RESUMO
BACKGROUND: Most patients with estrogen receptor positive (ER+) breast cancer do not respond to immune checkpoint inhibition (ICI); the tumor microenvironment (TME) of these cancers is generally immunosuppressive and contains few tumor-infiltrating lymphocytes. Radiation therapy (RT) can increase tumor inflammation and infiltration by lymphocytes but does not improve responses to ICIs in these patients. This may result, in part, from additional effects of RT that suppress anti-tumor immunity, including increased tumor infiltration by myeloid-derived suppressor cells and regulatory T cells. We hypothesized that anti-estrogens, which are a standard of care for ER+ breast cancer, may ameliorate these detrimental effects of RT by reducing the recruitment/ activation of suppressive immune populations in the radiated TME, increasing anti-tumor immunity and responsiveness to ICIs. METHODS: To interrogate the effect of the selective estrogen receptor downregulator, fulvestrant, on the irradiated TME in the absence of confounding growth inhibition by fulvestrant on tumor cells, we used the TC11 murine model of anti-estrogen resistant ER+ breast cancer. Tumors were orthotopically transplanted into immunocompetent syngeneic mice. Once tumors were established, we initiated treatment with fulvestrant or vehicle, followed by external beam RT one week later. We examined the number and activity of tumor infiltrating immune cells using flow cytometry, microscopy, transcript levels, and cytokine profiles. We tested whether fulvestrant improved tumor response and animal survival when added to the combination of RT and ICI. RESULTS: Despite resistance of TC11 tumors to anti-estrogen therapy alone, fulvestrant slowed tumor regrowth following RT, and significantly altered multiple immune populations in the irradiated TME. Fulvestrant reduced the influx of Ly6C+Ly6G+ cells, increased markers of pro-inflammatory myeloid cells and activated T cells, and augmented the ratio of CD8+: FOXP3+ T cells. In contrast to the minimal effects of ICIs when co-treated with either fulvestrant or RT alone, combinatorial treatment with fulvestrant, RT and ICIs significantly reduced tumor growth and prolonged survival. CONCLUSIONS: A combination of RT and fulvestrant can overcome the immunosuppressive TME in a preclinical model of ER+ breast cancer, enhancing the anti-tumor response and increasing the response to ICIs, even when growth of tumor cells is no longer estrogen sensitive.
Assuntos
Neoplasias , Receptores de Estrogênio , Animais , Camundongos , Fulvestranto/farmacologia , Imunoterapia , Estrogênios , Antagonistas de Estrogênios , ImunossupressoresRESUMO
BACKGROUND: 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. METHODS: In 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 (col1a1 tm1Jae/+ ) 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. RESULTS: ERα + primary tumors did not differ in growth rate, histologic type, ERα, or prolactin receptor (PRLR) expression between col1a1 tm1Jae/+ and WT recipients. However, the col1a1 tm1Jae/+ environment significantly increased circulating tumor cells and the number and size of lung metastases at end stage. Tumors in col1a1 tm1Jae/+ recipients displayed reduced STAT5 activation, and higher phosphorylation of ERK1/2 and AKT. Moreover, intratumoral collagen fibers in col1a1 tm1Jae/+ 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. CONCLUSIONS: A collagen-dense extracellular matrix can potently interact with hormonal signals to drive metastasis of ERα + breast cancers.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Colágeno Tipo I/metabolismo , Receptor alfa de Estrogênio/metabolismo , Prolactina/metabolismo , Transdução de Sinais , Animais , Biomarcadores , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células , Colágeno Tipo I/genética , Progressão da Doença , Matriz Extracelular/metabolismo , Feminino , Imuno-Histoquímica , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Transgênicos , Metástase Neoplásica , Estadiamento de Neoplasias , Células Neoplásicas Circulantes , Proteínas Proto-Oncogênicas c-akt , Fator de Transcrição STAT5/metabolismo , Carga TumoralRESUMO
The circadian clock plays a significant role in many aspects of female reproductive biology, including estrous cycling, ovulation, embryonic implantation, onset of puberty, and parturition. In an effort to link cell-specific circadian clocks to their specific roles in female reproduction, we used the promoter that controls expression of Steroidogenic Factor-1 (SF1) to drive Cre-recombinase-mediated deletion of the brain muscle arnt-like 1 (Bmal1) gene, known to encode an essential component of the circadian clock (SF1-Bmal1(-/-)). The resultant SF1-Bmal1(-/-) females display embryonic implantation failure, which is rescued by progesterone supplementation, or bilateral or unilateral transplantation of wild-type ovaries into SF1-Bmal1(-/-) dams. The observation that the central clock, and many other peripheral clocks, are fully functional in this model allows the assignment of the implantation phenotype to the clock in ovarian steroidogenic cells and distinguishes it from more general circadian related systemic pathology (e.g., early onset arthropathy, premature aging, ovulation, late onset of puberty, and abnormal estrous cycle). Our ovarian transcriptome analysis reveals that deletion of ovarian Bmal1 disrupts expression of transcripts associated with the circadian machinery and also genes critical for regulation of progesterone production, such as steroidogenic acute regulatory factor (Star). Overall, these data provide a powerful model to probe the interlocking and synergistic network of the circadian clock and reproductive systems.
