Debra E. Rugowski

Stat5 activation inhibits prolactin-induced AP-1 activity: distinct prolactin-initiated signals in tumorigenesis dependent on cell context

The essential role of prolactin (PRL) in normal mammary gland growth and differentiation has implicated this hormone in the development and progression of breast cancer. Although Stat5 is the best-characterized mediator of PRL signals, PRL also activates multiple other signals, whose roles in normal and pathologic processes are not well understood. We have shown that PRL stimulates activating protein-1 (AP-1) activity in breast cancer cells, and can cooperate with estradiol in this pathway. AP-1 modulates many processes critical for carcinogenesis, including cell proliferation, survival, transformation, invasion and angiogenesis, and is elevated in many neoplasms, including breast tumors. Here, we investigated the relationship between PRL signals to AP-1 and Stat5. We found that PRL activation of Stat5a and Stat5b, but not Stat1 or Stat3, reduced PRL signals to AP-1, without altering estradiol-induced AP-1 activity. The truncation mutant, Stat5/Δ53C, but not Stat5Y699F, was an effective inhibitor, consistent with a requirement for Stat5 dimerization and nuclear accumulation, but not its C-terminal transactivation activity. The association of Stat5 with AP-1 proteins suggests that this underlies the inhibition. Predictably, the ability of PRL to activate Stat5 and AP-1 was inversely related in mammary cell lines. Further, reduction of Stat5 protein with siRNA in T47D cells, which contain elevated Stat5, increased PRL-induced AP-1 signals, transcripts for the AP-1 target, matrix metalloproteinase-2 and associated invasive behavior. This study points to the importance of cell context in determining the spectrum of PRL-induced actions, which is critical for understanding the contributions of PRL to breast cancer.

Ovarian Hormones Are Not Required for PRL-induced Mammary Tumorigenesis, But Estrogen Enhances Neoplastic Processes

Epidemiologic studies have demonstrated that increased prolactin (PRL) exposure raises the risk of invasive estrogen receptor alpha (ERalpha)-positive breast cancer in women. However, the mechanism(s) whereby this occurs and the interactions with estrogen itself in this disease remain poorly understood. In order to investigate the role of ovarian hormones in the disease process, we employed a transgenic model neu-related lipocalin (NRL)-PRL in which transgenic PRL is directed to mammary epithelial cells by the PRL- and estrogen-insensitive NRL promoter, mimicking the endogenous PRL expression within the breast observed in women. This high local exposure leads to mammary lesion development and eventually carcinomas. Ovariectomy (ovx), shortly after puberty, did not alter the incidence or latency of PRL-induced mammary carcinomas, consistent with the independence of PRL from circulating estrogens as a risk factor for invasive breast cancer in women. However, chronic estrogen administration to ovx NRL-PRL females decreased the latency of both ERalpha-positive and -negative tumors. We identified multiple mechanisms that may underlie this observation. Elevated estrogen exposure cooperated with PRL to increase epithelial proliferation and myoepithelial abnormalities, increasing the incidence of preneoplastic lesions. Critical components of the extracellular matrix secreted by the myoepithelium were reduced with age, and transgenic PRL raised transcripts for tenascin-C and maspin, both associated with tumor progression and poor prognosis in subclasses of clinical breast tumors. Mammary pERK1/2 and pAkt, but not phosphorylated Stat5, were markedly elevated by local PRL. Together, these findings indicate that PRL employs multiple mechanisms to promote mammary tumorigenesis.

