3040 Background: Cancer is the 2 nd leading cause of mortality in the US, with an estimated 1.96M new cases expected in 2023. With guideline recommended screening unavailable for most cancers, multicancer early detection (MCED) tests in development may extend the reach of cancer screening. As part of an ongoing program examining multiple biomarker classes in cancer early detection, we examined the feasibility of combining blood DNA methylation and cancer protein markers for the detection of multiple cancer types. Methods: 236 samples from subjects with cancer (13 tumor organ types) were retrospectively collected and gender/age matched with 146 unscreened non-cancer control samples. Plasma and serum were used for cfDNA methylation analysis of 36 differentially methylated regions and 8 cancer-associated protein markers in a randomized, blinded fashion. Methylated DNA markers (MDMs) were assayed using target enrichment long-probe quantitative-amplified signal assays. Protein markers were assayed with commercial kits. Normalized methylation values/protein concentrations were used to develop a logistic regression model for cancer detection. The model was built with 5 random 0.7/0.3 training/validation splits, keeping case/control, cancer types, gender, and age distributions similar to the original data set. Models for MDMs and protein markers were created, and a combined model including both MDMs and protein markers was also evaluated for performance improvement. Results: A reduced subset of MDM and protein markers was identified to have robust detection of several cancer types. At 99% specificity, overall sensitivity for cancer detection was 68% (95% CI: 62%-74%) for MDMs, 43% (37%-50%) for proteins, and 75% (69%-80%) for MDMs and proteins combined. Conclusions: Building on promising results with mutation and protein biomarker analysis (Science 369:6499, 2020) this pilot study demonstrates the potential of combining DNA methylation and protein biomarkers and contributes to a larger scale effort to develop a multi-biomarker class MCED test (Ann Oncol 2022 Vol. 33:S575). [Table: see text]

Purpose: Many cancer deaths could be avoided, and survival could be improved with detection of cancer at an earlier stage. Screening programs for breast, colon, and cervical cancer as well as the NLST trial for lung cancer screening demonstrated significant improvement in survival rates with screening. However, for 2021, the American Cancer Society estimates these screened cancers represent less than ~40% of cancer incidence and deaths. The remainder of cancer deaths occur because of tumors in unscreened organs and, therefore, a multicancer test that detects cancer in these organs can increase survival rates. By focusing on detection of unscreened cancers, this type of multicancer test would be complementary to current screening procedures. Here we describe the validation of a combined panel of methylated DNA markers (MDMs) and proteins for multicancer detection through testing an independent set of case/control samples. Experimental Procedures: In this study, we further evaluate the performance of our previously identified panel of 15 MDMs and 5 proteins for multicancer detection (Allawi et al., 2021 AACR Annual Meeting) by testing 315 controls and 160 cases encompassing 6 cancer types (liver, esophageal, lung, ovarian, pancreatic, and stomach). All samples used in the study were case-control collections with smoking status, age, and gender matching between cases and asymptomatic controls. Testing was performed in blinded fashion using multiplex PCR followed by LQAS (Long probe Quantitative Amplified Signal) assay on bisulfite converted DNA extracted from 3 mL of plasma collected in LB Gard® blood tubes. Protein concentrations were determined from paired serum aliquots and combined with MDMs for a multi-analyte analysis. The subjects were divided into training and validation with equal representation of cancer type, staging, gender, and age. Two thirds of the cases and controls were used to train with a logistic prediction algorithm, and the remaining 1/3 were used to validate the model. Results: Using stepwise logistic regression, a training model of MDMs and protein markers resulted in an area under the receiver operating characteristics curve (AUC) of 0.97 and cancer sensitivity of 89% at 98% specificity. The same model predicted the validation set with an AUC of 0.96 and cancer sensitivity of 85% at a specificity of 95%. Applying the algorithm to the combined training and validation sets resulted in sensitivities and specificities of 88% and 97%, respectively. The sensitivities per cancer type ranged from 73% for pancreatic cancer to 97% for lung cancer. Conclusion and Next Steps: This study demonstrates the performance of our MDMs and protein markers and their importance as components in our multi-omics strategy for multicancer detection. The next steps would be to expand the testing to include additional cancer types and combine with NGS-based methods to improve performance and optimize workflow. Citation Format: Hatim T. Allawi, Slava Katerov, Abram Vaccaro, Harrison L. Fleming, Debra E. Rugowski, Brittany Otto, Justin Heilberger, Jillian Cassel, Jacquelyn Hennek, William Taylor, Graham Lidgard. Validation of a panel of methylated DNA and protein markers for multi-cancer detection in plasma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 631.