Assuntos
Fatores de Transcrição ARNTL/deficiência , Fatores de Transcrição ARNTL/fisiologia , Implantação do Embrião/fisiologia , Ovário/citologia , Ovário/fisiologia , Esteroides/biossíntese , Fatores de Transcrição ARNTL/genética , Envelhecimento/genética , Envelhecimento/fisiologia , Animais , Ritmo Circadiano/genética , Ritmo Circadiano/fisiologia , Implantação do Embrião/efeitos dos fármacos , Implantação do Embrião/genética , Estro/genética , Estro/fisiologia , Feminino , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovário/transplante , Gravidez , Progesterona/administração & dosagem , Regiões Promotoras Genéticas , Maturidade Sexual/genética , Maturidade Sexual/fisiologia , Fator Esteroidogênico 1/genéticaRESUMO
Elevated exposure to prolactin (PRL) is epidemiologically associated with an increased risk of aggressive ER+ breast cancer. To understand the underlying mechanisms and crosstalk with other oncogenic factors, we developed the NRL-PRL mouse. In this model, mammary expression of a rat prolactin transgene raises local exposure to PRL without altering estrous cycling. Nulliparous females develop metastatic, histotypically diverse mammary carcinomas independent from ovarian steroids, and most are ER+. These characteristics resemble the human clinical disease, facilitating study of tumorigenesis, and identification of novel preventive and therapeutic approaches.
Assuntos
Neoplasias da Mama/patologia , Neoplasias Mamárias Experimentais/patologia , Prolactina/fisiologia , Animais , Neoplasias da Mama/genética , Estradiol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Neoplasias Mamárias Experimentais/genética , Camundongos , Camundongos Transgênicos , Prolactina/sangue , Prolactina/genética , Ratos , Receptor Cross-Talk/fisiologiaRESUMO
Clinically, circulating prolactin levels and density of the extracellular matrix (ECM) are individual risk factors for breast cancer. As tumors develop, the surrounding stroma responds with increased deposition and cross-linking of the collagen matrix (desmoplasia). In mouse models, prolactin promotes mammary carcinomas that resemble luminal breast cancers in women, and increased collagen density promotes tumor metastasis and progression. Although the contributions of the ECM to the physiologic actions of prolactin are increasingly understood, little is known about the functional relationship between the ECM and prolactin signaling in breast cancer. Here, we examined consequences of increased ECM stiffness on prolactin signals to luminal breast cancer cells in three-dimensional collagen I matrices in vitro. We showed that matrix stiffness potently regulates a switch in prolactin signals from physiologic to protumorigenic outcomes. Compliant matrices promoted physiological prolactin actions and activation of STAT5, whereas stiff matrices promoted protumorigenic outcomes, including increased matrix metalloproteinase-dependent invasion and collagen scaffold realignment. In stiff matrices, prolactin increased SRC family kinase-dependent phosphorylation of focal adhesion kinase (FAK) at tyrosine 925, FAK association with the mitogen-activated protein kinase mediator GRB2, and pERK1/2. Stiff matrices also increased co-localization of prolactin receptors and integrin-activated FAK, implicating altered spatial relationships. Together, these results demonstrate that ECM stiffness is a powerful regulator of the spectrum of prolactin signals and that stiff matrices and prolactin interact in a feed-forward loop in breast cancer progression. Our study is the first reported evidence of altered ECM-prolactin interactions in breast cancer, suggesting the potential for new therapeutic approaches.
