RESUMO
INTRODUCTION: Amplification of the HER-2 receptor tyrosine kinase has been implicated in the pathogenesis and aggressive behavior of approximately 25% of invasive human breast cancers. Clinical and experimental evidence suggest that aberrant HER-2 signaling contributes to tumor initiation and disease progression. Transforming growth factor beta (TGF-beta) is the dominant factor opposing growth stimulatory factors and early oncogene activation in many tissues, including the mammary gland. Thus, to better understand the mechanisms by which HER-2 overexpression promotes the early stages of breast cancer, we directly assayed the cellular and molecular effects of TGF-beta1 on breast cancer cells in the presence or absence of overexpressed HER-2. METHODS: Cell proliferation assays were used to determine the effect of TGF-beta on the growth of breast cancer cells with normal or high level expression of HER-2. Affymetrix microarrays combined with Northern and western blot analysis were used to monitor the transcriptional responses to exogenous TGF-beta1 in luminal and mesenchymal-like breast cancer cells. The activity of the core TGF-beta signaling pathway was assessed using TGF-beta1 binding assays, phospho-specific Smad antibodies, immunofluorescent staining of Smad and Smad DNA binding assays. RESULTS: We demonstrate that cells engineered to over-express HER-2 are resistant to the anti-proliferative effect of TGF-beta1. HER-2 overexpression profoundly diminishes the transcriptional responses induced by TGF-beta in the luminal MCF-7 breast cancer cell line and prevents target gene induction by a novel mechanism that does not involve the abrogation of Smad nuclear accumulation, DNA binding or changes in c-myc repression. Conversely, HER-2 overexpression in the context of the mesenchymal MDA-MB-231 breast cell line potentiated the TGF-beta induced pro-invasive and pro-metastatic gene signature. CONCLUSION: HER-2 overexpression promotes the growth and malignancy of mammary epithelial cells, in part, by conferring resistance to the growth inhibitory effects of TGF-beta. In contrast, HER-2 and TGF-beta signaling pathways can cooperate to promote especially aggressive disease behavior in the context of a highly invasive breast tumor model.
Assuntos
Neoplasias da Mama/genética , Receptor ErbB-2/biossíntese , Fator de Crescimento Transformador beta/fisiologia , Northern Blotting , Western Blotting , Neoplasias da Mama/patologia , Proliferação de Células , Progressão da Doença , Células Epiteliais , Feminino , Perfilação da Expressão Gênica , Engenharia Genética , Humanos , Glândulas Mamárias Humanas/citologia , Mesoderma , Invasividade Neoplásica , Transdução de Sinais , Fator de Crescimento Transformador beta1RESUMO
Genomic expression profiling has greatly improved our ability to subclassify human breast cancers according to shared molecular characteristics and clinical behavior. The logical next question is whether this technology will be similarly useful for identifying the dominant signaling pathways that drive tumor initiation and progression within each breast cancer subtype. A major challenge will be to integrate data generated from the experimental manipulation of model systems with expression profiles obtained from primary tumors. We highlight some recent progress and discuss several obstacles in the use of expression profiling to identify pathway signatures in human breast cancer.
Assuntos
Neoplasias da Mama/genética , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Humanos , Prognóstico , Receptores de Estrogênio , Transdução de SinaisRESUMO
Dasatinib is an orally active small molecule kinase inhibitor of both the src and abl proteins. To evaluate the potential role of dasatinib in breast cancer we used 39 human breast cancer cell lines that have been molecular profiled using Agilent Microarrays. They represent both luminal and basal breast cancer subtypes based on the relative gene expression of cytokeratin (CK) 8/CK18 and CK5/CK17, respectively, and those that have undergone an epithelial-to-mesenchymal transition (post-EMT) based on their expression of vimentin and the loss of CKs. When treated with 1 mICROM dasatinib in vitro 8 of them were highly sensitive (>60% growth inhibition), 10 of them were moderately sensitive (40-59% growth inhibition), and 21 were resistant to dasatinib. A highly significant relationship between breast cancer subtype and sensitivity to dasatinib was observed (chi2 = 9.66 and P = 0.008). Specifically, basal-type and post-EMT breast cancer cell lines were most sensitive to growth inhibition by dasatinib. In an attempt to identify potential predictive markers of dasatinib response other than breast cancer subtype we analyzed the baseline gene expression profiles for differentially expressed genes. We identified a set of three biologically relevant genes whose elevated expression is associated with dasatinib inhibition including moesin, caveolin-1, and yes-associated protein-1 with a sensitivity and specificity of 88 and 86%, respectively. Importantly, these data provide scientific rationale for the clinical development of dasatinib in the treatment of women with "triple-negative" breast cancer, a subtype that is categorized as being aggressive and lacking effective treatments (i.e. hormonal manipulation or trastuzumab).
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Pirimidinas/farmacologia , Tiazóis/farmacologia , Quinases da Família src/antagonistas & inibidores , Administração Oral , Neoplasias da Mama/classificação , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dasatinibe , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Queratinas/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Pirimidinas/administração & dosagem , Sensibilidade e Especificidade , Tiazóis/administração & dosagem , Vimentina/genética , Quinases da Família src/metabolismoRESUMO
Increasing evidence continues to emerge supporting the early hypothesis that BRCA1 might be involved in transcriptional processes. BRCA1 physically associates with more than 15 different proteins involved in transcription and is paradoxically involved in both transcriptional activation and repression. However, the underlying mechanism by which BRCA1 affects the gene expression of various genes remains speculative. In this study, we provide evidence that BRCA1 protein complexes interact with specific DNA sequences. We provide data showing that the upstream stimulatory factor 2 (USF2) physically associates with BRCA1 and is a component of this DNA-binding complex. Interestingly, these DNA-binding complexes are downregulated in breast cancer cell lines containing wild-type BRCA1, providing a critical link between modulations of BRCA1 function in sporadic breast cancers that do not involve germline BRCA1 mutations. The functional specificity of BRCA1 tumor suppression for breast and ovarian tissues is supported by our experiments, which demonstrate that BRCA1 DNA-binding complexes are modulated by serum and estrogen. Finally, functional analysis indicates that missense mutations in BRCA1 that lead to subsequent cancer susceptibility may result in improper gene activation. In summary, these findings establish a role for endogenous BRCA1 protein complexes in transcription via a defined DNA-binding sequence and indicate that one function of BRCA1 is to co-regulate the expression of genes involved in various cellular processes.