Abstract
BackgroundNon-small cell lung cancer (NSCLC) is a devastating disease with a heterogeneous prognosis, and the molecular mechanisms underlying tumor progression remain elusive. Mammalian Eps15 homology domain 1 (EHD1) plays a promotive role in tumor progression, but its role in cancer angiogenesis remains unknown. This study thus explored the role of EHD1 in angiogenesis in NSCLC.MethodsThe changes in angiogenesis were evaluated through human umbilical vein endothelial cell (HUVEC) proliferation, migration and tube formation assays. The impact of EHD1 on β2-adrenoceptor (β2AR) signaling was evaluated by Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR) analysis, and enzyme-linked immunosorbent assay (ELISA). The interaction between EHD1 and β2AR was confirmed by immunofluorescence (IF) and coimmunoprecipitation (Co-IP) experiments, and confocal microscopy immunofluorescence studies revealed that β2AR colocalized with the recycling endosome marker Rab11, which indicated β2AR endocytosis. Xenograft tumor models were used to investigate the role of EHD1 in NSCLC tumor growth.ResultsThe microarray analysis revealed that EHD1 was significantly correlated with tumor angiogenesis, and loss- and gain-of-function experiments demonstrated that EHD1 potentiates HUVEC proliferation, migration and tube formation. EHD1 knockdown inhibited β2AR signaling activity, and EHD1 upregulation promoted vascular endothelial growth factor A (VEGFA) and β2AR expression. Interestingly, EHD1 interacted with β2AR and played a novel and critical role in β2AR endocytic recycling to prevent receptor degradation. Aberrant VEGFA or β2AR expression significantly affected EHD1-mediated tumor angiogenesis. The proangiogenic role of EHD1 was confirmed in xenograft tumor models, and immunohistochemistry (IHC) analysis confirmed that EHD1 expression was positively correlated with VEGFA expression, microvessel density (MVD) and β2AR expression in patient specimens.ConclusionCollectively, the data obtained in this study suggest that EHD1 plays a critical role in NSCLC angiogenesis via β2AR signaling and highlight a potential target for antiangiogenic therapy.
Highlights
Non-small cell lung cancer (NSCLC) is a devastating disease with a heterogeneous prognosis, and the molecular mechanisms underlying tumor progression remain elusive
Over the past five years, we have revealed that Eps15 homology domain 1 (EHD1) overexpression in NSCLC predicts poor prognosis for patients and that EHD1 might play a pivotal role in tumor metastasis, stemness, chemotherapy resistance and epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) resistance [13,14,15]
We treated human umbilical vein endothelial cell (HUVEC) with conditioned medium (CM) from untreated cells (UT), CM from control cells transfected with scrambled shRNA (Ctrl) or CM from EHD1downregulated cells (Sh)
Summary
Non-small cell lung cancer (NSCLC) is a devastating disease with a heterogeneous prognosis, and the molecular mechanisms underlying tumor progression remain elusive. Mammalian Eps homology domain 1 (EHD1) plays a promotive role in tumor progression, but its role in cancer angiogenesis remains unknown. C-terminal Eps15-homology (EH) domain-containing protein (EHD1) regulates cellular receptor recycling from the endocytic recycling compartment to the plasma membrane [5, 6]. Over the past five years, we have revealed that EHD1 overexpression in NSCLC predicts poor prognosis for patients and that EHD1 might play a pivotal role in tumor metastasis, stemness, chemotherapy resistance and epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) resistance [13,14,15]. The involvement of EHD1 in tumor angiogenesis is unknown
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