Prolactin (PRL) cooperates with other factors to orchestrate mammary development and lactation, and is epidemiologically linked to higher risk for breast cancer. However, how PRL collaborates with oncogenes to foster tumorigenesis and influence breast cancer phenotype is not well understood. To understand its interactions with canonical Wnt signals, which elevate mammary stem cell activity, we crossed heterozygous NRL-PRL mice with ApcMin/+ mice and treated pubertal females with a single dose of mutagen. PRL in the context of ApcMin/+ fueled a dramatic increase in tumor incidence in nulliparous mice, compared to ApcMin/+ alone. Although carcinomas in both NRL-PRL/ApcMin/+ and ApcMin/+ females acquired a mutation in the remaining wildtype Apc allele and expressed abundant β-catenin, PRL-promoted tumors displayed higher levels of Notch-driven target genes and Notch-dependent cancer stem cell activity, compared to β-catenin-driven activity in ApcMin/+ tumors. This PRL-induced shift to dominant Notch signals was evident in preneoplastic epithelial hyperplasias at 120 days of age. In NRL-PRL/ApcMin/+ females, rapidly proliferating hyperplasias, characterized by β-catenin at cell junctions and high NOTCH1 expression, contrasted with slower growing lesions with nuclear β-catenin in ApcMin/+ females. These studies demonstrate that PRL can powerfully modulate the incidence and phenotype of mammary tumors, shedding light on mechanisms whereby PRL elevates risk of breast cancer.

Metastatic, antiestrogen resistant estrogen receptor α positive (ER+) breast cancer is the leading cause of breast cancer deaths in USA women. While studies have demonstrated the importance of the stromal tumor microenvironment in cancer progression and therapeutic responses, effects on the responses of ER+ cancers to estrogen and antiestrogens are poorly understood, particularly in the complex in vivo environment. In this study, we used an estrogen responsive syngeneic mouse model to interrogate how a COL1A1-enriched fibrotic ECM modulates integrated hormonal responses in cancer progression. We orthotopically transplanted the ER+ TC11 cell line into wild-type (WT) or collagen-dense (Col1a1tm1Jae/+, mCol1a1) syngeneic FVB/N female mice. Once tumors were established, recipients were supplemented with 17β-estradiol (E2), tamoxifen, or left untreated. Although the dense/stiff environment in mCol1a1 recipients did not alter the rate of E2-induced proliferation of the primary tumor, it fostered the agonist activity of tamoxifen to increase proliferation and AP-1 activity. Manipulation of estrogen activity did not alter the incidence of lung lesions in either WT or mCol1a1 hosts. However, the mCol1a1 environment enabled tamoxifen-stimulated growth of pulmonary metastases and further fueled estrogen-driven growth. Moreover, E2 remodeled peritumoral ECM architecture in WT animals, modifying alignment of collagen fibers and altering synthesis of ECM components associated with increased alignment and stiffness, and increasing FN1 and POSTN expression in the pulmonary metastatic niche. These studies demonstrate dynamic interactions between ECM properties and estrogen activity in progression of ER+ breast cancer, and support the need for therapeutics that target both ER and the tumor microenvironment.

The NRL-PRL murine model, defined by mammary-selective transgenic rat prolactin ligand rPrl expression, establishes spontaneous ER+ mammary tumors in nulliparous females, 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% of tumors (23 of 29). Transcriptome analyses over the course of oncogenesis revealed marked alterations associated with Ras activity in established tumors compared with preneoplastic tissues; in cell-intrinsic processes associated with mitosis, cell adhesion, and invasion; as well as in the surrounding tumor environment. These genomic analyses suggest that PRL induces a selective bottleneck for spontaneous Ras-driven tumors that may model a subset of aggressive clinical ER+ breast cancers.

