar. The stroma surrounding these lesions in bitransgenic mice had significantly increased density (Figure 3A)
, indicated by increased numbers of fibroblast nuclei surrounding the macrocyst lining, compared with stroma adjacent to NRL-TGF macrocysts. Two bitransgenic females developed squamous adenocarcinomas (classified based on recommendations of the Annapolis Pathology Panel32
). The majority of mammary tumors and macrocysts developed in the cranial glands, although preneoplastic lesions were found in all mammary glands examined. Unlike NRL-TGF females, virgin NRL-PRL females did not develop these macrocysts but instead developed adenocarcinomas of varying histotype at a longer latency, as described previously.8
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Expression
PRL has been shown to regulate responsiveness of multiple reproductive tissues to ovarian hormones in vivo,33
and endogenously synthesized PRL increases ER in vitro.34
In contrast, elevated TGF, EGFR, and ErbB2 are generally associated with ER loss in breast tumors. Therefore, we were interested in examining the net outcome of PRL and TGF interaction on ER expression. Within the cells lining the macrocysts, ER expression varied widely among individual cysts in both bitransgenic and single transgenic NRL-TGF females (Figures 2, F and G, and 3B)
. Furthermore, despite the decreased latency to macrocyst development in bitransgenic females, the proliferation rates in the macrocysts of these females did not differ from NRL-TGF females (Figure 3C)
. Apoptosis levels in macrocysts of bitransgenics were significantly greater than NRL-TGF females (P < 0.05; Figure 3D
), which may reflect the influence of PRL on apoptosis. Overall, these results suggest that once the macrocysts have developed, the transgenes do not strongly modulate proliferation or ER expression.
PRL and TGF Cooperatively Induce Other Preneoplastic Lesions
Although the most common preneoplastic lesion in the bitransgenic animals was the macrocyst, other lesions were present in all mammary glands examined. Lesions were present with an increased incidence in end-stage glands of bitransgenic females compared with end-stage glands from NRL-TGF females, even though the latter were twice as old (5.6 versus 11.2 months; Table 1
). Epithelial hyperplasias (EHs), common in the older single transgenic females, were present in a majority of the bitransgenic glands. In addition, many of these glands also exhibited dilated ducts, associated with expression of PRL alone, and adenosis lesions (Figure 2C)
, found at a lower frequency with monotransgenic TGF expression (Table 1)
. The decreased latency of these abnormalities demonstrates strong cooperation between PRL and TGF in development of these lesions.
PRL Increases Proliferation and Apoptosis in Morphologically Normal Structures of Bitransgenic Females
To determine the cellular events underlying the interaction between the transgenes, we examined the rates of proliferation and apoptosis in normal appearing mammary structures. Glands of 6-month-old NRL-TGF females exhibited increased rates of proliferation in both alveoli and ducts (Figure 4, A and B)
and decreased rates of apoptosis in ducts (Figure 4, C and D)
compared with nontransgenic controls, similar to WAP-TGF animals.35
Bitransgenic females demonstrated significantly higher proliferation in both alveoli (Figure 4A)
and ducts (Figure 4B)
compared with the NRL-TGF glands, although not different from NRL-PRL animals.8
Bitransgenic animals also exhibited significantly more alveolar apoptosis than NRL-TGF mice (Figure 4C)
, comparable with NRL-PRL lines. Similarly, EHs in bitransgenic females showed significantly increased proliferation and apoptosis compared with hyperplasias in NRL-TGF females (Figure 5, A and B)
. NRL-PRL females had very few EHs at this age.
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Lowers ER Expression in Morphologically Normal Structures
Transgenic mammary PRL elevates ER expression in mammary structures.8
In contrast, glands of NRL-TGF females displayed significantly reduced ER expression in alveoli and ducts compared with nontransgenic controls at 6 months of age (Figure 4, E and F)
. Interestingly, age-matched bitransgenic females had more ER, particularly in ducts, than both NRL-PRL and nontransgenic animals (Figure 4, E and F)
. Furthermore, ER expression in EH was more than three times higher in bitransgenic glands than in NRL-TGF females (Figure 5C)
. These data indicate that PRL can modulate the effect of TGF on ER expression in morphologically normal structures and early lesions.
