RESUMO
BACKGROUND: Triple Negative subset of (TN) Breast Cancers (BC), a close associate of the basal-like subtype (with limited discordance) is an aggressive form of the disease which convey unpredictable, and poor prognosis due to limited treatment options and lack of proven effective targeted therapies. METHODS: We conducted an expression study of 240 formalin-fixed, paraffin-embedded (FFPE) primary biopsies from two cohorts, including 130 TN tumors, to identify molecular mechanisms of TN disease. RESULTS: The annotation of differentially expressed genes in TN tumors contained an overrepresentation of canonical Wnt signaling components in our cohort and others. These observations were supported by upregulation of experimentally induced oncogenic Wnt/ß-catenin genes in TN tumors, recapitulated using targets induced by Wnt3A. A functional blockade of Wnt/ß-catenin pathway by either a pharmacological Wnt-antagonist, WntC59, sulidac sulfide, or ß-catenin (functional read out of Wnt/ß-catenin pathway) SiRNA mediated genetic manipulation demonstrated that a functional perturbation of the pathway is causal to the metastasis- associated phenotypes including fibronectin-directed migration, F-actin organization, and invasion in TNBC cells. A classifier, trained on microarray data from ß-catenin transfected mammary cells, identified a disproportionate number of TNBC breast tumors as compared to other breast cancer subtypes in a meta-analysis of 11 studies and 1,878 breast cancer patients, including the two cohorts published here. Patients identified by the Wnt/ß-catenin classifier had a greater risk of lung and brain, but not bone metastases. CONCLUSION: These data implicate transcriptional Wnt signaling as a hallmark of TNBC disease associated with specific metastatic pathways.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Ósseas/secundário , Neoplasias Encefálicas/secundário , Neoplasias Pulmonares/secundário , Neoplasias de Mama Triplo Negativas/patologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Biomarcadores Tumorais/metabolismo , Western Blotting , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Movimento Celular , Proliferação de Células , Estudos de Coortes , Feminino , Seguimentos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Inclusão em Parafina , Prognóstico , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Taxa de Sobrevida , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Células Tumorais Cultivadas , Proteínas Wnt/genética , beta Catenina/antagonistas & inibidores , beta Catenina/genéticaRESUMO
Formalin-fixed paraffin-embedded (FFPE) breast tumor tissues are readily available and represent a largely untapped, vast resource for molecular profiling of clinical samples with long-term follow-up data. We have optimized the conditions and parameters that result in the preparation of total RNA that is of the necessary quality for use in the DASL (cDNA-mediated annealing, selection, extension, and ligation) assay in which expression of 502 genes are analyzed simultaneously using as little as 100 ng of input RNA.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Perfilação da Expressão Gênica/métodos , Proteínas de Neoplasias/análise , Inclusão em Parafina , RNA/genética , RNA/isolamento & purificação , Formaldeído , Humanos , Proteínas de Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Manejo de Espécimes/métodosRESUMO
Mechanisms linking mitogenic and growth inhibitory cytokine signaling and the cell cycle have not been fully elucidated in either cancer or in normal cells. Here we show that activation of protein kinase B (PKB)/Akt, contributes to resistance to antiproliferative signals and breast cancer progression in part by impairing the nuclear import and action of p27. Akt transfection caused cytoplasmic p27 accumulation and resistance to cytokine-mediated G1 arrest. The nuclear localization signal of p27 contains an Akt consensus site at threonine 157, and p27 phosphorylation by Akt impaired its nuclear import in vitro. Akt phosphorylated wild-type p27 but not p27T157A. In cells transfected with constitutively active Akt(T308DS473D)(PKB(DD)), p27WT mislocalized to the cytoplasm, but p27T157A was nuclear. In cells with activated Akt, p27WT failed to cause G1 arrest, while the antiproliferative effect of p27T157A was not impaired. Cytoplasmic p27 was seen in 41% (52 of 128) of primary human breast cancers in conjunction with Akt activation and was correlated with a poor patient prognosis. Thus, we show a novel mechanism whereby Akt impairs p27 function that is associated with an aggressive phenotype in human breast cancer.