Abstract Metastatic therapy-resistant breast cancers expressing estrogen receptor alpha (ER+) account for the majority of breast cancer deaths. Understanding of the underlying biology of aggressive luminal B cancers would improve preventive/treatment strategies. However, this has been limited by the paucity of preclinical immunocompetent models. Strong epidemiologic data link prolactin (PRL) exposure to development of ER+ metastatic breast cancer. We have capitalized on this relationship to generate a murine model which overexpresses PRL in mammary epithelial cells (NRL-PRL). Transgenic mammary PRL expression leads to the development of spontaneous metastatic ER+ carcinomas with characteristics of luminal B human cancers. In order to understand the mechanisms that underlie the ability of PRL to drive the development of cancers in this model, we used functional genomic analyses to identify genomic alterations and patterns of gene expression with cancer progression. Following development of spontaneous tumors, ER+ tumors and matched adjacent mammary glands and tail samples (N=5) were collected and examined by whole genome sequencing and RNA-seq analysis. A set of mammary cell preparations from the caudal glands of 12-week-old NRL-PRL females (N=5) were also analyzed. Unexpectedly, we found that all of the tumors contained somatic alterations of Kras, including four with canonical hotspot mutations (G12 and Q61) and one with an amplification of the Kras locus. In contrast to the tumors, no Kras mutations were detected in the matched mammary glands and tails, or the cell preparations. Our findings were further validated by targeted sequencing of an extension set consisting of frozen archived spontaneous ER+ tumors from NRL-PRL females (N=15), additional samples from mice with matched tumor, adjacent mammary gland and tails (N=7) and ER+ cell lines generated from PRL-induced spontaneous tumors (N=4). The Kras hotspot mutations were detected in 14 of 22 of the extension tumors and all four cell lines. One additional tumor contained a hotspot activating mutation in Nras. Immunohistochemistry demonstrated that RAS downstream effectors, pAKT and pERK, were upregulated, whereas nuclear STAT5A was decreased, in the tumors compared to the adjacent normal gland. Moreover, RNA-Seq analyses showed that acquisition of RAS mutations by the tumors was associated with alterations in multiple pathways compared to non-tumor glands, including distinct immune cell subpopulations. Although few clinical breast cancers display RAS protein mutations, studies have shown that the RAS pathway is aberrantly activated in more than 50% of all breast lesions. This enhanced RAS activity confers many benefits to these tumors resulting in a poor prognosis. The NRL-PRL model provides a valuable tool to dissect mechanisms that drive metastatic luminal B breast cancer and identify therapeutic opportunities. Citation Format: Kathleen A. O'Leary, Katie M. Campbell, Debra E. Rugowski, Kilannin Krysiak, William A. Mulligan, Malachi Griffith, Obi L. Griffith, Linda A. Schuler. Prolactin-induced mammary tumors: A preclinical model of luminal B breast cancer which exhibits mutations in the RAS pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5104.

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.

The 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.

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.

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.

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 post-transcriptionally increased nuclear cyclin D3 protein in D1(-/-) lunainal cells, indicating one compensatory mechanism. Consistently, pregnancy induced extensive lobuloalveolar growth in the absence of cyclin Dl. 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. (Am J Pathol 2012, 181:294-302; http://dx.doi.org/10.1016/j.ajpath.2012.03.041)

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.