Assuntos
Neoplasias da Mama/metabolismo , Colágeno Tipo I/metabolismo , Matriz Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Neoplasias Mamárias Animais/metabolismo , Proteínas de Neoplasias/metabolismo , Prolactina/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Colágeno Tipo I/genética , Matriz Extracelular/genética , Matriz Extracelular/patologia , Feminino , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/metabolismo , Humanos , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/patologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas de Neoplasias/genética , Fosforilação/genética , Prolactina/genética , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismoRESUMO
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.
Assuntos
Adenocarcinoma/fisiopatologia , Ciclina D1/fisiologia , Neoplasias Mamárias Experimentais/fisiopatologia , Prolactina/fisiologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Ciclina D1/deficiência , Ciclina D1/metabolismo , Ciclina D3/metabolismo , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Feminino , Lactação/fisiologia , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Neoplasias Mamárias Experimentais/prevenção & controle , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiologia , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Gravidez , Prolactina/metabolismoRESUMO
Prolactin coordinates with the ovarian steroids to orchestrate mammary development and lactation, culminating in nourishment and an increasingly appreciated array of other benefits for neonates. Its central activities in mammary epithelial growth and differentiation suggest that it plays a role(s) in breast cancer, but it has been challenging to identify its contributions, essential for incorporation into prevention and treatment approaches. Large prospective epidemiologic studies have linked higher prolactin exposure to increased risk, particularly for ER+ breast cancer in postmenopausal women. However, it has been more difficult to determine its actions and clinical consequences in established tumors. Here we review experimental data implicating multiple mechanisms by which prolactin may increase the risk of breast cancer. We then consider the evidence for role(s) of prolactin and its downstream signaling cascades in disease progression and treatment responses, and discuss how new approaches are beginning to illuminate the biology behind the seemingly conflicting epidemiologic and experimental studies of prolactin actions across diverse breast cancers.
Assuntos
Neoplasias da Mama , Prolactina , Neoplasias da Mama/epidemiologia , Progressão da Doença , Feminino , Humanos , Recém-Nascido , Lactação , Estudos ProspectivosRESUMO
Radiation therapy (RT) activates an in situ vaccine effect when combined with immune checkpoint blockade (ICB), yet this effect may be limited because RT does not fully optimize tumor antigen presentation or fully overcome suppressive mechanisms in the tumor-immune microenvironment. To overcome this, we develop a multifunctional nanoparticle composed of polylysine, iron oxide, and CpG (PIC) to increase tumor antigen presentation, increase the ratio of M1:M2 tumor-associated macrophages, and enhance stimulation of a type I interferon response in conjunction with RT. In syngeneic immunologically "cold" murine tumor models, the combination of RT, PIC, and ICB significantly improves tumor response and overall survival resulting in cure of many mice and consistent activation of tumor-specific immune memory. Combining RT with PIC to elicit a robust in situ vaccine effect presents a simple and readily translatable strategy to potentiate adaptive anti-tumor immunity and augment response to ICB or potentially other immunotherapies.
Assuntos
Nanopartículas Multifuncionais , Neoplasias , Animais , Antígenos de Neoplasias , Linhagem Celular Tumoral , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia/métodos , Camundongos , Neoplasias/radioterapia , Microambiente Tumoral , VacinaçãoRESUMO
Normal mammary development requires coordinated interactions of numerous factors, including prolactin (PRL) and insulin-like growth factor I (IGF-I), both of which have also been implicated in breast cancer pathogenesis and progression. We previously reported that PRL and IGF-I synergize in breast cancer cells to activate ERK1/2 and AKT, leading to increased proliferation, survival, and invasion. Intriguingly, PRL co-treatment with IGF-I augments IGF-I receptor (IGF-IR) phosphorylation 2-fold higher than IGF-I alone. Here, we showed the importance of the tyrosine phosphatase SHP-2 in this cross-talk using pharmacological inhibition and small interfering RNA. SHP-2 recruitment to IGF-IR was significantly attenuated by PRL co-treatment. Src family kinase activity was required for IGF-IR association with SHP-2, ligand-induced IGF-IR internalization, and PRL-enhanced IGF-IR phosphorylation. Inhibition of internalization, via knockdown of the GTPase, dynamin-2, prevented not only IGF-IR dephosphorylation, but also PRL-enhanced IGF-IR phosphorylation. Consistently, PRL diminished IGF-I-induced IGF-IR internalization, which may result from reduced SHP-2 association with IGF-IR, because we demonstrated an essential role for SHP-2 in IGF-IR internalization. Together, these findings describe a novel mechanism of cross-talk between PRL and IGF-I in breast cancer cells, with implications for our understanding of tumor progression and potential therapeutic strategies.