Prolactin Activates ERα in the Absence of Ligand in Female Mammary Development and Carcinogenesis in Vivo

Resistance of estrogen receptor positive (ERα+) breast cancers to antiestrogens is a major factor in the mortality of this disease. Although activation of ERα in the absence of ligand is hypothesized to contribute to this resistance, the potency of this mechanism in vivo is not clear. Epidemiologic studies have strongly linked prolactin (PRL) to both development of ERα+ breast cancer and resistance to endocrine therapies. Here we employed genetically modified mouse models to examine the ability of PRL and cross talk with TGFα to activate ERα, using a mutated ERα, ERα(G525L), which is refractory to endogenous estrogens. We demonstrate that PRL promotes pubertal ERα-dependent mammary ductal elongation and gene expression in the absence of estrogen, which are abrogated by the antiestrogen, ICI 182,780 (ICI). PRL and TGFα together reduce sensitivity to estrogen, and 30% of their combined stimulation of ductal proliferation is inhibited by ICI, implicating ligand-independent activation of ERα as a component of their interaction. However, PRL/TGFα-induced heterogeneous ERα+ tumors developed more rapidly in the presence of ICI and contained altered transcripts for surface markers associated with epithelial subpopulations and increased signal transducer and activator of transcription 5b expression. Together, these data support strong interactions between PRL and estrogen on multiple levels. Ligand-independent activation of ERα suggests that PRL may contribute to resistance to antiestrogen therapies. However, these studies also underscore ERα-mediated moderation of tumor phenotype. In light of the high expression of PRL receptors in ERα+ cancers, understanding the actions of PRL and cross talk with other oncogenic factors and ERα itself has important implications for therapeutic strategies.

Elevated collagen-I augments tumor progressive signals, intravasation and metastasis of prolactin-induced estrogen receptor alpha positive mammary tumor cells

BackgroundThe development and progression of estrogen receptor alpha positive (ERα+) breast cancer has been linked epidemiologically to prolactin. However, activation of the canonical mediator of prolactin, STAT5, is associated with more differentiated cancers and better prognoses. We have reported that density/stiffness of the extracellular matrix potently modulates the repertoire of prolactin signals in human ERα + breast cancer cells in vitro: stiff matrices shift the balance from the Janus kinase (JAK)2/STAT5 cascade toward pro-tumor progressive extracellular regulated kinase (ERK)1/2 signals, driving invasion. However, the consequences for behavior of ERα + cancers in vivo are not known.MethodsIn order to investigate the importance of matrix density/stiffness in progression of ERα + cancers, we examined tumor development and progression following orthotopic transplantation of two clonal green fluorescent protein (GFP) + ERα + tumor cell lines derived from prolactin-induced tumors to 8-week-old wild-type FVB/N (WT) or collagen-dense (col1a1tm1Jae/+) female mice. The latter express a mutant non-cleavable allele of collagen 1a1 “knocked-in” to the col1a1 gene locus, permitting COL1A1 accumulation. We evaluated the effect of the collagen environment on tumor progression by examining circulating tumor cells and lung metastases, activated signaling pathways by immunohistochemistry analysis and immunoblotting, and collagen structure by second harmonic generation microscopy.ResultsERα + primary tumors did not differ in growth rate, histologic type, ERα, or prolactin receptor (PRLR) expression between col1a1tm1Jae/+ and WT recipients. However, the col1a1tm1Jae/+ environment significantly increased circulating tumor cells and the number and size of lung metastases at end stage. Tumors in col1a1tm1Jae/+ recipients displayed reduced STAT5 activation, and higher phosphorylation of ERK1/2 and AKT. Moreover, intratumoral collagen fibers in col1a1tm1Jae/+ recipients were aligned with tumor projections into the adjacent fat pad, perpendicular to the bulk of the tumor, in contrast to the collagen fibers wrapped around the more uniformly expansive tumors in WT recipients.ConclusionsA collagen-dense extracellular matrix can potently interact with hormonal signals to drive metastasis of ERα + breast cancers.

