RESUMO
CCT251236 1, a potent chemical probe, was previously developed from a cell-based phenotypic high-throughput screen (HTS) to discover inhibitors of transcription mediated by HSF1, a transcription factor that supports malignancy. Owing to its activity against models of refractory human ovarian cancer, 1 was progressed into lead optimization. The reduction of P-glycoprotein efflux became a focus of early compound optimization; central ring halogen substitution was demonstrated by matched molecular pair analysis to be an effective strategy to mitigate this liability. Further multiparameter optimization led to the design of the clinical candidate, CCT361814/NXP800 22, a potent and orally bioavailable fluorobisamide, which caused tumor regression in a human ovarian adenocarcinoma xenograft model with on-pathway biomarker modulation and a clean in vitro safety profile. Following its favorable dose prediction to human, 22 has now progressed to phase 1 clinical trial as a potential future treatment for refractory ovarian cancer and other malignancies.
Assuntos
Antineoplásicos , Neoplasias Ovarianas , Humanos , Feminino , Fatores de Transcrição/metabolismo , Neoplasias Ovarianas/patologia , Linhagem Celular Tumoral , Antineoplásicos/farmacologiaRESUMO
A number of Gram-negative bacteria such as Pseudomonas aeruginosa are becoming resistant to front-line antibiotics. Consequently, there is a pressing need to find alternative bio-molecular targets for the development of new drugs. Since non-canonical DNA structures such as guanine-quadruplexes (G4s) have been implicated in regulating transcription, we were interested in determining whether there are putative quadruplex-forming sequences (PQS) in the genome of Pseudomonas aeruginosa. Using bioinformatic tools, we screened 36 genes potentially relevant to drug resistance for the presence of PQS and 10 of these were selected for biophysical characterisation (i.e. circular dichroism and thermal difference UV/Vis spectroscopy). These studies showed that three of these G-rich sequences (linked to murE, ftsB and mexC genes) form stable guanine-quadruplexes which were studied by NMR spectroscopy; detailed analysis of one of the sequences (mexC) confirmed that it adopts a two-quartet antiparallel quadruplex structure in the presence of K+ ions. We also show by FRET melting assays that small molecules can stabilise these three new G4 DNA structures under physiological conditions. These initial results could be of future interest in the development of new antibiotics with alternative bio-molecular targets which in turn would help tackle antimicrobial resistance.
RESUMO
Glucose metabolism modulates the islet ß cell responses to diabetogenic stress, including inflammation. Here, we probed the metabolic mechanisms that underlie the protective effect of glucose in inflammation by interrogating the metabolite profiles of primary islets from human donors and identified de novo glutathione synthesis as a prominent glucose-driven pro-survival pathway. We find that pyruvate carboxylase is required for glutathione synthesis in islets and promotes their antioxidant capacity to counter inflammation and nitrosative stress. Loss- and gain-of-function studies indicate that pyruvate carboxylase is necessary and sufficient to mediate the metabolic input from glucose into glutathione synthesis and the oxidative stress response. Altered redox metabolism and cellular capacity to replenish glutathione pools are relevant in multiple pathologies beyond obesity and diabetes. Our findings reveal a direct interplay between glucose metabolism and glutathione biosynthesis via pyruvate carboxylase. This metabolic axis may also have implications in other settings where sustaining glutathione is essential.
Assuntos
Glucose/metabolismo , Glutationa/biossíntese , Piruvato Carboxilase/metabolismo , Adulto , Animais , Antioxidantes/fisiologia , Feminino , Glutationa/metabolismo , Humanos , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Oxirredução , Estresse Oxidativo/fisiologia , Cultura Primária de CélulasRESUMO
Colistin is an antibiotic of last resort, but has poor efficacy and resistance is a growing problem. Whilst it is well established that colistin disrupts the bacterial outer membrane (OM) by selectively targeting lipopolysaccharide (LPS), it was unclear how this led to bacterial killing. We discovered that MCR-1 mediated colistin resistance in Escherichia coli is due to modified LPS at the cytoplasmic rather than OM. In doing so, we also demonstrated that colistin exerts bactericidal activity by targeting LPS in the cytoplasmic membrane (CM). We then exploited this information to devise a new therapeutic approach. Using the LPS transport inhibitor murepavadin, we were able to cause LPS accumulation in the CM of Pseudomonas aeruginosa, which resulted in increased susceptibility to colistin in vitro and improved treatment efficacy in vivo. These findings reveal new insight into the mechanism by which colistin kills bacteria, providing the foundations for novel approaches to enhance therapeutic outcomes.