INTRODUCTION:Tumors that express estrogen receptor alpha (ERα+) comprise 75% of breast cancers in women. While treatments directed against this receptor have successfully lowered mortality rates, many primary tumors initially or later exhibit resistance. The paucity of murine models of this "luminal" tumor subtype has hindered studies of factors that promote their pathogenesis and modulate responsiveness to estrogen-directed therapeutics. Since epidemiologic studies closely link prolactin and the development of ERα+ tumors in women, we examined characteristics of the aggressive ERα+ and ERα- carcinomas which develop in response to mammary prolactin in a murine transgenic model (neu-related lipocalin- prolactin (NRL-PRL)). To evaluate their relationship to clinical tumors, we determined phenotypic relationships among these carcinomas, other murine models of breast cancer, and features of luminal tumors in women.METHODS:We examined a panel of prolactin-induced tumors for characteristics relevant to clinical tumors: histotype, ERα/progesterone receptor (PR) expression and estrogen responsiveness, Activating Protein 1 (AP-1) components, and phosphorylation of signal transducer and activator of transcription 5 (Stat5), extracellular signal regulated kinase (ERK) 1/2 and AKT. We compared levels of transcripts in the ERα-associated "luminal" signature that defines this subtype of tumors in women and transcripts enriched in various mammary epithelial lineages to other well-studied genetically modified murine models of breast cancer. Finally, we used microarray analyses to compare prolactin-induced ERα+ and ERα- tumors, and examined responsiveness to estrogen and the anti-estrogen, Faslodex, in vivo.RESULTS:Prolactin-induced carcinomas were markedly diverse with respect to histotype, ERα/PR expression, and activated signaling cascades. They constituted a heterogeneous, but distinct group of murine mammary tumors, with molecular features of the luminal subtype of human breast cancer. In contrast to morphologically normal and hyperplastic structures in NRL-PRL females, carcinomas were insensitive to ERα-mediated signals. These tumors were distinct from mouse mammary tumor virus (MMTV)-neu tumors, and contained elevated transcripts for factors associated with luminal/alveolar expansion and differentiation, suggesting that they arose from physiologic targets of prolactin. These features were shared by ERα+ and ERα- tumors, suggesting a common origin, although the former exhibited transcript profiles reflecting greater differentiation.CONCLUSIONS:Our studies demonstrate that prolactin can promote diverse carcinomas in mice, many of which resemble luminal breast cancers, providing a novel experimental model to examine the pathogenesis, progression and treatment responsiveness of this tumor subtype.

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.

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.

Despite the important roles of both prolactin (PRL) and 17beta-estradiol (E2) in normal mammary development as well as in breast cancer, and coexpression of the estrogen receptor (ER) and PRL receptor in many mammary tumors, the interactions between PRL and E2 in breast cancer have not been well studied. The activating protein 1 (AP-1) transcription factor, a known regulator of processes essential for normal growth and development as well as carcinogenesis, is a potential site for cross-talk between these hormones in breast cancer cells. Here we demonstrate that PRL and E2 cooperatively enhance the activity of AP-1 in MCF-7-derived cells. In addition to the acute PRL-induced ERK1/2 activation, PRL and E2 also individually elicited delayed, sustained rises in levels of phosphorylated p38 and especially ERK1/2. Together, these hormones increased the dynamic phosphorylation of ERK1/2 and c-Fos, and induced c-fos promoter activity. Synergistic activation of the transcription factor, Elk-1, reflected the PRL-E2 interaction at ERK1/2 and is a likely mechanism for activation of the c-fos promoter via the serum response element. The enhanced AP-1 activity resulting from the interaction of these hormones may increase expression of many target genes that are critical for oncogenesis and may contribute to neoplastic progression.

The importance of prolactin (PRL) in physiological proliferation and differentiation of the mammary gland, together with high levels of PRL receptors in breast tumors, the association of circulating PRL with incidence of breast cancer, and the recognition of locally produced PRL, point to the need for greater understanding of PRL actions in mammary disease. Although PRL has been shown to activate multiple kinase cascades in various target cells, relatively little is known of its signaling pathways in the mammary gland apart from the Janus kinase 2/ signal transducer and activator of transcription 5 pathway, particularly in tumor cells. Another potential effector is activating protein-1 (AP-1), a transcription complex that regulates processes essential for neoplastic progression, including proliferation, survival and invasion. We demonstrate that PRL activates AP-1 in MCF-7 cells, detectable at 4 h and sustained for at least 24 h. Although Janus kinase 2 and ERK1/2 are the primary mediators of PRL-induced signals, c-Src, phosphatidylinositol 3'-kinase, protein kinase C, and other MAPKs contribute to maximal activity. PRL activation of these pathways leads to increased c-Jun protein and phosphorylation, JunB protein, and phosphorylation of c-Fos, elevating the levels of AP-1 complexes able to bind DNA. These active AP-1 dimers may direct expression of multiple target genes, mediating some of PRL's actions in mammary disease.