PRL and TGF Induce Developmental Abnormalities
To assess interactions of these transgenes during mammary development, we evaluated mammary whole mounts at 13 weeks of age and at end stage (Figure 6)
. In normal female mice, ducts in the mammary gland grow from the nipple past a central lymph node by 4 weeks of age, reach the border of the mammary fat pad with some remaining terminal end buds by 6 weeks, and have filled the fat pad with a ductal network by 12 weeks of age.35,36
Glands of NRL-TGF females (Figure 6, C and D)
demonstrated ductal development similar to nontransgenic females (Figures 6, G and H)
but also displayed widespread alveolar development, as reported for MMTV-TGF females.19
In contrast, NRL-PRL females demonstrated retarded ductal growth into the mammary fat pad compared with nontransgenic glands (Figure 6E
; Table 2
). These glands also exhibited dilated ducts and hyperplasias, which were more prominent by end stage (Figure 6F)
, consistent with abnormalities observed histologically (Table 1)
.
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. Similar to glands of NRL-PRL females, bitransgenic glands displayed decreased (P = 0.07) ductal penetration into the mammary gland (Table 2)
and increased epithelial development and hyperplasias, apparent by 13 weeks of age. These hyperplasias appeared to be enlarged compared with NRL-TGF females (compare Figure 6, A and B
versus C and D). Mammary Glands from Bitransgenic Females Contain Elevated ERK1/2 Activity
Although both transgenic PRL and TGF are produced by mammary epithelial cells, these ligands are secreted and their respective receptors are present on stromal and epithelial cells.37-39
In addition, both ligands can initiate multiple signaling cascades in their targets. Therefore many potential mechanism(s) may mediate the interaction between these transgenes to create the mammary phenotype described here. ERK1/2 is an important kinase in the proliferative response to many growth factors, including PRL and TGF.40-42
To examine the net effect on this kinase cascade in glands destined to develop macrocysts, but before morphological abnormalities, we compared levels of phosphorylated ERK1/2 in glands of 3-month-old NRL-TGF and NRL-TGF/PRL mice. As shown in Figure 7, A and B
, relative levels of activated ERK1/2 were higher in bitransgenic than single TGF transgenic glands (P < 0.02), demonstrating positive crosstalk to this end point. In contrast, levels of EGFR were similar at this time (Figure 7C)
; levels of ErbB2 were very low in both genotypes (not shown). To determine whether crosstalk to ERK1/2 could occur within breast cancer cells, we acutely stimulated MCF-7-derived cells with PRL and/or TGF. Each ligand alone transiently increased phosphorylated ERK1/2 as expected (Figure 8A)
. Although no interaction between them was observed after 15 or 45 minutes of stimulation, these factors together significantly prolonged phosphorylation of these kinases, evident after 120 minutes (Figure 8B
, P < 0.01). The importance of signal duration, including phosphorylation of mitogen-activated protein kinases, is increasingly recognized for multiple activities, including proliferation and apoptosis.40,43
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Discussion |
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to promote development of locally aggressive, highly secretory mammary lesions with greatly decreased latency compared with single transgenic animals. Furthermore, transgenic PRL reduced the latency of other mammary abnormalities and countered the fall in ER expression induced by transgenic TGF. Importantly, the two transgenic PRL lines exhibited similar cooperation with TGF, demonstrating that mammary PRL with or without elevated circulating PRL is sufficient for this synergy. These findings show that local PRL can powerfully potentiate the carcinogenic actions of another oncogene and modify apparent ovarian hormonal responsiveness. Together, they suggest that PRL may play an important role in mammary tumorigenesis and may be an effective therapeutic target.