Antibiotics are life-saving medicines, but many bacteria now have the ability to resist their effects. For some infections, all frontline antibiotics are now ineffective. To treat infections caused by these highly resistant bacteria, clinicians must use so-called 'antibiotics of last resort'. These antibiotics include a drug called colistin, which is moderately effective, but often fails to eradicate the infection. One of the challenges to making colistin more effective is that its mechanism is poorly understood. Bacteria have two layers of protection against the outside world: an outer cell membrane and an inner cell membrane. To kill them, colistin must punch holes in both. First, it disrupts the outer membrane by interacting with molecules called lipopolysaccharides. But how it disrupts the inner membrane was unclear. Bacteria have evolved several different mechanisms that make them resistant to the effects of colistin. Sabnis et al. reasoned that understanding how these mechanisms protected bacteria could reveal how the antibiotic works to damage the inner cell membrane. Sabnis et al. examined the effects of colistin on Escherichia coli bacteria with and without resistance to the antibiotic. Exposing these bacteria to colistin revealed that the antibiotic damages both layers of the cell surface in the same way, targeting lipopolysaccharide in the inner membrane as well as the outer membrane. Next, Sabnis et al. used this new information to make colistin work better. They found that the effects of colistin were magnified when it was combined with the experimental antibiotic murepavadin, which caused lipopolysaccharide to build up at the inner membrane. This allowed colistin to punch more holes through the inner membrane, making colistin more effective at killing bacteria. To find out whether this combination of colistin and murepavadin could work as a clinical treatment, Sabnis et al. tested it on mice with Pseudomonas aeruginosa infections in their lungs. Colistin was much better at killing Pseudomonas aeruginosa and treating infections when combined with murepavadin than it was on its own. Pseudomonas aeruginosa bacteria can cause infections in the lungs of people with cystic fibrosis. At the moment, patients receive colistin in an inhaled form to treat these infections, but it is not always successful. The second drug used in this study, murepavadin, is about to enter clinical trials as an inhaled treatment for lung infections too. If the trial is successful, it may be possible to use both drugs in combination to treat lung infections in people with cystic fibrosis.
Assuntos
Antibacterianos/farmacologia , Membrana Celular/efeitos dos fármacos , Colistina/farmacologia , Escherichia coli/efeitos dos fármacos , Lipopolissacarídeos/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Infecções Respiratórias/tratamento farmacológico , Animais , Membrana Celular/metabolismo , Modelos Animais de Doenças , Farmacorresistência Bacteriana , Quimioterapia Combinada , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Feminino , Humanos , Fluidez de Membrana/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Infecções Respiratórias/microbiologiaRESUMO
Chronic inflammation is linked to diverse disease processes, but the intrinsic mechanisms that determine cellular sensitivity to inflammation are incompletely understood. Here, we show the contribution of glucose metabolism to inflammation-induced changes in the survival of pancreatic islet ß-cells. Using metabolomic, biochemical and functional analyses, we investigate the protective versus non-protective effects of glucose in the presence of pro-inflammatory cytokines. When protective, glucose metabolism augments anaplerotic input into the TCA cycle via pyruvate carboxylase (PC) activity, leading to increased aspartate levels. This metabolic mechanism supports the argininosuccinate shunt, which fuels ureagenesis from arginine and conversely diminishes arginine utilization for production of nitric oxide (NO), a chief mediator of inflammatory cytotoxicity. Activation of the PC-urea cycle axis is sufficient to suppress NO synthesis and shield cells from death in the context of inflammation and other stress paradigms. Overall, these studies uncover a previously unappreciated link between glucose metabolism and arginine-utilizing pathways via PC-directed ureagenesis as a protective mechanism.