Assuntos
Neoplasias da Mama/metabolismo , Prolactina/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais/fisiologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Proteínas de Membrana/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Prolactina/farmacologia , RNA Interferente Pequeno , Receptor Cross-Talk/fisiologia , Receptor IGF Tipo 1/genéticaRESUMO
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.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Estrogênios/fisiologia , Neoplasias Mamárias Experimentais/metabolismo , Prolactina/genética , Animais , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Carcinoma/genética , Análise por Conglomerados , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Neoplasias Mamárias Experimentais/etiologia , Neoplasias Mamárias Experimentais/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Prolactina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT5/metabolismo , Fator de Transcrição AP-1/metabolismoRESUMO
Estrogen receptor alpha (ERα) marks heterogeneous breast cancers which display a repertoire of somatic genomic mutations and an immune environment that differs from other breast cancer subtypes. These cancers also exhibit distinct biological behaviors; despite an overall better prognosis than HER2+ or triple negative breast cancers, disseminated dormant cells can lead to disease recurrence decades after the initial diagnosis and treatment. Estrogen is the best studied driver of these cancers, and antagonism or reduction of estrogen activity is the cornerstone of therapeutic approaches. In addition to reducing proliferation of ERα+ cancer cells, these treatments also alter signals to multiple other target cells in the environment, including immune cell subpopulations, cancer-associated fibroblasts, and endothelial cells via several distinct estrogen receptors. In this review, we update progress in our understanding of the stromal cells populating the microenvironments of primary and metastatic ER+ tumors, the effects of estrogen on tumor and stromal cells to modulate immune activity and the extracellular matrix, and net outcomes in experimental and clinical studies. We highlight new approaches that will illuminate the unique biology of these cancers, provide the foundation for developing new treatment and prevention strategies, and reduce mortality of this disease.
RESUMO
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 ß-catenin, PRL-promoted tumors displayed higher levels of Notch-driven target genes and Notch-dependent cancer stem cell activity, compared to ß-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 ß-catenin at cell junctions and high NOTCH1 expression, contrasted with slower growing lesions with nuclear ß-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.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Neoplasias Mamárias Experimentais/patologia , Mutagênicos/toxicidade , Prolactina/genética , Animais , Núcleo Celular/metabolismo , Proliferação de Células , Feminino , Neoplasias Mamárias Experimentais/induzido quimicamente , Neoplasias Mamárias Experimentais/genética , Camundongos , Camundongos Transgênicos , Receptores Notch/metabolismo , Via de Sinalização WntRESUMO
Most breast cancers that occur in women express estrogen receptor alpha (ERalpha). However, a large subset of these cancers either does not initially respond to anti-estrogen therapy or develops resistance to such treatment modalities. One postulated mechanism of this failure is signaling cross talk between hormones and local growth factors. To examine these complex interactions in vivo, we assessed the effects of estrogen on transforming growth factor alpha (TGFalpha)- and prolactin (PRL)-induced mammary tumorigenesis in transgenic mice. Both PRL and estrogen reduced the latency of TGFalpha-induced oncogenesis, resulting in tumors that were variably ERalpha-positive, but were progesterone receptor-negative. However, despite elevated ERalpha levels in NRL-PRL/TGFalpha glands, tumor latency was not reduced with increasing estrogen levels, nor increased after ovariectomy. Furthermore, PRL and TGFalpha in combination blocked the mitogenic effects of estrogen, dramatically reduced progesterone receptor levels, and diminished ERalpha down-regulation in response to circulating estrogen levels, in contrast to the other genotypes. Notably, however, ductal morphology remained responsive to estrogen, indicating that TGFalpha and PRL in combination can inhibit some, but not all, estrogenic signals. Both in vitro and in vivo, PRL and TGFalpha cooperatively enhanced Akt phosphorylation, which is associated with endocrine resistance in human disease. These findings provide insight into the interactions of PRL with growth factors during mammary oncogenesis and suggest combinatorial approaches that may result in improved therapeutic efficacy.
