RESUMO
Clinical studies in breast cancer suggest important associations between intratumoral hypoxia, the upregulation of epidermal growth factor receptor (EGFR or HER1), hypoxia-inducible factor 1α (HIF-1α), and reduced patient survival. However, direct molecular links between EGFR and the hypoxia signaling system are not yet established. Since the oxygen sensor hypoxia-inducible factor prolyl hydroxylase 2 (PHD2) is considered to be the main HIF-1α regulator, we hypothesized that PHD2 and EGFR may be interconnected at the molecular level. By analyzing samples from 313 breast cancer patients, we found that EGFR is a first clinicopathological parameter positively correlating with PHD2. Mechanistically, we identified PHD2 as a direct binding partner of EGFR and show that PHD2 regulates EGFR stability as well as its subsequent signaling in breast carcinoma cells. Overall, we introduce for the first time the direct crosstalk between the oxygen sensor PHD2 and EGFR-mediated tumorigenesis in breast cancer.
Assuntos
Neoplasias da Mama/enzimologia , Receptores ErbB/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Transdução de Sinais , Hipóxia Tumoral , Microambiente Tumoral , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/agonistas , Receptores ErbB/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Fosforilação , Ligação Proteica , Estabilidade Proteica , Proteólise , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , TransfecçãoRESUMO
The prolyl-4-hydroxylase domain 1-3 (PHD1-3) enzymes are regulating the protein stability of the α-subunit of the hypoxia-inducible factor-1 (HIF-1), which mediates oxygen-dependent gene expression. PHD2 is the main isoform regulating HIF-1α hydroxylation and thus stability in normoxia. In human cancers, HIF-1α is overexpressed as a result of intratumoral hypoxia which in turn promotes tumor progression. The role of PHD2 for tumor progression is in contrast far from being thoroughly understood. Therefore, we established PHD2 knockdown clones of MDA-MB-231 breast cancer cells and analyzed their tumor-forming potential in a SCID mouse model. Tumor progression was significantly impaired in the PHD2 knockdown MDA-MB-231 cells, which could be partially rescued by re-establishing PHD2 expression. In a RNA profile screen, we identified the secreted phosphoprotein 1 (SPP1) as one target, which is differentially regulated as a consequence of the PHD2 knockdown. Knockdown of PHD2 drastically reduced the SPP1 expression in MDA-MB-231 cells. A correlation of SPP1 and PHD2 expression was additionally verified in 294 invasive breast cancer biopsies. In subsequent analyses, we identified that PHD2 alters the processing of transforming growth factor (TGF)-ß1, which is highly involved in SPP1 expression. The altered processing capacity was associated with a dislocation of the pro-protein convertase furin. Thus, our data demonstrate that in MDA-MB-231 cells PHD2 might affect tumor-relevant TGF-ß1 target gene expression by altering the TGF-ß1 processing capacity.