Assuntos
Arginina/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Inflamação/prevenção & controle , Células Secretoras de Insulina/efeitos dos fármacos , Distúrbios Congênitos do Ciclo da Ureia/patologia , Ureia/metabolismo , Adolescente , Adulto , Idoso , Ácido Aspártico/metabolismo , Sobrevivência Celular , Ciclo do Ácido Cítrico/efeitos dos fármacos , Feminino , Humanos , Inflamação/patologia , Células Secretoras de Insulina/patologia , Masculino , Metabolômica , Pessoa de Meia-Idade , Óxido Nítrico/metabolismo , Piruvato Carboxilase/metabolismo , Distúrbios Congênitos do Ciclo da Ureia/metabolismo , Adulto JovemRESUMO
The global emergence of antibiotic resistance is one of the most serious challenges facing modern medicine. There is an urgent need for validation of new drug targets and the development of small molecules with novel mechanisms of action. We therefore sought to inhibit bacterial DNA repair mediated by the AddAB/RecBCD protein complexes as a means to sensitize bacteria to DNA damage caused by the host immune system or quinolone antibiotics. A rational, hypothesis-driven compound optimization identified IMP-1700 as a cell-active, nanomolar potency compound. IMP-1700 sensitized multidrug-resistant Staphylococcus aureus to the fluoroquinolone antibiotic ciprofloxacin, where resistance results from a point mutation in the fluoroquinolone target, DNA gyrase. Cellular reporter assays indicated IMP-1700 inhibited the bacterial SOS-response to DNA damage, and compound-functionalized Sepharose successfully pulled-down the AddAB repair complex. This work provides validation of bacterial DNA repair as a novel therapeutic target and delivers IMP-1700 as a tool molecule and starting point for therapeutic development to address the pressing challenge of antibiotic resistance.
Assuntos
Antibacterianos/farmacologia , DNA Bacteriano/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Quinolonas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Reparo do DNA , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Quinolonas/síntese química , Quinolonas/química , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Expression of ß-lactamase is the single most prevalent determinant of antibiotic resistance, rendering bacteria resistant to ß-lactam antibiotics. In this article, we describe the development of an antibiotic prodrug that combines ciprofloxacin with a ß-lactamase-cleavable motif. The prodrug is only bactericidal after activation by ß-lactamase. Bactericidal activity comparable to ciprofloxacin is demonstrated against clinically relevant E. coli isolates expressing diverse ß-lactamases; bactericidal activity was not observed in strains without ß-lactamase. These findings demonstrate that it is possible to exploit antibiotic resistance to selectively target ß-lactamase-producing bacteria using our prodrug approach, without adversely affecting bacteria that do not produce ß-lactamase. This paves the way for selective targeting of drug-resistant pathogens without disrupting or selecting for resistance within the microbiota, reducing the rate of secondary infections and subsequent antibiotic use.
Assuntos
Antibacterianos/farmacologia , Cefalosporinas/farmacologia , Ciprofloxacina/análogos & derivados , Ciprofloxacina/farmacologia , Pró-Fármacos/farmacologia , beta-Lactamases/metabolismo , Antibacterianos/síntese química , Antibacterianos/metabolismo , Cefalosporinas/síntese química , Cefalosporinas/metabolismo , Ciprofloxacina/metabolismo , Resistência Microbiana a Medicamentos/fisiologia , Escherichia coli/efeitos dos fármacos , Hidrólise , Testes de Sensibilidade Microbiana , Estrutura Molecular , Pró-Fármacos/síntese química , Pró-Fármacos/metabolismo , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/síntese química , Inibidores da Topoisomerase II/metabolismo , Inibidores da Topoisomerase II/farmacologiaRESUMO
Polypharmacology is often a key contributor to the efficacy of a drug, but is also a potential risk. We investigated two hits discovered via a cell-based phenotypic screen, the CDK9 inhibitor CCT250006 (1) and the pirin ligand CCT245232 (2), to establish methodology to elucidate their secondary protein targets. Using computational pocket-based analysis, we discovered intrafamily polypharmacology for our kinase inhibitor, despite little overall sequence identity. The interfamily polypharmacology of 2 with B-Raf was used to discover a novel pirin ligand from a very small but privileged compound library despite no apparent ligand or binding site similarity. Our data demonstrates that in areas of drug discovery where intrafamily polypharmacology is often an issue, ligand dissimilarity cannot necessarily be used to assume different off-target profiles and that understanding interfamily polypharmacology will be important in the future to reduce the risk of idiopathic toxicity and in the design of screening libraries.
RESUMO
Proteins typically carry out their biological functions as multi-protein complexes, which can significantly affect the affinity of small-molecule inhibitors. HSP70 is an important target in oncology, so to study its chemical biology and the drug discovery potential of the HSP70/BAG1 complex, we designed a high-affinity non-nucleotide fluorescence polarisation probe.
Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Descoberta de Drogas , Corantes Fluorescentes/farmacologia , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Polarização de Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismoRESUMO
Phenotypic screens, which focus on measuring and quantifying discrete cellular changes rather than affinity for individual recombinant proteins, have recently attracted renewed interest as an efficient strategy for drug discovery. In this article, we describe the discovery of a new chemical probe, bisamide (CCT251236), identified using an unbiased phenotypic screen to detect inhibitors of the HSF1 stress pathway. The chemical probe is orally bioavailable and displays efficacy in a human ovarian carcinoma xenograft model. By developing cell-based SAR and using chemical proteomics, we identified pirin as a high affinity molecular target, which was confirmed by SPR and crystallography.
Assuntos
Amidas/química , Proteínas de Transporte/química , Proteínas de Ligação a DNA/química , Proteínas Nucleares/química , Quinolinas/química , Fatores de Transcrição/química , Administração Oral , Amidas/administração & dosagem , Amidas/farmacologia , Disponibilidade Biológica , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Dioxigenases , Descoberta de Drogas , Fatores de Transcrição de Choque Térmico , Ligantes , Espectroscopia de Prótons por Ressonância Magnética , Quinolinas/administração & dosagem , Quinolinas/farmacologia , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The present short-term longitudinal study examined the concurrent and prospective relations among executive functioning (i.e., working memory and cognitive flexibility), coping (primary and secondary control coping), and depressive symptoms in children. Participants were 192 children between 9 and 15 years old (M age = 12.36 years, SD = 1.77) recruited from the community. Youth were individually administered neuropsychological measures of executive functioning and intelligence and completed self-report measures of executive dysfunction, coping, and depressive symptoms in small groups; the latter two measures were completed again 4 months later (Time 2 [T2]). Linear regression analyses were used to examine direct associations among executive functions, coping, and depressive symptoms, and a bootstrapping procedure was used to test indirect effects of executive functioning on depressive symptoms through coping. Significant prospective relations were found between working memory measured at Time 1 (T1) and both primary and secondary control coping measured at T2, controlling for T1 coping. T1 cognitive flexibility significantly predicted T2 secondary control coping, controlling for T1 coping. Working memory deficits significantly predicted increases in depressive symptoms 4 months later, controlling for T1 depressive symptoms. Bootstrap analyses revealed that primary and secondary control coping each partially mediated the relation between working memory and depressive symptoms; secondary control coping partially mediated the relation between cognitive flexibility and depressive symptoms. Coping may be one pathway through which deficits in executive functioning contribute to children's symptoms of depression.
Assuntos
Adaptação Psicológica , Comportamento do Adolescente/psicologia , Comportamento Infantil/psicologia , Depressão/psicologia , Função Executiva , Adolescente , Criança , Feminino , Humanos , Estudos Longitudinais , Masculino , Memória de Curto Prazo , Estudos Prospectivos , Análise de Regressão , Autorrelato , Inquéritos e QuestionáriosRESUMO
The use of chemical tools to validate clinical targets has gained in popularity over recent years and the importance of understanding the activity, selectivity and mechanism of action of these compounds is well recognized. Dysregulation of the HSP70 protein family has been linked to multiple cancer types and drug resistance, highlighting their importance as popular targets for anti-cancer drug development. Apoptozole is a recently identified small molecule, which has been reported to possess strong affinity for the HSP70 isoforms HSP72 and HSC70. We investigated apoptozole as a potential chemical tool for HSP70 inhibition. Unfortunately, using both biochemical and biophysical techniques, we were unable to find any experimental evidence that apoptozole binds to HSP70 in a specific and developable way. Instead, we provide experimental evidence that apoptozole forms aggregates under aqueous conditions that could interact with HSP70 proteins in a non-specific manner.
Assuntos
Antineoplásicos/química , Antineoplásicos/metabolismo , Benzamidas/química , Proteínas de Choque Térmico HSP70/metabolismo , Imidazóis/química , Trifosfato de Adenosina/química , Animais , Benzamidas/metabolismo , Sítios de Ligação , Difusão Dinâmica da Luz , Corantes Fluorescentes/química , Proteínas de Choque Térmico HSC70/química , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP70/química , Humanos , Imidazóis/metabolismo , Ligação Proteica , Isoformas de Proteínas/metabolismo , Ratos , Ressonância de Plasmônio de SuperfícieRESUMO
The present prospective study examined the relations among stressful life events, coping, and depressive symptoms in children at varied risk for depression. Participants were 227 children between 7 and 17 years old (mean age = 12.13 years, SD = 2.31, 54.6 % female) who were part of a longitudinal study of depressed and nondepressed parents and their children. Youth completed measures assessing stressful life events and coping strategies at four time points over 22 months. Children's depressive symptoms were assessed at each time point by clinical interviews of parents and children, and children's self-report. Structural equation modeling indicated that stressful life events significantly predicted subsequent depressive symptoms. Bootstrap analyses of the indirect effects in three different models revealed that primary control engagement coping and disengagement coping strategies partially mediated the relation between stressful life events and children's depressive symptoms across time. Regarding the direction of effects, more consistent relations were found for coping as a mediator of the link from stress to depressive symptoms than from symptoms to stress. Thus, one potential mechanism by which stressful life events may contribute to depressive symptoms in children is through less use of primary control coping and greater use of disengagement coping strategies. This is consistent with the view that the adverse effects of stress may contribute to impairments in the ability to cope effectively.