The shortened latency to a similar dominant lesion in the bitransgenic and TGF monotransgenic gland, albeit with a more complex histotype and surrounding reactive stroma, indicates that PRL potentiates TGF signals at some mammary targets. Our data demonstrate that crosstalk to ERK1/2 is one mechanism that may mediate this interaction and that this can occur within epithelial cells. These kinases have been shown to be important for neoplasia in several transgenic models, including MMTV-ErbB2/TGF,41
in part by their signals to cell cycle progression.40,44
However, PRL and TGF share other signaling cascades observed in various model systems, many of which have been implicated in mammary carcinogenesis, including other mitogen-activated protein kinases, phosphatidylinositol 3-kinase-AKT, and STATs.1,9,45,46
These pathways, as well as proximal cross-activation of the other’s receptor and modulation of distal feedback loops, present multiple opportunities for interactions within common target cells. In addition, PRL and TGF may also target distinct cell subpopulations, permitting collaboration in lesion development via secondary secreted factors. Interestingly, some morphological abnormalities, such as ductal dilation, were common in NRL-PRL mammary glands but not those expressing transgenic TGF and were observed in many but not all bitransgenic glands. This suggests that PRL exerts some actions that are only modestly modulated by TGF and may reflect a different receptor distribution and/or use of distinct signaling pathways.
The positive interaction between PRL and TGF in proliferation of morphologically normal structures and hyperplasias contrasts with the opposing effect of these oncogenes on apoptosis. Whereas TGF alone decreased apoptosis, PRL in combination with TGF restored apoptosis to that of nontransgenic controls. This PRL-induced increase in cell turnover is further evidence of distinct activities of these two factors. The apparent inability of PRL to promote survival in vivo is consistent with the similar level of apoptosis in transplanted PRL+/+ and PRL–/– epithelium47
but differs from effects observed in vitro,1,48
underscoring the importance of in vivo studies.
In addition to these observations in adult glands, cooperation between transgenic TGF and PRL was also evident in pubertal ductal morphogenesis. In the normal mouse, paracrine stromal/epithelial signals elicit ductal elongation at adolescence, orchestrated by estrogen, growth hormone, and local growth factors, including EGF family members.12,49
Deficits in EGFR, ErbB2, and amphiregulin all result in profound defects in ductal growth and defective terminal end buds. Interestingly, transgenic overexpression of TGF alone also slightly retards ductal penetration.21
TGF levels are tightly controlled in the peri-pubertal murine gland50
; overexpression may disrupt appropriate signaling patterns at critical times, perhaps by competing with amphiregulin for the EGFR. The latter is considered to be the critical EGFR ligand at puberty51,52
and binds EGFR with a lower affinity than TGF.53
In the current study, transgenic PRL also delayed ductal morphogenesis, perhaps reflecting PRL suppression of estrogen-induced ductal proliferation, as reported in studies of ovariectomized mice by Hovey et al.38
The accentuated defective ductal elongation observed in the current study indicates that PRL crosstalk with TGF further confounds the precise temporal and spatial signals that regulate this event.
Our data also demonstrate the robust ability of PRL to elevate ER levels in vivo. Estrogen, EGFR, and TGF have complex interactions in the normal mammary gland as well as breast tumors. Although estrogen increases the secretion of TGF and TGF can at least partially mediate estradiol-stimulated growth of mammary epithelial cells,54-57
estrogen and another EGFR ligand, EGF, oppose one another’s signals via decreasing expression of their respective receptors in vitro.58
EGFR and ER also are inversely related in human breast tumors.15-17
In our model, transgenic TGF reduced ER expression in morphologically normal structures, consistent with these clinical findings. However, PRL overexpression in combination with TGF was able to restore ER levels in these structures and dramatically elevated expression in EH, overriding this aspect of the transgenic TGF phenotype. Recent studies have demonstrated that estrogen hastens progression from premalignant EH to ER-negative tumors.59,60
Our data suggest that PRL may be an important modulator of ER expression in vivo. Interestingly, ER expression varied widely among individual macrocysts at end stage in both NRL-TGF and bitransgenic females in our model. This may reflect loss of the normal regulation of ER by some subset of lesions, similar to that occurring in human disease. Studies in progress will determine the functional significance of the PRL-induced increase of these receptors in lesion development and tumor progression.
