RESUMO
The extracellular matrix is a major component of the local environment-that is, the niche-that determines cell behaviour1. During metastatic growth, cancer cells shape the extracellular matrix of the metastatic niche by hydroxylating collagen to promote their own metastatic growth2,3. However, only particular nutrients might support the ability of cancer cells to hydroxylate collagen, because nutrients dictate which enzymatic reactions are active in cancer cells4,5. Here we show that breast cancer cells rely on the nutrient pyruvate to drive collagen-based remodelling of the extracellular matrix in the lung metastatic niche. Specifically, we discovered that pyruvate uptake induces the production of α-ketoglutarate. This metabolite in turn activates collagen hydroxylation by increasing the activity of the enzyme collagen prolyl-4-hydroxylase (P4HA). Inhibition of pyruvate metabolism was sufficient to impair collagen hydroxylation and consequently the growth of breast-cancer-derived lung metastases in different mouse models. In summary, we provide a mechanistic understanding of the link between collagen remodelling and the nutrient environment in the metastatic niche.
Assuntos
Neoplasias da Mama/patologia , Metástase Neoplásica/patologia , Ácido Pirúvico/metabolismo , Animais , Neoplasias da Mama/enzimologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Colágeno/química , Colágeno/metabolismo , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feminino , Humanos , Hidroxilação/efeitos dos fármacos , Ácidos Cetoglutáricos/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Camundongos , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Ácido Pirúvico/farmacologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
Metabolomics and 13C tracer analysis are state-of-the-art techniques that allow determining the concentration of metabolites and the activity of metabolic pathways, respectively. Three dimensional (3D) cultures of cancer cells constitute an enriched in vitro environment that can be used to assay anchorage-independent growth, spheroid formation, and extracellular matrix production by (cancer) cells. Here, we describe how to perform metabolomics and 13C tracer analysis in 3D cultures of cancer cells.
Assuntos
Isótopos de Carbono/análise , Técnicas de Cultura de Células/métodos , Metabolômica/métodos , Técnicas de Cultura de Células/instrumentação , Cromatografia Líquida/instrumentação , Cromatografia Líquida/métodos , Meios de Cultura/química , Matriz Extracelular/metabolismo , Cromatografia Gasosa-Espectrometria de Massas/instrumentação , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Metabolômica/instrumentação , Esferoides Celulares/metabolismo , Células Tumorais CultivadasRESUMO
TMPRSS2/ERG is the most common type of gene fusions found in human prostate cancer. There are two important features of TMPRSS2/ERG fusions. One is that these gene fusions lead to ectopic expression of ERG, an ETS family transcription factor, in prostate epithelial cells from the 5' control region of an androgen/estrogen dual-responsive gene, TMPRSS2; the other is that ~60% of these fusions are generated via intrachromosomal deletion of the interstitial region between TMPRSS2 and ERG. To recapitulate these important aspects of TMPRSS2/ERG fusions, we generated several TMPRSS2/ERG knockin mouse models based on the endogenous Tmprss2 locus. We found that TMPRSS2/ERG represents an early event in prostate tumorigenesis, by sensitizing prostate cells for cooperation with other oncogenic events, such as PTEN-deficiency. We also found that the interstitial region between TMPRSS2 and ERG harbors at least one prostate tumor suppressor, ETS2, whose loss contributes to prostate cancer progression. In this protocol, we describe how these knockin mouse models can be utilized to study roles of TMPRSS2/ERG fusions in prostate cancer development both in vivo and in vitro.