Prolactin cooperates with loss of p53 to promote claudin-low mammary carcinomas

TP53 is one of the most commonly mutated genes in cancer. In breast cancer, it is mutated in about 40% of primary clinical tumors and is associated with poor survival. The mammotrophic hormone, prolactin (PRL), and/or its receptor are also expressed in many breast cancers, and accumulating epidemiologic data link PRL to breast cancer development and progression. Like TP53 mutations, evidence for PRL activity is evident across several molecular cancer subtypes, and elevated PRL expression and loss of p53 have been observed in some of the same clinical tumors. In order to examine the interaction of these factors, we used genetically modified mouse models of mammary-specific p53 loss and local overexpression of PRL. We demonstrated that mammary PRL decreased the latency of tumors in the absence of p53, and increased the proportion of triple-negative claudin-low carcinomas, which display similarities to human clinical metaplastic carcinomas. Moreover, PRL/p53−/− carcinomas displayed higher rates of proliferation and more aggressive behavior. Transcripts associated with cell cycle progression, invasion and stromal reactivity were differentially expressed in carcinomas that developed in the presence of elevated PRL. PRL/p53−/− carcinomas also exhibited selectively altered expression of activating protein-1 components, including higher levels of c-Jun and FosL1, which can drive transcription of many of these genes and the epithelial–mesenchymal transition. The ability of PRL to promote claudin-low carcinomas demonstrates that PRL can influence this subset of triple-negative breast cancers, which may have been obscured by the relative infrequency of this cancer subtype. Our findings suggest novel therapeutic approaches, and provide a preclinical model to develop possible agents.

Prolactin Promotes Mammary Pathogenesis Independently from Cyclin D1

Epidemiological and experimental studies have revealed an important role for prolactin (PRL) in breast cancer. Cyclin D1 is a major downstream target of PRL in lobuloalveolar development during pregnancy and is amplified and/or overexpressed in many breast carcinomas. To examine the importance of cyclin D1 in PRL-induced pathogenesis, we generated transgenic mice (NRL-PRL) that overexpress PRL in mammary epithelial cells, with wild-type, heterozygous, or genetically ablated cyclin D1 in the FVB/N genetic background. Although loss of one cyclin D1 allele did not affect PRL-induced mammary lesions in nonparous females, the complete absence of cyclin D1 (D1(-/-)) markedly decreased tumor incidence. Nevertheless, NRL-PRL/D1(-/-) females developed significantly more preneoplastic lesions (eg, epithelial hyperplasias and mammary intraepithelial neoplasias) than D1(-/-) females. Moreover, although lack of cyclin D1 reduced proliferation of morphologically normal mammary epithelium, transgenic PRL restored it to rates of wild-type females. PRL posttranscriptionally increased nuclear cyclin D3 protein in D1(-/-) luminal cells, indicating one compensatory mechanism. Consistently, pregnancy induced extensive lobuloalveolar growth in the absence of cyclin D1. However, transcripts for milk proteins were reduced, and pups failed to survive, suggesting that mammary differentiation was inadequate. Together, these results indicate that cyclin D1 is an important, but not essential, mediator of PRL-induced mammary proliferation and pathology in FVB/N mice and is critical for differentiation and lactation.

17β-Estradiol and ICI182,780 Differentially Regulate STAT5 Isoforms in Female Mammary Epithelium, With Distinct Outcomes

Abstract Prolactin (PRL) and estrogen cooperate in lobuloalveolar development of the mammary gland and jointly regulate gene expression in breast cancer cells in vitro. Canonical PRL signaling activates STAT5A/B, homologous proteins that have different target genes and functions. Although STAT5A/B are important for physiological mammary function and tumor pathophysiology, little is known about regulation of their expression, particularly of STAT5B, and the consequences for hormone action. In this study, we examined the effect of two estrogenic ligands, 17β-estradiol (E2) and the clinical antiestrogen, ICI182,780 (ICI, fulvestrant) on expression of STAT5 isoforms and resulting crosstalk with PRL in normal and tumor murine mammary epithelial cell lines. In all cell lines, E2 and ICI significantly increased protein and corresponding nascent and mature transcripts for STAT5A and STAT5B, respectively. Transcriptional regulation of STAT5A and STAT5B by E2 and ICI, respectively, is associated with recruitment of estrogen receptor alpha and increased H3K27Ac at a common intronic enhancer 10 kb downstream of the Stat5a transcription start site. Further, E2 and ICI induced different transcripts associated with differentiation and tumor behavior. In tumor cells, E2 also significantly increased proliferation, invasion, and stem cell-like activity, whereas ICI had no effect. To evaluate the role of STAT5B in these responses, we reduced STAT5B expression using short hairpin (sh) RNA. shSTAT5B blocked ICI-induced transcripts associated with metastasis and the epithelial mesenchymal transition in both cell types. shSTAT5B also blocked E2-induced invasion of tumor epithelium without altering E2-induced transcripts. Together, these studies indicate that STAT5B mediates a subset of protumorigenic responses to both E2 and ICI, underscoring the need to understand regulation of its expression and suggesting exploration as a possible therapeutic target in breast cancer.