Assuntos
Estradiol/farmacologia , Receptor alfa de Estrogênio/genética , Estrogênios/farmacologia , Substâncias de Crescimento/fisiologia , Glândulas Mamárias Animais/fisiologia , Prolactina/fisiologia , Receptor Cross-Talk/fisiologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Glândulas Mamárias Animais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Ovariectomia , Prolactina/farmacologia , Receptor Cross-Talk/efeitos dos fármacos , Fator de Crescimento Transformador alfa/farmacologia , Células Tumorais CultivadasRESUMO
Despite the growing body of evidence supporting prolactin (PRL) actions in human breast cancer, little is known regarding PRL regulation of its own receptor in these cells. Ligand-initiated endocytosis is a key process in the regulation of receptor availability and signaling cascades that may lead to oncogenic actions. Although exposure to exogenous PRL accelerates degradation of the long isoform of the PRL receptor (lPRLR), neither the signals initiated by PRL that lead to lPRLR internalization and subsequent down-regulation, nor the relationship to downstream pathways are understood in breast cancer cells. In this study, we showed that PRL-induced down-regulation of the lPRLR was reduced by inhibition of src family kinases (SFKs), but not Janus kinase 2, in MCF-7 cells. Inhibition of SFKs also resulted in accumulation of a PRL-induced PRLR fragment containing the extracellular domain, which appeared to be generated from newly synthesized PRLR. lPRLR was constitutively associated with SFKs in lipid rafts. PRL-induced SFK activation led to recruitment of the guanosine triphosphatase, dynamin-2, to an internalization complex, resulting in endocytosis. Inhibition of endocytosis by small interfering RNA-mediated knockdown of dynamin-2 blocked PRL-induced down-regulation of lPRLR, confirming that internalization is essential for this process. Endocytosis also was required for optimal phosphorylation of ERK1/2 and Akt, but not for Janus kinase 2 or signal transducer and activator of transcription 5, indicating that internalization selectively modulates signaling cascades. Together, these data indicate that SFKs are key mediators of ligand-initiated lPRLR internalization, down-regulation, and signal transduction in breast cancer cells, and underscore the importance of target cell context in receptor trafficking and signal transduction.
Assuntos
Neoplasias da Mama , Endocitose/fisiologia , Receptores da Prolactina/metabolismo , Transdução de Sinais/fisiologia , Quinases da Família src/metabolismo , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Dinamina II/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Microdomínios da Membrana/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , Quinases da Família src/genéticaRESUMO
Accumulating evidence suggests that our ability to predict chemical effects on breast cancer is limited by a lack of physiologically relevant in vitro models; the typical in vitro breast cancer model consists of the cancer cell and excludes the mammary microenvironment. As the effects of the microenvironment on cancer cell behavior becomes more understood, researchers have called for the integration of the microenvironment into in vitro chemical testing systems. However, given the complexity of the microenvironment and the variety of platforms to choose from, identifying the essential parameters to include in a chemical testing platform is challenging. This review discusses the need for more complex in vitro breast cancer models and outlines different approaches used to model breast cancer in vitro. We provide examples of the microenvironment modulating breast cancer cell responses to chemicals and discuss strategies to help pinpoint what components should be included in a model.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Microambiente Tumoral/efeitos dos fármacos , Carcinoma Intraductal não Infiltrante/diagnóstico , Linhagem Celular Tumoral , Progressão da Doença , Ensaios de Seleção de Medicamentos Antitumorais , Epitélio/patologia , Matriz Extracelular/metabolismo , Feminino , Humanos , Inflamação , Modelos Estatísticos , Neoplasias , Fenótipo , Engenharia TecidualRESUMO
Polydimethylsiloxane (PDMS) has become a staple of the microfluidics community by virtue of its simple fabrication process and material attributes, such as gas permeability, optical transparency, and flexibility. As microfluidic systems are put toward biological problems and increasingly utilized as cell culture platforms, the material properties of PDMS must be considered in a biological context. Two properties of PDMS were addressed in this study: the leaching of uncured oligomers from the polymer network into microchannel media, and the absorption of small, hydrophobic molecules (i.e. estrogen) from serum-containing media into the polymer bulk. Uncured PDMS oligomers were detectable via MALDI-MS in microchannel media both before and after Soxhlet extraction of PDMS devices in ethanol. Additionally, PDMS oligomers were identified in the plasma membranes of NMuMG cells cultured in PDMS microchannels for 24 hours. Cells cultured in extracted microchannels also contained a detectable amount of uncured PDMS. It was shown that MCF-7 cells seeded directly on PDMS inserts were responsive to hydrophilic prolactin but not hydrophobic estrogen, reflecting its specificity for absorbing small, hydrophobic molecules; and the presence of PDMS floating in wells significantly reduced cellular response to estrogen in a serum-dependent manner. Quantification of estrogen via ELISA revealed that microchannel estrogen partitioned rapidly into the surrounding PDMS to a ratio of approximately 9:1. Pretreatments such as blocking with serum or pre-absorbing estrogen for 24 hours did not affect estrogen loss from PDMS-based microchannels. These findings highlight the importance of careful consideration of culture system properties when determining an appropriate environment for biological experiments.