Assuntos
Adaptação Psicológica , Depressão/psicologia , Estresse Psicológico/psicologia , Adolescente , Criança , Filho de Pais com Deficiência/psicologia , Feminino , Humanos , Acontecimentos que Mudam a Vida , Estudos Longitudinais , Masculino , Fatores Sexuais , Inquéritos e QuestionáriosRESUMO
BACKGROUND AND OBJECTIVES: Identifying risk factors early in the course of depression has important implications for prevention, given that the likelihood of recurrence increases with each successive episode. DESIGN: This study examined relations among coping, executive functioning, and depressive symptom trajectories in a sample of remitted-depressed (n = 32) and never-depressed (ND; n = 36) young adults (aged 18-31). METHODS: Participants completed a clinical interview, a measure of coping, and tasks assessing two components of executive function - inhibition and cognitive flexibility. Participants were reassessed regarding the timing and severity of depressive symptoms that had occurred during the interval period (mean = 35.16 weeks, SD = 9.03). RESULTS: Among ND individuals, less primary control coping (e.g., problem-solving) and greater disengagement coping (e.g., avoidance) predicted increases in depressive symptoms. Greater secondary control coping (e.g., acceptance) predicted decreases in depressive symptoms and was unrelated to depression history. Higher inhibition scores predicted less increase in depressive symptoms for individuals reporting less primary control coping or more disengagement coping. Higher cognitive flexibility scores predicted less increase in depressive symptoms among individuals reporting less secondary control coping. CONCLUSIONS: Interventions aiming to enhance either coping strategies or executive functions may reduce risk of depression recurrence.
Assuntos
Adaptação Psicológica , Depressão/psicologia , Função Executiva , Adolescente , Adulto , Depressão/etiologia , Feminino , Humanos , Masculino , Escalas de Graduação Psiquiátrica , Inquéritos e Questionários , Adulto JovemRESUMO
Functional analyses of elopement (i.e., leaving a specific area without permission) are challenging to conduct because clients must have repeated opportunities to elope from one room (or area) to another safely. These analyses often require two or more adjoining rooms and retrieval of the client following each instance of elopement (e.g., Piazza et al., 1997). These room arrangements may be impractical in some settings, and therapist delivery of attention or demands during retrieval may confound the results. To address these issues, we evaluated the viability of conducting a functional analysis (FA) of elopement within a single room. Participants were 2 children and 2 adults with developmental disabilities who eloped from rooms at their day programs. Results of the single-room assessments were compared to those of a second FA that was conducted using methods similar to those described in previous studies. Function-based treatments were implemented for each participant. Results suggest that the single-room assessment may be a viable alternative for identifying the function of elopement.
Assuntos
Terapia Comportamental/métodos , Deficiências do Desenvolvimento , Avaliação de Resultados em Cuidados de Saúde , Recusa do Paciente ao Tratamento/psicologia , Adulto , Criança , Deficiências do Desenvolvimento/diagnóstico , Deficiências do Desenvolvimento/psicologia , Deficiências do Desenvolvimento/terapia , Feminino , Humanos , Masculino , Reprodutibilidade dos TestesRESUMO
An efficient one-pot synthesis of N-aryl[3,4-d]pyrazolopyrimidines in good yield and under mild reaction conditions is described. By exploiting electron-deficient hydroxylamines, the substituted oxime products were formed with very high E-diastereoselectivity. The key step utilizes a cyclization reaction upon an oxime derived from hydroxylamine-O-sulfonic acid to form the N-N bond of the product.
Assuntos
Pirimidinas/síntese química , Ciclização , Estrutura Molecular , Pirimidinas/química , EstereoisomerismoRESUMO
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.