Despite the established role of PRL in mammary gland development and lactation, attention has only been recently focused on its actions in breast cancer and the contribution of locally synthesized PRL to oncogenic processes in this gland. Our data demonstrate that prolonged exposure to increased mammary PRL cannot only lead to neoplasia but also potentiation of processes driven by other mammary oncogenes such as TGF, decreasing the latency of lesion development and increasing the apparent estrogenic responsiveness of the early lesions. Understanding the actions of this hormone in tumorigenesis and interactions with characterized players in breast cancer will direct efforts toward improved diagnostic and treatment modalities for human disease.
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Acknowledgements |
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Footnotes |
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Supported in part by the National Institutes of Health (grants R01 CA78312 to L.A.S.; K01 RR00145 to T.A.R.-H.; and T32 AG00265), the University of Wisconsin Center for Women’s Health and Women’s Health Research; and the University of Wisconsin School of Veterinary Medicine.
Current address of T.A.R.-H.: Department of Physiology, School of Medicine, University of Minnesota-Duluth, Duluth, MN.
Accepted for publication December 28, 2005.
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References |
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-positive and ER-negative mammary cancer in transgenic mice. Oncogene 2003, 22:4664-4674[CrossRef][Medline] in transgenic mice: induction of epithelial hyperplasia, pancreatic metaplasia, and carcinoma of the breast. Cell 1990, 61:1121-1135[CrossRef][Medline] overexpression in transgenic mice induces liver neoplasia and abnormal development of the mammary gland and pancreas. Cell 1990, 61:1137-1146[CrossRef][Medline] promotes mammary tumorigenesis through selective survival and growth of secretory epithelial cells. Am J Pathol 1995, 147:1081-1096[Abstract] and PRLR expression and increases estrogen responsiveness in breast cancer cells. J Steroid Biochem Mol Biol 2004, 88:69-77[CrossRef][Medline] (TGF-) in basal and hormone-stimulated growth by estradiol, prolactin and progesterone in human and rat mammary tumor cells: studies using TGF and EGF receptor antibodies. J Steroid Biochem Mol Biol 1991, 38:687-693[CrossRef][Medline]This article has been cited by other articles:
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  L. M. Arendt, T. L. Grafwallner-Huseth, and L. A. Schuler Prolactin-Growth Factor Crosstalk Reduces Mammary Estrogen Responsiveness Despite Elevated ER{alpha} Expression Am. J. Pathol., March 1, 2009; 174(3): 1065 - 1074. [Abstract] [Full Text] [PDF]
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 T. M. Piazza, J.-C. Lu, K. C. Carver, and L. A. Schuler Src Family Kinases Accelerate Prolactin Receptor Internalization, Modulating Trafficking and Signaling in Breast Cancer Cells Mol. Endocrinol., February 1, 2009; 23(2): 202 - 212. [Abstract] [Full Text] [PDF]
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 K. C. Carver and L. A. Schuler Prolactin Does Not Require Insulin-Like Growth Factor Intermediates but Synergizes with Insulin-Like Growth Factor I in Human Breast Cancer Cells Mol. Cancer Res., April 1, 2008; 6(4): 634 - 643. [Abstract] [Full Text] [PDF]
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L. M. Arendt and L. A. Schuler Prolactin Drives Estrogen Receptor-{alpha}-Dependent Ductal Expansion and Synergizes with Transforming Growth Factor-{alpha} to Induce Mammary Tumors in Males Am. J. Pathol., January 1, 2008; 172(1): 194 - 202. [Abstract] [Full Text] [PDF]
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T. A. Rose-Hellekant, K. M. Wentworth, S. Nikolai, D. W. Kundel, and E. P. Sandgren Mammary Carcinogenesis Is Preceded by Altered Epithelial Cell Turnover in Transforming Growth Factor-{alpha} and c-myc Transgenic Mice Am. J. Pathol., November 1, 2006; 169(5): 1821 - 1832. [Abstract] [Full Text] [PDF]
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19 months of age. The latencies were compared by Kaplan-Meier analysis, and differences were detected using the Mantel-Haenszel test. NRL-PRL lines have a significantly longer tumor latency than NRL-TGF