Assuntos
Membrana Celular/efeitos dos fármacos , Dimetilpolisiloxanos/farmacologia , Técnicas Analíticas Microfluídicas/métodos , Animais , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Técnicas de Cultura de Células/normas , Linhagem Celular , Membrana Celular/metabolismo , Meios de Cultura/química , Dimetilpolisiloxanos/análise , Dimetilpolisiloxanos/química , Ensaio de Imunoadsorção Enzimática , Estrogênios/química , Humanos , Luciferases/genética , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/normas , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismoRESUMO
Insulin-like growth factor (IGF)-II is a required intermediate for prolactin-induced up-regulation of cyclin D1 and proliferation in normal murine mammary epithelial cells in vivo and in vitro. However, we have recently shown that prolactin can rapidly induce cyclin D1 protein expression and subsequent proliferation in the MCF-7 human breast cancer cell line, suggesting that prolactin actions can be independent of IGFs in breast disease. Here, we investigate the relationship between these factors and show that prolactin up-regulated transcript levels of both IGF-I and IGF-II, but only after increases in cyclin D1 protein were observed. Moreover, prolactin increased cyclin D1 in the presence of the IGF-I receptor neutralizing antibody alphaIR3. However, on cotreatment, IGF-I and prolactin elicited cooperative phosphorylation of extracellular signal-regulated kinases 1 and 2 and protein kinase B/AKT, but not signal transducer and activator of transcription 5. This interaction extended to increased activation of activating protein-1 enhancer elements, phosphorylation of glycogen synthase kinase 3beta, induction of cyclin D1, and ultimately, increased cell number. It also increased invasive behavior, which correlated with elevated matrix metalloproteinase-2 transcript levels. Interestingly, prolactin augmented phosphorylation at Tyr(1135) and Tyr(1136) of IGF-I receptor on cotreatment with IGF-I, although prolactin alone had no effect. Together, these data indicate that strong cooperative cross talk between prolactin and IGF-I augments biological processes associated with neoplastic progression, with implications for therapeutic strategies.
Assuntos
Neoplasias da Mama/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Prolactina/farmacologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ciclina D1/metabolismo , Progressão da Doença , Elementos Facilitadores Genéticos , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Fosforilação/efeitos dos fármacos , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição AP-1/genéticaRESUMO
Transgenic models to explore the role of prolactin and its interactions with other factors in mammary oncogenesis have begun to reveal the dynamic contributions of prolactin to the development and progression of this disease. Targeting prolactin to mammary epithelial cells mimics the local production of this hormone that is prominent in women, and permits studies in the absence of effects on the ovarian steroid milieu. These models have demonstrated that local production of prolactin is sufficient to induce mammary tumors after a long latency. Prolactin also can potentiate actions of other oncogenic stimuli, decreasing tumor latency and increasing incidence in several models. Augmented proliferation, without alteration of apoptosis, is a consistent feature. Pathways in addition to the well-characterized Jak2-Stat5 pathway, including ERK1/2 and Akt1/2, are implicated in these actions. These studies have also revealed a complex relationship with estrogen; while prolactin increases ERalpha expression, it does not require estrogenic ligand for lesion development, and indeed, in combination with the EGFR ligand, TGFalpha, prolactin can contribute to estrogen insensitivity. These studies highlight the utility of these models to decipher the interplay between prolactin and other oncogenic factors in breast cancer, with implications for preventative and therapeutic strategies.
Assuntos
Neoplasias da Mama/metabolismo , Transformação Celular Neoplásica/metabolismo , Prolactina/metabolismo , Animais , Animais Geneticamente Modificados , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Estrogênios/metabolismo , Humanos , Proteínas Oncogênicas v-erbB/classificação , Proteínas Oncogênicas v-erbB/metabolismoRESUMO
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 17ß-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.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Receptor alfa de Estrogênio/metabolismo , Estrogênios/metabolismo , Matriz Extracelular/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Estradiol/metabolismo , Antagonistas de Estrogênios/farmacologia , Matriz Extracelular/efeitos dos fármacos , Feminino , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Camundongos , Tamoxifeno/farmacologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
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.