RESUMO
Systemic hormones and local growth factor-mediated tissue interactions are essential for mammary gland development. Using phenotypic and transplantation analyses of mice carrying the mesenchymal dysplasia (mes) allele of patched 1 (Ptch1(mes)), we found that Ptch1(mes) homozygosity led to either complete failure of gland development, failure of post-pubertal ductal elongation, or delayed growth with ductal dysplasia. All ductal phenotypes could be present in the same animal. Whole gland and epithelial fragment transplantation each yielded unique morphological defects indicating both epithelial and stromal functions for Ptch1. However, ductal elongation was rescued in all cases, suggesting an additional systemic function. Epithelial function was confirmed using a conditional null Ptch1 allele via MMTV-Cre-mediated disruption. In Ptch1(mes) homozygotes, failure of ductal elongation correlated with diminished estrogen and progesterone receptor expression, but could not be rescued by exogenous ovarian hormone treatment. By contrast, pituitary isografts were able to rescue the ductal elongation phenotype. Thus, Ptch1 functions in the mammary epithelium and stroma to regulate ductal morphogenesis, and in the pituitary to regulate ductal elongation and ovarian hormone responsiveness.
Assuntos
Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Morfogênese , Receptores de Superfície Celular/metabolismo , Animais , Sequência de Bases , Epitélio/metabolismo , Estrogênios/farmacologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter/genética , Heterozigoto , Homozigoto , Humanos , Masculino , Glândulas Mamárias Animais/anormalidades , Glândulas Mamárias Animais/efeitos dos fármacos , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Camundongos Transgênicos , Mutação/genética , Receptores Patched , Receptor Patched-1 , Polimorfismo Genético/genética , Progesterona/farmacologia , Receptores de Superfície Celular/genética , Células Estromais/metabolismoRESUMO
Altered hedgehog signaling is implicated in the development of approximately 20-25% of all cancers, especially those of soft tissues. Genetic evidence in mice as well as immunolocalization studies in human breast cancer specimens suggest that deregulated hedgehog signaling may contribute to breast cancer development. Indeed, two recent studies demonstrated that anchorage-dependent growth of some human breast cancer cell lines is impaired by cyclopamine, a potent hedgehog signaling antagonist targeting the Smoothened (SMO) protein. However, specificity of cyclopamine at the dosage required for growth inhibition (> or =10 microM) remained an open question. In this paper we demonstrate that hedgehog signaling antagonists, including cyclopamine, and a second compound, CUR0199691, can inhibit growth of estrogen receptor (ER)-positive and ER-negative tumorigenic breast cancer cells at elevated doses. However, our results indicate that, for most breast cancer cell lines, growth inhibition by these compounds can be independent of detectable Smo gene expression. Rather, our results suggest that cyclopamine and CUR0199691 have unique secondary molecular targets at the dosages required for growth inhibition that are unrelated to hedgehog signaling.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Alcaloides de Veratrum/farmacologia , Apoptose/efeitos dos fármacos , Ligação Competitiva , Neoplasias da Mama/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Colesterol/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Citometria de Fluxo , Imunofluorescência , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Receptor SmoothenedRESUMO
The hedgehog signaling network regulates pattern formation, proliferation, cell fate and stem/progenitor cell self-renewal in many organs. Altered hedgehog signaling is implicated in 20-25% of all cancers, including breast cancer. We demonstrated previously that heterozygous disruption of the gene encoding the patched-1 (PTCH1) hedgehog receptor, a negative regulator of smoothened (Smo) in the absence of ligand, led to mammary ductal dysplasia in virgin mice. We now show that expression of activated human SMO (SmoM2) under the mouse mammary tumor virus (MMTV) promoter in transgenic mice leads to increased proliferation, altered differentiation, and ductal dysplasias distinct from those caused by Ptch1 heterozygosity. SMO activation also increased the mammosphere-forming efficiency of primary mammary epithelial cells. However, limiting-dilution transplantation showed a decrease in the frequency of regenerative stem cells in MMTV-SmoM2 epithelium relative to wild type, suggesting enhanced mammosphere-forming efficiency was due to increased survival or activity of division-competent cell types under anchorage-independent growth conditions, rather than an increase in the proportion of regenerative stem cells per se. In human clinical samples, altered hedgehog signaling occurs early in breast cancer development, with PTCH1 expression reduced in approximately 50% of ductal carcinoma in situ (DCIS) and invasive breast cancers (IBC). Conversely, SMO is ectopically expressed in 70% of DCIS and 30% of IBC. Surprisingly, in both human tumors and MMTV-SmoM2 mice, SMO rarely colocalized with the Ki67 proliferation marker. Our data suggest that altered hedgehog signaling may contribute to breast cancer development by stimulating proliferation, and by increasing the pool of division-competent cells capable of anchorage-independent growth.