RESUMEN
Epithelial-to-mesenchymal transition (EMT) regulates tumour initiation, progression, metastasis and resistance to anti-cancer therapy1-7. Although great progress has been made in understanding the role of EMT and its regulatory mechanisms in cancer, no therapeutic strategy to pharmacologically target EMT has been identified. Here we found that netrin-1 is upregulated in a primary mouse model of skin squamous cell carcinoma (SCC) exhibiting spontaneous EMT. Pharmacological inhibition of netrin-1 by administration of NP137, a netrin-1-blocking monoclonal antibody currently used in clinical trials in human cancer (ClinicalTrials.gov identifier NCT02977195 ), decreased the proportion of EMT tumour cells in skin SCC, decreased the number of metastases and increased the sensitivity of tumour cells to chemotherapy. Single-cell RNA sequencing revealed the presence of different EMT states, including epithelial, early and late hybrid EMT, and full EMT states, in control SCC. By contrast, administration of NP137 prevented the progression of cancer cells towards a late EMT state and sustained tumour epithelial states. Short hairpin RNA knockdown of netrin-1 and its receptor UNC5B in EPCAM+ tumour cells inhibited EMT in vitro in the absence of stromal cells and regulated a common gene signature that promotes tumour epithelial state and restricts EMT. To assess the relevance of these findings to human cancers, we treated mice transplanted with the A549 human cancer cell line-which undergoes EMT following TGFß1 administration8,9-with NP137. Netrin-1 inhibition decreased EMT in these transplanted A549 cells. Together, our results identify a pharmacological strategy for targeting EMT in cancer, opening up novel therapeutic interventions for anti-cancer therapy.
Asunto(s)
Anticuerpos Monoclonales , Carcinoma de Células Escamosas , Transición Epitelial-Mesenquimal , Netrina-1 , Neoplasias Cutáneas , Animales , Humanos , Ratones , Células A549 , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/efectos de los fármacos , Receptores de Netrina/antagonistas & inhibidores , Receptores de Netrina/deficiencia , Receptores de Netrina/genética , Netrina-1/antagonistas & inhibidores , Netrina-1/deficiencia , Netrina-1/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Modelos Animales de Enfermedad , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Metástasis de la Neoplasia/tratamiento farmacológico , Análisis de Expresión Génica de una Sola Célula , RNA-Seq , Molécula de Adhesión Celular Epitelial/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
Unc5 Netrin Receptor C (UNC5C) is a netrin1 dependence receptor that mediates the induction of apoptosis in the absence of netrin1. The present study found that UNC5C is heterogeneously expressed in breast cancer cell lines. By knocking down UNC5C in SKBR3 and ZR7530 cells and overexpressing UNC5c in MDAMB231 cells, it was demonstrated that UNC5C exerts an inhibitory effect on the growth and metastasis of breast cancer cells. The mechanism involved a UNC5Cknockdowninduced enhancement of matrix metalloproteinase (MMP)3, MMP7, MMP9 and MMP10 expression via activation of the PI3K/AKT, ERK and p38 MAPK signaling pathways. Notably, UNC5C directly interacted with integrin α6, which is involved in the growth and metastasis of breast cancer cells. Additionally, UNC5Cknockdown enhanced the phosphorylation of FAK and SRC, which are key kinases in the netrin1/Unc5C and netrin1/integrin α6/ß4 signaling pathways. This suggests that netrin1 functions as an integrator for both the netrin1/Unc5C and netrin1/integrin α6/ß4 signaling pathways. UNC5Cknockdown potentiated netrin1/integrin α6/ß4 signaling. Given that UNC5Cknockdown inhibited integrinliked protein kinase phosphorylation at Thr173, at least in SKBR3 cells, this may be an inhibitory phosphorylation site rather than activating phosphorylation site for relaying integrin signaling.