Spontaneous aggressive ERα+ mammary tumor model is driven by Kras activation

The NRL-PRL murine model, defined by mammary-selective transgenic rat prolactin ligand rPrl expression, establishes spontaneous ER+ mammary tumors, mimicking the association between elevated prolactin (PRL) and risk for development of ER+ breast cancer in postmenopausal women. Whole genome and exome sequencing in a discovery cohort (n=5) of end stage tumors revealed canonical activating mutations and copy number amplifications of Kras. The frequent mutations in this pathway were validated in an extension cohort, identifying activating Ras alterations in 79% (23/29) of tumors. Transcriptome analyses over the course of oncogenesis revealed marked alterations associated with Ras activity in established tumors, compared to preneoplastic tissues, in cell-intrinsic processes associated with mitosis, cell adhesion and invasion, as well as in the tumor microenvironment, including immune activity. These genomic analyses suggest that PRL induces a selective bottleneck for spontaneous Ras-driven tumors which may model a subset of aggressive clinical ER+ breast cancers.

Abstract P2-04-04: Prolactin cooperates with loss of p53 to promote mammary tumorigenesis.

Prolactin (PRL) is critical for mammary development and lactation. Epidemiological studies also support a role for PRL in breast cancer. In order to study the contributions of PRL to tumor development and progression, we developed a transgenic mouse model that expresses PRL in the mammary epithelial cells (NRL-PRL), mimicking the mammary PRL production in women. Loss of function of the tumor suppressor p53 is a common occurrence in many human cancers including breast tumors. Interactions between PRL and p53 were examined by crossing the NRL-PRL mouse to p53 knockout mice which were made congenic on the FVB/N background. To circumvent the problem that p53 −/− mice are prone to multiple nonmammary tumors, mammary cells from 10–12 week old wild-type, NRL-PRL, p53 −/− , and NRL-PRL/ p53 −/− were transplanted to wild type mammary glands. Histological analysis of the donor glands showed that NRL-PRL glands displayed marked epithelial proliferation and focally dilated ducts, whereas p53 −/− glands exhibited irregular ductal epithelium with increased stromal density compared to wild type mice. In contrast, glands of donor PRL/ p53−/− females showed widespread epithelial hyperplasias and highly irregular ductal epithelium often surrounded by dense stroma. By one year of age, no tumors had developed from either wild-type or NRL-PRL transplanted epithelium. However, recipients of either p53 −/− or NRL-PRL/ p53 −/− mammary epithelium developed histologically similar anaplastic carcinomas. The presence of transgenic PRL decreased tumor latency (205 vs 244 days) and significantly increased tumor cell proliferation. In addition, these carcinomas appeared to be more aggressive: 5/16 (32%) of the NRL-PRL/ p53 −/− tumors invaded into the peritoneal cavity, compared to 0/10 p53 −/− tumors. Expression of matrix metalloproteinase 9 (MMP-9), but not MMP-2, was found to be significantly higher in the NRL-PRL/ p53 −/− , compared to p53 −/− tumors. MMP-9 is regulated by the transcription factor, AP-1, and PRL can signal through AP-1. NRL-PRL/ p53 −/− tumors displayed significantly increased expression of the AP-1 family members, c-Jun and FosL, but not JunD or c-Fos compared to tumors from p53 −/− mice. In addition, levels of c-Jun and FosL proteins increased in a similar pattern. In summary, interactions between PRL and loss of p53 promote breast cancer by increasing proliferation and invasiveness, potentially via AP-1 target genes. Supported by CDMRP BC053412. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-04-04.